儿茶素没食子酸酯对人视网膜色素上皮细胞氧化应激损伤的机制研究

Inhibitory effect of epigallocatechin gallate on oxidative stress injury induced by H_2O_2 in human retinal pigment epithelial cells

目的探讨儿茶素没食子酸酯(EGCG)对过氧化氢(H_2O_2)诱导人视网膜色素上皮(ARPE-19)细胞氧化应激损伤及TRAF6/TAK1信号通路活化的影响。方法采用H_2O_2(100μmol/L)体外诱导ARPE-19细胞氧化应激损伤模型,将ARPE-19细胞分为正常对照组、H_2O_2诱导组、H_2O_2+低剂量(40μmol/L)EGCG组、H_2O_2+中剂量(80μmol/L)EGCG组、H_2O_2+高剂量(160μmol/L)EGCG组;CCK8法检测ARPE-19细胞相对存活率,相关试剂盒检测氧化应激产物MDA、NO水平及SOD1活性,Western Blot方法分析HO-1、SOD1、TRAF6、TAK1及p-TAK1蛋白表达。结果与H_2O_2诱导组比较,不同浓度EGCG干预的ARPE-19细胞相对存活率明显升高(P<0.05),细胞匀浆氧化应激产物MDA、NO水平明显降低(P<0.05),匀浆中SOD1活性显著升高(P<0.05),细胞中HO-1及SOD1表达水平显著升高(P<0.05),细胞中TRAF6及p-TAK1蛋白表达显著降低(P<0.05),并表现出剂量依赖性。结论 EGCG能明显抑制H_2O_2诱导的ARPE-19细胞氧化应激损伤,其作用机制可能与抑制TRAF6/TAK1信号通路活化有关。

OBJECTIVE To investigate the inhibitory effects of epigallocatechin gallate(EGCG) on oxidative stress injury induced by H2O2 in human retinal pigment epithelial(ARPE-19)cells and the activation of TRAF6/TAK1 signaling pathway. METHODS The H2O2(100 μmol/L)was used to induce the model of oxidative stress injury in ARPE-19 cells in vitro. All ARPE-19 cells were divided into five groups, including control group, H2O2-induced group, H2O2+low dose(40 μmol/L) EGCG group, H2O2+medium dose(80 μmol/L) EGCG group, and H2O2+high dose(160μmol/L) EGCG group. And CCK8 assay was used to measure the relative survival rates of ARPE-19 cells. Related kits were used to detect the levels of MDA, NO and the activity of SOD1. Western Blot was used to determine the expression levels of HO-1, SOD1, TRAF6, TAK1 and p-TAK1 proteins. RESULTS Compared with H2O2 induced group, the relative survival rates of ARPE-19 cells were greatly increased after treatment with different concentrations of EGCG(P<0.05). The levels of oxidative stress productions(MDA and NO) were significantly decreased, while the activity of SOD1 was greatly increased(P<0.05). The expression levels of HO-1 and SOD1 were also greatly increased, while the expression levels of TRAF6 and p-TAK1 were greatly decreased(P <0.05) with a dose-dependent manner.CONCLUSIONS The EGCG significantly inhibited the oxidative stress injury of ARPE-19 cells induced by H2O2 via suppressing the activation of TRAF6/TAK1 signaling pathway.