自噬在双酚A诱导小鼠睾丸支持细胞凋亡中的作用  

Effect of autophagy on apoptosis induced by bisphenol A in TM4 cells

作　　者：刘暑霞 柳赟昊 张玲[1] 汪利芳 全超 LIU Shu-xia;LIU Yun-hao;ZHANG Ling;WANG Li-fang;QUAN Chao(School of Public Health,Medical College,Hubei Province Key Laboratory of Occupational Hazard Identification and Control,Wuhan University of Science and Technology,Wuhan,Hubei 430065,China;不详)

机构地区：[1]武汉科技大学公共卫生学院,职业危害识别与控制湖北省重点实验室,湖北武汉430065 [2]武汉市肺科医院中心实验室

出　　处：《环境与健康杂志》2024年第7期571-578,共8页Journal of Environment and Health

基　　金：湖北省教育厅科学技术研究项目(D20221101);武汉预防医学科研专项重大项目(WY22M01)。

摘　　要：目的 探究自噬在双酚A(bisphenol A,BPA)诱导的小鼠睾丸支持细胞株(TM4细胞)凋亡中的作用。方法 用不同浓度的BPA(0、20、40、80、160、320μmol/L)暴露TM4细胞24 h后,采用CCK-8法检测细胞活性。用不同浓度的BPA(0、25、50、100μmol/L)、100μmol/L BPA联合自噬诱导剂雷帕霉素(rapamycin,Rap)或抑制剂氯喹(chloroquine,CQ)暴露TM4细胞24 h后,采用MDC染色法和TUNEL染色法分别观察TM4细胞自噬和凋亡情况,采用Western blot法检测细胞内LC3-Ⅱ、Atg5、P62、Bax、Bcl-2及Cleaved Caspase-3等自噬和凋亡相关蛋白的表达情况。结果 与对照组比较,不同浓度BPA暴露TM4细胞的存活率和Bcl-2蛋白的表达量均降低,而LC3-Ⅱ、Atg5、P62、Bax、Cleaved Caspase-3蛋白的表达量及Bax/Bcl-2值均升高,差异有统计学意义(P<0.05);且随着BPA暴露浓度的升高,TM4细胞的存活率和Bcl-2蛋白的表达量均呈下降趋势,而LC3-Ⅱ、Atg5、P62、Bax、Cleaved Caspase-3蛋白的表达量及Bax/Bcl-2值均呈上升趋势。与对照组比较,BPA组、BPA+CQ组及BPA+Rap组TM4细胞的Bcl-2蛋白表达量显著降低,而LC3-Ⅱ、P62、Bax、Cleaved Caspase-3蛋白表达量及Bax/Bcl-2值显著升高,同时TM4细胞的凋亡率显著升高,差异有统计学意义(P<0.05);与BPA组比较,BPA+CQ组TM4细胞的Bcl-2蛋白表达量显著降低,而LC3-II、P62、Bax、Cleaved Caspase-3蛋白表达量及Bax/Bcl-2值显著升高,同时TM4细胞的凋亡率显著升高,差异有统计学意义(P<0.05);与BPA组比较,BPA+Rap组TM4细胞的Bcl-2蛋白表达量显著升高,而LC3-II、P62、Bax、Cleaved Caspase-3蛋白表达量及Bax/Bcl-2值显著降低,同时TM4细胞的凋亡率显著降低,差异有统计学意义(P<0.05)。结论 BPA诱导的TM4细胞凋亡可能是自噬流阻滞导致的,自噬流的通畅在BPA对TM4细胞的毒性损伤中可能发挥拮抗作用。Objective To research the role of autophagy in the apoptosis of mouse testicular support cell line(TM4 cells)induced by bisphenol A(BPA).Methods TM4 cells were exposed to different concentrations of BPA(0,20,40,80,160,320µmol/L)for 24 h,the cell activity was detected by CCK-8 method.TM4 cells were exposed to different concentrations of BPA(0,25,50,100 pmol/L)and 100µmol/L BPA combined with the autophagy inducer rapamycin(Rap)or the inhibitor chloroquine(CQ)for 24 h,the autophagy and apoptosis of TM4 cells were observed by MDC staining and TUNEL staining,respectively.The expressions of autophagy and apoptosis-related proteins such as LC3-Ⅱ,Atg5,P62,Bax,Bcl-2 and Cleaved Caspase-3 were detected by Western blot.Results Compared with the control group,the survival rate and Bel-2 protein expression of TM4 cells were decreased at different concentrations of BPA exposure,while LC3-Ⅱ,Atg5,P62,Bax,Cleaved Caspase-3 protein expression and Bax/Bcl-2 value were increased,with statistical significance(P<0.05).With the increase of BPA exposure concentration,TM4 cell survival rate and Bcl-2 protein expression decreased,while LC3-Ⅱ,Atg5,P62,Bax,Cleaved Caspase-3 protein expression and Bax/Bcl-2 value increased.Compared with the control group,the Bcl-2 protein expression of TM4 cells in the BPA,BPA+CQ and BPA+Rap groups was significantly decreased,while the expression levels of LC3-Ⅱ,P62,Bax,Cleaved Caspase-3 protein and Bax/Bcl-2 value were significantly increased,and the apoptosis rate of TM4 cells was significantly increased.The difference was statistically significant(P<0.05).Compared with the BPA group,the expression of Bcl-2 protein in TM4 cells in the BPA+CQ group was significantly decreased,while the expression levels of LC3-Ⅱ,P62,Bax,Cleaved Caspase-3 protein and Bax/Bcl-2 value were significantly increased,and the apoptosis rate of TM4 cells was significantly increased.The difference was statistically significant(P<0.05).Compared with the BPA group,the expression of Bel-2 protein in TM4 cells in the BPA+Rap

关 键 词：自噬 细胞凋亡 双酚A TM4细胞 

分 类 号：R994.6[医药卫生—毒理学]