menoprogen, a tcm herbal formula for menopause, …...2 biomedresearchinternational one of the most...

Research ArticleMenoprogen a TCM Herbal Formula for MenopauseIncreases Endogenous E2 in an Aged Rat Model of Menopause byReducing Ovarian Granulosa Cell Apoptosis

Yu Li1 Hong Ma1 Ye Lu2 B J Tan3 L Xu1 Temitope O Lawal4

Gail B Mahady4 and Daniel Liu5

1Traditional Chinese Medicine Department Nanjing University of Chinese Medicine Nanjing 210046 China2Institute of Botany Chinese Academy of Science Nanjing Jiangsu 210014 China3Hainan Medical College Haikou Hainan 571101 China4Department of Pharmacy Practice College of Pharmacy University of Illinois at Chicago Chicago IL 60612 USA5Beijing Clinical Services Center No 103 Chaoyang North Road Beijing 100123 China

Correspondence should be addressed to Daniel Liu chuanlmsncom

Received 9 November 2015 Revised 12 January 2016 Accepted 17 January 2016

Academic Editor Rosaria Scudiero

Copyright copy 2016 Yu Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The effect ofMenoprogen (MPG) on ovarian granulosa cell (GC) apoptosis was investigated in vitro and in vivo in an aged ratmodelof menopause Intragastric administration of Menoprogen or estradiol valerate to 14-month-old senile female rats for eight weeksincreased plasma E

2levels as well as the weight of both ovarian and uterine tissues Flow cytometric (FCM) analysis of isolated GCs

from MPG-treated aged rats showed reductions in the G0G1ratio and apoptotic peaks Isolated GCs also exhibited an increase

in cell size and the number of cytoplastic organelles and intracellular gap junctions the reappearance of secretory granules and alack of apoptotic bodies as determined by TEM Results from a TdT-mediated dUTP nick end-labeling (TUNEL) assay revealed areduction in TUNEL-positive GCs afterMPG treatment Immunohistochemical analysis showed a downregulation of proapoptoticBax proteins and an upregulation of antiapoptotic Bcl-2 proteins The addition of MPG-medicated serum to the media of culturedGCs also reduced cadmium chloride-induced apoptosis and downregulated caspase-3 protein expressionThis work demonstratesthatMenoprogen inhibits GC apoptosis in aged female rats and thereby increases E

2productionThis represents a novelmechanism

of action for this herbal medicine in the treatment of menopausal symptoms

1 Introduction

Menopause is defined as the permanent cessation of men-struation (ge12 months) resulting from the loss of ovarianfollicular activity [1 2] It is preceded by themenopausal tran-sition (perimenopause) which is associated with erratic hor-mone levels and diverse clinical symptoms [3]The symptomsassociated with the menopause and its transition are due toalterations within the ovary of which the most prominentchange is a dramatic decline in follicle numbers due to ageing[4 5] At the cellular level ovarian follicle ageing andreproductive senescence are characterized by a decline inspecific functions of oocytes and granulosa cells (GCs) due

to generalized cellular dysfunction including a reduction inmitochondrial activity and energy failure as well as increasesin the expression of senescence genes and proteins [4 5]Thedegree of cellular decline that occurs with ageing is sufficientto increase the susceptibility of the ovarian follicles ovulatedoocytes and granulosa cells to apoptosis [4 5] and it is GCapoptosis that triggers follicular atresia [6] Thus apoptosisis a primary driving force behind follicle loss during ageingcausing the age-related decline in the function of oocytes andgranulosa cells [4ndash6] Since GCs are the primary source ofestradiol and progesterone synthesis in the ovary a reductionin their number and function leads to reduced levels of endo-genous hormones and hence menopausal symptoms [5 6]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 2574637 12 pageshttpdxdoiorg10115520162574637

2 BioMed Research International

One of the most problematic issues associated withdeclining ovarian function is that of lowered estrogen(E2) levels [1] The decline in E

2causes a neuroendocrine

imbalance and menopausal symptoms such as hot flashesheadaches nocturnal sweating vaginal atrophy and anxietyChronically lower E

2levels also lead to significantly higher

incidences of cardiocerebrovascular diseases osteoporosisand senile dementia in postmenopausal women [1] In thepast hormone therapy (HT) has been the ldquogold standardrdquofor the treatment of menopause However the use of HTworldwide has declined due to an increased risk of adverseevents including a transitory increased risk of heart diseaseand increased risk of endometrial or breast cancer [7] Onthe basis of the findings and risk analyses of the WomenrsquosHealth Initiative the US Preventive Services Task Force hasrecommended against the routine use of HT for the preven-tion of chronic conditions in postmenopausal women [7] Asa consequence many women have been looking for com-plementary and alternative medicines including TraditionalChinese Medicines (TCM) to manage their menopausalsymptoms

One TCM formula Menoprogen has been marketed asan herbal supplement in China and Europe for over 15 yearsand has been available in the USA for about ten years forthe management of menopausal symptoms [8 9] Meno-progen is an ancient TCM formula containing semipurifiedextracts of five well-known ldquoherbal foodrdquo plants whichhave been used as dietetic and medical herbs in China forthousands of years [8ndash11] The herbal formula is comprisedof extracts of the herbs Fructus Lycii (Lycium barbarum Lfruits Chinese goji berries Solanaceae) Semen Cuscutae(Cuscuta chinensis Lam ripe seeds Convolvulaceae) RadixRehmanniae (Rehmannia glutinosa Libosch roots Scrophu-lariaceae) Fructus Mori (Morus alba L white mulberryfruits Moraceae) and Flos Carthami (Carthamus tinctoriusL safflower Asteraceae) [8ndash11] In two pilot clinical trialsMenoprogen (MPG) was shown to be effective for the man-agement of menopausal symptoms and reduced the Kupper-man index in treated women [8 9] In animal studies oraladministration of MPG to aged female rats restored ovarianfunction [12 13] Furthermore oral administration of MPGto female rats also increased blood circulation to the ovaryand uterine tissues as well as improved the quality of ovarianfollicles [14] however the mechanism of action for MPG hasnot been fully elucidated In this work we investigate theeffects of Menoprogen in aged female rats and provide evi-dence that this herbal formula reduces GC apoptosis througha mechanism involving the Bcl-2Bax family of proteinsand caspase-3 The reduction in GC apoptosis increases theproduction of endogenous E

2 thereby demonstrating a novel

mechanism of action for this herbal formula

2 Materials and Methods

21 Drugs and Reagents The MPG herbal formula wasprovided by Nanjing Mayflower Pharmaceutical TechnologyCorporation Ltd (Nanjing China) and contains five Chineseherbal extracts as described above [8 9] An optimizedhigh-performance liquid chromatography-photodiode array

detection (HPLC-PDA) fingerprinting method for MPGwas developed and has shown that some of the primarychemical constituents of MPG are flavonoids [9] Progy-nova (estradiol valerate tablets) were used as a positivecontrol and were obtained from Schering SA CompanyFrance Pregnant mare serum gonadotropin (PMSG) waspurchased from Chuangrui Biological Reagent Company(Nanjing China) cadmium chloride from Tingxin ChemicalPharmaceutical Company (Shanghai China) glutaraldehydephosphate buffered solution (PBS) from Boster Company(Wuhan China) lead citrate trihydrate and uranyl acetatedyeing solution from China National Pharmaceutical GroupCorp (Shanghai China) PI dyeing solution from Sigma (StLouis MO USA) and fetal bovine serum fromGibco (USA)

The radioimmunoassay kit for the serum estrogen assaywas obtained from Diagnostic Systems Laboratories Inc(Webster TX USA) TdT-mediated dUTP nick end-labeling(TUNEL) kit was obtained from Roche Diagnostic GmbH(Sandhofer Mannheim Germany) immunochemical assaykit for Bax and Bcl-2 from Santa Cruz Biotechnology (SantaCruz CA USA) and enzyme-linked immunosorbent assay(ELISA) kit for caspase-3 from Biyuntian Biological ReagentCompany (Nantong China)

22 In Vivo Rat Model of Naturally Aged Female Rats (NAM)A total of 60 Sprague-Dawley female rats (SPF grade) andfeed were purchased from BampK Universal Group Limited(Shanghai China) All rats were fed an aseptic diet ad libitumunder controlled conditions 24∘C 50ndash55 humidity anda 12-hour lightdark cycle The animal care protocols wereapproved in accordance with the UK Animal Scientific Pro-cedures Act of 1986 and associated guidelines All protocolswere approved by the ACC prior to initiation of the researchstudy For two continuous estrous cycles fifty 14-month SDfemale rats were monitored by a smear test of exfoliated vagi-nal epithelial cells and the blood was drawn intraorbitally forthe determination of E

2in the serum Rats with continuously

irregular estrous cycles and a significant decrease in E2levels

were regarded as naturally aged model (NAM) The NAMrats were randomly divided into 5 groups NAM group (119899 =10)MPG low-dose treatment group (162mgkg bodyweight119899 = 10) MPG middle-dose treatment group (324mgkg119899 = 10) MPG high-dose treatment group (648mgkg 119899 =10) and positive estradiol valerate control (Progynova 119899 =10) group In addition 4-month-old female rats (119899 = 10)were selected as a normal control group Rats in the MPGtreatment groups were treated with an aqueous extract ofMenoprogen at equivalent clinical dosages (648 324 and162mgkg po resp) while rats in the positive control groupwere treated orally with estradiol valerate (Progynova dose018mgkg) All rats underwent a smear test of exfoliatedvaginal epithelial cells after 8-week treatment Followingtreatment after being weighed but before estrous cycle andblood sampling the rats were sacrificed after anesthetizationThe bilateral ovaries and uterus were removed and the wetweight was recorded The comparison of organ weight wasperformed using an organ coefficient which was calculatedaccording to following formula organ coefficient = organ wetweightbody weight times 100 One of the bilateral ovaries was

BioMed Research International 3

used for the analyses by transmission electron microscope(TEM) and flow cytometry (FCM) and another was fixedin 10 neutral formalin for 24 h gradually dehydrated byalcohol transparentized with xylene and then embeddedin paraffin for the TUNEL assay and the Bcl-2 and Baximmunohistochemical assay

23 Analysis of Serum E2 Serum samples were processed

by a centrifugation at 3000 rpm for 15min followed by themeasurement of E

2by radioimmunoassay (Diagnostic Sys-

tems Laboratories IncWebster TX USA) using the protocoldescribed by the manufacturer

24 Transmission Electron Microscopy (TEM) One to threeturbid follicles were removed from a unilateral ovary (soakedin saline) using an ophthalmic scissor and then the follicleswere pricked using a 28-gauge needle to drain follicle fluidsThe follicles were first fixed in 5 glutaraldehyde and then in1 PBS (pH = 74 4∘C) and were processed under gradientdehydration with acetone and ethanol and embedded usingEpon 812 and epoxy resins The embedded blocks weretrimmed and located by a repairing instrument and thensliced in 50 nm thickness by ultramicrotome The slices werepolymerized and stained with lead citrate and uranyl acetateThe observation was conducted and photographed using aTECNAI 12 TEM (Philips Netherlands)

25 Assessment of Apoptosis by Flow Cytometry (FCM) Newturbid follicles were excised from the remaining unilateralovary and were washed twice with sterile normal saline (NS)at 37∘C Granulosa cells were isolated from the medium(2ndash5mm diameter in size) antral follicles by fine-needleaspiration for FCM analysis as described The saline solutioncontaining the GCs was centrifuged at 1000 rpm for 10minThe pellet was rinsed twice with NS and fixed in 75 ice-cold ethyl alcohol After the fixed GCs were centrifuged at1000 rpm for 10min the supernatants were removed Thesediment was rinsed with NS and then resuspended in05mLNS and filtered through 400-mesh nylon membraneThe resulting solution was stained with propidium iodide indark at 4∘C for 30min and then assayed for DNA contents incells at each stage by FCM analysis The data was analyzed byCell Quest and ModiFit 20 (USA) and described in a DNAhistogram

26 Assessment of Apoptosis Using the TUNEL Assay TheTdT-mediated dUTP nick end-labeling (TUNEL) assay wasperformed using the protocol of themanufacturer (describedabove) Briefly paraffin slices in 4120583mthickness were dried for2 h in oven at 80∘CAfter conventional dewaxing the sampleswere set with citrate buffer (1 2000) in microwave oven for8min and washed with PBS (001M pH 74) three times for2min each time All slices were dried digested with proteaseK 50120583L for 15min washed by PBS and then dried Theslices were then placed in 01 triton at room temperaturefor 8min washed and dried again an enzyme solutionand a nucleotide reaction solution were added Having beensubsequently incubated at 37∘C for one hour the sliceswere washed dried and then incubated with enzyme-labeled

anti-fluorescence antibody at 37∘C for an additional 30minAfter DAB color development for 10min the reacted sliceswere then washed with distilled water transparentized withxylene sealed by resin adhesive and finally measured underlight microscope

27 Immunohistochemistry Paraffin slices were dewaxedtrimmed washed with PBS and sealed by 10 normal goatserum The slices were rinsed in buffer before the primarybcl-2 (Cat number M0887 clone 124 Dako CorporationCA 1 100 dilution) or bax (Cat number 18-0218 clone 2D2Zymed Laboratories Inc San Francisco CA 1 200 dilution)antibodies were overlaid at 37∘C as previously described[15] The reacted slices were washed and incubated with theimmunohistochemical kit MaxVisiontrade for additional 15minat room temperature according to the instructions of themanufacturer Counterstaining was then performed usingDAB (331015840-diaminobenzidine) which is the most commonlyused amine substrate for histochemical detection of cell sur-face and intracellular antigens on frozen or embedded tissuesections and routinely used to stain rodent tissue sectionsDAB solutionwas prepared by adding 5mLof double distilledwater (ddH

2O) 3 drops of Tris buffer (ca 150 120583L) 2 drops of

DAB solution (ca 100 120583L) and 2 drops of peroxide solutionand kept in the dark The DAB solution was applied to thesection and incubated for 3ndash5min under a cover of aluminumfoil The development of the color was then observed under amicroscope and immediately stopped by removing the DABand rinsing the section with ddH

2O for at least 1min The

sections were counterstained with hematoxylin andmountedwith xylene After DAB color development and hematoxylincounterstaining the slices became blue following washingwith distilled water After the gradient dehydration withalcohol the slices were transparentized by xylene and thensealed by resin adhesive

28 Preparation of Serum for In Vitro Model of GCs Meno-progen was administered daily to Sprague-Dawley rats (200plusmn20 g 119899 = 8) by gavage at a clinical dosage of 648mgkg bodyweight (representing 10 times the dose for an adult of 60 kgwhich equals the everyday therapeutic dosage of an adult timesanimal-human dosage exchange ratio times 10) [16] The controlgroup was treated with equal volume of saline each timefor three days After three days of treatment abdominalaortic blood was collected before the rats had fasted for 12hours Then blood samples were centrifuged for 15 minutesat 3000 rpm and inactivated in 56∘C for 30 minutes Non-medicated serum and medicated serums were sterilized bymicroporous membrane and stored at minus80∘C until used

29 In Vitro Induction of Apoptosis in Granulosa Cells (GCs)by Cadmium Chloride Cadmium chloride (CdCl) is a heavymetal that can induce cellular apoptosis via the caspase andBcl-2 pathways [17 18] We have used this heavy metal toinduce apoptosis in GCs and then determine the effect ofMPGonCdCl-induced apoptosisThree SD female rats at ageof 22ndash27 days were sacrificed 48 hours after injection with50 IU pregnant marersquos serum gonadotropin (PMSG) Thebilateral ovaries split from capsules and peripheral adipose


tissues were obtained and washed twice with normal salineIn the DMEM-F12 culture media containing 10 calf serumthe follicular membrane was broken by ophthalmologicalscissor to release the GCs [19] The cell number was countedusing a hemocytometer under phase-contrast microscopyand viability was assessed using trypan blue dye exclusionThe solution with GCs was stirred and filtered through400-mesh nylon filter membrane After centrifugation at1000 rpm for 5min the GCs solutions were rinsed twiceand resuspended in the culture media The GCs suspensionwas adjusted to 2 times 106 cellsmL in the culture media andincubated under 5 CO

2 at 37∘C for 2 hours Cells in

the low-dose group middle-dose group high-dose groupserum control group and model group were cultured withvarious concentrations ofMPG-containing serum (25 50and 100) normal mouse serum and 40 120583molL cadmiumchloride (CdCl) for 24 hours [19] respectively

210MTTAssay of CulturedGCs AnMTT (3-(45-dimethyl-thiazol-2-yl)-25-diphenyltetrazolium bromide) cell viabilityassay kit was used to measure GC proliferation (Sigma-Aldrich USA) After incubation under 5 CO

2at 37∘C for

24 h GCs (105 cells per well) were subsequently inoculatedonto 96-well plates After treatment for 24 h the supernatantwas discarded 100120583L DMSO was added the 96-well platewas mixed on microvibrator for additional 10min and theoptical density of each well was measured at 120582570 nm usingan enzyme-immunoassay plate reader (Biorad USA) Theexperiment was performed in triplicate and an average valuewas used to determine the final result after the measurementwas repeated three times

211 TEM of Cultured GCs For TEM observation culturedGCs were collected and fixed with 2 (vv) glutaraldehydein PBS for 2 h and then with 2 (vv) osmium tetroxidein phosphate buffer for additional 2 h Thin sections wereprepared and stained as described in a previous study [20]The sections were observed using a JEM1230 TEM (JEOLUSA Peabody MA)

212 Assessment of Apoptosis in Cultured GCs by Flow Cytom-etry Cultured GCs were fixed in 75 ice ethyl alcohol andcentrifuged for 10min at 2000 rpm After the supernatantwas discarded the pellets were rinsed twice with NS andthen resuspended in 05mL NS The suspension was filteredthrough 400-mesh nylon filtermembrane and stainedwith PIdyeing solution in dark for 30min FCM was used to analyzethe DNA content of cells at different stages and data wasanalyzed as described above

213 Caspase-3 Analysis in Cultured GCs The Capase-3kit was used according to the manufacturerrsquos instructions(Biyuntian Nantong China) Briefly a 119901-nitroanilide (pNA)standard curvewas generated then increasing concentrationsof the drug-containing serum and CdCl were added to thecultured GCs After 2 hours of treatment the cells weredigested and centrifuged (1000 rpm 10min) after 24 h super-natant was removed while the cell pellet was washed twicewith PBS and centrifuged again The pellets were suspended

Table 1 Effects of Menoprogen and estradiol valerate (Progynova)on ovarian and uterine weight of aged rats (119909 plusmn sd)

Rat group Ovarian coefficient (10minus1)a Uterine coefficient(10minus1)a

Normal control 279 plusmn 038lowastlowast 206 plusmn 055lowastlowast

NAM 215 plusmn 046 147 plusmn 023Low-dose 270 plusmn 034lowastlowast 172 plusmn 025lowast

Middle-dose 256 plusmn 037lowast 199 plusmn 023lowastlowastlowast

High-dose 263 plusmn 037lowast 197 plusmn 048lowastlowast

Progynova 251 plusmn 058 194 plusmn 053lowastlowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus NAM group

aOrgan coefficient = organ wet weightbody weight times 100

in the lysate solution at a ratio of 100120583L lysate to 2 millioncells The GCs were then lysed in an ice bath for 15minThe supernatants which were obtained by centrifugation at14000 rpm at 4∘C for 15min were placed on ice bath forfurther analysis The caspase-3 concentration was measuredusing the protocol described by themanufacturer After colordevelopment the OD value wasmeasured at 405 nm using anenzyme immunoassay plate reader The concentrations werederived from the predefined standard curve The immuno-chemical assay kits for Bax and Bcl-2 (described above) wereused according to the instructions of the manufacturer

214 Statistical Analysis All of the data were expressed asmean (119909) and standard deviation (sd) and were analyzedusing one-way ANOVA followed by Dunnettrsquos test for thepost hoc analysis using the statistical software SPSS 120 (CAUSA)

3 Results

31 Effects of Menoprogen on Ovarian and Uterine Weightsof Aged Female Rats (NAM) Compared with normal four-month-old control female rats the 14-month-old NAMrats exhibited ovarian and uterine atrophy and exhibitedsignificant decreases in the ovarian and uterine coefficients(described in Section 2) respectively (Table 1) Both theovarian and uterine coefficients in Menoprogen-treated agedrats significantly increased while only the uterine coefficientincreased in the Progynova-fed rats

32 Menoprogen Enhances Serum E2Concentrations As

shown in Table 2 the serum E2concentrations of the 14-

month-old NAM rats were significantly lower than that of 4-month-old control rats indicating a decline of reproductiveendocrine functions that is commonly observed duringthe natural ageing process The serum E

2levels showed

significant increases after oral administration of Progynovaor Menoprogen to the NAM rats (Table 2)

33 Effects of Menoprogen on Ovarian GC Ultrastructure fromNAM Rats A series of electron micrographs showing theovarian GC ultrastructure pattern as observed under TEMare presented in Figure 1 As seen in Figure 1(a) the nucleus


(a) (b)

(c) (d)

Figure 1 The effects of Menoprogen and Progynova on ovarian GC ultrastructure (a) Euchromatins were observed in the normal controlgroup and all cell organs were morphologically normal (b) In the NAM group GC nuclei exhibited smaller cell bodies concentratedchromatins and apoptotic cells (c) GCs in the Menoprogen group also had increased euchromatins as compared with the NAM controlrats cellular organs were increased and there were no apoptotic bodies observed (d) In the Progynova group the GCs displayed primarilyeuchromatin and a few apoptotic bodies were observed

Table 2 Effects of treatments on serum E2levels in NAM rats

as compared with normal 4-month-old female rats and untreatedNAM rats (119909 plusmn sd) (119899 = 10 per arm)

Rat group E2(pgmL)

Before treatment After treatmentNormal control 7280 plusmn 2829 6978 plusmn 1968lowastlowastlowast

NAM (untreated) 3823 plusmn 1150lowastlowast 3968 plusmn 1332Low-dose 4517 plusmn 1563lowastlowast 6641 plusmn 1427lowastlowastlowast

Middle-dose 4881 plusmn 2763lowastlowast 9250 plusmn 2388lowastlowastlowast

High-dose 4084 plusmn 1017lowastlowast 7050 plusmn 934lowastlowastlowast

Progynova 4201 plusmn 1307lowastlowast 7136 plusmn 896lowastlowastlowastlowastlowast119875 lt 001 versus normal control group lowastlowastlowast119875 lt 0001 versus NAM group

of GCs isolated from normal control rats is round or ovaland contained more euchromatin indicating higher genetranscriptionThese normalGCs also exhibited an increase invesicle-like smooth endoplasmic reticulum (SER) scatteringdistribution ofmitochondriawith normal tubular cristae andmore Golgi complexes and round lipid droplets

In contrast to the normal controls the nucleus ofGCs from NAM rats exhibited more heterochromatin withmarginal concentration agglomeration and pyknosis increscent or fragment and higher nucleocytoplasmic ratio(Figure 1(b)) The abnormally concentrated cytoplasms con-tained fewer SERs and mitochondrion smaller mitochon-drion disorganized structures of Golgi complexes highernumber of apoptotic cells and bodies and few secretory gran-ules Furthermore there was a broadening of sinusoidal gapsand certain myelin-like deformation between and withinGCs were found (Figure 1(b))

The ovarian GC nuclei from the Menoprogen-treatedNAM rats exhibited increased quantities of euchromatinand cellular organelles including more SERs with rounddilation redundant mitochondria and more normally struc-tured Golgi complexes Some secretory granules and sparseapoptotic cells were seen but no apoptotic bodies wereobserved (Figure 1(c))

The GC nucleus of Progynova-fed rats primarily dis-played euchromatin abundant cytoplasmic mitochondriaSERs Golgi complexes and secretory granules and a fewof apoptotic cells and apoptotic bodies Moreover more


(a) (b)

(c) (d)

Figure 2 Effects of Menoprogen on GC apoptosis using the TdT-mediated dUTP nick end-labeling (TUNEL) assay Apoptotic cells weredetected as TUNEL-positive cells (brown staining) (a) Light microscopy showed many developing follicles at different stages and corporalutea in normal control group (b) In the NAM group there were more atretic follicles and apoptotic expression was prominent (c d) Therewere more developing follicles at different stages in the Menoprogen-treated (c) group and the Progynova group (d) (200x)

Table 3 Effects of Menoprogen on GC cell cycle (119909 plusmn sd) (119899 = 10rats per arm) as determined by FCM

Rats group G0G1

S G2M

Normal control 8260 plusmn 456lowast 980 plusmn 283lowast 840 plusmn 200lowast

NAM 9118 plusmn 144 470 plusmn 207 413 plusmn 119Low-dose 8772 plusmn 129 1108 plusmn 468lowast 378 plusmn 110Middle-dose 8365 plusmn 157lowast 807 plusmn 196lowast 486 plusmn 102High-dose 8707 plusmn 097lowast 596 plusmn 236 527 plusmn 275Progynova 8883 plusmn 366 767 plusmn 124 461 plusmn 075lowast119875 lt 005 versus NAM group

cytoplasmic residual vacuoles and wider sinusoidal gapbetween GCs were observed (Figure 1(d))

34 Effects of Menoprogen and Estradiol Valerate on theGC Cell Cycle Isolated ovarian GCs from untreated anduntreated NAM rats were subjected to FCM The resultsdemonstrated that when compared with the untreated NAMrats both the normal control animals andMPG-treatedNAMrats (treated with the middle or high dose of MPG or E

2) had

a lower percentage of GC cells in the G0G1stage (Table 3) In

addition more GCs were in the S stage in normal control andMPG-treated rats (low andmiddle dose) More GCs from thenormal control rats were in the G

2M phase of the cell cycle

(Table 3)

35 TUNEL and TEM Assessment of Follicles TUNEL andTEM assessment of follicles from normal control rats showedmany developing follicles at different stages and corporalutea including large mature follicles full of follicular fluids(Figure 2(a)) Protuberant oocytes shaped cumulus oophorusin follicular cavity and the follicles were hunching on theovarian surface The NAM rats had fewer developing folliclesand corpora lutea but more atretic follicles (Figure 2(b))Apoptotic expression in atretic follicles and corpora lutea wasmuch more prominent in the untreated NAM rats than thoseof the normal control rats The MPG-treated rats showedan increased restoration of developing follicles at differentstages and numbers of corpora lutea and less atretic follicles(Figure 2(c)) The Progynova-treated NAM rats exhibitedhigher quantities of both developing follicles and atreticfollicles (Figure 2(d))

36 Effects ofMenoprogen on Expression of BaxBcl-2 ProteinsBcl-2 is an antiapoptotic gene and its protein is presentas a buffy particle with strong expression in normal cellsbut is present in very low concentrations or absent in thecytoplasm of apoptotic cells [21] A significant increase inthe positive expression of Bcl-2 proteins (darker color) wasobserved in the GCs of normal control rats (Figure 3(a))the Menoprogen group (Figure 3(c)) and Progynova group(Figure 3(h)) compared with the negative expression inNAM group (Figure 3(b)) Moreover Bcl-2 expression in theProgynova group was higher than in the Menoprogen group


(a) (b) (c)

(d) (e) (f)

(g) (h)

Figure 3 Effects ofMenoprogen on expression of Bcl-2 and Bax proteins (a)There was higher Bcl-2 protein expression in the normal controlgroup (b) and lower Bcl-2 expression in the untreated NAM group (c) The expression of Bcl-2 was increased in NAM rats after treatmentwith Menoprogen (d) When compared with the Menoprogen treated rats the expression of Bcl-2 was higher in the Progynova treated rats(e) In addition the normal control group showed a weak expression of Bax protein (f) while the untreated NAM group showed stronglypositive expression of Bax (g h)The expression of Bax protein decreased in both the Menoprogen treated (g) and the Progynova treated (h)groups (200x)

showing that Progynova reduced GC apoptosis better thanMenoprogen (Figure 3(d)) Bax is a proapoptotic moleculeand Bax proteins are more highly expressed in apoptoticcells than in normal cells [21] In this study the GCs fromnormal control rats weakly expressed Bax proteins as shownin Figure 3(e) by a lighter color The NAM rats showed astrong positive expression of Bax as shown with a darkercolor (Figure 3(f))The expression of Bax proteinswasweaklyobserved in both MPG-fed (Figure 3(g)) and Progynova-treated rats (Figure 3(h)) respectively However Bax proteinexpression in the Progynova-treated rats declined more thanin the Menoprogen-treated animals again suggesting thatestrogen valerate was slightly better at reducing apoptosisthan MPG

37 Effects of MPG-Medicated Serums on CdCl-Induced Apop-tosis inCulturedGCs Theoptical density (OD)measurementof GCs isolated and cultured from the NAM group wassignificantly lower than that of the GCs isolated from normalcontrol rats or those treatedwithMPG respectively (Table 4)The results indicate that serum from MPG-fed rats (atthe three different doses) protects isolated rat ovarian GCsfrom CdCl-induced apoptosis Based on the most optimallyprotective effect the middle dose of MPG was used as themedicated serum sample in further experiments

38 Effect of Menoprogen Treated Serum on Cultured GCUltrastructure As assessed by TEM the GCs had a nor-mal ultrastructure in normal control group (Figure 4(a))


(a) (b) (c)

Figure 4 The in vitro effect of Menoprogen-medicated serum on GC ultrastructure (a) The GCs showed a normal ultrastructure in normalcontrol rats (b) In the MPG-medicated serum group both cellular organelles and secretary granule were enriched (c) In the NAM groupit was observed that chromatin within nucleus was in shifting and there was marginal aggregation endocytoplasmic reticulum becamevacuolated and the number of secretary granules was decreased

Table 4 The effect of serum from Menoprogen-fed rats on in vitroGC apoptosis (119909 plusmn sd) (119899 = 8 per arm) using the MTT method Thefinal results were performed in triplicate and repeated three times

Rat group Optical densityNormal control 054 plusmn 004lowastlowast

NAM 045 plusmn 004Low-dose medicated serum 062 plusmn 007lowastlowast

Middle-dose medicated serum 072 plusmn 007lowastlowast

High-dose medicated serum 051 plusmn 004lowastlowastlowastlowast119875 lt 001 versus NAM group

However the membranes fromGCs obtained from untreatedNAM rats showed declining or fading microvilli and visiblefoaming phenomena In addition the chromatin within thenucleus showed marginal aggregation with partial fragmentsor pyknosis The endocytoplasmic reticulum was vacuo-lated and the number of secretary granules was reduced(Figure 4(c)) When the GCs were cultured with the MPG-medicated serum the nucleus exhibited primarily euchro-matin suggesting an increase in active gene transcriptionThe cellular organelles and secretory granules were enrichedand the tumidity of endocytoplasmic reticulum was alsoreduced (Figure 4(b))

39 FCMAnalysis of GCApoptosis after Treatment withMPG-Medicated Serum In Figures 5(a)ndash5(c) the large red peakrepresents the G

1peak and the blue-green peak is the sub-

G1peak In theDNAhistogram the sub-G

1presenting before

the G1peak is considered the apoptotic peak In GCs isolated

from normal control rats FCM analysis shows a very smallapoptotic peak and only one main G

1peak in the GCs

of normal control rats (Figure 5(a)) However GCs isolatedfrom NAM rats show a large apoptotic peak (Figure 5(b))

Table 5 Effect of MPG-medicated serum on caspase-3 proteinexpression in rat GCs (120583molL) (119909 plusmn sd) (119899 = 3)

Rat group Caspase-3Normal control 7255 plusmn 170lowast

NAM rats 8214 plusmn 345Menoprogen-fed serum 5646 plusmn 749lowastlowast119875 lt 005 versus model group

which decreased significantly (gt50 reduction) after theaddition of the MPG-medicated serum to the cultured GCs(Figure 5(c))

310 Effects of MPG-Medicated Serum on Rat GC Caspase-3 Protein Expression Caspase-3 protein (C3P) is a memberof the cysteine-aspartic acid protease (caspase) family andplays a central role in cellular apoptosisThe expression of theC3P was significantly higher in the untreated NAM rats butC3P levels were significantly reduced in cultured GCs aftertreatment with MPG-medicated serum (Table 5)

4 Discussion

As women worldwide look for alternatives to hormone ther-apy to manage their menopausal symptoms many questionsarise as to the safety and efficacy of these herbal therapiesas well as their potential mechanisms of action [22] Whilemany plausible mechanisms of action for herbals used formenopause have been proposed such as the ldquophytoestro-genrdquo hypothesis the mechanisms of action for most herbalmedicines have remained elusive [22]

The Menoprogen herbal formula tested in this study isan ancient TCM formula that has been used for the man-agement of menopausal symptoms in China for many years


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Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)

Figure 5 Effect of Menoprogen-medicated serum on peak of CdCl-induced GC apoptosis in vitro (a) FCM analysis displayed a smallapoptotic peak in the cultured GCs isolated from the normal control rats (b) an obvious apoptotic peak in the GCs isolated from the NAMmodel and (c) a gt50 reduction in peak size after addition of MPG-medicated serum

[8 9] Two preliminary clinical trials have demonstrated thatthis formula reduced the Kupperman index in menopausalChinese women and increased endogenous E

2levels [8 9]

However its mechanism of action has remained unknownPrevious animal studies have shown that MPG increasesendogenous E

2levels in rats but further ER-binding studies

have shown that MPG does not have direct estrogenic effectsin that it does not act as a phytoestrogen [14 23 24] Sinceovarian granulosa cells are the primary source of estradiol inmammals we hypothesized that themechanismof this herbalformula may directly involve the ovary and granulosa cellsapoptosis

Granulosa cells play a crucial role in female reproductionby synthesizing sex hormones including E

2 as well as

interacting with the oocytes [4 5] Granulosa cells surroundthe oocyte and play an important role in its developmentReducing GC apoptosis favors follicular survival [25 26]However as women age there is a significant reductionin both follicular number and function through follicularatresia leading to reduction in E

2levels and culminating

in the menopause [1] It is this significant reduction of E2

levels that precipitates vasomotor and other menopausalsymptoms Since apoptosis is the driving force behind follicleatresia during ageing and causes an age related decline in


the function of oocytes and granulosa cells [4ndash6] we hypoth-esized that MPG may have antiapoptotic effects on ovarianGCs and thereby improve the function and E

2producing

capacity of these cellsOur results demonstrated that when compared with the

normalmorphology observed in theGCs isolated fromyoungfemale rats the nuclei of GCs from NAM rats exhibitedsmaller cell bodies and more concentrated heterochromatinsand apoptotic cells These results were consistent with otherstudies in mammalian species showing GC apoptosis as aresult of ageing [6 25] There was a significant reductionin GC apoptosis in NAM rats treated with varying doses ofMPG or estradiol valerate and more abundant and maturesmooth endocytoplasmic reticulum (SERs) were apparentin the GCs of MPG-treated rats As a comparison onestudy involving young women with active estrogen synthesisshowed that their GCs typically featured steroidal-secretingcells that is less rough endocytoplasmic reticulum (RER)and more SERs as well as more and larger mitochondria[27] Furthermore in the TUNEL assay untreated NAM ratsshowed a greater number of atretic follicles as well as moreprominent TUNEL-positive GCs indicating a significantlevel of GC apoptosis However in the MPG and estradiolvalerate treated groups there were more developing folliclesat different stages indicating a significant reduction in GCapoptosis The results also showed an abundance of SERswith evident dilation as well as increased numbers and largermitochondria in theGCcytoplasmandhigher E

2levels in rats

treated withMPGor ProgynovaThese data demonstrate thatthe reduction of GC apoptosis by both treatments involvesnot only an improvement inGCmorphology but also in theirfunction as there is an increase in E

2synthesis and secretion

Further in vitro data supported the in vivo studies andshowed thatMPG treatments also reducedCdCl-inducedGCapoptosis CdCl was used as a pathologicalmodel based on itsability to induce GC apoptosis and cause harmful effects onthe mammalian reproductive system [28] Thus in additionto the management of menopausal symptoms MPG may beuseful for the treatment of women who have been exposed toreproductive toxins

Apoptosis and the regulation of the cell cycle are asso-ciated with key control points during different phases ofcell cycle (G

1 S G

2 and M) [29] It has previously been

reported that several Traditional Chinese Medicines (TCM)alter apoptosis in various cell lines through the regulationof the cell cycle [30ndash32] When we compared untreatedNAM rats with those treated with MPG or Progynovathe treated animals exhibited significant decreases in thenumber of GCs in the G

0G1stage of the cell cycle and also

showed a progression from G0G1into S stage indicating

increased cell division In the TUNEL assay the observedincrease in apoptotic expression of GCs from NAM ratswas almost identical to that observed in other studies [33]further supporting the relationship between follicular atresiaand apoptosis Taken together the data demonstrate thattreatment of the NAM rats with MPG or estradiol valeratereducesGC apoptosis and thereby improves the structure andfunction of GCs (endogenous E

2levels are increased after

treatment)

Inasmuch as apoptosis is a highly regulated process inwhich cells activate signaling pathways that lead to pro-gramed cell death we also investigated some of the signalingfactors well known to be involved in apoptosis namely theBcl-2 and caspase family proteins [6 34] The Bcl-2 familyproteins are located in the outer mitochondrial membranethe endocytoplasmic reticulum membrane and the nuclearmembrane and are key regulators of apoptosis whose actionsite is the mitochondrial membrane [6 34 35] This familyincludes the antiapoptotic proteins Bcl-2 and Bcl-x with Bcl-2 being the first cell survival factor discovered in mammals[34 35] A reduction of Bcl-2 expression results in cell apop-tosis thereby demonstrating that it plays a critical role in itsregulation [34] Sasson et al [35] also found that Bcl-2 levelswere up to 3ndash76 times higher when humanGC apoptosis wasinhibited by corticosteroids They further demonstrated thatinhibition of Bcl-2 leads to the inhibition of GC apoptosis aswell as the stimulation of follicular growth and ovulation byBcl-2 [35] In another study Kugu et al [36] demonstrated anincrease in the expression of the proapoptotic protein Baxin the GCs of atretic follicles at an early stage but a lowerexpression of Bax in normal follicles or in old atretic follicleslinking the stimulation of Bax proteins to the early stages ofGC apoptosis in atretic follicles [6 36] In addition the ratioof Bcl-2 toBax in the dimer impacts the apoptotic status If theratio of Bcl-2 gtBax the dimer prefers cell survival if the ratioof Bcl-2 lt Bax the apoptosis would be dominant [37 38]In this work we demonstrate that in vitro cultured GCstreated withMPGor estradiol valerate (Progynova) exhibitedan increased expression of Bcl-2 proteins and reduced theexpression of proapoptotic Bax proteins thereby shifting thebalance to favor GC survival

Finally in addition to Bcl-2 family of proteins thecaspases are also known to play a central role in the process ofapoptosis and are members of the cysteine-aspartic acid pro-tease (caspase) family [39] During apoptosis after the releaseof cytochromeC from themitochondria apoptosis activatingfactor and caspase-9mediate the signal that activates caspase-3 [6] The activation of caspase-3 leads to the alteration inthe expression of the Bcl-2 family proteins [6]The sequentialactivation of caspases is critical for the execution phase ofcell apoptosis in GCs [39] Several previous reproductiveendocrinology studies have demonstrated that there is aninvolvement of caspase proteins in GC apoptosis [39 40] Forexample activated caspase-3 is present in oocytes and GCsof rats and plays a role in GC apoptosis and follicular atresia[39 40] Lzawa et al [41] confirmed the presence of genomictranscripts and precursor proteins of caspase-3 in GCs byPCR and Western blotting Our results found a significantincrease of caspase-3 levels in the GCs isolated from NAMrats as compared with normal young rats further supportingthe relationship between caspase-3 and GC apoptosis Basedon our results showing a decrease in the caspase-3 concentra-tion inGCs fromMenoprogen-treated animals it is suggestedthat Menoprogen may reduce GC apoptosis by blocking thecaspase cascade and altering the expression of Bcl-2 and Baxproteins

In terms of going forward with future clinical studies theresults presented here indicate that the low or middle doses


of 162ndash324mg may have a more optimal effect than the highdose which is not uncommon for herbal medicines The lowdose had a more significant effect on the uterine and ovariancoefficients as well as increasing the number of GCs in theS phase of the cell cycle while the middle dose had the besteffect on E

2levels Since the increase of endogenous E

2would

most likely lead to menopausal symptom reduction the useof the middle dose would likely be logical Further supportfor the use of the middle dose comes from a previous clinicalstudy that treated menopausal women with 200mg of MPGand demonstrated a significant reduction in the KMI andmenopausal symptoms as well as increases in serum E

2and

P4 [8] However this study was not a randomized controlledclinical trial (RCT) and a larger RCT should be performed todetermine safety and efficacy

In conclusion Menoprogen a TCM formula for treatingmenopausal symptoms appears to have a novel mechanismof action in that it is not directly estrogenic but instead worksindirectly by reducing GC apoptosis which in turn improvesthe function of GCs thereby enhancing their capacity to syn-thesize and secrete E

2 Unlike estradiol valerate Menoprogen

had no direct estrogenic effects and does not stimulate theproliferation of breast cancer cells in vitro [23] Thus thisrepresents a very novel mechanism of action for this herbalformula

Disclaimer

The contents are solely the responsibility of the authors anddo not represent the official views of the NCCIH or NIH

Conflict of Interests

The authors have no conflict of interests connected to thework reported in this paper

Acknowledgments

This study was supported by a project funded by the PriorityAcademic Program Development (PAPD) of Jiangsu HigherEducation Institutions supported by the Open ProjectProgram of Traditional Chinese Medicine Department ofNanjing University of Chinese Medicine Nanjing 210046China (YS2012ZYX304) supported by the Fund of Ministryof Science Technology of China for Special Project beforeMajor Basic Research (no 2003CCA01700) This publicationwas supported in part by Grant no R21-AT02381 from theNational Center for Complementary and Integrative Health(previously NCCAM) at NIH The authors would like tothank Dr M Y Wang for his assistance in this work

References

[1] E B Gold ldquoThe Timing of the age at which natural menopauseoccursrdquoObstetrics andGynecology Clinics of North America vol38 no 3 pp 425ndash440 2011

[2] S D Harlow and P Paramsothy ldquoMenstruation and themenopause transitionrdquoObstetrics ampGynecology Clinics of NorthAmerica vol 38 pp 595ndash607 2011

[3] J C Prior ldquoPerimenopause the complex endocrinology of themenopausal transitionrdquo Endocrine Reviews vol 19 no 4 pp397ndash428 1998

[4] A L Johnson ldquoIntracellular mechanisms regulating cell sur-vival in ovarian folliclesrdquo Animal Reproduction Science vol 78no 3-4 pp 185ndash201 2003

[5] C Tatone F Amicarelli M C Carbone et al ldquoCellular andmolecular aspects of ovarian follicle ageingrdquoHuman Reproduc-tion Update vol 14 no 2 pp 131ndash142 2008

[6] F Matsuda N Inoue N Manabe and S Ohkura ldquoFolliculargrowth and atresia in mammalian ovaries regulation by sur-vival and death of granulosa cellsrdquo Journal of Reproduction andDevelopment vol 58 no 1 pp 44ndash50 2012

[7] J E Rossouw G L Anderson R L Prentice et al ldquoRisks andbenefits of estrogen plus progestin in healthy postmenopausalwomen principal results from the womenrsquos health initiativerandomized controlled trialrdquo The Journal of the AmericanMedical Association vol 288 no 3 pp 321ndash333 2002

[8] D L Liu Y Lu H Ma et al ldquoA pilot observational study toassess the safety and efficacy of menoprogen for the manage-ment of menopausal symptoms in Chinese womenrdquo Journal ofAlternative amp ComplementaryMedicine vol 15 no 1 pp 79ndash852009

[9] G L Wang M Wei J Wang Y Lu G B Mahady and D LiuldquoHigh-performance liquid chromatography with photodiodearray (HPLC-PAD)quality control ofmenoprogen a traditionalChinese medicine (TCM) formula used for the management ofmenopauserdquo International Journal of Medicinal Plants Researchvol 2 pp 146ndash151 2013

[10] HMa Y Lu H Sun andHWang ldquoClinical research of Lichuncapsule on treating perimenopausal syndrome in 74 casesrdquoTianjin Journal of TCM vol 19 pp 55ndash56 2002

[11] M Wei S Z Zheng Y Lu D Liu H Ma and G B MahadyldquoHerbal formula menoprogen alters insulin-like growth factor-1 and insulin-like growth factor binding protein-1 levels in theserum and ovaries of an aged female rat model of menopauserdquoMenopause vol 22 no 10 pp 1125ndash1133 2015

[12] J Wu L Xu and H Ma ldquoEffect of ningxin hongqi capsule onfunction of pituitary gland and ovary axis of female senile ratsrdquoJournal of Nanjing University of Traditional Chinese Medicinevol 23 pp 33ndash35 2007

[13] L Xie R S Lai L Wang H Ma and Y Lu ldquoRegulatory effectof Ningxin Hongqi capsule on local ovarian autocrine andparacrine factors in rats during peri-menopausal periodrdquo Chi-nese Journal of Integrated Traditional andWesternMedicine vol28 no 3 pp 242ndash244 2008

[14] H Ma M H Chung Y Lu T Nishihara and M HattorildquoEstrogenic effects of the herbal formula menoprogen inovariectomized ratsrdquo Biological amp Pharmaceutical Bulletin vol33 no 3 pp 455ndash460 2010

[15] L-Y Khor M Desilvio R Li et al ldquoBcl-2 and bax expressionand prostate cancer outcome in men treated with radiotherapyin RadiationTherapyOncology Group protocol 86-10rdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 66 no1 pp 25ndash30 2006

[16] X-L Tong J Song L-H Zhao and H-Y Ji ldquoKaiyuqingreformula improves insulin secretion via regulating uncouplingprotein-2 and kATP channelrdquo Chinese Medical Journal vol 124no 17 pp 2746ndash2750 2011


[17] A A Baiomy and A A Mansour ldquoGenetic and histopatho-logical responses to cadmium toxicity in rabbitrsquoskidney andliver protection by ginger (Zingiber officinale)rdquo Biological TraceElement Research 2015

[18] H M Jia W L Zhang X Chen and J L Jiang ldquoEffects ofcadmium on the progesterone synthesis in rat ovary granulosacellsrdquo China Preventive Medicine vol 8 pp 345ndash348 2007

[19] Y Li H Ma and M Y Wang ldquoThe effect of ovarian granulosacell apoptosis with cadmiumrdquo Chinese Journal of Public Healthvol 9 pp 1171ndash1175 2010

[20] S Sakon X Xue M Takekawa et al ldquoNF-120581B inhibits TNF-induced accumulation of ROS that mediate prolonged MAPKactivation and necrotic cell deathrdquo The EMBO Journal vol 22no 15 pp 3898ndash3909 2003

[21] M Kuerban M Naito S Hirai et al ldquoInvolvement of FasFas-L and BaxBcl-2 systems in germ cell death following immu-nization with syngeneic testicular germ cells in micerdquo Journalof Andrology vol 33 no 5 pp 824ndash831 2012

[22] A Hajirahimkhan B M Dietz and J L Bolton ldquoBotanicalmodulation of menopausal symptoms mechanisms of actionrdquoPlanta Medica vol 79 no 7 pp 538ndash553 2013

[23] G B Mahady H Ma L Yu and D Liu ldquoSafety of Menoprogena TCM formula for the treatment of menopausal symptomsrdquoJournal of Womenrsquos Health vol 22 no 3 p 14 2013

[24] G B Mahady H Ma L Yu and D Liu ldquoNovel mechanism ofaction for Menoprogen a traditional Chinese Formula for thetreatment ofmenopauserdquo Journal ofWomenrsquos Health vol 22 no3 p 14 2013

[25] S Banerjee S Banerjee G Saraswat S A Bandyopadhyay andS N Kabir ldquoFemale reproductive aging ismaster-planned at thelevel of ovaryrdquo PLoS ONE vol 9 no 5 Article ID e96210 2014

[26] M Tiwari S Prasad A Tripathi et al ldquoApoptosis in mam-malian oocytes a reviewrdquoApoptosis vol 20 no 8 pp 1019ndash10252015

[27] J Guo Y J Zhang andW X Tan ldquoEffect of herbs for tonifyingfluid and Qi of Yang Ming to the ultrastructure of primarysenile rat ovary granular cellrdquoChengdu University of TraditionalChinese Medicine vol 4 no 30 pp 48ndash51 2008

[28] M C Henson and P J Chedrese ldquoEndocrine disruption bycadmium a common environmental toxicant with paradoxicaleffects on reproductionrdquo Experimental Biology Medicine vol229 no 5 pp 383ndash392 2004

[29] L L Rubin K L Philpott and S F Brooks ldquoApoptosis the cellcycle and cell deathrdquo Current Biology vol 3 no 6 pp 391ndash3941993

[30] M W Chen A Q Ma and L Ni ldquoComparative study of theeffects of matrine on the proliferation and apoptosis of humanoral epidermoid carcinomaKB cells and itrsquos moutidrug resistantcounterpart KBv200 cellsrdquoChinese Journal of Clinical Oncologyvol 32 no 9 pp 520ndash523 2005

[31] X X Wang Y H Qu J C Yao W Liu and J Y Gu ldquoExperi-mental study on the apoptosis of human gastric cancer BAC823cells induced by120573-elemenerdquoCentral South Pharmacy vol 6 pp329ndash331 2005

[32] Y Y Zhao F Jin J Liang et al ldquoReversing effect of arsenictrioxide on drug-resistance of human gastric cancer cell lineSGC-7901ADM and its apoptosis-inducing effectrdquo ChineseJournal of Clinical Oncology vol 32 pp 611ndash613 2005

[33] A L L Durlinger P Kramer B Karels J A Grootegoed J T JUilenbroek andA P NThemmen ldquoApoptotic and proliferativechanges during induced atresia of pre-ovulatory follicles in theratrdquo Human Reproduction vol 15 no 12 pp 2504ndash2511 2000

[34] F X Liu and Y Song ldquoStichopus Japonicus Acidic Mucopoly-saccharide induces apoptosis in HeLa cell via altering Bax andBcl-2 expressionrdquoThe Journal of Practical Medicine vol 26 pp2089ndash2091 2010

[35] R Sasson K Tajima and A Amsterdam ldquoGlucocorticoidsprotect against apoptosis induced by serum deprivationcyclic adenosine 3101584051015840-monophosphate and p53 activation inimmortalized human granulosa cells involvement of Bcl-2rdquoEndocrinology vol 142 no 2 pp 802ndash811 2001

[36] K Kugu V S Ratts G N Piquette et al ldquoAnalysis of apoptosisand expression of Bcl-2 gene family members in the human andbaboon ovaryrdquo Cell Death and Differentiation vol 5 no 1 pp67ndash76 1998

[37] G I Perez R Robles C M Knudson J A Flaws S JKorsmeyer and J L Tilly ldquoProlongation of ovarian lifespaninto advanced chronological age by Bax-deficiencyrdquo NatureGenetics vol 21 no 2 pp 200ndash203 1999

[38] J L Tilly K I Tilly andM L Keton ldquoExpression ofmembers ofthe bcl-2 gene family in the immature rat ovaryrdquo Endocrinologyvol 136 pp 230ndash236 1995

[39] MA Fenwick andP RHurst ldquoImmunohistochemical localiza-tion of active caspase-3 in the mouse ovary growth and atresiaof small folliclesrdquo Reproduction vol 124 no 5 pp 659ndash6652002

[40] D L Boone and B K Tsang ldquoCaspase-3 in the rat ovary local-ization and possible role in follicular atresia and luteal regres-sionrdquo Biology of Reproduction vol 58 no 6 pp 1533ndash1539 1998

[41] M Izawa P H Nguyen H H Kim and J Yeh ldquoExpressionof the apoptosis-related genes caspase-1 caspase-3 DNA frag-mentation factor and apoptotic protease activating factor-1 inhuman granulosa cellsrdquo Fertility and Sterility vol 70 no 3 pp549ndash552 1998


One of the most problematic issues associated withdeclining ovarian function is that of lowered estrogen(E2) levels [1] The decline in E

2causes a neuroendocrine

imbalance and menopausal symptoms such as hot flashesheadaches nocturnal sweating vaginal atrophy and anxietyChronically lower E

2levels also lead to significantly higher

incidences of cardiocerebrovascular diseases osteoporosisand senile dementia in postmenopausal women [1] In thepast hormone therapy (HT) has been the ldquogold standardrdquofor the treatment of menopause However the use of HTworldwide has declined due to an increased risk of adverseevents including a transitory increased risk of heart diseaseand increased risk of endometrial or breast cancer [7] Onthe basis of the findings and risk analyses of the WomenrsquosHealth Initiative the US Preventive Services Task Force hasrecommended against the routine use of HT for the preven-tion of chronic conditions in postmenopausal women [7] Asa consequence many women have been looking for com-plementary and alternative medicines including TraditionalChinese Medicines (TCM) to manage their menopausalsymptoms

One TCM formula Menoprogen has been marketed asan herbal supplement in China and Europe for over 15 yearsand has been available in the USA for about ten years forthe management of menopausal symptoms [8 9] Meno-progen is an ancient TCM formula containing semipurifiedextracts of five well-known ldquoherbal foodrdquo plants whichhave been used as dietetic and medical herbs in China forthousands of years [8ndash11] The herbal formula is comprisedof extracts of the herbs Fructus Lycii (Lycium barbarum Lfruits Chinese goji berries Solanaceae) Semen Cuscutae(Cuscuta chinensis Lam ripe seeds Convolvulaceae) RadixRehmanniae (Rehmannia glutinosa Libosch roots Scrophu-lariaceae) Fructus Mori (Morus alba L white mulberryfruits Moraceae) and Flos Carthami (Carthamus tinctoriusL safflower Asteraceae) [8ndash11] In two pilot clinical trialsMenoprogen (MPG) was shown to be effective for the man-agement of menopausal symptoms and reduced the Kupper-man index in treated women [8 9] In animal studies oraladministration of MPG to aged female rats restored ovarianfunction [12 13] Furthermore oral administration of MPGto female rats also increased blood circulation to the ovaryand uterine tissues as well as improved the quality of ovarianfollicles [14] however the mechanism of action for MPG hasnot been fully elucidated In this work we investigate theeffects of Menoprogen in aged female rats and provide evi-dence that this herbal formula reduces GC apoptosis througha mechanism involving the Bcl-2Bax family of proteinsand caspase-3 The reduction in GC apoptosis increases theproduction of endogenous E

2 thereby demonstrating a novel

mechanism of action for this herbal formula

2 Materials and Methods

21 Drugs and Reagents The MPG herbal formula wasprovided by Nanjing Mayflower Pharmaceutical TechnologyCorporation Ltd (Nanjing China) and contains five Chineseherbal extracts as described above [8 9] An optimizedhigh-performance liquid chromatography-photodiode array

detection (HPLC-PDA) fingerprinting method for MPGwas developed and has shown that some of the primarychemical constituents of MPG are flavonoids [9] Progy-nova (estradiol valerate tablets) were used as a positivecontrol and were obtained from Schering SA CompanyFrance Pregnant mare serum gonadotropin (PMSG) waspurchased from Chuangrui Biological Reagent Company(Nanjing China) cadmium chloride from Tingxin ChemicalPharmaceutical Company (Shanghai China) glutaraldehydephosphate buffered solution (PBS) from Boster Company(Wuhan China) lead citrate trihydrate and uranyl acetatedyeing solution from China National Pharmaceutical GroupCorp (Shanghai China) PI dyeing solution from Sigma (StLouis MO USA) and fetal bovine serum fromGibco (USA)

The radioimmunoassay kit for the serum estrogen assaywas obtained from Diagnostic Systems Laboratories Inc(Webster TX USA) TdT-mediated dUTP nick end-labeling(TUNEL) kit was obtained from Roche Diagnostic GmbH(Sandhofer Mannheim Germany) immunochemical assaykit for Bax and Bcl-2 from Santa Cruz Biotechnology (SantaCruz CA USA) and enzyme-linked immunosorbent assay(ELISA) kit for caspase-3 from Biyuntian Biological ReagentCompany (Nantong China)

22 In Vivo Rat Model of Naturally Aged Female Rats (NAM)A total of 60 Sprague-Dawley female rats (SPF grade) andfeed were purchased from BampK Universal Group Limited(Shanghai China) All rats were fed an aseptic diet ad libitumunder controlled conditions 24∘C 50ndash55 humidity anda 12-hour lightdark cycle The animal care protocols wereapproved in accordance with the UK Animal Scientific Pro-cedures Act of 1986 and associated guidelines All protocolswere approved by the ACC prior to initiation of the researchstudy For two continuous estrous cycles fifty 14-month SDfemale rats were monitored by a smear test of exfoliated vagi-nal epithelial cells and the blood was drawn intraorbitally forthe determination of E

2in the serum Rats with continuously

irregular estrous cycles and a significant decrease in E2levels

were regarded as naturally aged model (NAM) The NAMrats were randomly divided into 5 groups NAM group (119899 =10)MPG low-dose treatment group (162mgkg bodyweight119899 = 10) MPG middle-dose treatment group (324mgkg119899 = 10) MPG high-dose treatment group (648mgkg 119899 =10) and positive estradiol valerate control (Progynova 119899 =10) group In addition 4-month-old female rats (119899 = 10)were selected as a normal control group Rats in the MPGtreatment groups were treated with an aqueous extract ofMenoprogen at equivalent clinical dosages (648 324 and162mgkg po resp) while rats in the positive control groupwere treated orally with estradiol valerate (Progynova dose018mgkg) All rats underwent a smear test of exfoliatedvaginal epithelial cells after 8-week treatment Followingtreatment after being weighed but before estrous cycle andblood sampling the rats were sacrificed after anesthetizationThe bilateral ovaries and uterus were removed and the wetweight was recorded The comparison of organ weight wasperformed using an organ coefficient which was calculatedaccording to following formula organ coefficient = organ wetweightbody weight times 100 One of the bilateral ovaries was























2at 37∘C for















3 Results





2levels showed




(a) (b)

(c) (d)




Rat group E2(pgmL)











(a) (b)

(c) (d)



Rats group G0G1

S G2M





2) had




(Table 3)




(a) (b) (c)

(d) (e) (f)

(g) (h)






(a) (b) (c)











1presenting before



1peak in the GCs







4 Discussion




0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



2levels [8 9]





2 as well as







2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References

































































2at 37∘C for















3 Results





2levels showed




(a) (b)

(c) (d)




Rat group E2(pgmL)











(a) (b)

(c) (d)



Rats group G0G1

S G2M





2) had




(Table 3)




(a) (b) (c)

(d) (e) (f)

(g) (h)






(a) (b) (c)











1presenting before



1peak in the GCs







4 Discussion




0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



2levels [8 9]





2 as well as







2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References












































(a) (b)

(c) (d)




Rat group E2(pgmL)











(a) (b)

(c) (d)



Rats group G0G1

S G2M





2) had




(Table 3)




(a) (b) (c)

(d) (e) (f)

(g) (h)






(a) (b) (c)











1presenting before



1peak in the GCs







4 Discussion




0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



2levels [8 9]





2 as well as







2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References












































(a) (b)

(c) (d)



Rats group G0G1

S G2M





2) had




(Table 3)




(a) (b) (c)

(d) (e) (f)

(g) (h)






(a) (b) (c)











1presenting before



1peak in the GCs







4 Discussion




0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



2levels [8 9]





2 as well as







2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References












































(a) (b) (c)

(d) (e) (f)

(g) (h)






(a) (b) (c)











1presenting before



1peak in the GCs







4 Discussion




0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



2levels [8 9]





2 as well as







2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References












































(a) (b) (c)











1presenting before



1peak in the GCs







4 Discussion




0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



2levels [8 9]





2 as well as







2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References












































0

100

200

300

400

500

Num

ber

50 100 150 200 2500Channels (FL2-A)

(a)

50 100 150 200 2500Channels (FL2-A)

0

90

180

270

360

Num

ber

(b)

50 100 150 2000Channels (FL2-A)

0

80

160

240

320

Num

ber

(c)



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2producing



2levels in rats





1 S G







treatment)







2would


2and





Disclaimer




Acknowledgments


References














































2would


2and





Disclaimer




Acknowledgments


References











































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