The Role Of PERK-ATF4-CHOP Pathway Mediating Autophagosome Accumulation In The Apoptosis Of TM4 Cells Induced By Bisphenol A

Background Bisphenol A(BPA)has been proved to be an environmental endocrine disruptor.BPA,as an important raw material for the synthesis of polycarbonate plastics and epoxy resins,inevitably escapes into the environment during the production and use process.Therefore,the exposure of BPA in the environment and the population is very common.There have been a lot of studies on the mechanism of BPA in male reproductive toxicity,but the focus is mainly on spermatogenic cells.Testis sertoli cells are important part of testis and play a crucial role in spermatogenesis.Whether BPA affects spermatogenesis by affecting the function of testis sertoli cells is worthy of further exploration.It has been reported that BPA can induce autophagy and apoptosis in goat testes sertoli cells,but the relationship between autophagy and apoptosis and the mechanism of action need further study.Objective This study aimed to observe the effects of BPA on autophagy and apoptosis of mouse testicular Sertoli cells(TM4),so as to explore the relationship between autophagy and apoptosis and the role of endoplasmic reticulum stress-related PERK-ATF4-CHOP pathway in regulation of autophagy and apoptosis.Methods The effects of BPA on TM4 cells viability were detected using the CCK-8assay.TM4 cells were treated with different concentrations of BPA(0,25,50,100μmol/L)or 100 μmol/L BPA combined with Rapamycin(Rap,an autophagy inducer,300 nmol/L)or Chloroquine(CQ,an autophagy inhibitor,5 μmol/L),respectively,for 24 h.The autophagy and apoptosis of TM4 cells were observed by MDC staining and TUNEL staining,respectively,and the expression of autophagy-related proteins and apoptosis-related proteins such as LC3,Atg5,Atg7,Beclin1,P62,Bax,Bcl-2,and Cleaved Caspase-3 was detected by Western blot.Different concentrations of BPA(0,25,50,and 100 μmol/L)were used to treat TM4 cells for 24 h,and the expression of endoplasmic reticulum stress-related protein GRP78 and PERK-ATF4-CHOP pathway-related proteins was detected by Western blot.TM4 cells were treated with 100 μmol/L BPA combined with the PERK inhibitor GSK2656157 for 24 h,and the autophagy and apoptosis of TM4 cells were observed by MDC staining and TUNEL staining,respectively.The expression of autophagy-related proteins and apoptosis-related proteins such as LC3,P62,Bax,Bcl-2,Cleaved Caspase-3 and PERK-ATF4-CHOP pathway-related proteins was detected by Western blot.Results Compared with the control group,the survival rate of TM4 cells decreased with the increase of BPA exposure concentration(P<0.05);the average fluorescence intensity of MDC staining in the BPA treatment group significantly increased and showed an upward trend with the increase of BPA concentration(P<0.05);the apoptosis rate of TM4 cells increased with the increase of BPA concentration(P<0.05);the protein expression levels of LC3-II,Atg5,Atg7,Beclin1,P62,Cleaved Caspase-3,and the Bax/Bcl-2 ratio in the BPA treatment group were significantly increased(P<0.05).Compared with the 100 μmol/L BPA group,the average fluorescence intensity of MDC staining significantly increased in the 100 μmol/L BPA+CQ group(P<0.05);100 μmol/L BPA+CQ significantly increased the protein expression levels of LC3-II,P62,Cleaved Caspase-3,and the Bax/Bcl-2 ratio(P<0.05),and the apoptosis rate of TM4 cells significantly increased(P<0.05).Compared with the 100 μmol/L BPA group,the average fluorescence intensity of MDC staining significantly decreased in the 100 μmol/L BPA+Rap group(P<0.05);100 μmol/L BPA+Rap significantly decreased the protein expression levels of LC3-II,P62,Cleaved Caspase-3,and the Bax/Bcl-2 ratio(P<0.05),and the apoptosis rate of TM4 cells significantly decreased(P<0.05).The expression levels of GRP78 protein in each treatment group increased with the increase of BPA concentration and the difference was statistically significant compared with the control group(P<0.05).Compared with the control group,the expression of PERK-ATF4-CHOP pathway related proteins(p-PERK,p-e IF2α,ATF4 and CHOP)increased with the increase of BPA exposure concentration(P<0.05).Compared with the 100 μmol/L BPA group,the protein expression levels of p-PERK,p-e IF2α,ATF4,CHOP,LC3-II,P62,Cleaved Caspase-3,and the Bax/Bcl-2 ratio significantly decreased(P<0.05),and the apoptosis rate of TM4 cells significantly decreased(P<0.05)in the 100 μmol/L BPA+GSK2656157 group.Meanwhile,Compared with the 100 μmol/L BPA group,the average fluorescence intensity of MDC staining also significantly decreased in the 100 μmol/L BPA+GSK2656157 group(P<0.05).Conclusions BPA can activate endoplasmic reticulum stress and induce apoptosis of TM4 cells by regulating the PERK-ATF4-CHOP pathway mediating abnormal accumulation of autophagosomes.The patency of autophagic flow may play a protective role in the toxic injury of BPA on TM4 cells.