research article antidepressant-like effects of cordycepin in a...

Research ArticleAntidepressant-Like Effects of Cordycepin in a Mice Model ofChronic Unpredictable Mild Stress

Zhang Tianzhu1 Yang Shihai2 and Du Juan3

1Changchun University of Chinese Medicine Changhcun 130117 China2Jilin Agricultural University Changchun 130118 China3School of Life Science Peking University Beijing 100871 China

Correspondence should be addressed to Yang Shihai yangshihaibest126com

Received 22 August 2014 Revised 31 October 2014 Accepted 3 December 2014 Published 23 December 2014

Academic Editor Xiu-Min Li

Copyright copy 2014 Zhang Tianzhu 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

Cordycepin (31015840-deoxyadenosine) a major bioactive component isolated from Cordyceps militaris has multiple pharmacologicalactivities This study is attempted to investigate whether cordycepin (COR) possesses beneficial effects on chronic unpredictablemild stress- (CUMS-) induced behavioral deficits (depression-like behaviors) and explore the possible mechanisms ICRmice weresubjected to chronic unpredictable mild stress for 42 consecutive days Then COR and fluoxetine (FLU positive control drug)were administered for 21 consecutive days at the last three weeks of CUMS procedure The classical behavioral tests open fieldtest (OFT) sucrose preference test (SPT) tail suspension test (TST) and forced swimming test (FST) were applied to evaluate theantidepressant effects of COR Then the serotonin (5-HT) and noradrenaline (NE) concentrations in hippocampal were evaluatedby HPLC tumor necrosis factor-120572 (TNF-120572) and interleukin-6 (IL-6) in hippocampal were evaluated and the proteins of TNF-120572 IL-6 NF-120581BP65 5-HT2A receptor (5-HT2AR) and brain-derived neurotrophic factor (BDNF) in hippocampal were evaluatedby Western blot Our results indicated that 6 weeks of CUMS exposure induced significant depression-like behavior with low 5-HT and NE levels high TNF-120572 and IL-6 in brain and high hippocampal TNF-120572 IL-6 P-NF-120581BP65 and 5-HT2AR levels and lowBDNF expression levels Whereas chronic COR (20 40mgkg) treatments reversed the behavioral deficiency induced by CUMSexposure treatment with COR normalized the change of TNF-120572 IL-6 5-HT and NE levels which demonstrated that COR couldpartially restore CUMS-induced 5-HT receptor impairments and inflammation Besides hippocampal BDNF expressions werealso upregulated after COR treatments In conclusion COR remarkably improved depression-like behavior in CUMS mice and itsantidepressant activity ismediated at least in part by the upregulatingBDNFanddownregulating 5-HT2AR levels and inflammationin hippocampus

1 Introduction

Depression is one of the most common mental disorderscharacterized by feelings of sadness and major depressivedisorder is a primary cause of disability [1] It is the fourthmajor cause of morbidity worldwide at present and it willbecome the second by 2020 according to the World HealthOrganization [2] Although there are many clinical effectiveantidepressant medications most of them generate severeside effects Therefore it is necessary to develop new antide-pressant drugs with lower adverse effects and better efficacy

Plenty of clinical reports suggest that prolonged expo-sure to life stressful episodes as a common risk factor

could provoke the development of major depression [3ndash5]Currently scientists adopt chronic unpredictable mild stress(CUMS) procedure which is performed such that animalsare consecutively exposed to a series of unpredictable mildstressors to simulate a series of life stress events [6] Indeeda large number of ethological symptoms and neurobiologicalabnormalities found in CUMS-induced animals are similarto those exhibited in human depressed patients [7] ThusCUMS-induced depressive animal model has good validityand reliable predictability to screen new antidepressantsthrough a series of behavioral tests

The 5-HT2receptors in the CNS are thought to be

involved in psychiatric disorders such as depression anxiety

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 438506 9 pageshttpdxdoiorg1011552014438506

2 Evidence-Based Complementary and Alternative Medicine

Experimentalgroups

Week 0 Week 3 Week 6Killed

No CUMS

CUMS

Control

Model

COR

FLU

Time

CUMS + COR (20mgkg)

CUMS + COR (40mgkg)

CUMS + FLU (15mgkg)

mdash

Figure 1 The protocol in this study

sleep disorders and hallucinations [8 9] Increased densitiesof cortical 5-HT

2A receptors are observed upon postmortemexamination of depressed patients [10]

An increasing body of evidence presented in recent yearshas revealed that activation of the inflammatory response sys-tem plays a role in the pathophysiology of depression [11 12]Several studies reported increased levels of proinflammatorycytokines for example tumor necrosis factor-alpha (TNF-120572)and interleukin-6 (IL-6) in depressive disorders [11 12]

Cordycepin (31015840-deoxyadenosine) a major bioactive com-ponent isolated from Cordyceps militaris has multiple phar-macological activities such as anti-inflammatory andimmunomodulatory effects Cordycepin prevents lipopol-ysaccharide- (LPS-) induced airway neutrophilia in miceand effectively blocks LPS-induced expression of vascularadhesion molecule-1 (VCAM-1) in the human epithelialcell line A549 [13] Cordycepin inhibits interleukin-1120573-(IL-1120573-) induced matrix metalloproteinase-1 (MMP-1)and MMP-3 expressions in rheumatoid arthritis synovialfibroblasts (RASFs) and significantly inhibits AP-1 activation[14] While there is little evidence regarding the relevanceof COR on CUMS-induced depression in this study wehypothesized that COR would improve CUMS-induceddepression through regulation of r5-HT

2A receptor andBDNF

2 Materials and Methods

21 Animals Male ICR mice weighing 18ndash22 g (same batch)were purchased from Experimental Animal Center in JilinUniversity (Changchun China SCXK-0003) Prior to anyexperimentation the mice were allowed to have one weekto acclimatize to the laboratories And during the wholestudy the mice were housed in group in constant labora-tory conditions at a temperature of 22∘C and 60 relativehumidity under a 12 h light12 h dark cycle In our studyall the experimental procedures and laboratory animal carewere performed in accordance with the National Institutes ofHealth (NIH) Guide

22 Drug and Treatment COR was purchased from NanjingJiancheng Co Ltd Fluoxetine hydrochloride (positive con-trol drug) was obtained from Jinlin drugstore (ChanghunChina) COR and fluoxetine were dissolved in 003 sodiumcarboxymethyl cellulose (CMC-Na) Mice were randomlydivided into five different groups one control group oneCUMS-vehicle (003 CMC-Na) group one CUMS-FLU(15mgkg) group and three CUMS-COR (20 40mgkg)groups the doses selection of COR was according to Zhanget alrsquos report [15] Each group included 20 mice Everymorning all drug treatment groups were administered orallyvia intragastric gavage in a dose of 10mLkg body weight

23 CUMS Procedure The CUMS procedure was performedas described byWillner et al [16] with amodification Brieflymice in the CUMS groups were exposed to different stressorsnamely food deprivation water deprivation empty waterbottles (after water deprivation) cage tilt grouped housingsoiled cage stroboscopic lighting restricted access to food(only give a small amount of food after food deprivation)5 min cold swimming (at 15∘C) 1 min tail pinch (1 cm fromthe end of the tail) physical restraint illumination and whitenoise One of these stressors (in random order) was givenevery day for 6 weeks The control group mice were leftundisturbed except for necessary procedures such as routinecage cleaning and the protocol of the study can be seen inFigure 1

24 Sucrose Preference Test (SPT) Sucrose preference testwas carried out at the end of week 0 week 3 and week 6respectively The method was performed as previous report[17] In brief 72 h before the test a mouse was individuallyplaced into cage with two bottles of sucrose solution toadapt to sucrose solution (1 wv) for 24 h then one bottleof sucrose solution was replaced with water (24 h) afterthe adaptation laboratory mice were deprived of water andfood (24 h) Sucrose preference test was conducted at 930am Each mouse was housed in individual cage and wasfree to access two bottles containing 100mL of sucrose

Evidence-Based Complementary and Alternative Medicine 3

solution (1wv) and 100mLofwater respectively After 12 hthe consumed weights of sucrose solution and water wererecordedThe sucrose preference valuewas obtained from thefollowing formula sucrose preference () = sucrose intake(g)[sucrose intake (g) + water intake (g)] times 100

25 Open Field Test (OFT) The open field test was used toevaluate the locomotor activity of mice (crossing horizontalmovement scores reflect range of motion rearing verticalmovement scores reflect exploratory behaviors) Mice wereplaced individually in the middle of an open field apparatusin a wooden box (40 times 60 times 50 cm) with the floor of thearena divided into 12 equal squares [18] Following 2 minacclimatized to the apparatus the numbers of crossings andrearings were counted in a 3 min session After each trial theopen field apparatus was cleaned

26 Tail Suspension Test (TST) At the end of week 0 week3 and week 6 respectively the tail suspension test wasperformed based on the previous method that the mouse washung 15 cm above the floor by the tip of the tail (1 cm) andwasadhered to the lever [19] The total test procedure of mouseimmobility time was counted during a test period of 6min(prior 2 min to adapt and recorded the last 4 min) And onlywhen themouse was in a passively suspended and completelymotionless status it could be regarded as immobile

27 Forced Swimming Test (FST) Forced swim test wascarried out at the end of the 6-week CUMS procedureThe test session was similar to that described in a previousreport [20] Briefly each mouse was placed individually inan open cylindrical vitreous tank (height 20 cm diameter14 cm) containing 15 cm depth of water at 25 plusmn 2∘C All micewere forced to swim for 6min and the total duration ofimmobility was recorded during the last 4min of the testThedefinition of immobile status was that the mouse was floatingin the water without any movement only small motions arerequired to maintain its head above the water

28 Measurement of 5-HT and DA Levels in the Hippocam-pus Twenty-four hours after completion of the final foodconsumption test six of the mice in each group were usedfor tissue assays of 5-HT and DA The animals were decap-itated and their brains rapidly were removed and dissectedon an ice-chilled glass plate to obtain the hippocampusaccording to Franklin and Herberg [21] and Paxinos et al[22] Each tissue sample was weighed and homogenized bysonication in 200120583L 04M perchloric acid The homogenatewas kept on ice for 1 h and then centrifuged at 12000 rpm(4∘C) for 20min The supernatant was preserved and theconcentrations of 5-HT and DA were measured using highperformance liquid chromatography with electrochemicaldetection (HPLC-ECD) as described by Qi et al [23] withminor modifications The mobile phase consisted of 85mMcitrate 100mMsodiumacetate 09mMoctyl-sodium sulfate02mM EDTA and 12 methanol pH 37 An LC-10A pump(Shimadzu Kyoto Japan) was operated at 10mLmin Thedetector (L-ECD-6A Shimadzu) was set at thorn060V Externalstandard curves were used to quantify the amounts of 5-HT

and DA in each sample calculated using the area under thecurve The injection volume was 20120583L

29 Measurement of TNF-120572 and IL-6 Levels in the Hip-pocampus IL-6 and TNF-120572 levels in the hippocampus weremeasured by ELISA kits (DRG international USA) accordingto the manufacturerrsquos instructions

210 Western Blot Analysis Mouse hippocampus waschopped into small pieces and homogenized in ice-cold RIPAbuffer containing 01 phenylmethylsulfonyl fluoride Thedissolved proteins were collected from the supernatant aftercentrifugation at 12000 g for 20min Protein concentrationswere determined using Coomassie blue based assay reagentProtein extracts were separated by a SDS-polyacrylamidegel electrophoresis and then transferred onto a PVDFmembrane The membrane was blocked with 5 skim milkin tris buffer saline and then incubated at 4∘C overnight withrespective primary antibodies for anti-5-HT

2AR antibody(1 1000) anti-BDNF antibody (1 1000) anti-P-NF-120581BP65(1 1000) anti-NF-120581BP65 (1 1000) anti-TNF-120572 (1 1000)anti-IL-6 (1 1000) and GAPDH (inner control 1 1000)After washing with tris buffered saline tween 20 (TBST) themembranes were incubated with a horseradish peroxidaseconjugated secondary antibody (1 12000) for 15 h at roomtemperature The antibody-reactive bands were visualized byusing enhanced chemiluminescence detection reagents anda gel imaging system (Tanon Science amp Technology Co LtdChina)

211 Statistical Analysis All data were normally distributedand are presented asmean plusmn SEM In the case of singlemeancomparison data were analyzed by a Studentrsquos 119905-test In thecase of multiple mean comparisons the data were analyzedby ANOVA and the Newman-Keuls post-test or two-wayrepeated measures ANOVA followed by Bonferroni multiplecomparison tests 119875 values less than 005 were regarded toreflect a significant difference

3 Results

31 Effects of COR on the Percentages of Sucrose ConsumptionAs shown in Figure 2 the value of sucrose consumption wasmeasured three times during the experiment At the begin-ning of the experiment (week 0) there were no significantdifferences among the 6 groups However after 3 weeks ofCUMS periods the sucrose consumption of stressed micewas lower than the control group At the end of CUMSregimen (week 6) sucrose consumption in CUMS group wassignificantly reduced compared with that in control groupHowever the sucrose consumption of CUMS-exposed micetreated for 3 weeks with COR treatment (20 and 40mgkg) orFLU (15mgkg) increased significantly compared with that inCUMS-vehicle group

32 Effects of COR in the Open Field Test Locomotor activitywas presented in Figure 3 The results showed that thenumber of crossings inCUMS-vehicle groupmice exhibited a


80

60

40

20

0

Sucr

ose p

refe

renc

e (

)

0 3 6

lowastlowast

lowastlowast

lowastlowast

Time (weeks)

CUMS + COR (20mgkg)CUMS + COR (40mgkg)

CUMS + FLU (15mgkg)

ControlCUMS

Figure 2 Effect of chronic COR administration on sucrose pref-erence in CUMS exposure mouseThree-time sucrose consumptiontest was carried out at the end of 0-week 3-week and 6-week CUMSexposure All values given are the mean plusmn SEM

119875 lt 005 and119875 lt 001 versus control group lowast119875 lt 005 and lowastlowast119875 lt 001 versus

CUMS-vehicle group

significant reduction versus the control groupmiceHoweverCOR (20 40mgkg) or FLU (15mgkg) treatments reversedthese effects on crossing CUMS procedure also decreasedthe number of rearing and COR or FLU could improve therearing number

33 Effects of COR in the Tail Suspension Test Figure 4showed the effect of COR treatments on the duration ofimmobility in tail suspension test At the end of week 0no significant difference was seen between the 6 groupsHowever 5 groups subjected to CUMS appeared to increaseimmobility time after arriving at the middle of the CUMSprocedure (week 3) Finally (week 6) CUMS-vehicle groupsignificantly increased immobility time versus the controlgroup COR at dose of 20mgkg and 40mgkg or FLU at doseof 20mgkg markedly decreased the immobility time in thetail suspension test compared with the CUMS-vehicle

34 Effects of COR in the Forced Swimming Test The resultsof forced swimming test (Figure 5) revealed that CUMS expo-sure significantly increased in immobility duration comparedwith control group COR at dose of 20mgkg and 40mgkg orthe positive control FLU at dose of 20mgkg treatments sig-nificantly reduced immobility time in the forced swimmingtest versus CUMS-vehicle group

35 Effects of COR on TNF-120572 and IL-6 Levels in the Hip-pocampus in CUMS Mice The effect of COR on TNF-120572 andIL-6 levels in the hippocampus in CUMS mice is shownin Figure 6 CUMS significantly increased TNF-120572 and IL-6 levels concentrations in the hippocampus compared withthat in control animals Administration of COR (20 and40mgkg) was able to reverse the effects of CUMS on TNF-120572and IL-6 levels concentrations FLU was able to reverse theeffects of CUMS on both DA and 5-HT

36 Effects of COR on 5-TH andDALevels in theHippocampusin CUMS Mice The effect of COR on 5-HT and DA levelsin the hippocampus in CUMS mice is shown in Figure 7CUMS significantly reduced 5-HT and DA concentrationsin the hippocampus compared with that in control animalsAdministration of COR (20 and 40mgkg)was able to reversethe effects of CUMS on 5-HT and DA concentrations FLUwas able to reverse the effects of CUMS on both DA and 5-HT

37 Effects of COR on NF-120581BP65 TNF-120572 IL-6 5-HT2119860R

and BDNF Proteins Expressions in the Hippocampus Theeffect of COR on NF-120581BP65 TNF-120572 IL-6 5-HT

2AR andBDNF proteins levels in the hippocampus in CUMS mice isshown in Figure 8 CUMS significantly increased 5-HT

2ARP-NF-120581BP65 TNF-120572 and IL-6 and decreased BDNF in thehippocampus compared with that in control animals admin-istration of COR (20 and 40mgkg) and FLU (15mgkg) wasable to reverse the effects of CUMS onNF-120581BP65 TNF-120572 IL-6 5-HT

2AR and BDNF

4 Discussion

Currently we primarily tested the antidepressant-like effectsof oral SKA treatments in mice subjected to CUMS In thiswork the CUMS mice model satisfactorily mimicked thedepressive status which was described as the reduction ofthe sucrose solution consumption and open field activityand increased 5-TH and DA levels 5-HT

2AR and BDNFrespectively However COR (20 and 40mgkg) administra-tion could oppose the anomalous behavioral changes causedby CUMS process This study provided new experimentalevidence for the antidepressant efficacy of COR in mousemodel of CUMS-induced depression

Chronic stress-induced depression model is a reliablemodel for studying depression and has been widely uti-lized in probing the pathological mechanism of depressionand screening antidepressant drugs [6] And CUMS modelcan mimic the core symptoms of depression includingdecreases in sucrose consumption and open field activitiesand increases in immobility durations (TST and FST) [7]

The sucrose preference test represents the anhedonia-likebehavioral change [24] Anhedonia wasmodeled by inducinga decrease in responsiveness to rewards reflected by a reducedconsumption of sucrose solutions it is a core symptom ofhuman major depression [25] In this study CUMS-exposedmice showed a reducedpreference of sucrose solution as com-pared to control group mice This result is in agreement with


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

80

60

40

20

0

Cros

sings

num

bers

Con

trol

CUM

S

Con

trol

CUM

S

Rear

ings

num

bers

30

20

10

0

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Figure 3 Effect of COR treatment on the number of crossings and rearings in the open field test All values given are the mean plusmn SEM119875 lt 005 and

119875 lt 001 versus control group lowast119875 lt 005 and lowastlowast119875 lt 001 versus CUMS-vehicle group

80

60

40

20

0

lowastlowast

lowastlowastlowastlowast

0 3 6

Time (weeks)

Imm

obili

ty ti

me i

n th

e tal

l sus

pens

ion

test

(s)


CUMS + FLU (15mgkg)

ControlCUMS

Figure 4 Effects of COR treatment on immobility time in thetail suspension test in mice Three times sucrose consumption testwas carried out at the end of 0-week 3-week and 6-week CUMSexposure All values given are the mean plusmn SEM


CUMS-vehicle group

previous findings that mice exposed to CUMS consumedless sucrose solution as compared to control group miceTreatment with COR significantly reversed this behavioralchange which represents the antidepressant-like effect ofCOR in CUMS model of depression

Open field test is widely used to evaluate locomotor andexploratory behaviors in experimental animals [18] in theopen field test CUMS mice exhibited decreased crossingand rearing which indicated reduced exploration and apathyrespectively in these animals COR has a significant amelio-rative effect on locomotor behavior in CUMS mice

Imm

obili

ty ti

mes

in F

ST (s

)

Con

trol

CUM

S

150

100

50

0

lowastlowast

lowastlowast

lowast

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Figure 5 Effect of COR treatment on the immobility times of micein the forced swimming test All values given are the mean plusmn SEM119875 lt 005 and

119875 lt 001 versus control group lowast119875 lt 005 andlowastlowast119875 lt 001 versus CUMS-vehicle group

The tail suspension test and forced swimming test arebehavioral despair tests and had been frequently used todetermine depressant-like behavior in rodents after exposureto stressThe immobility time of TSTFST reflects ldquobehavioraldespairrdquo as seen in human depressionThedata of this investi-gation showed that mice subjected to chronic stress exhibiteda significant prolongation of immobility time in TSTFSTChronic COR administration significantly decreased theduration of immobility in mice TSTFST which indicates theantidepressant-like effect

Several lines of evidence suggest that inflammation playsa role in the pathophysiology of major depression and thatanti-inflammatory drugs have antidepressant-like effects [26


Con

trol

CUM

S

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

50

40

30

20

10

0

80

60

40

20

0

IL-6

in h

ippo

cam

pus (

pgm

g tis

sue)

TNF-120572

in h

ippo

cam

pus (

pgm

g tis

sue)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Figure 6 Effects of COR on IL-6 and TNF-120572 levels in the hippocampus in CUMSmice All values given are the mean plusmn SEM 119875 lt 005 and119875 lt 001 versus control group lowast119875 lt 005 and lowastlowast119875 lt 001 versus CUMS-vehicle group

200

150

100

50

0

5-H

T (n

gg

tissu

e)

150

100

50

0

DA

(ng

g tis

sue)

Con

trol

CUM

S

Con

trol

CUM

Slowastlowast

lowastlowast



CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Figure 7 Effects of COR on 5-TH and DA levels in the hippocampus in CUMS mice All values given are the mean plusmn SEM 119875 lt 005 and119875 lt 001 versus control group lowast119875 lt 005 and lowastlowast119875 lt 001 versus CUMS-vehicle group

27]The data of this investigation showed that mice subjectedto chronic stress had increased NF-120581BP65 TNF-120572 andIL-6 levels in hippocampus Chronic COR administrationsignificantly decreased the duration of NF-120581BP65 TNF-120572and IL-6 in mice which indicates the antidepressant-likeeffect


involved in psychiatric disorders such as depression anxietysleep disorders and hallucinations The data of this inves-tigation showed that mice subjected to chronic stress hadincreased 5-HT

2AR Chronic COR administration signifi-cantly decreased the duration of 5-HT

2AR in mice whichindicates the antidepressant-like effect

BDNF a kind of the nerve growth factor possessesthe ability to support neuronal survival differentiation

function and plasticity [28] Many types of insults inducemodifications in brain BDNF expression and chronic stressapplication markedly reduces BDNF in hippocampus tissue[29] CUMS exposure was found to decrease BDNF inthe hippocampus of mice and COR treatments reversedthe CUMS-induced decreases in BDNF which provide amechanism of action for the antidepressant-like effect ofCOR

Our primary findings concluded that COR could improvethe depressive-like symptoms induced by CUMS that maybe related to regulation of hippocampal 5-HT

2AR and BDNFproteins expressions Hence we consider COR as a potentialantidepressant and its antidepressant activity is partiallymediated by the alteration of 5-HT

2AR and BDNF proteinsexpressions


GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM

SFigure 8 Effects of COR onNF-120581BP65 TNF-120572 IL-6 5-HT

2AR and BDNF proteins expressions in the hippocampus All values given are themean plusmn SEM 119875 lt 005 and

119875 lt 001 versus control group lowast119875 lt 005 and lowastlowast119875 lt 001 versus CUMS-vehicle group A Control B CUMSC CUMS + FLU (15mgkg) D CUMS + COR (20mgkg) and E CUMS + COR (40mgkg)

Conflict of Interests

The authors declare that they have no conflict of interests inthe matter and no direct financial relation with the commer-cial identity mentioned in this work

References

[1] M Peet B Murphy J Shay and D Horrobin ldquoDepletionof omega-3 fatty acid levels in red blood cell membranes ofdepressive patientsrdquo Biological Psychiatry vol 43 no 5 pp 315ndash319 1998

[2] R C Kessler P Berglund O Demler et al ldquoThe epidemiologyof major depressive disorder results from the national comor-bidity survey replication (NCS-R)rdquoThe Journal of the AmericanMedical Association vol 289 no 23 pp 3095ndash3105 2003

[3] C Lloyd ldquoLife events and depressive disorder reviewed IIEvents as precipitating factorsrdquo Archives of General Psychiatryvol 37 no 5 pp 541ndash548 1980

[4] M N Hill K G C Hellemans P Verma B B Gorzalka andJ Weinberg ldquoNeurobiology of chronic mild stress parallels tomajor depressionrdquo Neuroscience amp Biobehavioral Reviews vol36 no 9 pp 2085ndash2117 2012

[5] S Claes ldquoGlucocorticoid receptor polymorphisms in majordepressionrdquo Annals of the New York Academy of Sciences vol1179 pp 216ndash228 2009

[6] P Willner ldquoValidity reliability and utility of the chronic mildstress model of depression a 10-year review and evaluationrdquoPsychopharmacology vol 134 no 4 pp 319ndash329 1997

[7] R K Farooq E Isingrini A Tanti et al ldquoIs unpredictablechronic mild stress (UCMS) a reliable model to studydepression-induced neuroinflammationrdquo Behavioural BrainResearch vol 231 no 1 pp 130ndash137 2012

[8] A M Redmond J P Kelly and B E Leonard ldquoEffect ofparoxetine and fluvoxamine on behavioural changes in a num-ber of paradigms in the olfactory bulbectomized rat model ofdepressionrdquo Journal of Serotonin Research vol 1 no 3 pp 199ndash205 1994

[9] G Baxter G Kennett T Blackburn and F Blaney ldquo5-HT2receptor subtypes a family re-unitedrdquo Trends in Pharmacolog-ical Sciences vol 16 no 3 pp 105ndash110 1995

[10] P D Hrdina E Demeter T B Vu P Sotonyi and MPalkovits ldquo5-HT uptake sites and 5-HT2 receptors in brain ofantidepressant-free suicide victimsdepressives increase in 5-HT2 sites in cortex and amygdalardquo Brain Research vol 614 no1-2 pp 37ndash44 1993

[11] M Maes ldquoDepression is an inflammatory disease but cell-mediated immune activation is the key component of depres-sionrdquo Progress in Neuro-Psychopharmacology and BiologicalPsychiatry vol 35 no 3 pp 664ndash675 2011

[12] B Leonard and M Maes ldquoMechanistic explanations how cell-mediated immune activation inflammation and oxidative andnitrosative stress pathways and their sequels and concomitantsplay a role in the pathophysiology of unipolar depressionrdquoNeuroscience amp Biobehavioral Reviews vol 36 no 2 pp 764ndash785 2012

[13] H Kim A S Naura Y Errami J Ju and A H BoularesldquoCordycepin blocks lung injury-associated inflammation andpromotes BRCA1-deficient breast cancer cell killing by effec-tively inhibiting PARPrdquoMolecular Medicine vol 17 no 9-10 pp893ndash900 2011

[14] E-M Noh J-S Kim H Hur et al ldquoCordycepin inhibits IL-1120573-inducedMMP-1 andMMP-3 expression in rheumatoid arthritissynovial fibroblastsrdquo Rheumatology vol 48 no 1 pp 45ndash482009

[15] D-W Zhang Z-L Wang W Qi W Lei and G-Y ZhaoldquoCordycepin (31015840-deoxyadenosine) down-regulates the proin-flammatory cytokines in inflammation-induced osteoporosismodelrdquo Inflammation vol 37 no 4 pp 1044ndash1049 2014

[16] P Willner A Towell D Sampson S Sophokleous and R Mus-cat ldquoReduction of sucrose preference by chronic unpredictablemild stress and its restoration by a tricyclic antidepressantrdquoPsychopharmacology vol 93 no 3 pp 358ndash364 1987

[17] DD Luo S CAn andX Zhang ldquoInvolvement of hippocampalserotonin and neuropeptide Y in depression induced by chronicunpredicted mild stressrdquo Brain Research Bulletin vol 77 no 1pp 8ndash12 2008


[18] J C Capra M P Cunha D G Machado et al ldquoAntidepressant-like effect of scopoletin a coumarin isolated from Polygalasabulosa (Polygalaceae) in mice evidence for the involvementofmonoaminergic systemsrdquo European Journal of Pharmacologyvol 643 no 2-3 pp 232ndash238 2010

[19] L Steru R Chermat B Thierry and P Simon ldquoThe tailsuspension test a new method for screening antidepressants inmicerdquo Psychopharmacology vol 85 no 3 pp 367ndash370 1985

[20] R D Porsolt A Bertin and M Jalfre ldquoBehavioral despair inmice a primary screening test for antidepressantsrdquo ArchivesInternationales de Pharmacodynamie et deTherapie vol 229 no2 pp 327ndash336 1977

[21] K B J Franklin and L J Herberg ldquoNoncontingent displace-ment of catecholamines by intraventricular tyramine biphasicdose response effects on self stimulationrdquo Neuropharmacologyvol 16 no 1 pp 53ndash55 1977

[22] G Paxinos C Watson and P C Emson ldquoAChE-stainedhorizontal sections of the rat brain in stereotaxis coordinatesrdquoJournal of Neuroscience Methods vol 3 no 2 pp 129ndash149 1980

[23] J Qi J-Y Yang M Song Y Li F Wang and C-F Wu ldquoInhi-bition by oxytocin of methamphetamine-induced hyperactivityrelated to dopamine turnover in themesolimbic region inmicerdquoNaunyn-Schmiedebergrsquos Archives of Pharmacology vol 376 no6 pp 441ndash448 2008


[25] A Jindal RMahesh and S Bhatt ldquoEtazolate rescues behavioraldeficits in chronic unpredictable mild stress model modula-tion of hypothalamic-pituitary-adrenal axis activity and brain-derived neurotrophic factor levelrdquo Neurochemistry Interna-tional vol 63 no 5 pp 465ndash475 2013

[26] R Dantzer J C OrsquoConnor G G Freund R W Johnson andK W Kelley ldquoFrom inflammation to sickness and depressionwhen the immune system subjugates the brainrdquoNature ReviewsNeuroscience vol 9 no 1 pp 46ndash56 2008

[27] A H Miller V Maletic and C L Raison ldquoInflammation andits discontents the role of cytokines in the pathophysiology ofmajor depressionrdquo Biological Psychiatry vol 65 no 9 pp 732ndash741 2009

[28] E J Huang and L F Reichardt ldquoNeurotrophins roles inneuronal development and functionrdquo Annual Review of Neuro-science vol 24 pp 677ndash736 2001

[29] TNumakawaNAdachiM Richards S Chiba andHKunugildquoBrain-derived neurotrophic factor and glucocorticoids recip-rocal influence on the central nervous systemrdquo Neurosciencevol 239 pp 157ndash172 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Experimentalgroups

Week 0 Week 3 Week 6Killed

No CUMS

CUMS

Control

Model

COR

FLU

Time

CUMS + COR (20mgkg)

CUMS + COR (40mgkg)

CUMS + FLU (15mgkg)

mdash

Figure 1 The protocol in this study

sleep disorders and hallucinations [8 9] Increased densitiesof cortical 5-HT

2A receptors are observed upon postmortemexamination of depressed patients [10]

An increasing body of evidence presented in recent yearshas revealed that activation of the inflammatory response sys-tem plays a role in the pathophysiology of depression [11 12]Several studies reported increased levels of proinflammatorycytokines for example tumor necrosis factor-alpha (TNF-120572)and interleukin-6 (IL-6) in depressive disorders [11 12]

Cordycepin (31015840-deoxyadenosine) a major bioactive com-ponent isolated from Cordyceps militaris has multiple phar-macological activities such as anti-inflammatory andimmunomodulatory effects Cordycepin prevents lipopol-ysaccharide- (LPS-) induced airway neutrophilia in miceand effectively blocks LPS-induced expression of vascularadhesion molecule-1 (VCAM-1) in the human epithelialcell line A549 [13] Cordycepin inhibits interleukin-1120573-(IL-1120573-) induced matrix metalloproteinase-1 (MMP-1)and MMP-3 expressions in rheumatoid arthritis synovialfibroblasts (RASFs) and significantly inhibits AP-1 activation[14] While there is little evidence regarding the relevanceof COR on CUMS-induced depression in this study wehypothesized that COR would improve CUMS-induceddepression through regulation of r5-HT

2A receptor andBDNF

2 Materials and Methods

21 Animals Male ICR mice weighing 18ndash22 g (same batch)were purchased from Experimental Animal Center in JilinUniversity (Changchun China SCXK-0003) Prior to anyexperimentation the mice were allowed to have one weekto acclimatize to the laboratories And during the wholestudy the mice were housed in group in constant labora-tory conditions at a temperature of 22∘C and 60 relativehumidity under a 12 h light12 h dark cycle In our studyall the experimental procedures and laboratory animal carewere performed in accordance with the National Institutes ofHealth (NIH) Guide

22 Drug and Treatment COR was purchased from NanjingJiancheng Co Ltd Fluoxetine hydrochloride (positive con-trol drug) was obtained from Jinlin drugstore (ChanghunChina) COR and fluoxetine were dissolved in 003 sodiumcarboxymethyl cellulose (CMC-Na) Mice were randomlydivided into five different groups one control group oneCUMS-vehicle (003 CMC-Na) group one CUMS-FLU(15mgkg) group and three CUMS-COR (20 40mgkg)groups the doses selection of COR was according to Zhanget alrsquos report [15] Each group included 20 mice Everymorning all drug treatment groups were administered orallyvia intragastric gavage in a dose of 10mLkg body weight

23 CUMS Procedure The CUMS procedure was performedas described byWillner et al [16] with amodification Brieflymice in the CUMS groups were exposed to different stressorsnamely food deprivation water deprivation empty waterbottles (after water deprivation) cage tilt grouped housingsoiled cage stroboscopic lighting restricted access to food(only give a small amount of food after food deprivation)5 min cold swimming (at 15∘C) 1 min tail pinch (1 cm fromthe end of the tail) physical restraint illumination and whitenoise One of these stressors (in random order) was givenevery day for 6 weeks The control group mice were leftundisturbed except for necessary procedures such as routinecage cleaning and the protocol of the study can be seen inFigure 1

24 Sucrose Preference Test (SPT) Sucrose preference testwas carried out at the end of week 0 week 3 and week 6respectively The method was performed as previous report[17] In brief 72 h before the test a mouse was individuallyplaced into cage with two bottles of sucrose solution toadapt to sucrose solution (1 wv) for 24 h then one bottleof sucrose solution was replaced with water (24 h) afterthe adaptation laboratory mice were deprived of water andfood (24 h) Sucrose preference test was conducted at 930am Each mouse was housed in individual cage and wasfree to access two bottles containing 100mL of sucrose












3 Results




80

60

40

20

0

Sucr

ose p

refe

renc

e (

)

0 3 6

lowastlowast

lowastlowast

lowastlowast

Time (weeks)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group










2AR and BDNF

4 Discussion






lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

80

60

40

20

0

Cros

sings

num

bers

Con

trol

CUM

S

Con

trol

CUM

S

Rear

ings

num

bers

30

20

10

0

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)



80

60

40

20

0

lowastlowast


0 3 6

Time (weeks)

Imm

obili

ty ti

me i

n th

e tal

l sus

pens

ion

test

(s)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group



Imm

obili

ty ti

mes

in F

ST (s

)

Con

trol

CUM

S

150

100

50

0

lowastlowast

lowastlowast

lowast

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)






Con

trol

CUM

S

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

50

40

30

20

10

0

80

60

40

20

0

IL-6

in h

ippo

cam

pus (

pgm

g tis

sue)

TNF-120572

in h

ippo

cam

pus (

pgm

g tis

sue)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)


200

150

100

50

0

5-H

T (n

gg

tissu

e)

150

100

50

0

DA

(ng

g tis

sue)

Con

trol

CUM

S

Con

trol

CUM

Slowastlowast

lowastlowast



CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)













GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results




80

60

40

20

0

Sucr

ose p

refe

renc

e (

)

0 3 6

lowastlowast

lowastlowast

lowastlowast

Time (weeks)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group










2AR and BDNF

4 Discussion






lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

80

60

40

20

0

Cros

sings

num

bers

Con

trol

CUM

S

Con

trol

CUM

S

Rear

ings

num

bers

30

20

10

0

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)



80

60

40

20

0

lowastlowast


0 3 6

Time (weeks)

Imm

obili

ty ti

me i

n th

e tal

l sus

pens

ion

test

(s)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group



Imm

obili

ty ti

mes

in F

ST (s

)

Con

trol

CUM

S

150

100

50

0

lowastlowast

lowastlowast

lowast

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)






Con

trol

CUM

S

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

50

40

30

20

10

0

80

60

40

20

0

IL-6

in h

ippo

cam

pus (

pgm

g tis

sue)

TNF-120572

in h

ippo

cam

pus (

pgm

g tis

sue)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)


200

150

100

50

0

5-H

T (n

gg

tissu

e)

150

100

50

0

DA

(ng

g tis

sue)

Con

trol

CUM

S

Con

trol

CUM

Slowastlowast

lowastlowast



CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)













GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















80

60

40

20

0

Sucr

ose p

refe

renc

e (

)

0 3 6

lowastlowast

lowastlowast

lowastlowast

Time (weeks)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group










2AR and BDNF

4 Discussion






lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

80

60

40

20

0

Cros

sings

num

bers

Con

trol

CUM

S

Con

trol

CUM

S

Rear

ings

num

bers

30

20

10

0

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)



80

60

40

20

0

lowastlowast


0 3 6

Time (weeks)

Imm

obili

ty ti

me i

n th

e tal

l sus

pens

ion

test

(s)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group



Imm

obili

ty ti

mes

in F

ST (s

)

Con

trol

CUM

S

150

100

50

0

lowastlowast

lowastlowast

lowast

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)






Con

trol

CUM

S

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

50

40

30

20

10

0

80

60

40

20

0

IL-6

in h

ippo

cam

pus (

pgm

g tis

sue)

TNF-120572

in h

ippo

cam

pus (

pgm

g tis

sue)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)


200

150

100

50

0

5-H

T (n

gg

tissu

e)

150

100

50

0

DA

(ng

g tis

sue)

Con

trol

CUM

S

Con

trol

CUM

Slowastlowast

lowastlowast



CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)













GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

80

60

40

20

0

Cros

sings

num

bers

Con

trol

CUM

S

Con

trol

CUM

S

Rear

ings

num

bers

30

20

10

0

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)



80

60

40

20

0

lowastlowast


0 3 6

Time (weeks)

Imm

obili

ty ti

me i

n th

e tal

l sus

pens

ion

test

(s)


CUMS + FLU (15mgkg)

ControlCUMS



CUMS-vehicle group



Imm

obili

ty ti

mes

in F

ST (s

)

Con

trol

CUM

S

150

100

50

0

lowastlowast

lowastlowast

lowast

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)






Con

trol

CUM

S

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

50

40

30

20

10

0

80

60

40

20

0

IL-6

in h

ippo

cam

pus (

pgm

g tis

sue)

TNF-120572

in h

ippo

cam

pus (

pgm

g tis

sue)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)


200

150

100

50

0

5-H

T (n

gg

tissu

e)

150

100

50

0

DA

(ng

g tis

sue)

Con

trol

CUM

S

Con

trol

CUM

Slowastlowast

lowastlowast



CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)













GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

CUM

S

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

50

40

30

20

10

0

80

60

40

20

0

IL-6

in h

ippo

cam

pus (

pgm

g tis

sue)

TNF-120572

in h

ippo

cam

pus (

pgm

g tis

sue)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)


200

150

100

50

0

5-H

T (n

gg

tissu

e)

150

100

50

0

DA

(ng

g tis

sue)

Con

trol

CUM

S

Con

trol

CUM

Slowastlowast

lowastlowast



CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)













GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GAPDH

BDNF

A B C D E

5-HT2AR

GAPDH

05

04

03

02

01

BDN

FG

APD

H

06

04

02

00

06

04

02

00

06

04

02

00

00

Con

trol

CUM

S

Con

trol

CUM

S

Con

trol

Con

trol

CUM

S

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


08

IL-6

GA

PDH

A B C D E

TNF-120572

IL-6

P-NF-120581BP65

NF-120581BP65

P-N

F-120581

BP65

NF-120581

BP65

5-H

T 2A

RG

APD

H

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

CUM

S

Figure 8 Continued


lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowastlowast

lowastlowast

TNF-120572

GTA

PDH

05

04

03

02

01

00

CUM

S+

COR

(40

mg

kg)

CUM

S+

COR

(20

mg

kg)

CUM

S+

FLU

(15

mg

kg)

Con

trol

CUM






References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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