Effects Of Prenatal BPA Exposure On The Reproductive System Of Male Offspring And Its Epigenetic Mechanism

ObjectiveBisphenol A(BPA)is a widely used environmental endocrine disruptor that can enter the human body through a variety of ways,causing damage to the nervous,reproductive and immune systems.Studies have shown that BPA can induce apoptosis in immune cells,which in turn affects the body's normal immune function.Testicular macrophages(TM)are a type of immune cells present in the testicular stroma that play an important role in maintaining the entire testicular environment.In this study,rat TM cells were used as target cells,and lipopolysaccharide(LPS)was used as an activator to establish a BPA exposure model.Gene expression profiling chip technology was used to investigate the effect of BPA on TM cell gene expression.A BPA exposure model of rat TM cells was established,and its role in BPA-induced apoptosis of rat TM cells was investigated from the TGF-?/SMAD signaling pathway.On this basis,the model of BPA exposure in pregnant rats was established to explore the relationship between TGF-?/SMAD signaling pathway and testicular injury induced by BPA,which provided a theoretical basis for further study on the mechanism of BPA immune and reproductive toxicity.MethodTM cells were extracted from SD Rats.50 ?g/ml LPS as activator and 100?mol/L BPA in TM cells for 24h.Gene expression profiling technology was used to screen the most influential genes and signaling pathways;50 ?g/ml LPS was used as activator,and 100 ?mol/L BPA was used to treat TM cells for 4h and 24h.The apoptosis level of TM cells was detected by flow cytometry for 24h.RT-PCR was used to detect TGF-?2,TGF-?3,SMAD6,SMAD7,BAX and BCL-2 gene expression levels.A model of BPA exposure during pregnancy was established,and a control group(corn oil)andBPA group(25,50,100mg/kg)were established,respectively.from the 15th day of pregnancy to the 21st day after birth.epididymis,liver,kidney,adrenal gland were taken at 22 days after birth and 44 days after birth,weighed and frozen at-80?.The organ coefficients of each organ were calculated and compared.The apoptosis level of testis was detected by TUNEL staining.The expression levels of TGF-?2,TGF-?3,SMAD6,SMAD7,BAX and BCL-2 were detected by RT-PCR.The expression levels of BAX and BCL-2 protein were detected by Western Blot.Result1.Compared with the control group,LPS up-regulated the expression of 29 genes and reduced the expression of 3,525 genes.Compared with LPS,100 ?mol/L BPA increased the expression of 184 genes and decreased the expression of 154 genes.BPA affects biological processes such as the production and regulation of macrophage secreting factors,regulation of immune function,antigen presentation and apoptosis.The most obvious signal pathways for BPA up-regulation were TGF-?/SMAD signaling pathway,Rheumatoid arthritis(RA),Inflammatory bowel disease(IBD),Hippo signaling pathway,Graft-versus-host disease,etc.The most obvious down-regulation signal pathways wereRheumatoid arthritis,Amoebiasis,TNF signaling pathway,Pertussis,Jak-STAT signaling pathway.2.Compared with the 50 ?g/ml activator group,100?gmol/L BPA-treated TM cells for 24h had no significant effect on early apoptosis,!ate apoptosis and total apoptosis of TM cells(P>0.05).Compared with the 50 ?g/ml activator group,100 ?gmol/L BPA could significantly up-regulate BAX gene expression and down-regulate BCL-2 gene expression(P<0.05).At the same time,100 ?gmol/L BPA significantly up-regulated the expression of TGF-?3 and SMAD6 genes(P<0.05),but did not significantly change the expression of TGF-?2 and SMAD7(P>0.05).3.Compared with the control group,the adrenal organ coefficient of the 50 mg/kg BPA group was significantly increased at 22 days after birth,and the organ coefficients of other organs were not significantly changed(P>0.05).The testicular organ coefficient of the 25 mg/kg,50 mg/kg,100 mg/kg BPA group was significantly decreased(P<0.05),and the organ coefficient of other organs was not significantly changed(P>0.05).TUNEL staining of testicular tissue 22 days after birth showed that there were more brown-stained apoptotic cells in the 25 mg/kg,50 mg/kg,and 100 mg/kg BPA groups compared with the control group.PCR results showed that each group of BAX genes There was no significant change in expression,and the expression of BCL-2 gene was increased in 100 mg/kg BPA group(P<0.05).Compared with the control group,the expression of BAX and BCL-2 in the testis of 100 mg/kg BPA group increased at 44 days after birth(P<0.05),while the expression of BAX protein increased and the expression of BCL-2 protein did not change significantly-Compared with the control group,the expression of TGF-?2 and TGF-?3 in testis tissue of 100 mg/kg BPA group increased at 22 days after birth(P<0.05),and the expression of SMAD6 and SMAD7 genes not change significantly.The expression of TGF-?2 and TGF-?3 was increased in each group at 44 days after birth,and the expression of SMAD6 and SMAD7 gene was decreased(P<0.05).The gene expression difference was more significant with the increase of dose.Conclusion1.BPA can significantly affect TM cell gene expression.2.1n vitro studies indicate that BPA may induce apoptosis of TM cells by inhibiting TGF-?/SMAD signaling pathway.The vivo studies have shown that BPA exposure during pregnancy may induce testicular cell apoptosis by activating TGF-?/SMAD signaling pathway,causing damage to testis and affecting testicular development.In summary,the TGF-?/SMAD signaling pathway plays a role in BPA-induced apoptosis.