research article yangjing capsule ameliorates...

Research ArticleYangjing Capsule Ameliorates Spermatogenesis inMale Mice Exposed to Cyclophosphamide

Hongle Zhao1 Baofang Jin1 Xindong Zhang2 Yugui Cui3 Dalin Sun1

Chao Gao3 Yalong Gu1 and Bin Cai1

1Andrology Department of Integrative Medicine Zhongda Hospital School of Medicine Southeast University Nanjing 210009 China2Reproductive Medical Center Department of Obstetrics and Gynecology Nanjing Drum Tower HospitalNanjing University Medical School Nanjing 210008 China3State Key Laboratory of Reproductive Medicine Clinical Center of Reproductive Medicine First Affiliated HospitalNanjing Medical University Nanjing 210029 China

Correspondence should be addressed to Baofang Jin hexiking126com

Received 28 September 2015 Accepted 2 December 2015

Academic Editor Hyunsu Bae

Copyright copy 2015 Hongle Zhao et al This 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

Yangjing capsule (YC) a traditional Chinese compound herbal preparation has been proven as an effective drug to improvespermatogenesis in clinical practice However its pharmacological mechanisms were not fully clarified This study was designedto investigate the protective effects of YC on spermatogenesis in the mouse model of spermatogenesis dysfunction induced bycyclophosphamide (CP) The administration of YC significantly increased the epididymal index sperm count and sperm motilityof model mice Histopathological changes demonstrated that CP caused obvious structural damage to testis which were reversedby the administration of YC Results from TUNEL assay showed that treatment with YC dramatically decreased the apoptosis ofspermatogenic cell induced by CP Moreover YC treatment could inhibit the mRNA and protein expression of Bax to Bcl-2 andalso raised expression of AR at both mRNA and protein levels These data suggest that YC might ameliorate spermatogenesis inmale mice exposed to CP through inhibiting the apoptosis of spermatogenic cell and enhancing the actions of testosterone inspermatogenesis

1 Introduction

Infertility affects 15 of couples worldwide It is estimatedthat roughly half of the infertility cases are due tomale factors[1]Male infertility could be caused by various reasons includ-ing failure in spermatogenesis defects in sperm transporta-tion or accessory gland function genetic or environmentalfactors and sexual disorders [2 3] Among these causesspermatogenic defect is the primary one in male infertility[4]

Spermatogenesis is a sophisticated multistep processinvolving three major phases mitotic meiotic and postmei-otic phases Other cellular events such as apoptosis of sper-matogenic cell migration and differentiation also play vitalroles in the process of spermatogenesis [5] The dynamic bal-ance between cell proliferation and apoptosis determines thenumber of cells in the seminiferous tubules of the testis [6]

Excess apoptosis will result in depletion of sperm productionThe whole process of spermatogenesis is also controlled by aseries of hormones in which testosterone plays a vital rolein regulating spermatogenesis by binding to the androgenreceptor (AR) The absence of testosterone or AR in theprocesses of spermatogenesis will weaken the actions oftestosterone resulting in dysfunction of spermatogenesis [7]

The Yangjing capsule (YC) a traditional Chinese com-pound herbal preparation has been used for over ten yearsfor the treatment of male reproductive diseases in Chinaincluding male infertility and sexual dysfunction Clinicalpractice showed that YC could improve the density of spermits motility and DNA integrity in infertile men [8 9] Thesestudies indicated that YC is an effective drug to improvespermatogenesis in infertile men but the underlying mech-anisms are not well understood From in vitro experimentsit was found that YC could stimulate self-renewal of GC-1

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 980583 8 pageshttpdxdoiorg1011552015980583

2 Evidence-Based Complementary and Alternative Medicine

spermatogonia cells and protect GC-1 spermatogonia cellsfrom apoptosis [10] It was also found that YC could promotethe synthesis of testosterone in mouse Leydig tumor cells-1by increasing the expression of steroidogenic acute regulatoryprotein (StAR) cytochrome P450 family 11 subfamily Apolypeptide 1 (CYP11A1) and 3-hydroxysteroid dehydroge-naseisomerase (3-HSD)mRNAs andproteins [11]Thereforeit is assumed that YC treats the dysfunction of spermatogen-esis by inhibiting the apoptosis of sperm cells and enhancingthe synthesis and metabolism of testosterone However theeffects of YC on testosterone synthesis metabolism andspermatogenic cell apoptosis are still unclear in vivo

In recent years mouse models of spermatogenesis dys-function are used widely to investigate the mechanisms ofspermatogenesis Alkylating agents such as CP are the mostcommon agents implicated in inducing the mouse modelFurthermore it has been demonstrated that CP can inhibittestosterone synthesis and induce apoptosis of spermatogeniccells [12 13] Therefore CP was selected to obstruct thespermatogenesis ofmice in this projectThis study is aimed atinvestigating the protective effects of YC on the spermatoge-nesis testosterone synthesis and apoptosis of spermatogeniccells in a mouse model of spermatogenesis dysfunctioninduced by CP

2 Materials and Methods

21 Drugs and Chemicals CP was purchased from PudeMedicine Co Ltd (Shanxi China) Iodine [125I] Testos-terone Radioimmunoassay Kit was supplied by Beijing NorthInstitute of Biological Technology (Beijing China) Theprimers were synthesized by Invitrogen Life Tech (Carls-bad CA) The mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) and horseradishperoxidase-conjugated secondary antibodies were purchasedfrom Bioworld (St Louis Park MN) The rabbit monoclonalanti-tubulin and rabbit polyclonal anti-Bax antibodies wereprocured from Abcam (Cambridge MA) The rabbit poly-clonal anti-AR and rabbit polyclonal anti-B-cell lymphoma2 (anti-Bcl-2) antibodies goat anti-rabbit immunoglobulinG (120574-chain specific) (peroxidase conjugate) and In Situ CellApoptosis Detection Kit I (POD) were procured from BosterBiological Engineering Co Ltd (Wuhan China)

22 Preparation of the YC The YC was provided by Nan-jing General Hospital of Nanjing Military Region (NanjingChina) YC is composed of 11 types of traditional Chinesedrugs 133 Herba Epimedii Brevicornus 67 RhizomaPolygonati Sibirici 67 Radix Rehmanniae Preparata 10Radix Astragali Mongolici 67 Placenta Hominis 67Semen Astragali Complanati 10 Radix Angelicae Sinensis67 Hirudo 67 Semen Litchi 133 Semen VaccariaeSegetalis and 133 Concha Ostreae (calcined) In this studycontents of the dried capsule were diluted into differentconcentrations using distilled deionized water and the sus-pensions were stored at 4∘C

23 Animals and Treatments Mature male (8-week) Balbcmice (22ndash25 g) were procured from Yangzhou University

Comparative Medical Center (Yangzhou China) Mice werehoused under standard laboratory conditions and were pro-vided with free access to standard food and water ad libitumThis study was approved by the Animal and Human EthicsBoard of Southeast University After adapting to the envi-ronment for a week mice were randomly divided into fourgroups control (119899 = 9) CP (119899 = 9) CP plus YC (630mgkg)(119899 = 9) and CP plus YC (1260mgkg) (119899 = 9) For thefirst 7 days the mice of CP CP plus YC (630mgkg) and CPplus YC (1260mgkg) groups were injected intraperitoneally(ip) with 50mgkg of CP once a day This method has beenpreviously shown to induce dysfunction of spermatogenesis[14] For the subsequent 30 days themice in the YC treatmentgroups were administered with YC suspension once a day byoral gavage The animals were weighed weekly to adjust thegavage volume and their general health was monitored daily

24 Preparation of Serum and Tissue The animals wereweighed and anesthetizedwith pentobarbital sodium (50mgkg ip) at the end of the treatment Blood samples werecollected by extirpation of eyeball and allowed to clot andthe serum was separated at 3000 rpm for 15min and stored atminus80∘C to analyze the testosterone level

The testes and epididymis were rapidly excised andweighed The relative testicular or epididymal weight (thetestis index or the epididymal index) was calculated bydividing the weight of testis or epididymis by body weightOne testis and one epididymis from a mouse were fixedin Bouinrsquos solution for pathological examination and ter-minal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) analysisThe other testis was freshly washed in ice-cold saline and immediately stored in liquid nitrogen Thefresh testis frozen in liquid nitrogenwas then divided into twoparts for the isolation of RNA and extraction of protein

25 Epididymal Sperm Analysis The epididymal spermconcentrations and motility were evaluated as previouslydescribedwith somemodifications [15] An entire epididymisfrom a mouse was minced in 1mL saline which was pre-heated at 37∘C The epididymis was incubated for another15min at 37∘C to allow the sperm to swim out of the epi-didymal tubules The total number of sperm and the numberof motile sperms were determined using a hemocytometer(Anxin Shanghai China) the sperm motility was convertedinto a percentage

26 Histological Examination For histological studies tis-sues were initially fixed in Bouinrsquos fluid for 16 h and they werethen washed three times with 70 ethanol followed by dehy-dration in graded ethanol They were finally embedded inparaffin Five-micrometer-thick sections were stained usinghematoxylin-eosin and then examined under a light micro-scope at 200x or 400xmagnification A total of 200 seminifer-ous tubules fromeach testiswere examinedDamaged tubuleswhich showed severe hypocellularity (reduction in numberof spermatogenic cells) and thinned seminiferous epitheliumwere recorded

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Effects of YC on characteristics of sperm seminiferous tubules serum testosterone body weight and the relative testicular andepididymal weights

Parameters Control CP CP + YC (630mgkg) CP + YC (1260mgkg)Sperm count (times106mLminus1) 8256 plusmn 749 4394 plusmn 470lowastlowast 5131 plusmn 267 6511 plusmn 818

Sperm motility () 6900 plusmn 622 4576 plusmn 728lowastlowast 6222 plusmn 468 6525 plusmn 706

Proportion of damaged tubules () 29plusmn 23 751 plusmn 53lowastlowast 205 plusmn 76 133 plusmn 61

Body weight (g) 2911 plusmn 154 260 plusmn 141lowastlowast 2650 plusmn 141 2811 plusmn 176

Relative testicular weight (mgg bodyweight) 402 plusmn 028 275 plusmn 054lowastlowast 313 plusmn 029 332 plusmn 039

Relative epididymal weight (mgg bodyweight) 147 plusmn 020 110 plusmn 011lowastlowast 144 plusmn 013 137 plusmn 012

Testosterone (molL) 181 plusmn 086 056 plusmn 022lowast 061 plusmn 026 076 plusmn 035Data are expressed as the mean plusmn SD (119899 = 9) lowast119875 lt 005 lowastlowast119875 lt 001 compared to the control group 119875 lt 005 119875 lt 001 compared to the CP-treated groupCP cyclophosphamide SD standard deviation YC Yangjing capsule

27 Assessment of Testosterone Serum testosterone wasexamined using Iodine [125I] Testosterone Radioimmunoas-say Kit according to the protocol described by the manu-facturer The amount of testosterone was determined from acalibration curve

28 In Situ Detection of Apoptosis Five-micrometer-thicksections of the testis were used for TUNEL staining toexamine cell apoptosis The TUNEL method was performedaccording to themanufacturerrsquos instructionsThe numbers ofpositive and total cells were recorded and the apoptosis index[(number of positive cellstotal cells) times 100] of the tissue wascalculated as described by Yuan et al [14]

29 Isolation of RNA and Real-Time Quantitative PCR BaxBcl-2 and AR mRNA expression levels in the testes weredetermined using real-time quantitative PCRThe total RNAof the testes samples was extracted using Trizol reagent(TaKaRa Biotechnology Dalian China) Sample cDNAswereused as templates for amplification to quantify themRNA lev-els of target genes using SYBRGreen PCRMasterMix reagentkits (TaKaRa Biotechnology) GAPDH was selected as thecontrol The primer sequences were as follows GAPDHforward 51015840-TGG CCT TCC GTG TTC CTA C-31015840 reverse51015840-GAG TTG CTG TTG AAG TCG CA-31015840 AR forward 51015840-TCC AAG ACC TAT CGA GGA GCG-31015840 reverse 51015840-GTGGGC TTG AGG AGA ACC AT-31015840 Bax forward 51015840-AGACAG GGG CCT TTT TGC TAC-31015840 reverse 51015840-AAT TCGCCG GAG ACA CTC G-31015840 Bcl-2 forward 51015840-GCT ACCGTC GTG ACT TCG C-31015840 reverse 51015840-CCC CAC CGA ACTCAA AGA AGG-31015840 Relative quantification of expression oftarget genes was estimated using the 2minusΔΔCt method

210 Western Blot Analysis Total proteins extracted fromtestis were prepared following standard procedures andquantified using the bicinchoninic acid protein assay (Bey-otime Shanghai China) The proteins were separated by 12sodium dodecyl sulfate-polyacrylamide gel electrophoresisand subsequently transferred onto nitrocellulosemembranesThe membranes were blocked with 5 skim milk in Tris-buffer saline containing 01 Tween 20 at 37∘C for 1 h andthen incubated at 4∘C overnight with the primary antibodiesof Bax (1 200 dilution) Bcl-2 (1 300 dilution) and AR(1 300 dilution) Tubulin or GAPDH was used as an internal

control Densitometric analysis of the scanned immunoblot-ting images was performed using a Quantity One imagesystem

211 Statistical Analysis All quantitative data derived fromthis study were analyzed using the SPSS 200 statistical pack-age The results were expressed as the mean plusmn standard devi-ation The one-way analysis of variance was used to analyzethe difference between groups and post hoc least significantdifference test was used for intergroup comparisons119875 lt 005was considered as statistical significance

3 Results

31 Effects of YC on the RelativeWeights of Reproductive Organand the Sperm Parameters The relative weights of reproduc-tive organ and the sperm parameters were used to monitorthe damage to the male reproductive system As shown inTable 1 in model mice treated with CP there were significantdecreases in body weight the testis index and the epididymalindex when compared with the control group (119875 lt 001resp) CP treatment also decreased the sperm count andmotility by 47 and 34 respectively as compared to thecontrol group (119875 lt 001) After treatment with YC the bodyweight and the testis index in the YC (1260mgkg) groupsignificantly increased by 81 and 207 as compared to theCP group YC (630 and 1260mgkg) treatments also signif-icantly increased the epididymal index by 309 and 245(119875 lt 001) when compared with the CP-treated group YC(630 and 1260mgkg) treatment also notably increased thesperm count andmotility when compared with the CP group(Table 1)

32 Treatment with YC Reduced the CP-Induced Testicularand Epididymal Injury in Model Mice The histopathologyof the testis and epididymis was shown in Figure 1 Normalhistological features of testis were visible in the controlgroup the normal thickness of the seminiferous epitheliallayer a number of spermatogenic cells and interstitial cells(Figure 1(a)(A)) The epididymis tissues had normal densityof sperm and few premature sperm cells (Figure 1(b)(A))In contrast as shown in Figure 1(a)(B) the CP-treated micedisplayed the atrophied seminiferous tubules the thinnedseminiferous epithelium that was arrayed loosely the reduced


20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(b)

Figure 1The effects of treatmentwithYCon the testicular ((a)times400) and epididymal ((b)times400) damage in the spermatogenesis dysfunctionmice induced by CP (lesion site labeled with arrow heads) (A) control (B) CP (50mgkg) (C) CP + YC (630mgkg) (D) CP + YC(1260mgkg)

spermatogenic cells that were shed particularly in the post-meiotic stages and the reduced interstitial cellsThe adminis-tration of CP also lowered the density of sperm and increasedthe number of premature sperm cells which came off from theseminiferous tubules in cauda epididymisThese results werein agreement with a previous study [16] (Figure 1(b)(B)) Theabovementioned histological damage instances were restoredat least in some degree by treatment with YC

In the YC (630mgkg) group in spite of thinned seminif-erous epithelium many mature spermatids appeared in theseminiferous tubules (Figure 1(a)(C)) In the YC (1260mgkg) group the layer of seminiferous epithelium showed asignificant increase The sperm cells at different stages weredistributed regularly and many mature spermatids appearedin the seminiferous tubules (Figure 1(a)(D)) In addition thenumber of interstitial cells in theYC-treated groups presenteda significant increase (Figures 1(a)(C) and 1(a)(D)) YC (630and 1260mgkg) treatments also improved the density ofsperms and decreased the number of premature sperm cellsin cauda epididymis (Figures 1(b)(C) and 1(b)(D))

The mean percentage of damaged seminiferous tubulesin the CP-treated mice was significantly higher 751 plusmn 53which was 29 plusmn 23 in the control group (Table 1) Afterdaily administration of YC the rates of damaged tubulesdropped significantly 205 plusmn 76 in the YC (630mgkg) and133 plusmn 61 in the YC (1260mgkg) group

33 Effect of YC on Serum Testosterone Level The admin-istration of CP caused a significant decrease in the serumtestosterone level from 181plusmn086molL in the control groupto 056 plusmn 022molL in the CP-treated group (119875 lt 005)In the YC (630 and 1260mgkg) groups the values were061plusmn026 and 076plusmn035 (molL) respectively Although theserum testosterone level increased in the YC-treated groupsthe difference did not reach statistical significance whencompared with the CP group (Table 1)

34 Treatment with YC Inhibited CP-Induced Apoptosis ofSpermatogenic Cell TUNEL assay was performed to analyzethe apoptosis of spermatogenic cell After administration ofCP apoptotic cells were observed more frequently in thetestis of the CP group mice (Figure 2(a)(B)) than those inthe control group (Figure 2(a)(A)) The apoptotic cells in theCP group were mainly spermatocytes After treatment withYC fewer apoptotic cells were detected in the testis sections(Figures 2(a)(C) and 2(a)(D)) The administration of CP alsoresulted in a significant increase in the apoptotic index ofspermatogenic cell (134 plusmn 26) compared to 25 plusmn 10 inthe control group (Figure 2(b)) When treatment was carriedout with YC (630 and 1260mgkg) there was a remark-able decrease in the apoptotic index by 726 and 754respectively compared with the CP group (119875 lt 001)

35 Effects of YC on Bax Bcl-2mRNA and Protein ExpressionTo further study the potential mechanism of antiapoptosis ofYC the expressions of Bax and Bcl-2 at mRNA and proteinlevels in the testes of mice were examined using the quan-titative real-time PCR and Western blotting As shown inFigure 3 the testes from the CP group mice showed a signif-icant increase in the ratio of Bax to Bcl-2 mRNA and proteinexpression levels as compared to the control group (119875 lt 005resp) Treatment with YC significantly decreased the ratioof Bax mRNA to Bcl-2 mRNA in a dose-dependent manner(Figure 3(a)) while YC (1260mgkg) dramatically reducedthe ratio of Bax to Bcl-2 protein when compared with theCP-treated group (Figure 3(b)) (119875 lt 005)

36 Effects of YC on AR mRNA and Protein ExpressionTestosterone is the major androgen in the testis that regulatesspermatogenesis Effects of androgen aremediated by theARThe lowered testosterone or dysfunctional AR will result inthe impaired spermatogenesis In this study the expressionsof AR mRNA and protein in the testes of different groups


20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

Apop

totic

inde

x (

)

10

15

20

5

0CP (mgkg) 50 50 50YC (mgkg) 630

lowastlowast

minus

minusminus 1260

(b)

Figure 2 The effects of treatment with YC on apoptosis of spermatogenic cell in the spermatogenesis dysfunction mice induced by CP (a)The representative apoptosis of spermatogenic cell in the testes using TUNEL assay (times400) (b) The spermatogenic cell apoptotic index (A)Control (B) CP (50mgkg) (C) CP + YC (630mgkg) (D) CP + YC (1260mgkg) (119899 = 5) lowastlowast119875 lt 001 compared to the control group 119875 lt001 compared to the CP-treated group CP cyclophosphamide TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling YCYangjing capsule

were compared As shown in Figure 4 administration of CPsignificantly decreased the expressions of AR mRNA andprotein (119875 lt 005 resp) as compared with the control groupYC (630 and 1260mgkg) treatment significantly improvedthe ARmRNA and protein expression levels when comparedwith the CP group (Figures 4(a) and 4(b))

4 Discussion

Male infertility causes significant duress to couples Defectsin spermatogenesis are the most common reasons for maleinfertility At present hormonal treatment or empiricmedicaltreatment (eg aromatase inhibitor or selective estrogenreceptor modulator) is used to treat infertile men with sper-matogenic defects but clinical results are limited especiallyfor patients with idiopathic failure of spermatogenesis [17]Traditional Chinese formulations intended for kidney sup-port have shown remarkable advantages in the treatment ofmale infertility [18] YC a Chinese compound herbal prepa-ration has been proven as an effective drug to improve sper-matogenesis in the clinical practice for over ten years but theunderlying mechanisms are not well understood Thereforethe present study was performed to study the effects of YCon the spermatogenesis and the underlying mechanisms in amouse model of spermatogenesis dysfunction

CP as an alkylating agent is the most common agentimplicated in causing dysfunction of spermatogenesis Inthis study mature male Balbc mice were injected with CP(50mgkg) ip for 7 days to induce the dysfunction of sper-matogenesis For the next 30 days the mice received treat-ment with YC Animals were killed at 30 days after the lastinjection We found that administration of CP significantlydecreased the sperm count and motility as well as the relativetesticular weights and body weights The administration of

CP also caused histologic lesions of the testis and epididymisTesticular tissues from the CP-treatedmice showed atrophiedseminiferous tubules thinned seminiferous epithelium andreduced spermatogenic cells and interstitial cells We alsofound that the cauda epididymis from the CP-treated miceillustrated a lower density of sperm and an increase in thenumber of premature sperm cells

Interestingly we found that treatment with YC signif-icantly restored these CP damage instances YC (630 and1260mgkg) treatment not only notably increased the spermcount and motility but also increased seminiferous epitheliallayers and the number of mature spermatids and interstitialcells in testis YC treatment also increased the density ofsperm and decreased the deciduous premature sperm cells inthe ductus epididymis when compared with the CP groupThese results indicated that YC could ameliorate spermato-genesis in male mice exposed to CP

Mammalian spermatogenesis depends on the balanceamong proliferation differentiation and apoptosis of germcells Apoptosis of spermatogenic cell occurs during variousstages of mammalian spermatogenesis to remove abnormalspermatogenic cells [6] which maintains the ratio of germcell to Sertoli cell within a normal range so as to keep the con-tinual spermatogenesis [19] However excess apoptosis willbe followed by severe impairment in the production of spermAmong those modulators of apoptosis the Bcl-2 subfamily(Bcl-2 Bcl-x long Bcl-w Mcl-1 A1Bfl1 and Nr13) promotesthe survival of cell and the Bax subfamily (Bax Bcl-x shortBak and Bok) enhances the apoptosis of cell [19]The ratio ofBax to Bcl-2 has been implicated as a critical determinantof cell fate [20] It has been reported that acute or chronicexposure to CP results in elevated apoptotic rates of germ cell[21 22] Studies have revealed that the elevated apoptotic rateof germ cell induced by CPwas related to the elevated ratio of


Relat

ive m

RNA

expr

essio

n Ba

xBc

l-2

0

100

250

200

150

50

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

lowast

(a)

00

15

10

05

lowast

Relat

ive p

rote

in ex

pres

sion

Bax

Bcl-2

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

24kDa Bcl-2

20kDa Bax

55kDa tubulin

(b)

Figure 3 The effects of YC on the ratio of Bax to Bcl-2 mRNA expression (a) and protein expression (b) in the testes of spermatogenesisdysfunction mice induced by CP Data given are the mean plusmn SD (119899 = 4) lowast119875 lt 005 compared to the control group 119875 lt 005 119875 lt 001compared to the CP-treated group CP cyclophosphamide SD standard deviation YC Yangjing capsule

lowast

001

000

004

003

002

CP (mgkg) 50 50 50YC (mgkg) 630

minusminusminus 1260

Relat

ive m

RNA

expr

essio

n A

RG

APD

H

(a)

lowast

02

00

10

08

06

04

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive p

rote

in ex

pres

sion

AR

GA

PDH

110 kDa AR36kDa

GAPDH

(b)

Figure 4 The effects of YC on AR mRNA (a) and protein expressions (b) in the testes of spermatogenesis dysfunction mice induced by CPData are expressed as mean plusmn SD (119899 = 3) lowast119875 lt 005 compared to the control group 119875 lt 005 119875 lt 001 compared to the CP-treated groupAR androgen receptor CP cyclophosphamide GAPDH glyceraldehyde 3-phosphate dehydrogenase SD standard deviation YC Yangjingcapsule

Bax to Bcl-2 [14 23] In the present study administration ofCP significantly increased TUNEL-positive cells in the testissections Administration of CP also increased the ratio of Baxto Bcl-2 mRNA and protein expression levels in the testes ofmice After remedial treatment with YC the increased apop-totic sperm cells and apoptotic index by CP treatment weredramatically reduced accompanied with the reduction in theratio of Bax to Bcl-2 mRNA and protein expression levelswhen compared with the CP group These results indicated

that YC inhibited the CP-induced apoptosis of spermatogeniccells by normalizing the elevated ratio of Bax to Bcl-2

Testosterone is a major regulator in spermatogenesiswhich works by binding to AR In the absence of testosteroneor the dysfunctional AR spermatogenesis is halted duringmeiosis so that few germ cells develop to the haploidspermatid stage and the elongated spermatid can not form[24ndash26] Furthermore in testosterone-suppressed rats or ARhypomorphic mice the attachment of Sertoli cells to round


spermatids or elongated spermatids could not be maintainedand germ cells are released prematurely [27 28] In additionmature spermatids are retained and phagocytized by Sertolicells in the absence of testosterone signaling [24]The absenceof testosterone or AR in the processes of spermatogenesis willweaken the actions of testosterone resulting in the dysfunc-tion of spermatogenesis

Previous studies have shown that administration of CPcould inhibit the synthesis of testosterone [12 29 30] Inthis study administration of CP caused a significant decreasein the serum testosterone level which was consistent withprevious studies In addition CP also markedly lowered theexpressions of AR mRNA and protein in the testes whencompared with the control groupThe decreased testosteronelevel and AR expression level in the CP group probablycontributed to the morphological injuries of testis and epi-didymis After treatment with YC serum testosterone leveldisplayed a rising trend in the model mice although thisdifference did not reach statistical significance as comparedwith the CP group However treatment with YC substantiallyupregulated the expression of AR when compared with theCPgroupThese indicated that YCmay enhance the actions oftestosterone to improve spermatogenesis

As we know traditional Chinese medicine especiallythose compound recipes plays a role through multiwayand multitarget with its multicomponents YC is composedof 11 traditional Chinese drugs Of necessity YC containsmany active ingredients It has been demonstrated that totalflavonoids of Herba Epimedii Brevicornus and polysaccha-rides could not only enhance testosterone actions [11 31] butalso inhibit cell apoptosis [11 14] In addition it has beenreported that flavonoids and polysaccharides exerted clearantioxidative activities [11 14] Oxidative stress is an impor-tant cause ofmale infertility which can increase the apoptosisof germ cells and decrease the synthesis of testosterone[32] Therefore the protective effects of YC on spermatoge-nesis may be related to its antioxidant effect Based on theabove analysis we believe that the active ingredients in YCwhich ameliorate spermatogenesis could be total flavonoidsof Herba Epimedii Brevicornus and polysaccharides Addi-tional studies for the pharmacological mechanisms of YCare necessary because this innovative traditional Chinesemedicine has complex active ingredients

5 Conclusions

In conclusion the present study suggests that treatmentwith YC can ameliorate the impairment of spermatogenesisinduced by CP in male mice The effects of YC on improvingspermatogenesis are likely due to the inhibition of apoptosisof spermatogenic cell and enhancement in the actions oftestosterone in spermatogenesis Therefore YC might beconsidered as an alternative therapeutic remedy for maleinfertility

Abbreviations

YC Yangjing capsuleCP Cyclophosphamide

TUNEL Terminal deoxynucleotidyl transferase dUTPnick end labeling

AR Androgen receptorStAR Steroidogenic acute regulatory proteinCYP11A1 Cytochrome P450 family 11 subfamily A

polypeptide 13120573-HSD 3120573-Hydroxysteroid

dehydrogenaseisomeraseBax Bcl-2 Associated X Protein

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (Grants nos 81273760 and 81302969)and Postgraduate Innovation Program of the Jiangsu Prov-ince Education Department (SJLX 0430) The authors thankthe State Key Laboratory of Reproductive Medicine theClinical Center of Reproductive Medicine Nanjing MedicalUniversity for providing laboratory and technological assis-tance

References

[1] J Poongothai T S Gopenath and S Manonayaki ldquoGenetics ofhuman male infertilityrdquo Singapore Medical Journal vol 50 no4 pp 336ndash347 2009

[2] E Vicari A E Calogero R A Condorelli L O Vicari and S LaVignera ldquoMale accessory gland infection frequency in infertilepatients with chronic microbial prostatitis and irritable bowelsyndromerdquo International Journal of Andrology vol 35 no 2 pp183ndash189 2012

[3] K L OrsquoFlynn OrsquoBrien A C Varghese and A Agarwal ldquoThegenetic causes of male factor infertility a reviewrdquo Fertility andSterility vol 93 no 1 pp 1ndash12 2010

[4] S R Singh O Burnicka-Turek C Chauhan and S X HouldquoSpermatogonial stem cells infertility and testicular cancerrdquoJournal of Cellular and Molecular Medicine vol 15 no 3 pp468ndash483 2011

[5] K L Fok H Chen Y C Ruan and H C Chan ldquoNovel regu-lators of spermatogenesisrdquo Seminars in Cell and DevelopmentalBiology vol 29 pp 31ndash42 2014

[6] L D Russell H Chiarini-Garcia S J Korsmeyer and C MKnudson ldquoBax-dependent spermatogonia apoptosis is requiredfor testicular development and spermatogenesisrdquo Biology ofReproduction vol 66 no 4 pp 950ndash958 2002

[7] L B Smith andW H Walker ldquoThe regulation of spermatogen-esis by androgensrdquo Seminars in Cell and Developmental Biologyvol 30 pp 2ndash13 2014

[8] B Jin Y Huang X Yang X Shang X Lu and X Wang ldquoClin-ical observation on treatment of oligosperntatisn with yangjingcapsulerdquo Journal of Nanjing University of Traditional ChineseMedicine vol 22 no 5 pp 286ndash289 2006

[9] B Jin Y Huang X Xia X Wang X Yang and Z Zhou ldquoEffectof Yangjing capsule and Xinxibao on sperm DNA integrity ofpatients with male infertilityrdquo Chinese Journal of Andrology vol20 no 12 pp 45ndash49 2006


[10] ZWang B Jin X Zhang Y Cui D Sun and C Gao ldquoYangjingcapsule extract promotes proliferation of GC-1 Spg cellsrdquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 640857 9 pages 2014

[11] D Sun Y Cui B Jin X Zhang X Yang and C Gao ldquoEffects ofthe Yangjing capsule extract on steroidogenesis and apoptosisin mouse Leydig cellsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2012 Article ID 985457 10 pages 2012

[12] N Elangovan T-J Chiou W-F Tzeng and S-T ChuldquoCyclophosphamide treatment causes impairment of spermandits fertilizing ability in micerdquo Toxicology vol 222 no 1-2 pp60ndash70 2006

[13] D Anderson J B Bishop R C Garner P Ostrosky-Wegmanand P B Selby ldquoCyclophosphamide review of its mutagenicityfor an assessment of potential germ cell risksrdquo MutationResearchmdashFundamental and Molecular Mechanisms of Mutage-nesis vol 330 no 1-2 pp 115ndash181 1995

[14] D Yuan H Wang H He et al ldquoProtective effects of totalflavonoids from Epimedium on the male mouse reproductivesystem against cyclophosphamide-induced oxidative injury byup-regulating the expressions of SOD3 and GPX1rdquo Phytother-apy Research vol 28 no 1 pp 88ndash97 2014

[15] M S Oh M S Chang W Park et al ldquoYukmijihwang-tangprotects against cyclophosphamide-induced reproductive toxi-cityrdquoReproductive Toxicology vol 24 no 3-4 pp 365ndash370 2007

[16] S H Kim I C Lee H S Baek C Moon S H Kim andJ C Kim ldquoProtective effect of diallyl disulfide on cyclophos-phamide-induced testicular toxicity in ratsrdquo Laboratory AnimalResearch vol 29 no 4 pp 204ndash211 2013

[17] B D Anawalt ldquoApproach to male infertility and induction ofspermatogenesisrdquo Journal of Clinical Endocrinology and Metab-olism vol 98 no 9 pp 3532ndash3542 2013

[18] H Li and Y Li ldquoBushenshengjing pill treatment of spermabnormality of 220 cases of male infertilityrdquo Journal of Tradi-tional Chinese Medicine vol 44 no 10 pp 767ndash768 2003

[19] D Royere F Guerif V Laurent-Cadoret and M-T Hochereaude Reviers ldquoApoptosis in testicular germ cellsrdquo InternationalCongress Series vol 1266 pp 170ndash176 2004

[20] A P S Hikim and R S Swerdloff ldquoHormonal and genetic con-trol of germ cell apoptosis in the testisrdquoReviews of Reproductionvol 4 no 1 pp 38ndash47 1999

[21] D-W He X-L Li L-Q Yue G-H Wei T Lin and J-HLiu ldquoEffect of cyclophosphamide on spermatogonial stem cellsrdquoZhonghua Nan Ke Xue vol 12 no 5 pp 387ndash393 2006

[22] L Cai B F Hales and B Robaire ldquoInduction of apoptosis inthe germ cells of adult male rats after exposure to cyclophos-phamiderdquo Biology of Reproduction vol 56 no 6 pp 1490ndash14971997

[23] G Turk A O Ceribasi F Sakin M Sonmez and A AtessahinldquoAntiperoxidative and anti-apoptotic effects of lycopene andellagic acid on cyclophosphamide-induced testicular lipid per-oxidation and apoptosisrdquo Reproduction Fertility and Develop-ment vol 22 no 4 pp 587ndash596 2010

[24] K De Gendt J V Swinnen P T K Saunders et al ldquoASertoli cell-selective knockout of the androgen receptor causesspermatogenic arrest in meiosisrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no5 pp 1327ndash1332 2004

[25] C Chang Y-T Chen S-D Yeh et al ldquoInfertility with defectivespermatogenesis and hypotestosteronemia inmalemice lackingthe androgen receptor in Sertoli cellsrdquo Proceedings of the

National Academy of Sciences of the United States of Americavol 101 no 18 pp 6876ndash6881 2004

[26] S YehM-Y Tsai Q Xu et al ldquoGeneration and characterizationof androgen receptor knockout (ARKO) mice an in vivomodel for the study of androgen functions in selective tissuesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 99 no 21 pp 13498ndash13503 2002

[27] R W Holdcraft and R E Braun ldquoAndrogen receptor functionis required in Sertoli cells for the terminal differentiation ofhaploid spermatidsrdquo Development vol 131 no 2 pp 459ndash4672004

[28] L OrsquoDonnell R I McLachlan N GWreford D M de Kretserand D M Robertson ldquoTestosterone withdrawal promotesstage-specific detachment of round spermatids from the ratseminiferous epitheliumrdquo Biology of Reproduction vol 55 no4 pp 895ndash901 1996

[29] Y Zhang N Shen L Qi W Chen Z Dong and D-H ZhaoldquoEfficacy of Schizandra chinesis polysaccharide on cyclophos-phamide induced dyszoospermia of rats and its effects on repro-ductive hormonesrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol33 no 3 pp 361ndash364 2013

[30] U B Das MMallick J M Debnath and D Ghosh ldquoProtectiveeffect of ascorbic acid on cyclophosphamide-induced testiculargametogenic and androgenic disorders in male ratsrdquo AsianJournal of Andrology vol 4 no 3 pp 201ndash207 2002

[31] B She D Qin S Wang and Y She ldquoEffects of flavonoidsfrom herba epimedii on the reproductive system in male ratsrdquoChinese Journal of Andrology vol 17 no 5 pp 294ndash296 2003

[32] J S Aprioku ldquoPharmacology of free radicals and the impact ofreactive oxygen species on the testisrdquo Journal of Reproductionand Infertility vol 14 no 4 pp 158ndash172 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


spermatogonia cells and protect GC-1 spermatogonia cellsfrom apoptosis [10] It was also found that YC could promotethe synthesis of testosterone in mouse Leydig tumor cells-1by increasing the expression of steroidogenic acute regulatoryprotein (StAR) cytochrome P450 family 11 subfamily Apolypeptide 1 (CYP11A1) and 3-hydroxysteroid dehydroge-naseisomerase (3-HSD)mRNAs andproteins [11]Thereforeit is assumed that YC treats the dysfunction of spermatogen-esis by inhibiting the apoptosis of sperm cells and enhancingthe synthesis and metabolism of testosterone However theeffects of YC on testosterone synthesis metabolism andspermatogenic cell apoptosis are still unclear in vivo

In recent years mouse models of spermatogenesis dys-function are used widely to investigate the mechanisms ofspermatogenesis Alkylating agents such as CP are the mostcommon agents implicated in inducing the mouse modelFurthermore it has been demonstrated that CP can inhibittestosterone synthesis and induce apoptosis of spermatogeniccells [12 13] Therefore CP was selected to obstruct thespermatogenesis ofmice in this projectThis study is aimed atinvestigating the protective effects of YC on the spermatoge-nesis testosterone synthesis and apoptosis of spermatogeniccells in a mouse model of spermatogenesis dysfunctioninduced by CP

2 Materials and Methods

21 Drugs and Chemicals CP was purchased from PudeMedicine Co Ltd (Shanxi China) Iodine [125I] Testos-terone Radioimmunoassay Kit was supplied by Beijing NorthInstitute of Biological Technology (Beijing China) Theprimers were synthesized by Invitrogen Life Tech (Carls-bad CA) The mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) and horseradishperoxidase-conjugated secondary antibodies were purchasedfrom Bioworld (St Louis Park MN) The rabbit monoclonalanti-tubulin and rabbit polyclonal anti-Bax antibodies wereprocured from Abcam (Cambridge MA) The rabbit poly-clonal anti-AR and rabbit polyclonal anti-B-cell lymphoma2 (anti-Bcl-2) antibodies goat anti-rabbit immunoglobulinG (120574-chain specific) (peroxidase conjugate) and In Situ CellApoptosis Detection Kit I (POD) were procured from BosterBiological Engineering Co Ltd (Wuhan China)

22 Preparation of the YC The YC was provided by Nan-jing General Hospital of Nanjing Military Region (NanjingChina) YC is composed of 11 types of traditional Chinesedrugs 133 Herba Epimedii Brevicornus 67 RhizomaPolygonati Sibirici 67 Radix Rehmanniae Preparata 10Radix Astragali Mongolici 67 Placenta Hominis 67Semen Astragali Complanati 10 Radix Angelicae Sinensis67 Hirudo 67 Semen Litchi 133 Semen VaccariaeSegetalis and 133 Concha Ostreae (calcined) In this studycontents of the dried capsule were diluted into differentconcentrations using distilled deionized water and the sus-pensions were stored at 4∘C

23 Animals and Treatments Mature male (8-week) Balbcmice (22ndash25 g) were procured from Yangzhou University

Comparative Medical Center (Yangzhou China) Mice werehoused under standard laboratory conditions and were pro-vided with free access to standard food and water ad libitumThis study was approved by the Animal and Human EthicsBoard of Southeast University After adapting to the envi-ronment for a week mice were randomly divided into fourgroups control (119899 = 9) CP (119899 = 9) CP plus YC (630mgkg)(119899 = 9) and CP plus YC (1260mgkg) (119899 = 9) For thefirst 7 days the mice of CP CP plus YC (630mgkg) and CPplus YC (1260mgkg) groups were injected intraperitoneally(ip) with 50mgkg of CP once a day This method has beenpreviously shown to induce dysfunction of spermatogenesis[14] For the subsequent 30 days themice in the YC treatmentgroups were administered with YC suspension once a day byoral gavage The animals were weighed weekly to adjust thegavage volume and their general health was monitored daily

24 Preparation of Serum and Tissue The animals wereweighed and anesthetizedwith pentobarbital sodium (50mgkg ip) at the end of the treatment Blood samples werecollected by extirpation of eyeball and allowed to clot andthe serum was separated at 3000 rpm for 15min and stored atminus80∘C to analyze the testosterone level

The testes and epididymis were rapidly excised andweighed The relative testicular or epididymal weight (thetestis index or the epididymal index) was calculated bydividing the weight of testis or epididymis by body weightOne testis and one epididymis from a mouse were fixedin Bouinrsquos solution for pathological examination and ter-minal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) analysisThe other testis was freshly washed in ice-cold saline and immediately stored in liquid nitrogen Thefresh testis frozen in liquid nitrogenwas then divided into twoparts for the isolation of RNA and extraction of protein

25 Epididymal Sperm Analysis The epididymal spermconcentrations and motility were evaluated as previouslydescribedwith somemodifications [15] An entire epididymisfrom a mouse was minced in 1mL saline which was pre-heated at 37∘C The epididymis was incubated for another15min at 37∘C to allow the sperm to swim out of the epi-didymal tubules The total number of sperm and the numberof motile sperms were determined using a hemocytometer(Anxin Shanghai China) the sperm motility was convertedinto a percentage

26 Histological Examination For histological studies tis-sues were initially fixed in Bouinrsquos fluid for 16 h and they werethen washed three times with 70 ethanol followed by dehy-dration in graded ethanol They were finally embedded inparaffin Five-micrometer-thick sections were stained usinghematoxylin-eosin and then examined under a light micro-scope at 200x or 400xmagnification A total of 200 seminifer-ous tubules fromeach testiswere examinedDamaged tubuleswhich showed severe hypocellularity (reduction in numberof spermatogenic cells) and thinned seminiferous epitheliumwere recorded
















3 Results




20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(b)










20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

Apop

totic

inde

x (

)

10

15

20

5

0CP (mgkg) 50 50 50YC (mgkg) 630

lowastlowast

minus

minusminus 1260

(b)



4 Discussion







Relat

ive m

RNA

expr

essio

n Ba

xBc

l-2

0

100

250

200

150

50

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

lowast

(a)

00

15

10

05

lowast

Relat

ive p

rote

in ex

pres

sion

Bax

Bcl-2

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

24kDa Bcl-2

20kDa Bax

55kDa tubulin

(b)


lowast

001

000

004

003

002

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive m

RNA

expr

essio

n A

RG

APD

H

(a)

lowast

02

00

10

08

06

04

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive p

rote

in ex

pres

sion

AR

GA

PDH

110 kDa AR36kDa

GAPDH

(b)









5 Conclusions


Abbreviations








Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results




20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(b)










20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

Apop

totic

inde

x (

)

10

15

20

5

0CP (mgkg) 50 50 50YC (mgkg) 630

lowastlowast

minus

minusminus 1260

(b)



4 Discussion







Relat

ive m

RNA

expr

essio

n Ba

xBc

l-2

0

100

250

200

150

50

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

lowast

(a)

00

15

10

05

lowast

Relat

ive p

rote

in ex

pres

sion

Bax

Bcl-2

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

24kDa Bcl-2

20kDa Bax

55kDa tubulin

(b)


lowast

001

000

004

003

002

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive m

RNA

expr

essio

n A

RG

APD

H

(a)

lowast

02

00

10

08

06

04

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive p

rote

in ex

pres

sion

AR

GA

PDH

110 kDa AR36kDa

GAPDH

(b)









5 Conclusions


Abbreviations








Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(b)










20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

Apop

totic

inde

x (

)

10

15

20

5

0CP (mgkg) 50 50 50YC (mgkg) 630

lowastlowast

minus

minusminus 1260

(b)



4 Discussion







Relat

ive m

RNA

expr

essio

n Ba

xBc

l-2

0

100

250

200

150

50

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

lowast

(a)

00

15

10

05

lowast

Relat

ive p

rote

in ex

pres

sion

Bax

Bcl-2

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

24kDa Bcl-2

20kDa Bax

55kDa tubulin

(b)


lowast

001

000

004

003

002

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive m

RNA

expr

essio

n A

RG

APD

H

(a)

lowast

02

00

10

08

06

04

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive p

rote

in ex

pres

sion

AR

GA

PDH

110 kDa AR36kDa

GAPDH

(b)









5 Conclusions


Abbreviations








Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20120583m

(A)

20120583m

(B)

20120583m

(C)

20120583m

(D)

(a)

Apop

totic

inde

x (

)

10

15

20

5

0CP (mgkg) 50 50 50YC (mgkg) 630

lowastlowast

minus

minusminus 1260

(b)



4 Discussion







Relat

ive m

RNA

expr

essio

n Ba

xBc

l-2

0

100

250

200

150

50

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

lowast

(a)

00

15

10

05

lowast

Relat

ive p

rote

in ex

pres

sion

Bax

Bcl-2

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

24kDa Bcl-2

20kDa Bax

55kDa tubulin

(b)


lowast

001

000

004

003

002

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive m

RNA

expr

essio

n A

RG

APD

H

(a)

lowast

02

00

10

08

06

04

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive p

rote

in ex

pres

sion

AR

GA

PDH

110 kDa AR36kDa

GAPDH

(b)









5 Conclusions


Abbreviations








Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Relat

ive m

RNA

expr

essio

n Ba

xBc

l-2

0

100

250

200

150

50

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

lowast

(a)

00

15

10

05

lowast

Relat

ive p

rote

in ex

pres

sion

Bax

Bcl-2

CP (mgkg) 50 50 50YC (mgkg) 630

minus

minusminus 1260

24kDa Bcl-2

20kDa Bax

55kDa tubulin

(b)


lowast

001

000

004

003

002

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive m

RNA

expr

essio

n A

RG

APD

H

(a)

lowast

02

00

10

08

06

04

CP (mgkg) 50 50 50YC (mgkg) 630


Relat

ive p

rote

in ex

pres

sion

AR

GA

PDH

110 kDa AR36kDa

GAPDH

(b)









5 Conclusions


Abbreviations








Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusions


Abbreviations








Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article yangjing capsule ameliorates...

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