The Alleviative Effect Of Cuscuta Chinensis Flavonoids On Reproductive Damage Of Offspring Mice Induced By Bisphenol A During Pregnancy

Bisphenol A is not only one of the most common chemical materials added in many daily necessities,but also an environmental endocrine disruptor(EEDs),which can disturb the normal physiological activities,especially,exerts its profound effect on the reproductive function.Cuscuta chinensis nourishes the kidney and calms the fetus.Modern pharmacology research found that Cuscuta chinensis flavonoids(CCFs)are the main component of Cuscuta chinensis.CCFs have the function of improving male and female reproductive endocrine,but the mechanism of that has not been fully revealed.Therefore,the study on the alleviating effect of CCFs on BPA-induced damage contributes to reveal the molecular mechanism of CCFs in tonifying kidney and consolidating essence.Thereby protect the reproductive function of animals and improve the fecundity.It also provides theoretical reference for the further study of reducing the reproductive toxicity of environmental pollutants like BPA.Selected experiment of BPA dosage:The BPA exposure model of pregnant mice was prepared by intragastric administration of BPA at the doses of 0,2.5,5,10,20,40 mg/kg/d at gestation day(GD)0.5-17.5.Fifteen offspring mice randomly selected from each group were sacrificed after anesthesia and collect the blood,uterus,ovaries and testes.Then the organic index,pups'survival rate and dams'abortation rate were calculated.The ovarian and testicular histomorphology were observed with hematoxylin-eosin staining(H&E).The activity of follicle stimulating hormone(FSH),luteinizing hormone(LH)and the contents of estradiol(E2),progesterone(P4)and testosterone(T)in serum were detected by radioimmunoassay.The contents of ovarian and testicular estrogen receptor alpha(ER?)and estrogen receptor beta(ER?)were detected by enzyme-linked immunosorbent assay(ELISA).The expression of caspase-7,caspase-9,bcl-2 and bax in ovaries and testes of offspring mice were detected by Western blot and immunofluorescence,and apoptosis in ovaries and testes was detected by TUNEL.The mRNA relative transcription levels of ERa and progesterone receptor(PgR)were detected by real-time quantitative PCR(RT-qPCR).For female offspring:The results showed that BPA at 5 mg/kg,10 mg/kg,20 mg/kg and 40 mg/kg significantly increased the abortion rate of the pregnant mice,and each dose of BPA significantly reduced the survival rate of the pups(P<0.01 or P<0.05).Besides,there was a non-monotonic dose-response relationship between serum hormone,ovarian receptor levels and BPA in F1 females at both PND 21 and 56.BPA increased the ovarian/uterine index in F1 females at both PND 21 and 56,increased the mRNA relative transcript levels of ovarian ER? and PgR in F1 females at PND 21,while decreased it at PND 56(P<0.01 or P<0.05).BPA also increased the relative expression of caspase-7,caspase-9,bax,inhibited the relative expression of bcl-2 in F1 females at both PND 21 and 56,and increased the apoptosis rate in the ovaries in F1 mice at PND 56(P<0.01).The number of follicles in the ovary was increased in F1 females at PND 21,and the ovaries were significantly atrophied when sexual maturity(PND 56).Our results indicated that BPA can promote the expression of ER?,advance the development of ovary,but increase the apoptosis rate of ovarian cells thus hinder the development of ovary.For male offspring:The results showed that maternal exposure to 20 mg/kg BPA during pregnancy significantly increased the testicular index of F1 males at PND 21,and 40 mg/kg BPA significantly decreased the testicular index of F1 males at PND 56(P<0.01).BPA significantly reduced serum T and E2 levels,improved testicular ERa and ER? levels in F1 males at both PND 21 and 56.BPA exposure also increased the expression of testicular caspase-7,caspase-9,bax,and decreased the expression of bcl-2 at PND 56.Consistent with that,BPA improved the apoptosis rate in the testis at PND 56(P<0.01 or P<0.05).Our study indicates that BPA disrupts the secretion of T,E2 and estrogen receptors in offspring males,which damages testicular tissues and affects the potential reproductive function.Protection experiment of CCFs:Six groups(n=20)of pregnant mice were intragastrically administrated with BPA(5 mg/kg/d)and CCFs(20,30,40 mg/kg/d)at gestation day(GD)0.5-17.5.Fifteen offspring mice randomly selected from each group were sacrificed after anesthesia and collect the blood,uterus,ovaries and testes.Then the pups' survival rate,dams' abortation rate and the related indexes of ovaries and testes were counted.For female offspring:Our results showed that 40 mg/kg/d CCFs significantly reduced the abortion rate of the dams and increased the survival rate of F1 mice.It also significantly reduced the ovarian index of F1 females and the ovarian cytoapoptosis(P<0.01).Forty mg/kg CCFs significantly inhibited the hypermethylation of the H19/Igf2 imprinted gene induced by BPA(P<0.01).It indicated that CCFs adjusted H19/Igf2 methylation by increasing the expression of DNA methyltransferases(DNMTs),thereby increasing the levels of reproductive hormones and receptors along with reducing the cytoapoptosis.For male offspring:Our results showed that compared with the BPA group,CCFs could significantly increase the serum contents of T,E2 in males at PND 21 and PND 56,as well as the contents and transcript levels of DNA methyltransferase 3A(Dnmt3A),Dnmt3B in males at PND 21 and that of ERa at PND 56.The expression of Dnmt1 and ER? at PND 21,ER? at both PND 21 and PND 56 in males was significantly decreased with the administration of different concentrations of CCFs(P<0.01 or P<0.05).CCFs also significantly inhibited the BPA-induced hypermethylated status of ERa promoter and H19/Igf2 imprinting control region(ICR)in testis at PND 56.These results indicated that CCFs could decrease the methylation levels of ERa by inhibiting the expression of DNMTs,thereby decrease the levels of reproductive hormones and receptors in adult males,so that alleviate the negative effect of BPA on testicular development in male mice.Our results showed that compared with the BPA group,the testicular index in CCFs groups were significantly increased at PND 21(P<0.01).For the mice of CCFs groups,the expression levels of bax,caspase-9 and caspase-7 proteins were significantly decreased at PND 21 and PND 56,while the expression level of bcl-2 protein was significantly increased,and testicular apoptotic cells were also decreased significantly(P<0.01 or P<0.05).The results indicated that CCFs could alleviate the reproductive damage of BPA by alleviating the cytoapoptosis of testes,regulating the levels of reproductive hormone,hormone receptors,DNMTs and the methylation of ER?.ConclusionThis study preliminarily confirmed that CCFs can alleviate BPA induced reproductive damage in both male and female,inhibit cell apoptosis in ovary and testis,and inhibit hypermethylation of H19/Igf2 in ovary and ER?/H19/Igf2 in testis by regulating reproductive hormone,hormone receptors and DNMTs.Thereby alleviate the reproductive damage caused by BPA.