线粒体靶向性肽SS-31对H_2O_2诱导人晶状体上皮细胞凋亡的保护作用

Protective effect of mitochondria-targeted peptide SS-31 against H_2O_2-induced apoptosis in human lens epithelial cells

目的研究线粒体靶向性肽SS-31在H2O2诱导的人晶状体上皮细胞凋亡中的保护作用及其分子机制。方法体外培养人晶状体上皮细胞系SRA01/04,选取相同代次的SRA01/04细胞进行实验,应用100μmol/L H2O2构建凋亡模型,随机分为正常对照组、H2O2处理组、3个不同浓度的SS-31预处理组。应用MTT法检测各组细胞存活率;流式细胞术检测各组细胞凋亡率;Western blotting检测各组凋亡蛋白Bcl-2、Bax、Cleaved-Caspase-3的表达。结果H2O2处理24 h后,细胞存活率降低,凋亡率升高,促凋亡蛋白Bax的表达升高,抗凋亡蛋白Bcl-2的表达降低,Bax/Bcl-2比率升高,Cleaved-Caspase-3的表达升高。SS-31预处理能明显提高细胞的存活率,且与浓度呈正相关。同时,SS-31还能减少细胞的凋亡率,下调Bax的表达,上调Bcl-2的表达,减少Cleaved-Caspase-3的激活,从而发挥抗凋亡作用。结论SS-31能在H2O2诱导的人晶状体上皮细胞凋亡中有效地发挥保护作用,但能否应用于白内障治疗有待深入研究。

Objective To study the protective effect of SS-31 against H2O2-induced apoptosis in human lens epithelial cells and the possible mechanism. Methods SRA01 /04 cells,an immortal human lens epithelial cell line,were cultured in vitro. SRA01 /04 cells at the same generation were chosen and 100 μmol /L H2O2 was used to build the model of apoptosis.The cells were randomly allocated into five groups： the control group,the group treated by H2O2,and three groups pretreated by different concentrations of SS-31. The cell viabilities were monitored by using 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide（ MTT） assay. The cell apoptotic rates were detected with Annexin V-Propidium iodide double-staining flow cytometry. The expressions of Bcl-2,Bax and Cleaved-Caspase-3 were tested by using Western blotting. Results After exposure of 100 μmol /L H2O2 for 24 h,the cell viabilities decreased,cell apoptotic rates increased,expression of pro-apoptotic protein Bax increased,expression of anti-apoptotic protein Bcl-2 decreased,and expression of Cleaved-Caspase-3 increased. Pretreatment of SS-31 at different concentrations improved cell viabilities in a dose-dependent manner. Meanwhile,SS-31 plays an anti-apoptotic role by decreasing cell apoptotic rate,down-regulating the expression of Bax,up-regulating the expression of Bcl-2,and suppressing the activation of CleavedCaspase-3. Conclusion SS-31 efficiently protects human lens epithelial cells against H2O2-induced apoptosis. Further studies are needed on whether SS-31 can be used in medicine treatment for cataract.