基于线粒体损伤探讨双酚A诱导小鼠睾丸间质细胞凋亡的研究

Study on Apoptosis of Mouse Leydig Cells Induced by BPA Based on Mitochondrial Damage

【目的】从线粒体损伤角度探究双酚A(BPA)暴露引起小鼠睾丸间质细胞(TM3)凋亡的作用机制。【方法】利用不同浓度BPA(0、10、30、60、90、120、150μmol/L)处理TM3细胞24 h后,采用CCK-8法筛选出引起细胞凋亡的最低BPA浓度。将TM3细胞随机分为对照组(0μmol/L BPA)和BPA组(60μmol/L BPA),每组3个重复,各处理24 h。通过倒置光学显微镜观察细胞状态,TUNEL染色观察细胞凋亡。再将TM3细胞随机分为对照组(0μmol/L BPA)、BPA组(60μmol/L BPA)、BPA和N-乙酰-L-半胱氨酸(NAC)共处理组(60μmol/L BPA+5 mmol/L NAC),每组3个重复,各处理24 h。ELISA法检测细胞培养上清中丙二醛(MDA)含量和总超氧化物歧化酶(T-SOD)活性,试剂盒检测细胞内活性氧(ROS)水平,JC-1染色观察细胞线粒体膜电位,实时定量PCR检测细胞中谷胱甘肽过氧化物酶1(Gpx1)、谷氨酸-半胱氨酸连接酶修饰剂亚基(Gclm)、过氧化氢酶(Cat)、谷氨酸-半胱氨酸连接酶催化亚基(Gclc)、线粒体融合蛋白2(Mfn2)、NAD依赖性蛋白脱乙酰酶Sirtuin3(Sirt3)、半胱天冬酶3(Casp3)、细胞色素C(Cycs)、B淋巴细胞瘤-2(BCL-2)相关X蛋白(Bax)基因mRNA表达水平,Western blotting检测BAX和细胞凋亡调节因子BCL-2蛋白的相对表达量,流式细胞仪检测细胞凋亡率。【结果】TM3细胞在60μmol/L BPA处理24 h后,细胞相对存活率开始出现极显著下降(P<0.01),细胞凋亡率极显著增高(P<0.01),细胞数量明显减少。与对照组相比,60μmol/L BPA处理24 h后,TM3细胞中MDA含量极显著增加(P<0.01),T-SOD活性极显著下降(P<0.01),抗氧化相关基因(Gpx 1、Cat、Gclm、Gclc基因)mRNA表达水平显著或极显著降低(P<0.05或P<0.01),ROS生成极显著增加(P<0.01);细胞中红/绿荧光强度比值极显著下降(P<0.01),线粒体功能相关基因(Mfn 2、Sirt 3基因)mRNA表达水平极显著降低(P<0.01),线粒体凋亡通路相关基因(Casp 3、Cycs、Bax基因)mRNA表达水平极显著升高(P<0.01),BAX蛋白表达量极显著升高(P<0.01),BCL-2蛋白的相对表达量极显著降低(P<0.01),细胞凋亡率极显著升高(P<0.01)。与BPA处理组相比,BPA+NAC处理组TM3细胞中MDA含量显著降低(P<0.05),T-SOD活性显著升高(P<0.05),抗氧化相关基因(Gpx 1、Cat、Gclm、Gclc基因)mRNA表达水平显著升高(P<0.05),ROS生成极显著减少(P<0.01),细胞中绿色荧光极显著减弱,红色荧光极显著增强,红/绿荧光强度比值显著升高(P<0.05),线粒体功能相关基因(Mfn 2、Sirt 3基因)mRNA表达水平显著或极显著升高(P<0.05或P<0.01),线粒体凋亡通路相关基因(Casp 3、Cycs、Bax基因)mRNA表达水平极显著降低(P<0.01),BAX蛋白表达量极显著降低(P<0.01),BCL-2蛋白相对表达量极显著升高(P<0.01),细胞凋亡率极显著下降(P<0.01)。【结论】BPA暴露可导致TM3细胞发生氧化应激,引起线粒体功能受损,从而诱导细胞凋亡。

【Objective】The aim of this study was to investigate the mechanism of action of apoptosis induced by bisphenol A(BPA)exposure in mouse Leydig cells(TM3)from the perspective of mitochondrial damage.【Method】The TM3 cells were treated with different concentrations of BPA(0,10,30,60,90,120 and 150μmol/L)for 24 h,and the lowest BPA concentration causing apoptosis was screened by CCK-8 method.TM3 cells were randomly divided into control group(0μmol/L BPA)and BPA group(60μmol/L BPA)with three replicates in each group for 24 h.The state of cells was observed by inverted light microscope and apoptosis was observed by TUNEL staining.The TM3 cells were randomly divided into control group(0μmol/L BPA),BPA group(60μmol/L BPA),and BPA and N-acetyl-L-cysteine(NAC)co-treatment group(60μmol/L BPA+5 mmol/L NAC),with three replicates in each group for 24 h.The content of malondialdehyde(MDA)and the activity of total superoxide dismutase(T-SOD)in the supernatant of cell culture were detected by ELISA method,the level of intracellular reactive oxygen species(ROS)was detected by kit,and the mitochondrial membrane potential was observed by JC-1 staining.The mRNA expression levels of glutathione peroxidase 1(Gpx1),glutamate-cysteine ligase modifier subunit(Gclm),catalase(Cat),glutamate-cysteine ligase catalytic subunit(Gclc),mitochondrial fusion protein 2(Mfn2),NAD-dependent protein deacetylase Sirtuin3(Sirt3),caspase 3(Casp3),cytochrome C(Cycs),B-cell lymphoma-2(BCL-2)associated X protein(Bax)in cells were detected by Real-time quantitative PCR.The relative expression of BAX and apoptosis regulatory factor BCL-2 proteins were detected by Western blotting,and the cell apoptosis rate was detected by flow cytometry.【Result】After treated with 60μmol/L BPA for 24 h,the relative survival rate of TM3 cells began to decrease extremely significantly(P<0.01),the number of apoptosis was extremely significantly increased(P<0.01),and the number of cells were obviously decreased.Compared with control group,after 24 h of treatment with 60μmol/L BPA,MDA content in TM3 cells was significantly increased(P<0.01),T-SOD activity was decreased significantly(P<0.01),the mRNA expression of antioxidation related genes(Gpx1,Cat,Gclm and Gclc genes)were significantly or extremely significantly reduced(P<0.05 or P<0.01),ROS production was extremely significantly increased(P<0.01).The red/green fluorescence intensity ratio was extremely significantly decreased(P<0.01).The mRNA expression of mitochondrial function related genes(Mfn 2 and Sirt 3 genes)were extremely significantly decreased(P<0.01),while the mRNA expression of mitochondrial apoptosis pathway related genes(Casp 3,Cycs and Bax genes)were extremely significantly increased(P<0.01),the expression of BAX protein was extremely significantly increased(P<0.01),the relative expression of BCL-2 protein was extremely significantly decreased and the apoptosis rate was extremely significantly increased(P<0.01).Compared with the BPA-treated group,the MDA content in TM3 cells of the BPA+NAC treatment group was significantly reduced(P<0.05),T-SOD activity was significantly increased(P<0.05),the mRNA expression levels of antioxidant-related genes(Gpx1,Cat,Gclm and Gclc genes)were significantly increased(P<0.05),the ROS production was extremely significantly decreased(P<0.01).The red/green fluorescence intensity ratio was increased significantly(P<0.05).The mRNA expression levels of mitochondrial function-related genes(Mfn2 and Sirt3 genes)were significantly or extremely significantly increased(P<0.05 or P<0.01),and the mRNA expression of mitochondrial apoptosis pathway related genes(Casp 3,Cycs,Bax genes)were extremely significantly decreased(P<0.01).The expression of BAX protein was extremely significantly decreased(P<0.01),while the relative expression of BCL-2 extremely significantly increased(P<0.01),and apoptosis rate was extremely significantly decreased(P<0.01).【Conclusion】BPA exposure induced oxidative stress,resulting in mitochondrial dysfunction and subsequently triggering apoptosis in TM3 cells.