Study on Apoptosis of Mouse Leydig Cells Induced by BPA Based on Mitochondrial Damage
[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 150 μmol/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(0 μmol/L BPA)and BPA group(60 μmol/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(0 μmol/L BPA),BPA group(60 μmol/L BPA),and BPA and N-acetyl-L-cysteine(NAC)co-treatment group(60 μmol/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 60 μmol/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 60 μmol/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(Mfn2 and Sirt3 genes)were extremely significantly decreased(P＜0.01),while the mRNA expression of mitochondrial apoptosis pathway related genes(Casp3,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(Gpa 1,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(Casp3,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.
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维普
