PERK-eIF2α-ATF4-CHOP通路在双酚A诱导小鼠睾丸支持细胞凋亡中的作用

The role of PERK-eIF2α-ATF4-CHOP pathway in the apoptosis of TM4 cells induced by bisphenol A

目的探讨双酚A(bisphenol A,BPA)对小鼠睾丸支持细胞(mouse testicular sertoli cells,TM4细胞)增殖和凋亡的影响及PERK-eIF2α-ATF4-CHOP通路在其中的作用。方法用不同浓度BPA(0、25、50和100μmol/L)及100μmol/L BPA联合蛋白激酶R样内质网激酶(protein kinase R-like ER kinase,PERK)抑制剂GSK2656157处理TM4细胞24 h后,利用TUNEL染色观察TM4细胞凋亡情况,Western blot检测凋亡相关蛋白Bax、Bcl-2、cleaved Caspase-3,内质网应激相关蛋白GRP78及PERK-eIF2α-ATF4-CHOP通路相关蛋白的表达情况。结果25、50和100μmol/L BPA染毒组TM4细胞的凋亡率分别增加为3.31%±0.34%、7.51%±1.10%和14.58%±0.91%,显著高于对照组的0.73%±0.03%(P<0.05);与对照组(1.00)相比,25、50和100μmol/L BPA染毒组TM4细胞的cleaved Caspase-3蛋白表达量分别升高为1.49±0.11、1.59±0.12和2.42±0.24,Bax/Bcl-2比值分别升高为2.06±0.19、3.94±0.34和6.14±0.71,GRP78蛋白表达量分别升高为1.29±0.06、1.39±0.06和1.92±0.17,p-PERK蛋白表达量分别升高为1.64±0.03、2.52±0.09和2.80±0.11,p-eIF2α蛋白表达量分别升高为1.79±0.05、2.48±0.10和4.77±0.32,ATF4蛋白表达量分别升高为2.51±0.03、3.24±0.14和7.45±0.51,CHOP蛋白表达量分别升高为1.44±0.01、3.20±0.11和3.80±0.11,差异均具有统计学意义(P<0.05)。与100μmol/L BPA染毒组相比,100μmol/L BPA+10μmol/L GSK2656157组p-PERK、p-eIF2α、ATF4、CHOP、cleaved Caspase-3蛋白表达量及Bax/Bcl-2比值分别降低为2.17±0.11、1.81±0.13、1.71±0.23、2.18±0.22、1.43±0.03和2.22±0.13,TM4细胞的凋亡率也显著降低为7.28%±0.47%,差异均具有统计学意义(P<0.05)。结论BPA通过激活内质网应激并调控PERK-eIF2α-ATF4-CHOP通路诱导TM4细胞发生凋亡。

OBJECTIVE To investigate the effects of bisphenol A(BPA)on the proliferation and apoptosis of mouse testicular sertoli cells(TM4 cells)and the role of PERK-eIF2α-ATF4-CHOP pathway.METHODS TM4 cells were treated with different concentrations of BPA(0,25,50,100μmol/L)and 100μmol/L BPA combined with protein kinase R-like ER kinase(PERK)inhibitor GSK2656157 for 24 h,and the apoptosis of TM4 cells was observed by TUNEL staining.The expression levels of Bax,Bcl-2,cleaved Caspase-3,GRP78 and PERK-eIF2α-ATF4-CHOP pathway-related proteins were detected by Western blot.RESULTS The apoptosis rate of TM4 cells in 25,50 and 100μmol/L BPA exposed groups was increased to 3.31%±0.34%,7.51%±1.10%and 14.58%±0.91%,respectively,which was significantly higher than that in control group(0.73%±0.03%,P<0.05).Compared with the control group(1.00),cleaved Caspase-3 protein expression of TM4 cells in the 25,50 and 100μmol/L BPA exposed groups increased to 1.49±0.11,1.59±0.12,2.42±0.24,respectively;the ratio of Bax/Bcl-2 increased to 2.06±0.19,3.94±0.034,6.14±0.71,respectively;the protein expression of GRP78 increased to 1.29±0.06,1.39±0.06,1.92±0.17,respectively;the expression of p-PERK protein was increased to 1.64±0.03,2.52±0.09,2.80±0.11,respectively;the expression of p-eIF2αprotein was increased to 1.79±0.05,2.48±0.10,4.77±0.32,respectively;ATF4 protein expression was increased to 2.51±0.03,3.24±0.14 and 7.45±0.51,respectively;CHOP protein expression was increased to 1.44±0.01,3.20±0.11 and 3.80±0.11,respectively,and all the differences were statistically significant(P<0.05).Compared to 100μmol/L BPA group,the expression level of p-PERK,p-eIF2α,ATF4,CHOP,cleaved Caspase-3 protein and the ratio of Bax/Bcl-2 in 100μmol/L BPA+10μmol/L GSK2656157 group were decreased to 2.17±0.11,1.81±0.13,1.71±0.23,2.18±0.22,1.43±0.03,2.22±0.13,respectively;the apoptosis rate of TM4 cells was also decreased to 7.28%±0.47%,all the differences were statistically significant(P<0.05).CONCLUSIONBPA can induce apoptosis of TM4 cells by activating endoplasmic reticulum stress and regulating PERK-eIF2α-ATF4-CHOP pathway.