Research ArticleAntidepressant-Like Effects of Gyejibokryeong-hwan ina Mouse Model of Reserpine-Induced Depression

Bo-Kyung Park1 Yu Ri Kim1 Young Hwa Kim1 Changsop Yang 1 Chang-Seob Seo 2

In Chul Jung3 Ik-Soon Jang4 Seung-Hyung Kim5 andMi Young Lee 1

1Clinical Medicine Division Korea Institute of Oriental Medicine Daejeon 34054 Republic of Korea2Herbal Medicine Research Division Korea Institute of Oriental Medicine Daejeon 34054 Republic of Korea3Department of Oriental Neuropsychiatry College of Korean Medicine Daejeon University Daejeon 34520 Republic of Korea4Division of Bioconvergence Analysis Korea Basic Science Institute Daejeon 34133 Republic of Korea5Institute of Traditional Medicine and Bioscience Daejeon University Daejeon 34520 Republic of Korea

Correspondence should be addressed to Mi Young Lee myleekiomrekr

Received 21 February 2018 Revised 11 May 2018 Accepted 28 May 2018 Published 26 June 2018

Academic Editor Naohiko Masaki

Copyright copy 2018 Bo-Kyung Park et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Treatment with the antihypertensive agent reserpine depletes monoamine levels resulting in depression In the present study weevaluated the antidepressant effects ofGyejibokryeong-hwan (GBH) a traditional Korean medicine in a mouse model of reserpine-induced depression Mice were treated with reserpine (05 mgsdotkgminus1 ip) or phosphate-buffered saline (PBS ip normal) once dailyfor 10 days GBH (50 100 300 and 500 mgsdotkgminus1) PBS (normal control) fluoxetine (FXT 20 mgsdotkgminus1) or amitriptyline (AMT 30mgsdotkgminus1) was administered orally 1 h prior to reserpine treatment Mouse behavior was examined in the forced swim test (FST) tailsuspension test (TST) and open-field test (OFT) following completion of the treatment protocol Administration of GBH reducedimmobility time in the FST and TST and significantly increased the total distance traveled in the OFT Plasma serotonin levelswere significantly lower in control mice than in normal mice although these decreases were significantly attenuated to a similarextent by treatment with GBH FXT or AMT Reserpine-induced increases in plasma corticosterone were also attenuated by GBHtreatment Moreover GBH attenuated reserpine-induced increases in interleukin- (IL-) 1120573 IL-6 and tumor necrosis factor- (TNF-)120572 mRNA expression in the hippocampus In addition GBH mice exhibited increased levels of brain-derived neurotrophic factor(BDNF) and a higher ratio of phosphorylated cAMP response element-binding protein (p-CREB) to CREB (p-CREBCREB) in thehippocampus Our results indicated that GBH can ameliorate depressive-like behaviors affect the concentration of mood-relatedhormones and help to regulate immuneendocrine dysfunction in mice with reserpine-induced depression likely via activation ofthe BDNF-CREB pathway Taken together these findings indicate that GBH may be effective in treating patients with depression

1 Introduction

Depression is a mood disorder characterized by feelings ofunpleasantness helplessness sadness and despair [1] Suchfeelings are often accompanied by symptoms such as sleepdisturbance loss of appetite and decreased concentrationwhich can substantially impact the patientrsquos quality of lifeand social functioning Depression is also a main risk factorfor suicide representing a significant public health concern[2] World Health Organization (WHO) data have revealedthat depression is the fourth greatest contributor to disability-adjusted life years a parameter representing the number of

years lost due to accidents illness disability and prematuredeath Moreover researchers have projected that depressionwill become the second greatest contributor by 2020 [3]Depression can be divided into several types includingmajordepressive disorder (MDD) dysthymic disorder psychoticdepression postpartum depression seasonal affective dis-order (SAD) and bipolar disorder However MDD anddysthymic disorder are themost common types of depression[4]

Although various pathological causes of depression havebeen identified the monoamine hypothesis is the most welldescribed and accepted [5] In the 1960s a neurochemical

HindawiBioMed Research InternationalVolume 2018 Article ID 5845491 12 pageshttpsdoiorg10115520185845491

2 BioMed Research International

model of depression was proposed based on reports thatmonoamine depletion induced by reserpine a treatment forhypertension caused adverse effects in patients leading todepression [6] These data suggest that depression is asso-ciated with monoaminergic dysfunction in the central ner-vous system Many antidepressant therapies targeting mono-amines such as tricyclic antidepressants (TCAs) selectiveserotonin reuptake inhibitors (SSRIs) noradrenaline reup-take inhibitors (NRIs) serotonin and noradrenaline reup-take inhibitors (SNRIs) and monoamine oxidase inhibitors(MAOIs) have been developed based on this theory [7]However these antidepressants are often associated withanticholinergic or neurological side effects such as dizzinesssedation sexual dysfunction insomnia and anxiety There-fore much research has focused on identifying natural andalternative therapies for depressionwith fewer side effects [8]

Gyejibokryeong-hwan (GBH) is a traditional Koreanmedicine described in the Donguibogam a traditional text-book of Korean medicine [9] GBH is comprised of herbsincluding Ramulus Cinnamomi cassia Scierotium Poriaecocos Radix Albus paeoniae Lactiflorae Cortex Radicismoutan and Semen Pruni persicae [10] GBH has been usedextensively to treat blood stasis and climacteric syndromethroughout Asia [11 12] and has been approved by both theKorean Ministry of Food and Drug Safety (MFDS) and theUSFood andDrugAdministration (FDA) Additional studieshave investigated the safety and efficacy of GBH in variouspopulations [13 14]

Ramulus Cinnamomi cassia exhibits anti-inflammatoryeffects in murine BV2 microglia cells [15] while CortexRadicis moutan reduces oxidative stress in rat pheochromo-cytoma PC12 cells [16] Moreover amygdalin a componentof Semen Pruni Persicae induces neurotrophic effects via theactivation of the ERK12 pathway in PC12 cells [17] Howeverto the best of our knowledge no studies have investigated thetherapeutic potential of GBH for depression In the presentstudy we aimed to assess the antidepressant effect of GBH inanimalmodels of depression in whichmonoamine depletionwas induced using reserpine

2 Materials and Methods

21 Preparation of GBH GBH was purchased from Han-poong Pharm and Foods Co Ltd (Jeonju Korea) A voucherspecimen of the herb sample was deposited in the Herbariumof Hanpoong Pharm and Foods Co Ltd The componentherbs of GBH (ratio of five herbs = 11111 total weight = 17kg) were extracted in water after boiling for 3 h The GBHextract was then filtered and vacuum-concentrated resultingin a yield of 2953 The extract was stored at minus80∘C anddissolved in phosphate-buffered saline (PBS) before use

22 High-Performance Liquid Chromatography (HPLC) Anal-ysis of GBH The GBH extract was analyzed using a Shi-madzu Prominence LCndash20A system (Kyoto Japan) whichconsisted of a solvent delivery unit an online degassera column oven a sample autoinjector and a photodiodearray (PDA) detector The data were acquired and processedusing LCsolution software (Version 124 SP1 Kyoto Japan)

Amygdalin (PubChem CID 656516 purity 990) gallicacid (PubChem CID 370 purity 990) and coumarin(PubChem CID 323 purity 990) were purchased fromMerck KGaA (Darmstadt Germany) Albiflorin (PubChemCID 51346141 purity 998) paeoniflorin (PubChem CID442534 purity 988) cinnamic acid (PubChem CID444539 purity 990) and paeonol (PubChem CID 11092purity 999) were obtained fromWako (Osaka Japan) Theeightmarker componentswere separated on aWaters SunFireC18

column (250 times 46 mm 5 120583m Milford MA USA) andmaintained at 40∘CThemobile phases consisted of water (A)and acetonitrile (J T Baker Phillipsburg NJ USA) (B) bothcontaining 10 (vv) acetic acid (Merck KGaA DarmstadtGermany) The gradient elution of the mobile phase was asfollows 10ndash60 B for 0ndash30 min 60ndash100 B for 30ndash40 min100 B for 40ndash45 min and 100ndash10 B for 45ndash50 min Theflow-rate and injection volume were 10 mlmin and 10 120583lrespectively

23 Animal Experiments Seven-week-old male C57BL6mice were purchased from Daehan Biolink Co (ChungbukKorea) Animal experiments were performed in accordancewith the National Institutes of Health (NIH) Guide for theCare and Use of Laboratory Animals and approved by theKorean Institute of Oriental Medicine Institutional AnimalCare and Use Committee (written approval number 17-081) The mice were acclimated for 1 week following whichdepression was induced by administering reserpine (05mgsdotkgminus1 in PBS ip Sigma-Aldrich St Louis MO USA)once per day for 10 days Normal mice were injected withPBS alone The reserpine-treated mice were divided intoseven groups (n = 6 per group) and orally treated with PBS(Control) GBH (50 100 300 and 500 mgsdotkgminus1) the SSRIfluoxetine (FXT 20 mgsdotkgminus1 Sigma-Aldrich) or the TCAamitriptyline (AMT 30 mgsdotkgminus1 Sigma-Aldrich) Normalmice were treated with oral doses of PBS The experimentalschematic including reserpine induction and administrationschedules is presented in Figure 2(a)

24 Alterations in BodyWeight and Food Intake Body weightwas measured on days 1 5 and 10 Food intake was estimatedas the difference between the amount of food remaining inthe feeder on day 5 or 10 and the amount of food provided onday 1 [18]

25 Behavioral Tests The forced swim test (FST) is usedto evaluate learned helplessness in rodents and has oftenbeen used to examine the effect of antidepressants in animalmodels of depression Mice were allowed to swim for 15 min(session 1) on the day prior to the FST [19] On test dayindividual mice were introduced into a cylinder (height 45cm diameter 20 cm) containing tap water (25 plusmn 2∘C depth25 cm) from which they could not escape and had to swimto stay afloat Total immobility time was measured duringthe last 4 min of a 6 min trial using video tracking software(SMART 30 Panlab SI Barcelona Spain)

The tail suspension test (TST) is a useful behavioral toolfor examining the effects of antidepressant drugs [20] Micewere acoustically and visually isolated following which they

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Table 1 The sequences of the real-time PCR primers

Gene Sequence

mouse IL-1120573 forward 51015840- GCTGAAAGCTCTCCACCTCA -31015840reverse 51015840- AGGCCACAGGTATTTTGTCG -31015840

mouse IL-6 forward 51015840- GAGGATACCACTCCCAACAGACC -31015840reverse 51015840- AAGTGCATCATCGTTGTTCATACA -31015840

mouse TNF-120572 forward 51015840- AGACCCTCACACTCAGATCATCTTC -31015840reverse 51015840- CCACTTGGTGGTTTGCTACGA -31015840

mouse GAPDH forward 51015840- AAGGTGGTGAAGCAGGCAT -31015840reverse 51015840- GGTCCAGGGTTTCTTACTCCT -31015840

were suspended 50 cm above the floor using adhesive tapeplaced approximately 1 cm from the tip of the tail Immobilitytime was recorded during the last 4 min of a 6 min sessionusing the same video tracking software used in the FST

The open-field test (OFT) can be used to measure anxietyand locomotor behavior in rodents [21] In the presentstudy the open-field arena (30 times 30 cm) was constructedfrom acrylic sheets and mice were placed individually inthe center of the field Their behavior was recorded for 10min Recordings were analyzed using video tracking soft-ware (EthoVision XT 90 Noldus Information TechnologyWageningen Netherlands) as described by Deussing [22]

Mice underwent behavioral testing in the following orderwith a 6 h interval between each experiment OFT TST FST

26 Enzyme-Linked Immunosorbent Assay (ELISA) Mouseblood was collected into heparin coated tubes under anes-thesia with tiletaminezolazepam (25 mgsdotkgminus1 Zoletil 50Virbac Cedex France) For plasma collection the sampleswere centrifuged at 3000 rpm for 10 min at 4∘C followingwhich the supernatant was carefully transferred to a newtube Plasma samples were stored at minus80∘C before use Theconcentrations of plasma serotonin (Abcam CambridgeUK) and corticosterone (Cayman chemical company AnnArbor MI USA) were determined using ELISA kits inaccordance with the manufacturerrsquos protocols

27 Real-Time Polymerase Chain Reaction (PCR) Total RNAwas isolated using Trizol reagent (Invitrogen) while cDNAsynthesis was performed using the PrimeScript RT reagentkit (TaKaRa Shiga Japan) Interleukin- (IL-) 1120573 IL-6 tumornecrosis factor- (TNF-) 120572 and glyceraldehyde-3 phosphatedehydrogenase (GAPDH) mRNA was quantified using aQuantStudio 6 Flex real-time polymerase chain reaction(real-time PCR) system (Applied Biosystems CA USA) withPower SYBR Green PCR Master Mix (Applied Biosystems)[23] The sequences of the real-time PCR primers are pre-sented in Table 1

28 Western Blotting The hippocampus was homogenizedin 300 120583l lysis buffer (Pro-Prep Intron BiotechnologyKorea) containing 1 mM PMSF and 1 120583gsdotmlminus1 of proteaseinhibitor mix Equal amounts (20 120583g) of protein were sepa-rated using 10 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to polyvinyli-dene fluoride (PVDF) membranes (Amersham Biosciences

Piscataway NJ USA) The membranes were then blocked in5 skim milk in Tris-buffered saline with 01 TWEEN 20(TBST) for 1 h The membranes were probed overnight withantibodies for brain-derived neurotrophic factor (BDNFAbcam Cambridge UK) phosphorylated cAMP responseelement-binding protein (p-CREB) and CREB (Cell Sig-naling Technology Inc Danvers MA USA) at 4∘C Nextblots were incubated in horseradish peroxidase- (HRP-)conjugated secondary antibody for 1 h at 25∘C and HRPwas detected using a chemiluminescent detection reagent(Amersham Biosciences) 120573-Actin (Sigma-Aldrich) was usedas a loading control [24]

29 Immunofluorescence The brain was frozen at ndash20∘C andsections were cut to a thickness of 20 120583m using a CryostatMicrotome (CM 3050 S Leica Microsystems Wetzlar Ger-many) We performed double immunofluorescence stainingby incubating tissue sections with antibodies for BDNF(Novus Biologicals Inc Littleton CO) p-CREB and CREB(Cell Signaling Technology Inc) overnight at 4∘C Subse-quently FITC-conjugated secondary antibody was added for2 h and nuclear stainingwas performed usingDAPI Sectionswere observed using an Eclipse Ti-E inverted fluorescentmicroscope (Nikon Instruments Inc Mississauga Canada)

210 Statistical Analysis All data are expressed as the meanplusmn standard deviation (SD) One-way analysis of variance(ANOVA) was performed using GraphPad Prism version 7(GraphPad Software Inc San Diego CA USA) to assessbetween-group differences Comparisons among multiplegroups were performed using one-way ANOVAs followed bypost hocTukey testsThe level of statistical significancewas setat p lt 005

3 Results

31 HPLC Analysis of GBH The composition of GBH wasverified using HPLC This analysis provided chemical infor-mation regarding GBH guaranteeing the reproducibility ofour experiments using other batches of GBH Analyses wereperformed in accordance with chemical standards describedby Kim et al [14] Quantitative and qualitative analysesof the eight marker compounds (gallic acid amygdalinalbiflorin paeoniflorin benzoic acid coumarin cinnamicacid and paeonol) in GBH were conducted using the opti-mized HPLCndashphotodiode array (PDA) method Each GBH

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(a)

Marker compound

Retention time (min)

Amount (mgg)

Sources

Gallic acid 401 561 Radix Albus paeoniae LactifloraeAmygdalin 969 4904 Semen Pruni persicaeAlbiflorin 1164 506 Radix Albus paeoniae Lactiflorae

Paeoniflorin 1244 177Radix Albus paeoniae Lactiflorae

Cortex Radicis moutanBenzoic acid 1802 3196 Radix Albus paeoniae Lactiflorae

Coumarin 2011 021 Ramulus Cinnamomi cassiaCinnamic acid 2277 021 Ramulus Cinnamomi cassia

Paeonol 2727 002Radix Albus paeoniae Lactiflorae

Cortex Radicis moutan

(b)

Figure 1 Three-dimensional chromatogram of Gyejibokryeong-hwan (GBH) sample based on HPLCndashPDA analysis HLPC high-performance liquid chromatography PDA photodiode array

component was identified by comparing the retention timeand UV spectra with the respective reference standards Theretention times and amounts of the eight marker compoundsin GBH are shown in Figure 1

32 Effect of GBH on Body Weight and Food Intake Nosignificant differences in mean body weight were observedamong the groups on day 1 After 5 and 10 days of reserpineinjection body weight was significantly lower in control micethan in normal mice however oral GBH administrationameliorated this effect after 10 days of treatment (GBH 1002554 plusmn 071 g p lt 001 GBH 300 2535 plusmn 082 g p lt 005and GBH 500 2616 plusmn 085 g p lt 0001 Figure 2(b)) Ten daysof FXT administration (20 mgsdotkgminus1) significantly increasedbody weight (FXT 2553 plusmn 10 g p lt 001 Figure 2(b))

however there was no difference in body weight followingAMT administration (30 mgsdotkgminus1) relative to that observedin control mice

Food intake was significantly lower in control mice thanin normal mice on day 10 (185 plusmn 397 g p lt 0001)however administration of GBH FXT or AMT significantlyameliorated this effect (GBH 50 3049 plusmn 183 g GBH 1003102 plusmn 176 g GBH 300 3083 plusmn 214 g GBH 500 314 plusmn101 g FXT 2971 plusmn 233 g AMT 309 plusmn 013 g all p lt 0001Figure 2(c)) These results suggest that GBH counteracteddecreases in body weight and food intake in mice withreserpine-induced depression

33 Effect of GBH on Depressive-Like Behavior Wemeasuredimmobilization time in the FST (F[7 40] = 609 p lt 0001

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acclimation-Reserpine (05mgkgday)-Daily oral administration

Sacrifice

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minus7 0 11 12 days10Body weight Food intake

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lowastlowastlowastlowastlowastlowast

(c)

Figure 2 Effect of Gyejibokryeong-hwan (GBH) on development of reserpine-induced depression in mice Mice underwent oral treatmentwith vehicle (PBS) GBH (50 100 300 or 500 mgsdotkgminus1) fluoxetine (FXT 20 mgsdotkgminus1) or amitriptyline (AMT 30 mgsdotkgminus1) once per day for10 days (a) Schematic showing reserpine treatment oral administration schedule and behavioral testing timeline (b) Body weight and (c)food intake were measured on days 1 5 and 10 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

Figure 3(a)) 1 day after the completion of treatment (day 11Figure 2(a)) As expected immobilization time was signifi-cantly higher in control mice than in normal mice (control17568 plusmn 1087 s p lt 0001) Moreover immobilization timewas significantly lower in GBH mice than in control mice(GBH 100 14057 plusmn 1306 s p lt 001 GBH 300 13819 plusmn 153 sp lt 001 GBH 500 13388 plusmn 972 s p lt 0001 Figure 3(a))Furthermore treatment with FXT and AMT significantlydecreased immobilization time (FXT 14455 plusmn 1836 s p lt005 AMT 13426 plusmn 2063 s p lt 0001)

Mice treated with GBH exhibited significant decreases inimmobilization time during the TST (F[7 40] = 5754 plt 0001 Figure 3(b)) (GBH 100 16453 plusmn 3428 s p lt 001GBH 300 2104 plusmn 8154 s p lt 005) when compared withcontrol mice (control 3600 plusmn 1062 s p lt 001 versus normalFigure 3(b))

Distance traveled in the OFT (F[7 40] = 3641 p lt 0001Figure 3(c)) was significantly greater in mice treated withGBH (GBH 100 232564 plusmn 10226 cm p lt 0001 GBH 300215078 plusmn 15816 cm p lt 001 GBH 500 240132 plusmn 30127

cm p lt 0001) than in control mice (control 163727 plusmn 12165cm p lt 0001 versus normal) In addition FXT and AMTtreatment significantly increased the distance traveled (FXT214708 plusmn 897 cm p lt 005 AMT 252821 plusmn 28007 cm plt 0001 Figure 3(c)) These results suggest that GBH canameliorate depressive-like behaviors in mice with reserpine-induced depression

34 Effect of GBH on Mood-Related Hormones in Reserpine-Treated Mice The concentration of plasma serotonin akey regulator of emotions and mood disorders [25] wassignificantly decreased in control mice following reserpineadministration (control 3791 plusmn 273 ngml p lt 0001 versusnormal) However treatment with GBH FXT and AMTattenuated these decreases (GBH 100 6884 plusmn 142 ngml p lt005 GBH 300 7582 plusmn 1615 ngml p lt 001 GBH 500 14552plusmn 2096 ngml p lt 0001 FXT 723 plusmn 1565 ngml p lt 005AMT 8119 plusmn 1197 ngml p lt 0001 Figure 4(a)) Moreovertreatment with GBH FXT and AMT significantly attenuatedthe reserpine-induced increase in serum levels of the stress

6 BioMed Research International

0

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ile T

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lowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

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lowastlowastlowastlowastlowastlowast lowastlowastlowast

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(c)

Figure 3 Effect of Gyejibokryeong-hwan (GBH) on reserpine-induced depressive-like behaviors in mice (a) Immobility time in the forcedswim test (FST) and (b) tail suspension test (TST) (c) total distance traveled in the open-field test (OFT) All behavioral tests were conductedon day 11 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt 001 p lt 0001 versus normal lowastp lt005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

0

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gm

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GBHNor

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lowastlowastlowastlowast

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lowast

(b)

Figure 4 Effect ofGyejibokryeong-hwan (GBH) on the concentration of plasma serotonin and corticosterone inmice with reserpine-induceddepressionThe concentrations of plasma (a) serotonin and (b) corticosterone were determined via ELISA Data are presented as the mean plusmnstandard deviation (SD) (n = 6 one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

BioMed Research International 7

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-1b

(RQ

)

GBHNor

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trol

FXT

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lowastlowast lowast

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(a)

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(RQ

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lowast lowastlowast

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TNF-

a (R

Q)

GBHNor

mal

Con

trol

FXT

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lowast lowastlowast lowastlowast

lowastlowast

(c)

Figure 5 Effect of Gyejibokryeong-hwan (GBH) on IL-1120573 IL-6 and TNF-120572 mRNA expression in mice with reserpine-induced depressionThe expression of (a) IL-1120573 (b) IL-6 and (c) TNF-120572mRNA was determined via quantitative real-time PCR Data represent mean plusmn standarddeviation (SD) (n = 6 one-way ANOVA p lt 001 versus normal lowastp lt 005 lowastlowastp lt 001 versus control) IL-1120573 interleukin 1 beta IL-6interleukin 6 TNF-120572 tumor necrosis factor alpha PCR polymerase chain reaction

hormone corticosterone (GBH 100 101304 plusmn 30321 pgml plt 001 GBH 300 115506plusmn 32415 pgml plt 005 FXT 65259plusmn 16033 pgml p lt 0001 AMT 94741 plusmn 3778 pgml p lt001) [26] relative to levels observed in control mice (control172291 plusmn 18169 pgml p lt 0001 versus normal Figure 4(b))These data suggest that GBH can affect the concentrationof mood-related hormones in mice with reserpine-induceddepression Dopamine and norepinephrinewere not detectedin the plasma (data not shown)

35 Effect of GBH on Proinflammatory Cytokines in Reserpine-Induced Depression Chronic stress and the release of proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 lead tochronic neuroinflammation which contributes to depression[27 28] IL-1120573 IL-6 and TNF-120572 mRNA expression wassignificantly increased in the hippocampus of control micealthough this effect was ameliorated by treatment with GBHFXT and AMT (Figure 5) These data indicate that GBHmay help to regulate immune and endocrine dysfunctionassociated with depression

36 Effect of GBH on BDNF and p-CREB Expression in theBrain In order to determine the molecular mechanismsunderlying the antidepressant effects of GBH we examinedBDNF and p-CREB expression in the brain via Westernblot analysis Hippocampal BDNF levels were significantlylower in control mice than in normal mice indicative ofneuronal dysfunction in the brain [29] however mice inthe GBH 100 group exhibited significantly increased levels ofBDNF (Figure 6(a)) The BDNF-CREB pathway is associatedwith MDD [30] We observed a significant reduction inhippocampal p-CREB in control mice which was preventedin a dose-dependent manner in mice treated with GBHMoreover FXT and AMT treatment significantly increasedp-CREB expression (Figure 6(b)) These results suggest thatGBH can affect hippocampal neuronal activity in mice withreserpine-induced depression

To confirm the effect of GBH treatment in our modelof reserpine-induced depression we examined BDNF andp-CREB expression in the hippocampus via immunofluo-rescence analysis BDNF levels were significantly decreased

8 BioMed Research International

Normal Control FXT100 300 500

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-actin

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(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

BioMed Research International 3

Table 1 The sequences of the real-time PCR primers

Gene Sequence

mouse IL-1120573 forward 51015840- GCTGAAAGCTCTCCACCTCA -31015840reverse 51015840- AGGCCACAGGTATTTTGTCG -31015840

mouse IL-6 forward 51015840- GAGGATACCACTCCCAACAGACC -31015840reverse 51015840- AAGTGCATCATCGTTGTTCATACA -31015840

mouse TNF-120572 forward 51015840- AGACCCTCACACTCAGATCATCTTC -31015840reverse 51015840- CCACTTGGTGGTTTGCTACGA -31015840

mouse GAPDH forward 51015840- AAGGTGGTGAAGCAGGCAT -31015840reverse 51015840- GGTCCAGGGTTTCTTACTCCT -31015840

were suspended 50 cm above the floor using adhesive tapeplaced approximately 1 cm from the tip of the tail Immobilitytime was recorded during the last 4 min of a 6 min sessionusing the same video tracking software used in the FST

The open-field test (OFT) can be used to measure anxietyand locomotor behavior in rodents [21] In the presentstudy the open-field arena (30 times 30 cm) was constructedfrom acrylic sheets and mice were placed individually inthe center of the field Their behavior was recorded for 10min Recordings were analyzed using video tracking soft-ware (EthoVision XT 90 Noldus Information TechnologyWageningen Netherlands) as described by Deussing [22]

Mice underwent behavioral testing in the following orderwith a 6 h interval between each experiment OFT TST FST

26 Enzyme-Linked Immunosorbent Assay (ELISA) Mouseblood was collected into heparin coated tubes under anes-thesia with tiletaminezolazepam (25 mgsdotkgminus1 Zoletil 50Virbac Cedex France) For plasma collection the sampleswere centrifuged at 3000 rpm for 10 min at 4∘C followingwhich the supernatant was carefully transferred to a newtube Plasma samples were stored at minus80∘C before use Theconcentrations of plasma serotonin (Abcam CambridgeUK) and corticosterone (Cayman chemical company AnnArbor MI USA) were determined using ELISA kits inaccordance with the manufacturerrsquos protocols

27 Real-Time Polymerase Chain Reaction (PCR) Total RNAwas isolated using Trizol reagent (Invitrogen) while cDNAsynthesis was performed using the PrimeScript RT reagentkit (TaKaRa Shiga Japan) Interleukin- (IL-) 1120573 IL-6 tumornecrosis factor- (TNF-) 120572 and glyceraldehyde-3 phosphatedehydrogenase (GAPDH) mRNA was quantified using aQuantStudio 6 Flex real-time polymerase chain reaction(real-time PCR) system (Applied Biosystems CA USA) withPower SYBR Green PCR Master Mix (Applied Biosystems)[23] The sequences of the real-time PCR primers are pre-sented in Table 1

28 Western Blotting The hippocampus was homogenizedin 300 120583l lysis buffer (Pro-Prep Intron BiotechnologyKorea) containing 1 mM PMSF and 1 120583gsdotmlminus1 of proteaseinhibitor mix Equal amounts (20 120583g) of protein were sepa-rated using 10 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred to polyvinyli-dene fluoride (PVDF) membranes (Amersham Biosciences

Piscataway NJ USA) The membranes were then blocked in5 skim milk in Tris-buffered saline with 01 TWEEN 20(TBST) for 1 h The membranes were probed overnight withantibodies for brain-derived neurotrophic factor (BDNFAbcam Cambridge UK) phosphorylated cAMP responseelement-binding protein (p-CREB) and CREB (Cell Sig-naling Technology Inc Danvers MA USA) at 4∘C Nextblots were incubated in horseradish peroxidase- (HRP-)conjugated secondary antibody for 1 h at 25∘C and HRPwas detected using a chemiluminescent detection reagent(Amersham Biosciences) 120573-Actin (Sigma-Aldrich) was usedas a loading control [24]

29 Immunofluorescence The brain was frozen at ndash20∘C andsections were cut to a thickness of 20 120583m using a CryostatMicrotome (CM 3050 S Leica Microsystems Wetzlar Ger-many) We performed double immunofluorescence stainingby incubating tissue sections with antibodies for BDNF(Novus Biologicals Inc Littleton CO) p-CREB and CREB(Cell Signaling Technology Inc) overnight at 4∘C Subse-quently FITC-conjugated secondary antibody was added for2 h and nuclear stainingwas performed usingDAPI Sectionswere observed using an Eclipse Ti-E inverted fluorescentmicroscope (Nikon Instruments Inc Mississauga Canada)

210 Statistical Analysis All data are expressed as the meanplusmn standard deviation (SD) One-way analysis of variance(ANOVA) was performed using GraphPad Prism version 7(GraphPad Software Inc San Diego CA USA) to assessbetween-group differences Comparisons among multiplegroups were performed using one-way ANOVAs followed bypost hocTukey testsThe level of statistical significancewas setat p lt 005

3 Results

31 HPLC Analysis of GBH The composition of GBH wasverified using HPLC This analysis provided chemical infor-mation regarding GBH guaranteeing the reproducibility ofour experiments using other batches of GBH Analyses wereperformed in accordance with chemical standards describedby Kim et al [14] Quantitative and qualitative analysesof the eight marker compounds (gallic acid amygdalinalbiflorin paeoniflorin benzoic acid coumarin cinnamicacid and paeonol) in GBH were conducted using the opti-mized HPLCndashphotodiode array (PDA) method Each GBH

4 BioMed Research International

(a)

Marker compound

Retention time (min)

Amount (mgg)

Sources

Gallic acid 401 561 Radix Albus paeoniae LactifloraeAmygdalin 969 4904 Semen Pruni persicaeAlbiflorin 1164 506 Radix Albus paeoniae Lactiflorae

Paeoniflorin 1244 177Radix Albus paeoniae Lactiflorae

Cortex Radicis moutanBenzoic acid 1802 3196 Radix Albus paeoniae Lactiflorae

Coumarin 2011 021 Ramulus Cinnamomi cassiaCinnamic acid 2277 021 Ramulus Cinnamomi cassia

Paeonol 2727 002Radix Albus paeoniae Lactiflorae

Cortex Radicis moutan

(b)

Figure 1 Three-dimensional chromatogram of Gyejibokryeong-hwan (GBH) sample based on HPLCndashPDA analysis HLPC high-performance liquid chromatography PDA photodiode array

component was identified by comparing the retention timeand UV spectra with the respective reference standards Theretention times and amounts of the eight marker compoundsin GBH are shown in Figure 1

32 Effect of GBH on Body Weight and Food Intake Nosignificant differences in mean body weight were observedamong the groups on day 1 After 5 and 10 days of reserpineinjection body weight was significantly lower in control micethan in normal mice however oral GBH administrationameliorated this effect after 10 days of treatment (GBH 1002554 plusmn 071 g p lt 001 GBH 300 2535 plusmn 082 g p lt 005and GBH 500 2616 plusmn 085 g p lt 0001 Figure 2(b)) Ten daysof FXT administration (20 mgsdotkgminus1) significantly increasedbody weight (FXT 2553 plusmn 10 g p lt 001 Figure 2(b))

however there was no difference in body weight followingAMT administration (30 mgsdotkgminus1) relative to that observedin control mice

Food intake was significantly lower in control mice thanin normal mice on day 10 (185 plusmn 397 g p lt 0001)however administration of GBH FXT or AMT significantlyameliorated this effect (GBH 50 3049 plusmn 183 g GBH 1003102 plusmn 176 g GBH 300 3083 plusmn 214 g GBH 500 314 plusmn101 g FXT 2971 plusmn 233 g AMT 309 plusmn 013 g all p lt 0001Figure 2(c)) These results suggest that GBH counteracteddecreases in body weight and food intake in mice withreserpine-induced depression

33 Effect of GBH on Depressive-Like Behavior Wemeasuredimmobilization time in the FST (F[7 40] = 609 p lt 0001

BioMed Research International 5

acclimation-Reserpine (05mgkgday)-Daily oral administration

Sacrifice

Behavior test

minus7 0 11 12 days10Body weight Food intake

Pre-swim(a)

1 5 1023

24

25

26

27

28

Day

Body

Wei

ght (

gm

ouse

)

NormalControlGBH-50GBH-100

GBH-300GBH-500FXTAMT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)

5 10Day

0

10

20

30

40

Food

Inta

ke (g

mou

se)

NormalControlGBH-50GBH-100

GBH-300GBH-500FXTAMT

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 2 Effect of Gyejibokryeong-hwan (GBH) on development of reserpine-induced depression in mice Mice underwent oral treatmentwith vehicle (PBS) GBH (50 100 300 or 500 mgsdotkgminus1) fluoxetine (FXT 20 mgsdotkgminus1) or amitriptyline (AMT 30 mgsdotkgminus1) once per day for10 days (a) Schematic showing reserpine treatment oral administration schedule and behavioral testing timeline (b) Body weight and (c)food intake were measured on days 1 5 and 10 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

Figure 3(a)) 1 day after the completion of treatment (day 11Figure 2(a)) As expected immobilization time was signifi-cantly higher in control mice than in normal mice (control17568 plusmn 1087 s p lt 0001) Moreover immobilization timewas significantly lower in GBH mice than in control mice(GBH 100 14057 plusmn 1306 s p lt 001 GBH 300 13819 plusmn 153 sp lt 001 GBH 500 13388 plusmn 972 s p lt 0001 Figure 3(a))Furthermore treatment with FXT and AMT significantlydecreased immobilization time (FXT 14455 plusmn 1836 s p lt005 AMT 13426 plusmn 2063 s p lt 0001)

Mice treated with GBH exhibited significant decreases inimmobilization time during the TST (F[7 40] = 5754 plt 0001 Figure 3(b)) (GBH 100 16453 plusmn 3428 s p lt 001GBH 300 2104 plusmn 8154 s p lt 005) when compared withcontrol mice (control 3600 plusmn 1062 s p lt 001 versus normalFigure 3(b))

Distance traveled in the OFT (F[7 40] = 3641 p lt 0001Figure 3(c)) was significantly greater in mice treated withGBH (GBH 100 232564 plusmn 10226 cm p lt 0001 GBH 300215078 plusmn 15816 cm p lt 001 GBH 500 240132 plusmn 30127

cm p lt 0001) than in control mice (control 163727 plusmn 12165cm p lt 0001 versus normal) In addition FXT and AMTtreatment significantly increased the distance traveled (FXT214708 plusmn 897 cm p lt 005 AMT 252821 plusmn 28007 cm plt 0001 Figure 3(c)) These results suggest that GBH canameliorate depressive-like behaviors in mice with reserpine-induced depression

34 Effect of GBH on Mood-Related Hormones in Reserpine-Treated Mice The concentration of plasma serotonin akey regulator of emotions and mood disorders [25] wassignificantly decreased in control mice following reserpineadministration (control 3791 plusmn 273 ngml p lt 0001 versusnormal) However treatment with GBH FXT and AMTattenuated these decreases (GBH 100 6884 plusmn 142 ngml p lt005 GBH 300 7582 plusmn 1615 ngml p lt 001 GBH 500 14552plusmn 2096 ngml p lt 0001 FXT 723 plusmn 1565 ngml p lt 005AMT 8119 plusmn 1197 ngml p lt 0001 Figure 4(a)) Moreovertreatment with GBH FXT and AMT significantly attenuatedthe reserpine-induced increase in serum levels of the stress

6 BioMed Research International

0

50

100

150

200Im

mob

ile T

ime (

sec)

GBH

FST

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

(a)

0

100

200

300

400

500

Imm

obile

Tim

e (se

c)

GBH

TST

Nor

mal

Con

trol

FXT

AM

T

lowast

lowastlowast lowastlowastlowastlowastlowast

(b)

0

1000

2000

3000

4000

Tota

l dis

tanc

e (cm

)

GBH

OFT

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowast lowast

(c)

Figure 3 Effect of Gyejibokryeong-hwan (GBH) on reserpine-induced depressive-like behaviors in mice (a) Immobility time in the forcedswim test (FST) and (b) tail suspension test (TST) (c) total distance traveled in the open-field test (OFT) All behavioral tests were conductedon day 11 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt 001 p lt 0001 versus normal lowastp lt005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

0

50

100

150

200600

800

1000

Sero

toni

n (n

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast

lowastlowastlowast

lowastlowastlowastlowast lowast

(a)

0

500

1000

1500

2000

Cor

ticos

tero

ne (p

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowast

lowastlowastlowast

lowast

(b)

Figure 4 Effect ofGyejibokryeong-hwan (GBH) on the concentration of plasma serotonin and corticosterone inmice with reserpine-induceddepressionThe concentrations of plasma (a) serotonin and (b) corticosterone were determined via ELISA Data are presented as the mean plusmnstandard deviation (SD) (n = 6 one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

BioMed Research International 7

00

05

10

15

20

25IL

-1b

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast lowast

lowast lowast

(a)

00

05

10

15

20

IL-6

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast

lowastlowastlowastlowast

(b)

00

05

10

15

20

25

TNF-

a (R

Q)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast lowastlowast

lowastlowast

(c)

Figure 5 Effect of Gyejibokryeong-hwan (GBH) on IL-1120573 IL-6 and TNF-120572 mRNA expression in mice with reserpine-induced depressionThe expression of (a) IL-1120573 (b) IL-6 and (c) TNF-120572mRNA was determined via quantitative real-time PCR Data represent mean plusmn standarddeviation (SD) (n = 6 one-way ANOVA p lt 001 versus normal lowastp lt 005 lowastlowastp lt 001 versus control) IL-1120573 interleukin 1 beta IL-6interleukin 6 TNF-120572 tumor necrosis factor alpha PCR polymerase chain reaction

hormone corticosterone (GBH 100 101304 plusmn 30321 pgml plt 001 GBH 300 115506plusmn 32415 pgml plt 005 FXT 65259plusmn 16033 pgml p lt 0001 AMT 94741 plusmn 3778 pgml p lt001) [26] relative to levels observed in control mice (control172291 plusmn 18169 pgml p lt 0001 versus normal Figure 4(b))These data suggest that GBH can affect the concentrationof mood-related hormones in mice with reserpine-induceddepression Dopamine and norepinephrinewere not detectedin the plasma (data not shown)

35 Effect of GBH on Proinflammatory Cytokines in Reserpine-Induced Depression Chronic stress and the release of proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 lead tochronic neuroinflammation which contributes to depression[27 28] IL-1120573 IL-6 and TNF-120572 mRNA expression wassignificantly increased in the hippocampus of control micealthough this effect was ameliorated by treatment with GBHFXT and AMT (Figure 5) These data indicate that GBHmay help to regulate immune and endocrine dysfunctionassociated with depression

36 Effect of GBH on BDNF and p-CREB Expression in theBrain In order to determine the molecular mechanismsunderlying the antidepressant effects of GBH we examinedBDNF and p-CREB expression in the brain via Westernblot analysis Hippocampal BDNF levels were significantlylower in control mice than in normal mice indicative ofneuronal dysfunction in the brain [29] however mice inthe GBH 100 group exhibited significantly increased levels ofBDNF (Figure 6(a)) The BDNF-CREB pathway is associatedwith MDD [30] We observed a significant reduction inhippocampal p-CREB in control mice which was preventedin a dose-dependent manner in mice treated with GBHMoreover FXT and AMT treatment significantly increasedp-CREB expression (Figure 6(b)) These results suggest thatGBH can affect hippocampal neuronal activity in mice withreserpine-induced depression

To confirm the effect of GBH treatment in our modelof reserpine-induced depression we examined BDNF andp-CREB expression in the hippocampus via immunofluo-rescence analysis BDNF levels were significantly decreased

8 BioMed Research International

Normal Control FXT100 300 500

BDNF

-actin

GBH 50 AMT

00

05

10

15

BDN

F re

lativ

e int

ensit

y

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

Tlowast

lowastlowastlowastlowast

(a)

-actin

P-CREB

CREB

Normal Control FXT100 300 500

GBH 50 AMT

00

05

10

15

p-CR

EBC

REB

rela

tive i

nten

sity

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

T

lowast lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

4 BioMed Research International

(a)

Marker compound

Retention time (min)

Amount (mgg)

Sources

Gallic acid 401 561 Radix Albus paeoniae LactifloraeAmygdalin 969 4904 Semen Pruni persicaeAlbiflorin 1164 506 Radix Albus paeoniae Lactiflorae

Paeoniflorin 1244 177Radix Albus paeoniae Lactiflorae

Cortex Radicis moutanBenzoic acid 1802 3196 Radix Albus paeoniae Lactiflorae

Coumarin 2011 021 Ramulus Cinnamomi cassiaCinnamic acid 2277 021 Ramulus Cinnamomi cassia

Paeonol 2727 002Radix Albus paeoniae Lactiflorae

Cortex Radicis moutan

(b)

Figure 1 Three-dimensional chromatogram of Gyejibokryeong-hwan (GBH) sample based on HPLCndashPDA analysis HLPC high-performance liquid chromatography PDA photodiode array

component was identified by comparing the retention timeand UV spectra with the respective reference standards Theretention times and amounts of the eight marker compoundsin GBH are shown in Figure 1

32 Effect of GBH on Body Weight and Food Intake Nosignificant differences in mean body weight were observedamong the groups on day 1 After 5 and 10 days of reserpineinjection body weight was significantly lower in control micethan in normal mice however oral GBH administrationameliorated this effect after 10 days of treatment (GBH 1002554 plusmn 071 g p lt 001 GBH 300 2535 plusmn 082 g p lt 005and GBH 500 2616 plusmn 085 g p lt 0001 Figure 2(b)) Ten daysof FXT administration (20 mgsdotkgminus1) significantly increasedbody weight (FXT 2553 plusmn 10 g p lt 001 Figure 2(b))

however there was no difference in body weight followingAMT administration (30 mgsdotkgminus1) relative to that observedin control mice

Food intake was significantly lower in control mice thanin normal mice on day 10 (185 plusmn 397 g p lt 0001)however administration of GBH FXT or AMT significantlyameliorated this effect (GBH 50 3049 plusmn 183 g GBH 1003102 plusmn 176 g GBH 300 3083 plusmn 214 g GBH 500 314 plusmn101 g FXT 2971 plusmn 233 g AMT 309 plusmn 013 g all p lt 0001Figure 2(c)) These results suggest that GBH counteracteddecreases in body weight and food intake in mice withreserpine-induced depression

33 Effect of GBH on Depressive-Like Behavior Wemeasuredimmobilization time in the FST (F[7 40] = 609 p lt 0001

BioMed Research International 5

acclimation-Reserpine (05mgkgday)-Daily oral administration

Sacrifice

Behavior test

minus7 0 11 12 days10Body weight Food intake

Pre-swim(a)

1 5 1023

24

25

26

27

28

Day

Body

Wei

ght (

gm

ouse

)

NormalControlGBH-50GBH-100

GBH-300GBH-500FXTAMT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)

5 10Day

0

10

20

30

40

Food

Inta

ke (g

mou

se)

NormalControlGBH-50GBH-100

GBH-300GBH-500FXTAMT

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 2 Effect of Gyejibokryeong-hwan (GBH) on development of reserpine-induced depression in mice Mice underwent oral treatmentwith vehicle (PBS) GBH (50 100 300 or 500 mgsdotkgminus1) fluoxetine (FXT 20 mgsdotkgminus1) or amitriptyline (AMT 30 mgsdotkgminus1) once per day for10 days (a) Schematic showing reserpine treatment oral administration schedule and behavioral testing timeline (b) Body weight and (c)food intake were measured on days 1 5 and 10 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

Figure 3(a)) 1 day after the completion of treatment (day 11Figure 2(a)) As expected immobilization time was signifi-cantly higher in control mice than in normal mice (control17568 plusmn 1087 s p lt 0001) Moreover immobilization timewas significantly lower in GBH mice than in control mice(GBH 100 14057 plusmn 1306 s p lt 001 GBH 300 13819 plusmn 153 sp lt 001 GBH 500 13388 plusmn 972 s p lt 0001 Figure 3(a))Furthermore treatment with FXT and AMT significantlydecreased immobilization time (FXT 14455 plusmn 1836 s p lt005 AMT 13426 plusmn 2063 s p lt 0001)

Mice treated with GBH exhibited significant decreases inimmobilization time during the TST (F[7 40] = 5754 plt 0001 Figure 3(b)) (GBH 100 16453 plusmn 3428 s p lt 001GBH 300 2104 plusmn 8154 s p lt 005) when compared withcontrol mice (control 3600 plusmn 1062 s p lt 001 versus normalFigure 3(b))

Distance traveled in the OFT (F[7 40] = 3641 p lt 0001Figure 3(c)) was significantly greater in mice treated withGBH (GBH 100 232564 plusmn 10226 cm p lt 0001 GBH 300215078 plusmn 15816 cm p lt 001 GBH 500 240132 plusmn 30127

cm p lt 0001) than in control mice (control 163727 plusmn 12165cm p lt 0001 versus normal) In addition FXT and AMTtreatment significantly increased the distance traveled (FXT214708 plusmn 897 cm p lt 005 AMT 252821 plusmn 28007 cm plt 0001 Figure 3(c)) These results suggest that GBH canameliorate depressive-like behaviors in mice with reserpine-induced depression

34 Effect of GBH on Mood-Related Hormones in Reserpine-Treated Mice The concentration of plasma serotonin akey regulator of emotions and mood disorders [25] wassignificantly decreased in control mice following reserpineadministration (control 3791 plusmn 273 ngml p lt 0001 versusnormal) However treatment with GBH FXT and AMTattenuated these decreases (GBH 100 6884 plusmn 142 ngml p lt005 GBH 300 7582 plusmn 1615 ngml p lt 001 GBH 500 14552plusmn 2096 ngml p lt 0001 FXT 723 plusmn 1565 ngml p lt 005AMT 8119 plusmn 1197 ngml p lt 0001 Figure 4(a)) Moreovertreatment with GBH FXT and AMT significantly attenuatedthe reserpine-induced increase in serum levels of the stress

6 BioMed Research International

0

50

100

150

200Im

mob

ile T

ime (

sec)

GBH

FST

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

(a)

0

100

200

300

400

500

Imm

obile

Tim

e (se

c)

GBH

TST

Nor

mal

Con

trol

FXT

AM

T

lowast

lowastlowast lowastlowastlowastlowastlowast

(b)

0

1000

2000

3000

4000

Tota

l dis

tanc

e (cm

)

GBH

OFT

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowast lowast

(c)

Figure 3 Effect of Gyejibokryeong-hwan (GBH) on reserpine-induced depressive-like behaviors in mice (a) Immobility time in the forcedswim test (FST) and (b) tail suspension test (TST) (c) total distance traveled in the open-field test (OFT) All behavioral tests were conductedon day 11 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt 001 p lt 0001 versus normal lowastp lt005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

0

50

100

150

200600

800

1000

Sero

toni

n (n

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast

lowastlowastlowast

lowastlowastlowastlowast lowast

(a)

0

500

1000

1500

2000

Cor

ticos

tero

ne (p

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowast

lowastlowastlowast

lowast

(b)

Figure 4 Effect ofGyejibokryeong-hwan (GBH) on the concentration of plasma serotonin and corticosterone inmice with reserpine-induceddepressionThe concentrations of plasma (a) serotonin and (b) corticosterone were determined via ELISA Data are presented as the mean plusmnstandard deviation (SD) (n = 6 one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

BioMed Research International 7

00

05

10

15

20

25IL

-1b

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast lowast

lowast lowast

(a)

00

05

10

15

20

IL-6

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast

lowastlowastlowastlowast

(b)

00

05

10

15

20

25

TNF-

a (R

Q)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast lowastlowast

lowastlowast

(c)

Figure 5 Effect of Gyejibokryeong-hwan (GBH) on IL-1120573 IL-6 and TNF-120572 mRNA expression in mice with reserpine-induced depressionThe expression of (a) IL-1120573 (b) IL-6 and (c) TNF-120572mRNA was determined via quantitative real-time PCR Data represent mean plusmn standarddeviation (SD) (n = 6 one-way ANOVA p lt 001 versus normal lowastp lt 005 lowastlowastp lt 001 versus control) IL-1120573 interleukin 1 beta IL-6interleukin 6 TNF-120572 tumor necrosis factor alpha PCR polymerase chain reaction

hormone corticosterone (GBH 100 101304 plusmn 30321 pgml plt 001 GBH 300 115506plusmn 32415 pgml plt 005 FXT 65259plusmn 16033 pgml p lt 0001 AMT 94741 plusmn 3778 pgml p lt001) [26] relative to levels observed in control mice (control172291 plusmn 18169 pgml p lt 0001 versus normal Figure 4(b))These data suggest that GBH can affect the concentrationof mood-related hormones in mice with reserpine-induceddepression Dopamine and norepinephrinewere not detectedin the plasma (data not shown)

35 Effect of GBH on Proinflammatory Cytokines in Reserpine-Induced Depression Chronic stress and the release of proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 lead tochronic neuroinflammation which contributes to depression[27 28] IL-1120573 IL-6 and TNF-120572 mRNA expression wassignificantly increased in the hippocampus of control micealthough this effect was ameliorated by treatment with GBHFXT and AMT (Figure 5) These data indicate that GBHmay help to regulate immune and endocrine dysfunctionassociated with depression

36 Effect of GBH on BDNF and p-CREB Expression in theBrain In order to determine the molecular mechanismsunderlying the antidepressant effects of GBH we examinedBDNF and p-CREB expression in the brain via Westernblot analysis Hippocampal BDNF levels were significantlylower in control mice than in normal mice indicative ofneuronal dysfunction in the brain [29] however mice inthe GBH 100 group exhibited significantly increased levels ofBDNF (Figure 6(a)) The BDNF-CREB pathway is associatedwith MDD [30] We observed a significant reduction inhippocampal p-CREB in control mice which was preventedin a dose-dependent manner in mice treated with GBHMoreover FXT and AMT treatment significantly increasedp-CREB expression (Figure 6(b)) These results suggest thatGBH can affect hippocampal neuronal activity in mice withreserpine-induced depression

To confirm the effect of GBH treatment in our modelof reserpine-induced depression we examined BDNF andp-CREB expression in the hippocampus via immunofluo-rescence analysis BDNF levels were significantly decreased

8 BioMed Research International

Normal Control FXT100 300 500

BDNF

-actin

GBH 50 AMT

00

05

10

15

BDN

F re

lativ

e int

ensit

y

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

Tlowast

lowastlowastlowastlowast

(a)

-actin

P-CREB

CREB

Normal Control FXT100 300 500

GBH 50 AMT

00

05

10

15

p-CR

EBC

REB

rela

tive i

nten

sity

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

T

lowast lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

BioMed Research International 5

acclimation-Reserpine (05mgkgday)-Daily oral administration

Sacrifice

Behavior test

minus7 0 11 12 days10Body weight Food intake

Pre-swim(a)

1 5 1023

24

25

26

27

28

Day

Body

Wei

ght (

gm

ouse

)

NormalControlGBH-50GBH-100

GBH-300GBH-500FXTAMT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(b)

5 10Day

0

10

20

30

40

Food

Inta

ke (g

mou

se)

NormalControlGBH-50GBH-100

GBH-300GBH-500FXTAMT

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

(c)

Figure 2 Effect of Gyejibokryeong-hwan (GBH) on development of reserpine-induced depression in mice Mice underwent oral treatmentwith vehicle (PBS) GBH (50 100 300 or 500 mgsdotkgminus1) fluoxetine (FXT 20 mgsdotkgminus1) or amitriptyline (AMT 30 mgsdotkgminus1) once per day for10 days (a) Schematic showing reserpine treatment oral administration schedule and behavioral testing timeline (b) Body weight and (c)food intake were measured on days 1 5 and 10 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

Figure 3(a)) 1 day after the completion of treatment (day 11Figure 2(a)) As expected immobilization time was signifi-cantly higher in control mice than in normal mice (control17568 plusmn 1087 s p lt 0001) Moreover immobilization timewas significantly lower in GBH mice than in control mice(GBH 100 14057 plusmn 1306 s p lt 001 GBH 300 13819 plusmn 153 sp lt 001 GBH 500 13388 plusmn 972 s p lt 0001 Figure 3(a))Furthermore treatment with FXT and AMT significantlydecreased immobilization time (FXT 14455 plusmn 1836 s p lt005 AMT 13426 plusmn 2063 s p lt 0001)

Mice treated with GBH exhibited significant decreases inimmobilization time during the TST (F[7 40] = 5754 plt 0001 Figure 3(b)) (GBH 100 16453 plusmn 3428 s p lt 001GBH 300 2104 plusmn 8154 s p lt 005) when compared withcontrol mice (control 3600 plusmn 1062 s p lt 001 versus normalFigure 3(b))

Distance traveled in the OFT (F[7 40] = 3641 p lt 0001Figure 3(c)) was significantly greater in mice treated withGBH (GBH 100 232564 plusmn 10226 cm p lt 0001 GBH 300215078 plusmn 15816 cm p lt 001 GBH 500 240132 plusmn 30127

cm p lt 0001) than in control mice (control 163727 plusmn 12165cm p lt 0001 versus normal) In addition FXT and AMTtreatment significantly increased the distance traveled (FXT214708 plusmn 897 cm p lt 005 AMT 252821 plusmn 28007 cm plt 0001 Figure 3(c)) These results suggest that GBH canameliorate depressive-like behaviors in mice with reserpine-induced depression

34 Effect of GBH on Mood-Related Hormones in Reserpine-Treated Mice The concentration of plasma serotonin akey regulator of emotions and mood disorders [25] wassignificantly decreased in control mice following reserpineadministration (control 3791 plusmn 273 ngml p lt 0001 versusnormal) However treatment with GBH FXT and AMTattenuated these decreases (GBH 100 6884 plusmn 142 ngml p lt005 GBH 300 7582 plusmn 1615 ngml p lt 001 GBH 500 14552plusmn 2096 ngml p lt 0001 FXT 723 plusmn 1565 ngml p lt 005AMT 8119 plusmn 1197 ngml p lt 0001 Figure 4(a)) Moreovertreatment with GBH FXT and AMT significantly attenuatedthe reserpine-induced increase in serum levels of the stress

6 BioMed Research International

0

50

100

150

200Im

mob

ile T

ime (

sec)

GBH

FST

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

(a)

0

100

200

300

400

500

Imm

obile

Tim

e (se

c)

GBH

TST

Nor

mal

Con

trol

FXT

AM

T

lowast

lowastlowast lowastlowastlowastlowastlowast

(b)

0

1000

2000

3000

4000

Tota

l dis

tanc

e (cm

)

GBH

OFT

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowast lowast

(c)

Figure 3 Effect of Gyejibokryeong-hwan (GBH) on reserpine-induced depressive-like behaviors in mice (a) Immobility time in the forcedswim test (FST) and (b) tail suspension test (TST) (c) total distance traveled in the open-field test (OFT) All behavioral tests were conductedon day 11 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt 001 p lt 0001 versus normal lowastp lt005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

0

50

100

150

200600

800

1000

Sero

toni

n (n

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast

lowastlowastlowast

lowastlowastlowastlowast lowast

(a)

0

500

1000

1500

2000

Cor

ticos

tero

ne (p

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowast

lowastlowastlowast

lowast

(b)

Figure 4 Effect ofGyejibokryeong-hwan (GBH) on the concentration of plasma serotonin and corticosterone inmice with reserpine-induceddepressionThe concentrations of plasma (a) serotonin and (b) corticosterone were determined via ELISA Data are presented as the mean plusmnstandard deviation (SD) (n = 6 one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

BioMed Research International 7

00

05

10

15

20

25IL

-1b

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast lowast

lowast lowast

(a)

00

05

10

15

20

IL-6

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast

lowastlowastlowastlowast

(b)

00

05

10

15

20

25

TNF-

a (R

Q)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast lowastlowast

lowastlowast

(c)

Figure 5 Effect of Gyejibokryeong-hwan (GBH) on IL-1120573 IL-6 and TNF-120572 mRNA expression in mice with reserpine-induced depressionThe expression of (a) IL-1120573 (b) IL-6 and (c) TNF-120572mRNA was determined via quantitative real-time PCR Data represent mean plusmn standarddeviation (SD) (n = 6 one-way ANOVA p lt 001 versus normal lowastp lt 005 lowastlowastp lt 001 versus control) IL-1120573 interleukin 1 beta IL-6interleukin 6 TNF-120572 tumor necrosis factor alpha PCR polymerase chain reaction

hormone corticosterone (GBH 100 101304 plusmn 30321 pgml plt 001 GBH 300 115506plusmn 32415 pgml plt 005 FXT 65259plusmn 16033 pgml p lt 0001 AMT 94741 plusmn 3778 pgml p lt001) [26] relative to levels observed in control mice (control172291 plusmn 18169 pgml p lt 0001 versus normal Figure 4(b))These data suggest that GBH can affect the concentrationof mood-related hormones in mice with reserpine-induceddepression Dopamine and norepinephrinewere not detectedin the plasma (data not shown)

35 Effect of GBH on Proinflammatory Cytokines in Reserpine-Induced Depression Chronic stress and the release of proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 lead tochronic neuroinflammation which contributes to depression[27 28] IL-1120573 IL-6 and TNF-120572 mRNA expression wassignificantly increased in the hippocampus of control micealthough this effect was ameliorated by treatment with GBHFXT and AMT (Figure 5) These data indicate that GBHmay help to regulate immune and endocrine dysfunctionassociated with depression

36 Effect of GBH on BDNF and p-CREB Expression in theBrain In order to determine the molecular mechanismsunderlying the antidepressant effects of GBH we examinedBDNF and p-CREB expression in the brain via Westernblot analysis Hippocampal BDNF levels were significantlylower in control mice than in normal mice indicative ofneuronal dysfunction in the brain [29] however mice inthe GBH 100 group exhibited significantly increased levels ofBDNF (Figure 6(a)) The BDNF-CREB pathway is associatedwith MDD [30] We observed a significant reduction inhippocampal p-CREB in control mice which was preventedin a dose-dependent manner in mice treated with GBHMoreover FXT and AMT treatment significantly increasedp-CREB expression (Figure 6(b)) These results suggest thatGBH can affect hippocampal neuronal activity in mice withreserpine-induced depression

To confirm the effect of GBH treatment in our modelof reserpine-induced depression we examined BDNF andp-CREB expression in the hippocampus via immunofluo-rescence analysis BDNF levels were significantly decreased

8 BioMed Research International

Normal Control FXT100 300 500

BDNF

-actin

GBH 50 AMT

00

05

10

15

BDN

F re

lativ

e int

ensit

y

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

Tlowast

lowastlowastlowastlowast

(a)

-actin

P-CREB

CREB

Normal Control FXT100 300 500

GBH 50 AMT

00

05

10

15

p-CR

EBC

REB

rela

tive i

nten

sity

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

T

lowast lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

6 BioMed Research International

0

50

100

150

200Im

mob

ile T

ime (

sec)

GBH

FST

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowast

(a)

0

100

200

300

400

500

Imm

obile

Tim

e (se

c)

GBH

TST

Nor

mal

Con

trol

FXT

AM

T

lowast

lowastlowast lowastlowastlowastlowastlowast

(b)

0

1000

2000

3000

4000

Tota

l dis

tanc

e (cm

)

GBH

OFT

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowast lowastlowastlowast

lowastlowast lowast

(c)

Figure 3 Effect of Gyejibokryeong-hwan (GBH) on reserpine-induced depressive-like behaviors in mice (a) Immobility time in the forcedswim test (FST) and (b) tail suspension test (TST) (c) total distance traveled in the open-field test (OFT) All behavioral tests were conductedon day 11 Data are presented as the mean plusmn standard deviation (SD) (n = 6 one-way ANOVA p lt 001 p lt 0001 versus normal lowastp lt005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

0

50

100

150

200600

800

1000

Sero

toni

n (n

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast

lowastlowastlowast

lowastlowastlowastlowast lowast

(a)

0

500

1000

1500

2000

Cor

ticos

tero

ne (p

gm

l)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowast

lowastlowastlowast

lowast

(b)

Figure 4 Effect ofGyejibokryeong-hwan (GBH) on the concentration of plasma serotonin and corticosterone inmice with reserpine-induceddepressionThe concentrations of plasma (a) serotonin and (b) corticosterone were determined via ELISA Data are presented as the mean plusmnstandard deviation (SD) (n = 6 one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)

BioMed Research International 7

00

05

10

15

20

25IL

-1b

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast lowast

lowast lowast

(a)

00

05

10

15

20

IL-6

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast

lowastlowastlowastlowast

(b)

00

05

10

15

20

25

TNF-

a (R

Q)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast lowastlowast

lowastlowast

(c)

Figure 5 Effect of Gyejibokryeong-hwan (GBH) on IL-1120573 IL-6 and TNF-120572 mRNA expression in mice with reserpine-induced depressionThe expression of (a) IL-1120573 (b) IL-6 and (c) TNF-120572mRNA was determined via quantitative real-time PCR Data represent mean plusmn standarddeviation (SD) (n = 6 one-way ANOVA p lt 001 versus normal lowastp lt 005 lowastlowastp lt 001 versus control) IL-1120573 interleukin 1 beta IL-6interleukin 6 TNF-120572 tumor necrosis factor alpha PCR polymerase chain reaction

hormone corticosterone (GBH 100 101304 plusmn 30321 pgml plt 001 GBH 300 115506plusmn 32415 pgml plt 005 FXT 65259plusmn 16033 pgml p lt 0001 AMT 94741 plusmn 3778 pgml p lt001) [26] relative to levels observed in control mice (control172291 plusmn 18169 pgml p lt 0001 versus normal Figure 4(b))These data suggest that GBH can affect the concentrationof mood-related hormones in mice with reserpine-induceddepression Dopamine and norepinephrinewere not detectedin the plasma (data not shown)

35 Effect of GBH on Proinflammatory Cytokines in Reserpine-Induced Depression Chronic stress and the release of proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 lead tochronic neuroinflammation which contributes to depression[27 28] IL-1120573 IL-6 and TNF-120572 mRNA expression wassignificantly increased in the hippocampus of control micealthough this effect was ameliorated by treatment with GBHFXT and AMT (Figure 5) These data indicate that GBHmay help to regulate immune and endocrine dysfunctionassociated with depression

36 Effect of GBH on BDNF and p-CREB Expression in theBrain In order to determine the molecular mechanismsunderlying the antidepressant effects of GBH we examinedBDNF and p-CREB expression in the brain via Westernblot analysis Hippocampal BDNF levels were significantlylower in control mice than in normal mice indicative ofneuronal dysfunction in the brain [29] however mice inthe GBH 100 group exhibited significantly increased levels ofBDNF (Figure 6(a)) The BDNF-CREB pathway is associatedwith MDD [30] We observed a significant reduction inhippocampal p-CREB in control mice which was preventedin a dose-dependent manner in mice treated with GBHMoreover FXT and AMT treatment significantly increasedp-CREB expression (Figure 6(b)) These results suggest thatGBH can affect hippocampal neuronal activity in mice withreserpine-induced depression

To confirm the effect of GBH treatment in our modelof reserpine-induced depression we examined BDNF andp-CREB expression in the hippocampus via immunofluo-rescence analysis BDNF levels were significantly decreased

8 BioMed Research International

Normal Control FXT100 300 500

BDNF

-actin

GBH 50 AMT

00

05

10

15

BDN

F re

lativ

e int

ensit

y

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

Tlowast

lowastlowastlowastlowast

(a)

-actin

P-CREB

CREB

Normal Control FXT100 300 500

GBH 50 AMT

00

05

10

15

p-CR

EBC

REB

rela

tive i

nten

sity

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

T

lowast lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

BioMed Research International 7

00

05

10

15

20

25IL

-1b

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowastlowast lowast

lowast lowast

(a)

00

05

10

15

20

IL-6

(RQ

)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast

lowastlowastlowastlowast

(b)

00

05

10

15

20

25

TNF-

a (R

Q)

GBHNor

mal

Con

trol

FXT

AM

T

lowast lowastlowast lowastlowast

lowastlowast

(c)

Figure 5 Effect of Gyejibokryeong-hwan (GBH) on IL-1120573 IL-6 and TNF-120572 mRNA expression in mice with reserpine-induced depressionThe expression of (a) IL-1120573 (b) IL-6 and (c) TNF-120572mRNA was determined via quantitative real-time PCR Data represent mean plusmn standarddeviation (SD) (n = 6 one-way ANOVA p lt 001 versus normal lowastp lt 005 lowastlowastp lt 001 versus control) IL-1120573 interleukin 1 beta IL-6interleukin 6 TNF-120572 tumor necrosis factor alpha PCR polymerase chain reaction

hormone corticosterone (GBH 100 101304 plusmn 30321 pgml plt 001 GBH 300 115506plusmn 32415 pgml plt 005 FXT 65259plusmn 16033 pgml p lt 0001 AMT 94741 plusmn 3778 pgml p lt001) [26] relative to levels observed in control mice (control172291 plusmn 18169 pgml p lt 0001 versus normal Figure 4(b))These data suggest that GBH can affect the concentrationof mood-related hormones in mice with reserpine-induceddepression Dopamine and norepinephrinewere not detectedin the plasma (data not shown)

35 Effect of GBH on Proinflammatory Cytokines in Reserpine-Induced Depression Chronic stress and the release of proin-flammatory cytokines such as IL-1120573 IL-6 and TNF-120572 lead tochronic neuroinflammation which contributes to depression[27 28] IL-1120573 IL-6 and TNF-120572 mRNA expression wassignificantly increased in the hippocampus of control micealthough this effect was ameliorated by treatment with GBHFXT and AMT (Figure 5) These data indicate that GBHmay help to regulate immune and endocrine dysfunctionassociated with depression

36 Effect of GBH on BDNF and p-CREB Expression in theBrain In order to determine the molecular mechanismsunderlying the antidepressant effects of GBH we examinedBDNF and p-CREB expression in the brain via Westernblot analysis Hippocampal BDNF levels were significantlylower in control mice than in normal mice indicative ofneuronal dysfunction in the brain [29] however mice inthe GBH 100 group exhibited significantly increased levels ofBDNF (Figure 6(a)) The BDNF-CREB pathway is associatedwith MDD [30] We observed a significant reduction inhippocampal p-CREB in control mice which was preventedin a dose-dependent manner in mice treated with GBHMoreover FXT and AMT treatment significantly increasedp-CREB expression (Figure 6(b)) These results suggest thatGBH can affect hippocampal neuronal activity in mice withreserpine-induced depression

To confirm the effect of GBH treatment in our modelof reserpine-induced depression we examined BDNF andp-CREB expression in the hippocampus via immunofluo-rescence analysis BDNF levels were significantly decreased

8 BioMed Research International

Normal Control FXT100 300 500

BDNF

-actin

GBH 50 AMT

00

05

10

15

BDN

F re

lativ

e int

ensit

y

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

Tlowast

lowastlowastlowastlowast

(a)

-actin

P-CREB

CREB

Normal Control FXT100 300 500

GBH 50 AMT

00

05

10

15

p-CR

EBC

REB

rela

tive i

nten

sity

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

T

lowast lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

8 BioMed Research International

Normal Control FXT100 300 500

BDNF

-actin

GBH 50 AMT

00

05

10

15

BDN

F re

lativ

e int

ensit

y

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

Tlowast

lowastlowastlowastlowast

(a)

-actin

P-CREB

CREB

Normal Control FXT100 300 500

GBH 50 AMT

00

05

10

15

p-CR

EBC

REB

rela

tive i

nten

sity

GBH

Hippocampus

Nor

mal

Con

trol

FXT

AM

T

lowast lowast

lowastlowastlowast lowastlowastlowast

lowastlowast

(b)

Figure 6 Effect of Gyejibokryeong-hwan (GBH) on hippocampal levels of BDNF and p-CREBCREB as measured via Western blottingin mice with reserpine-induced depression Isolated hippocampal lysates were analyzed via Western blotting using (a) BDNF and (b) p-CREBCREB antibodies 120573-Actin was used as the loading control Data are representative of three independent experiments and are presentedas the mean plusmn standard deviation (SD) (one-way ANOVA p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt 0001 versus control)BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein p-CREB phosphorylated CREB

in the dentate gyrus of control mice although this decreasewas attenuated by treatment with GBH FXT and AMT (Fig-ure 7(a)) Similar results were observed when we examinedchanges in p-CREB expression (Figure 7(b)) These resultssuggest that GBH recovers reserpine-induced depressive-likebehaviors via activation of the BDNF-CREB pathway

4 Discussion

In the present study we examined the antidepressant effect ofGBH in mice with reserpine-induced depression Our resultsindicated that GBH can ameliorate depressive-like behaviorsaffect the concentration of mood-related hormones and helpto regulate immuneendocrine dysfunction in mice withreserpine-induced depression likely via activation of theBDNF-CREB pathway

As there are various causes of depression the patho-physiology of the disorder remains to be fully elucidatedSeveral hypotheses have been proposed regarding the basis ofdepression including hypothalamic-pituitary-adrenal (HPA)axis hyperactivity [31] disturbances in monoamine gluta-mate and gamma-aminobutyric acid (GABA) transmissionneurotrophic factor dysfunction neuroinflammation [32]and glial pathology [33 34]

Monoamines are transported into presynaptic vesiclesvia the vesicular monoamine transporter (VMAT) whichis blocked by reserpine [35] Mice with reserpine-induceddepression due to monoamine depletion exhibit anxiety-and depressive-like behaviors such as increased immobility

time and decreased locomotor activity in behavioral testsrelative to findings observed in controlmice [36] In additionprevious studies have reported that mice treated with reser-pine exhibit increased levels of plasma corticosterone [37]and proinflammatory cytokines in the brain [38]

Before initiating our study we optimized our mousemodel of reserpine-induced depression Our findings indi-cated that higher concentrations of reserpine administeredfor 3 consecutive days were lethal in some mice prior tothe 10-day GBH treatment period Furthermore some micerecovered from reserpine-induced depression 10 days afterdrug treatment while treatment at lower doses for 3 days wasnot sufficient for inducing depression In the present studywemodified the animal systems used in previous studies [36]by reducing the concentration of reserpine to 05 mgsdotkgminus1In addition the reserpine treatment time was extended to 10days and GBH was administered simultaneously

Our results indicated that GBH successfully ameliorateddepressive-like behaviors in reserpine-treated mice as indi-cated by significant reductions in immobility time in theFST and TST Furthermore our findings demonstrated thatsuch decreases were not due to increases in spontaneouslocomotor activity (Supplementary Material 1) HoweverGBHdid increase the distance traveled in theOFT indicatingthat GBH can suppress anxiety-related behavior in mice withreserpine-induced depression

Reserpine blocks amine storage processes which leadsto increased hippocampal excitability and blood corticoidlevels Previous studies have demonstrated that reserpine

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

BioMed Research International 9

Control FXTGBH-100 GBH-300 GBH-500GBH-50 AMTNormal BDNF

DAPI

merge

Control FXTGBH-100 GBH-300

(b)

(a)

GBH-500GBH-50 AMTNormal

pCREB

DAPI

merge

0

50

100

150

Mea

n IO

D o

f BD

NF

( o

f nor

mal

)

Hippocampus

0

50

100

150

200

Mea

n IO

D o

f pCR

EB (

of n

orm

al)

Hippocampus

GBH GBHNor

mal

Con

trol

FXT

AM

T

Nor

mal

Con

trol

FXT

AM

T

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowast

Figure 7 Effect of Gyejibokryeong-hwan (GBH) on hippocampal BDNF and p-CREB as measured using immunofluorescence in mice withreserpine-induced depression Frozen hippocampal sections were analyzed based on immunofluorescence using (a) BDNF (green) and (b)p-CREB (green) antibodies DAPI (blue) was used to visualize nuclei Data are representative of three independent experiments and arepresented as the mean plusmn standard deviation (SD) (one-way ANOVA p lt 005 p lt 0001 versus normal lowastp lt 005 lowastlowastp lt 001 lowastlowastlowastp lt0001 versus control) BDNF brain-derived neurotrophic factor p-CREB phosphorylated cAMP response element-binding protein

alters the secretion of 5-hydroxytryptamine (5-HT) in thebrain [37] In the present study we observed that GBH treat-ment significantly increased plasma serotonin levels whilesignificantly decreasing corticosterone levels in reserpine-treated mice relative to levels observed in control miceAlthough plasma serotonin levels may represent releasefrom both the central nervous system and platelets GBH

is known to suppress blood aggregationmdasha process thatactivates serotonin release [39 40] Thus it is unlikely thatplatelets represented the major source of serotonin release inour study Although the recovery of plasma serotonin levelswas significantly greater in theGBH-treated group than in thecontrol group the relative serotonin level in the GBH-treatedgroupwas still significantly lower than that in the naive group

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

10 BioMed Research International

The balance of extracellular catecholamines is a key mod-ulator of inflammatory mediator production [38 41] there-fore we assessed the expression of proinflammatory cytokinemRNA in the hippocampus Our findings indicated thatGBH treatment attenuated reserpine-induced increases in theexpression of IL-1120573 IL-6 and TNF-120572mRNA in control mice

An overproduction of proinflammatory cytokines canimpair neuronal structure and function leading to deficits inneuroplasticity [42] BDNF and CREB are involved in neu-ronal differentiation survival and synaptic plasticitymdashwhichare associated with learning and memorymdashand in manynervous systemdisorders including depression [30]Westernblot and immunofluorescence analyses revealed that BDNFand p-CREB expression in the hippocampus was greater inGBH-treated mice than in control mice Because treatmentwith GBH at 300 and 500 mgsdotkgminus1 resulted in gradualdecreases in BDNF levels our findings suggest that the opti-mal concentration of GBH for improving BDNF expression is100 mgsdotkgminus1 Such results may have been associated with thepharmacokinetic properties of GBH which is comprised ofextracts from five types of plants Indeed such properties areoften unpredictable inmulticompound herbalmedicines Forexample an unidentified chemical compound in GBH mayexert adverse effects on BDNF levels beyond a certain thresh-old decreasing the efficacy of GBH treatment Our data sug-gest that GBH not only exerts regulatory effects on neuroin-flammation but also influences BDNF expression in the brain

Recently developed antidepressants include modulatorsof neuroinflammation oxidative stress the hypothalamic-pituitary-adrenal (HPA) axis glutamate opioids the cholin-ergic system and neuropeptides (eg substance P neuropep-tide Y and galanin) In addition many studies have soughtto treat depression without side effects [43 44] Naturalmedicines with anti-inflammatory and antioxidant proper-ties are attractive targets because they have been effective intreating neurodegenerative and autoimmune diseases as wellas cancer Moreover they are associated with significantlyfewer side effects (eg St Johnrsquos wort [Hypericum perfora-tum]) [45 46] Toxicity studies have demonstrated that GBHis safe at dosages up to 500 mgsdotkgsdotdayminus2 regardless of genderFurthermore standardization results have been reported inaccordance with Ministry of Food and Drug Safety (MFDS)guidelines [47]

5 Conclusions

Taken together our results indicate that GBH treatment caninduce an antidepressant-like effect in mice with reserpine-induced depression GBH is composed of several herbstherefore our results suggest that a multitarget approach canbe effective in treating depression with fewer side effectsFurther studies are required to determine the active com-pound(s) in GBH as well as the molecular mechanismsunderlying its regulatory effects on neuroinflammation andHPA axis hyperactivity

Abbreviations

MDD Major depressive disorder

SAD Seasonal affective disorderTCAs Tricyclic antidepressantsSSRIs Selective serotonin reuptake inhibitorsNRIs Noradrenaline reuptake inhibitorsSNRIs Serotonin and noradrenaline reuptake

inhibitorsMAOIs Monoamine oxidase inhibitorsGBH Gyejibokryeong-hwanBDNF Brain-derived neurotrophic factorPBS Phosphate-buffered salineFST Forced swimming testTST Tail suspension testOFT Open-field testp-CREB Phosphorylated cAMP response

element-binding proteinHRP Horseradish peroxidaseIL InterleukinTNF Tumor necrosis factorHPA Hypothalamic-pituitary-adrenalWHO World Health OrganizationMFDS Ministry of Food and Drug SafetyFDA Food and Drug AdministrationIP IntraperitonealGABA Gamma-aminobutyric acid5-HT 5-hydroxytryptamineSD Standard deviationFXT FluoxetineAMT AmitriptylineVMAT Vesicular monoamine transporter

Data Availability

The datasets used andor analyzed in the current study areavailable from the corresponding author upon reasonablerequest The role of the funding body in the design of thestudy and collection analysis and interpretation of data andin writing the article should be declared in this request

Disclosure

The funding bodies played no role in the design of the studycollection analysis and interpretation of data or in writingthe manuscript

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by a grant from the Korea Instituteof Oriental Medicine (KIOM) (K17061) and by a grantfrom the National Research Foundation of Korea (NRF)funded by the Korean government (MSIP) (Grant no NRF-2016R1A2B4009614)

Supplementary Materials

We performed behavior test and ELISA in which normalmice were treated with GBH at dosages of 100 300 and

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

BioMed Research International 11

500 mgkg for 10 days The GBH did not show a significanteffect in these experiments Supplementary Material 1 effectof Gyejibokryeong-hwan (GBH) on depressive-like behaviorsand the concentration of plasma serotonin and corticosteroneinmice (a) Immobility time in the forced swim test (FST) and(b) tail suspension test (TST) (c) total distance traveled in theopen-field test (OFT) All behavioral tests were conducted onday 11The concentration of plasma (d) serotonin and (e) cor-ticosterone was determined by ELISA Data represent meanplusmn SD (n = 6 one-way ANOVA) (Supplementary Materials)

References

[1] W H Organization ldquoPharmacological Treatment of MentalDisordersrdquo in in Primary Health Care World Health Organi-zation Geneva 2009

[2] M Schwartz-Lifshitz G Zalsman L Giner and M AOquendo ldquoCan we really prevent suiciderdquo Current PsychiatryReports vol 14 no 6 pp 624ndash633 2012

[3] M Reddy ldquoDepression the disorder and the burdenrdquo IndianJournal of Psychological Medicine vol 32 no 1 pp 1-2 2010

[4] P GilbertDepression the evolution of powerlessness RoutledgeTaylor amp Francis Group London UK Classic edition edition2017 edition edition

[5] H Tiemeier ldquoBiological risk factors for late life depressionrdquoEuropean Journal of Epidemiology vol 18 no 8 pp 745ndash7502003

[6] J J Schildkraut ldquoThe catecholamine hypothesis of affective dis-orders a review of supporting evidencerdquoThe American Journalof Psychiatry vol 122 no 5 pp 509ndash522 1965

[7] G E Torres R R Gainetdinov and M G Caron ldquoPlasmamembrane monoamine transporters structure regulation andfunctionrdquo Nature Reviews Neuroscience vol 4 no 1 pp 13ndash252003

[8] J Sarko ldquoAntidepressants old and new A review of theiradverse effects and toxicity in overdoserdquo Emergency MedicineClinics of North America vol 18 no 4 pp 637ndash654 2000

[9] H JunDonguibogam Namsandang Seoul 1980 NamsandangSeoul

[10] L Han X Guo H Bian et al ldquoGuizhi fuling wan a tra-ditional chinese herbal formula sensitizes cisplatin-resistanthuman ovarian cancer cells through inactivation of the PI3KAKTmTOR pathwayrdquo Evidence-Based Complementary andAlternative Medicine vol 2016 Article ID 4651949 11 pages2016

[11] J-S Park S Park C-H Cheon et al ldquoEffects and safety of gyeji-bongnyeong-hwan on dysmenorrhea caused by blood stagna-tion a randomized controlled trialrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2013 Article ID 4247306 pages 2013

[12] S-J Jeong H-S Lim C-S Seo et al ldquoAnti-inflammatoryactions of herbal formula Gyejibokryeong-hwan regulated byinhibiting chemokine production and STAT1 activation inHaCaT cellsrdquo Biological amp Pharmaceutical Bulletin vol 38 no3 pp 425ndash434 2015

[13] X Lei J Chen C Liu J Lin J Lou and H Shang ldquoStatus andthoughts of Chinese patent medicines seeking approval in theUS marketrdquo Chinese Journal of Integrative Medicine vol 20 no6 pp 403ndash408 2014

[14] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds in

Gyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[15] S-H Hwang Y G Choi M-Y Jeong Y-M Hong J-H Leeand S Lim ldquoMicroarray analysis of gene expression profileby treatment of Cinnamomi Ramulus in lipopolysaccharide-stimulated BV-2 cellsrdquo Gene vol 443 no 1-2 pp 83ndash90 2009

[16] S Rho H-S Chung M Kang et al ldquoInhibition of productionof reactive oxygen species and gene expression profile by treat-ment of ethanol extract of Moutan Cortex Radicis in oxidativestressed PC12 cellsrdquo Biological amp Pharmaceutical Bulletin vol28 no 4 pp 661ndash666 2005

[17] C Yang J Zhao Y Cheng X Li and J Rong ldquoBioactivity-guided fractionation identifies amygdalin as a potent neu-rotrophic agent from herbal medicine semen Persicae extractrdquoBioMed Research International vol 2014 Article ID 3068572014

[18] B-K Park E Chun J J Choi et al ldquoAdministration ofWasabiakoreana Ameliorates Irritable Bowel Syndrome-Like Symptomsin a Zymosan-Induced Mouse Modelrdquo Journal of MedicinalFood vol 20 no 5 pp 474ndash484 2017

[19] A Can D T Dao M Arad C E Terrillion S C Piantadosiand T D Gould ldquoTheMouse Forced Swim Testrdquo Journal of Vis-ualized Experiments no 58 2011

[20] A Can D T Dao C E Terrillion S C Piantadosi S Bhatand T D Gould ldquoThe tail suspension testrdquo Journal of VisualizedExperiments JOVE no 59 Article ID e3769 2012

[21] M L Seibenhener and M C Wooten ldquoUse of the Open FieldMaze to measure locomotor and anxiety-like behavior in micerdquoJournal of Visualized Experiments no 96 Article ID e524342015

[22] J M Deussing ldquoAnimal models of depressionrdquo Drug DiscoveryToday Disease Models vol 3 no 4 pp 375ndash383 2007

[23] B-K Park Y-C Park I C Jung et al ldquoGamisasangja-tang sup-presses pruritus and atopic skin inflammation in the NCNgamurine model of atopic dermatitisrdquo Journal of Ethnopharma-cology vol 165 pp 54ndash60 2015

[24] B-K Park S Park J-B Park M C Park T S Min and MJin ldquoOmega-3 fatty acids suppressTh2-associated cytokine geneexpressions and GATA transcription factors in mast cellsrdquo TheJournal of Nutritional Biochemistry vol 24 no 5 pp 868ndash8762013

[25] K Martinowich and B Lu ldquoInteraction between BDNF andserotonin role in mood disordersrdquo Neuropsychopharmacologyvol 33 no 1 pp 73ndash83 2008

[26] W Liu E Y Yuen and Z Yan ldquoThe stress hormone cortico-sterone increases synaptic 120572-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- andglucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complexrdquoThe Journal of Biological Chemistry vol 285 no9 pp 6101ndash6108 2010

[27] V Krishnan and E J Nestler ldquoThe molecular neurobiology ofdepressionrdquo Nature vol 455 no 7215 pp 894ndash902 2008

[28] Y-K Kim K-S Na A-M Myint and B E Leonard ldquoThe roleof pro-inflammatory cytokines in neuroinflammation neuro-genesis and the neuroendocrine system in major depressionrdquoProgress in Neuro-Psychopharmacology amp Biological Psychiatryvol 64 pp 277ndash284 2016

[29] B Lu G Nagappan and Y Lu ldquoBDNF and synaptic plasticitycognitive function and dysfunctionrdquo Handbook of Experimen-tal Pharmacology vol 220 pp 223ndash250 2015

12 BioMed Research International

[30] A Nair and V A Vaidya ldquoCyclic AMP response element bind-ing protein and brain-derived neurotrophic factor Moleculesthat modulate our moodrdquo Journal of Biosciences vol 31 no 3pp 423ndash434 2006

[31] C Stetler and G E Miller ldquoDepression and hypothalamic-pituitary-adrenal activation a quantitative summary of fourdecades of researchrdquo Psychosomatic Medicine vol 73 no 2 pp114ndash126 2011

[32] O J SchiepersMCWichers andMMaes ldquoErratum to ldquoCyto-kines and major depressionrdquo [Prog NeuropsychopharmacolBiol Psychiat 29(2) (2005) 201ndash217]rdquo Progress in Neuro-Psy-chopharmacology amp Biological Psychiatry vol 29 no 4 pp 637-638 2005

[33] G Hasler ldquoPathophysiology of depression do we have any solidevidence of interest to cliniciansrdquo World Psychiatry vol 9 no3 pp 155ndash161 2010

[34] V Maletic M Robinson T Oakes S Iyengar S G Ball and JRussell ldquoNeurobiology of depression an integrated view of keyfindingsrdquo International Journal of Clinical Practice vol 61 no12 pp 2030ndash2040 2007

[35] L Luo Principles of neurobiology Garland Science New YorkNY USA 2015

[36] J Tian Y Cui L Hu et al ldquoAntidepressant-like effect of genipinin micerdquoNeuroscience Letters vol 479 no 3 pp 236ndash239 2010

[37] A M Revzin R P Maickel and E Costa ldquoEffects of reserpineand benzoquinolizines on limbic system excitability brainamine storage and plasma corticosterone level in ratsrdquo Life Sci-ences vol 1 no 12 pp 699ndash707 1962

[38] J Szelenyi and E S Vizi ldquoThe catecholamine-cytokine balanceInteraction between the brain and the immune systemrdquo Annalsof the New York Academy of Sciences vol 1113 pp 311ndash324 2007

[39] K Terawaki M Noguchi M Yuzurihara Y Omiya Y Ikarashiand Y Kase ldquoKeishibukuryogan a Traditional Japanese Medi-cine Inhibits Platelet Aggregation in Guinea PigWhole BloodrdquoEvidence-Based Complementary and Alternative Medicine vol2015 pp 1ndash8 2015

[40] M R Thomas and R F Storey ldquoThe role of platelets in inflam-mationrdquo Thrombosis and Haemostasis vol 114 no 3 pp 449ndash458 2015

[41] Q J Huang H Jiang X L Hao and T R Minor ldquoBrain IL-1beta was involved in reserpine-induced behavioral depressionin ratsrdquo Acta Pharmacol Sin vol 25 no 3 pp 293ndash296 2004

[42] F Calabrese A C Rossetti G Racagni P Gass M A Rivaand R Molteni ldquoBrain-derived neurotrophic factor a bridgebetween inflammation andneuroplasticityrdquoFrontiers in CellularNeuroscience vol 8 article 430 2014

[43] G I Papakostas and D F Ionescu ldquoTowards new mecha-nisms An update on therapeutics for treatment-resistant majordepressive disorderrdquo Molecular Psychiatry vol 20 no 10 pp1142ndash1150 2015

[44] J D Rosenblat R S McIntyre G S Alves K N Fountoulakisand A F Carvalho ldquoBeyond monoamines-novel targets fortreatment-resistant depression A comprehensive reviewrdquo Cur-rent Neuropharmacology vol 13 no 5 pp 636ndash655 2015

[45] C Grosso Herbal medicine in depression traditional medicineto innovative drug delivery Springer International PublishingSwitzerland 2016

[46] B Gaster and J Holroyd ldquoSt Johnrsquos wort for depression A sys-tematic reviewrdquo JAMA InternalMedicine vol 160 no 2 pp 152ndash156 2000

[47] Y Kobayashi S Minematsu S Maemura C Atkinson andM Mulhern ldquoA single oral dose toxicity study and a 13-week repeated dose study with a 4-week recovery periodof TSUMURA Keishi-bukuryo-gan (TJ-25) in ratsrdquo JapanesePharmacology andTherapeutics vol 23 no 7 pp 151ndash168 1995

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAutismDepression Research

and TreatmentHindawiwwwhindawicom Volume 2018

Neurology Research International

Hindawiwwwhindawicom Volume 2018

Alzheimerrsquos DiseaseHindawiwwwhindawicom Volume 2018

International Journal of

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Hindawiwwwhindawicom Volume 2018

Research and TreatmentSchizophrenia

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018Hindawiwwwhindawicom Volume 2018

Neural PlasticityScienticaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Sleep DisordersHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

MedicineAdvances in

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Psychiatry Journal

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Multiple Sclerosis InternationalHindawiwwwhindawicom Volume 2018

StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

Hindawiwwwhindawicom Volume 2018

Case Reports in Neurological Medicine

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAutismDepression Research

and TreatmentHindawiwwwhindawicom Volume 2018

Neurology Research International

Hindawiwwwhindawicom Volume 2018

Alzheimerrsquos DiseaseHindawiwwwhindawicom Volume 2018

International Journal of

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Hindawiwwwhindawicom Volume 2018

Research and TreatmentSchizophrenia

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018Hindawiwwwhindawicom Volume 2018

Neural PlasticityScienticaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Sleep DisordersHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

MedicineAdvances in

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Psychiatry Journal

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Multiple Sclerosis InternationalHindawiwwwhindawicom Volume 2018

StrokeResearch and TreatmentHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

Hindawiwwwhindawicom Volume 2018

Case Reports in Neurological Medicine

Submit your manuscripts atwwwhindawicom