KEAP1/PGAM5/AIFM1介导的氧死亡途径参与磷酸三(1,3-二氯-2-丙基)酯暴露致小鼠睾丸支持细胞活力降低的机制

KEAP1/PGAM5/AIFM1 mediated oxeiptosis pathway in TDCIPP-induced reduction of TM4 cell viability

目的探讨磷酸三(1,3-二氯-2-丙基)酯[tri(1,3-dichloro-2-propyl)phosphate,TDCIPP]暴露对小鼠睾丸支持细胞(TM4细胞)的毒性作用及其潜在作用机制。方法分别用不同浓度的TDCIPP(0、12.5、25和50μmol/L)以及50μmol/L TDCIPP联合抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)处理TM4细胞24 h,CCK8法检测细胞活力,DCFH-DA探针法检测细胞内ROS水平,Western blot法检测细胞内氧死亡通路相关蛋白如KEAP1、PGAM5、AIFM1和磷酸化AIFM1(p-AIFM1)蛋白质水平。结果TDCIPP以剂量依赖方式降低了TM4细胞活力(P<0.05);12.5、25和50μmol/L TDCIPP染毒组TM4细胞ROS水平分别为9.44±1.42、17.25±1.81和18.38±2.66,显著高于对照组的5.08±0.90(P<0.05);5 mmol/L NAC+50μmol/L TDCIPP组TM4细胞ROS水平为14.70±0.50,显著低于TDCIPP组的26.44±0.73(P<0.05)。KEAP1siRNA+TDCIPP组和PGAM5siRNA+TDCIPP组的TM4细胞活力分别为77.00±1.73和76.67±1.53,显著高于TDCIPP组的68.67±1.53(P<0.05)。25和50μmol/L TDCIPP染毒组TM4细胞的KEAP1蛋白相对表达量分别为0.77±0.04和0.82±0.02,显著高于对照组的0.57±0.01(P<0.05);TDCIPP染毒组TM4细胞PGAM5蛋白相对表达量分别为1.17±0.04、1.38±0.03和1.41±0.03,显著高于对照组的0.81±0.02(P<0.05);AIFM1蛋白相对表达量分别为0.42±0.01、0.63±0.01和0.68±0.02,显著高于对照组的0.34±0.02(P<0.05);p-AIFM1蛋白相对表达量分别为1.73±0.02、1.52±0.02和0.73±0.01,显著低于对照组的2.25±0.02(P<0.05)。5 mmol/L NAC+50μmol/L TDCIPP组TM4细胞的KEAP1、PGAM5和AIFM1蛋白相对表达量分别为0.61±0.01、0.58±0.01和0.48±0.03,显著低于TDCIPP组的0.86±0.12(P<0.05)、0.74±0.02(P<0.05)和0.92±0.01(P<0.05);p-AIFM1蛋白相对表达量为0.45±0.11,显著高于TDCIPP组的0.23±0.01(P<0.05)。结论TDCIPP诱导TM4细胞活力降低可能与ROS介导的KEAP1/PGAM5/AIFM1通路调控细胞发生氧死亡有关。

OBJECTIVE To investigate the toxic effects and potential mechanisms of tri(1,3-dichloro-2-propyl)phosphate(TDCIPP)exposure on the mouse testicular supporting cell line(TM4 cells).METHODS TM4 cells were treated with different concentrations of TDCIPP(0,12.5,25 and 50μmol/L),or 50μmol/L TDCIPP combined with antioxidant N-acetylcysteine(NAC)for 24 h.Cell viability was assessed using the CCK8 assay,intracellular ROS levels were detected using the DCFHDA probe,and the protein levels of oxeiptosis-related proteins,such as KEAP1,PGAM5,AIFM1 and phosphorylated AIFM1(p-AIFM1),were detected using Western blot.RESULTS TDCIPP dose-dependently reduced TM4 cell viability(P<0.05).ROS levels in TM4 cells treated with 12.5,25 and 50μmol/L TDCIPP were 9.44±1.42,17.25±1.81 and 18.38±2.66,respectively,significantly higher than the control group’s 5.08±0.90(P<0.05).ROS levels in the 5 mmol/L NAC+50μmol/L TDCIPP group were 14.70±0.50,significantly lower than the corresponding TDCIPP group’s 26.44±0.73(P<0.05).The activity of TM4 cells in KEAP1siRNA+TDCIPP group and PGAM5siRNA+TDCIPP group were 77.00±1.73 and 76.67±1.53,respectively,significantly higher than TDCIPP group 68.67±1.53(P<0.05).The relative expression of KEAP1 protein in TM4 cells treated with 25 and 50μmol/L TDCIPP were 0.77±0.04 and 0.82±0.02,respectively,significantly higher than the control group’s 0.57±0.01(P<0.05).The relative expression of PGAM5 protein in TDCIPP-treated TM4 cells were 1.17±0.04,1.38±0.03 and 1.41±0.03,respectively,significantly higher than the control group’s 0.81±0.02(P<0.05).The relative expression of AIFM1 protein were 0.42±0.01,0.63±0.01 and 0.68±0.02,respectively,significantly higher than the control group’s 0.34±0.02(P<0.05).The relative expression of p-AIFM1 protein were 1.73±0.02,1.52±0.02 and 0.73±0.01,respectively,significantly lower than the control group’s 2.25±0.02(P<0.05).In the 5 mmol/L NAC+50μmol/L TDCIPP group,the relative expression of KEAP1,PGAM5 and AIFM1 proteins in TM4 cells were 0.61±0.01,0.58±0.01 and 0.48±0.03,respectively,significantly lower than the TDCIPP group’s 0.86±0.12(P<0.05),0.74±0.02(P<0.05)and 0.92±0.01(P<0.05).The relative expression of p-AIFM1 protein in the 5 mmol/L NAC+50μmol/L TDCIPP group was 0.45±0.11,significantly higher than the TDCIPP group’s 0.23±0.01(P<0.05).CONCLUSION The reduction of TM4 cell viability induced by TDCIPP may be related to ROS-mediated regulation of the KEAP1/PGAM5/AIFM1 pathway,leading to oxeiptosis.