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.