黄芪甲苷通过调控线粒体功能抑制H_(2)O_(2)诱导的SH-SY5Y细胞凋亡

Astragaloside Ⅳ inhibits H_(2)O_(2)-induced apoptosis of SH-SY5Y cells by regulating mitochondrial function

目的:探讨黄芪甲苷(astragaloside Ⅳ,AST Ⅳ)对由氧化应激损伤引起的线粒体功能障碍以及细胞凋亡的作用及机制。方法:体外培养人神经母细胞瘤SH-SY5Y细胞,用过氧化氢(hydrogen peroxide,HO)诱导建立氧化应激模型,分为PBS组、模型组(HO组)和HO+AST Ⅳ组。应用ATP检测试剂盒检测细胞内ATP水平;用线粒体膜电位检测试剂盒JC-1检测细胞线粒体膜电位变化;应用Western blot法检测线粒体呼吸链上的complex Ⅰ~Ⅴ,分别为NADH:泛醌氧化还原酶亚基B8(NADH:ubiquinone oxidoreductase subunit B8,NDUFB8;complex Ⅰ)、琥珀酸脱氢酶B(succinate dehydrogenase B,SDHB;complex Ⅱ)、泛醇-细胞色素C还原酶核心蛋白2(ubiquinol-cytochrome C reductase core protein 2,UQCRC2;complex Ⅲ)、细胞色素C氧化酶Ⅰ(mitochondrially encoded cytochrome C oxidase Ⅰ,MTCO1;complex Ⅳ)和ATP合酶F1亚基α(ATP synthase F1 subunit alpha,ATP5A;complex Ⅴ),检测线粒体动力学分裂蛋白——磷酸化发动蛋白相关蛋白1(phosporylated dynamin-related protein 1,p-Drp1)和线粒体分裂蛋白1(mitochondrial fission protein 1,Fis1),以及融合蛋白——线粒体融合蛋白1(mitofusin 1,Mfn1)、Mfn2和视神经萎缩症蛋白1(optic atrophy protein 1,OPA1),检测凋亡相关蛋白Bcl-2、Bax和cleaved caspase-3;采用免疫荧光染色法检测NDUFB8和MTCO1表达;采用TUNEL染色检测细胞凋亡。结果:在200μmol/L H_(2)O_(2)诱导的SY5Y细胞氧化应激模型中,线粒体膜电位(P<0.01)和ATP水平(P<0.05)显著下调,呼吸链氧化磷酸化过程中NDUFB8、SDHB、ATP5A和MTCO1的表达均显著减少(P<0.05或P<0.01),线粒体分裂蛋白Fis1(P<0.05)和p-Drp1(P<0.01)蛋白水平显著升高,融合蛋白Mfn1、Mfn2和OPA1表达则显著降低(P<0.05或P<0.01),促凋亡蛋白Bax和cleaved caspase-3蛋白水平显著升高(P<0.01),抗凋亡蛋白Bcl-2表达量显著降低(P<0.01)。AST Ⅳ能够显著提高细胞线粒体膜电位(P<0.01)和ATP水平(P<0.01),显著促进呼吸链氧化磷酸化过程中NDUFB8、SDHB、MTCO1、ATP5A及UQCRC2的表达(P<0.05或P<0.01),显著降低p-Drp1和Fis1蛋白水平(P<0.01),显著增加OPA1、Mfn1和Mfn2的表达(P<0.05或P<0.01),显著增加Bcl-2表达(P<0.05),显著降低Bax和cleaved caspase-3蛋白水平(P<0.01)。结论:AST Ⅳ能够保护神经元,其可能的机制是通过改善线粒体功能,调控线粒体动力学分裂/融合平衡,从而减轻氧化应激损伤导致的神经元凋亡。

AIM:To investigate the effects of astragaloside Ⅳ(AST Ⅳ)on mitochondrial damage and cell apoptosis in human neuroblastoma SH-SY5Y cells induced by hydrogen peroxide(H_(2)O_(2)).METH_(2)O_(2)DS:The SH-SY5Y cells were treated with PBS,H_(2)O_(2)or H_(2)O_(2)+AST Ⅳ. Intracellular ATP level was measured by ATP detection kit. Mitochondrial membrane potential was measured by mitochondrial membrane potential assay kit with JC-1. Mitochondrial respiratory chain-related proteins NADH:ubiquinone oxidoreductase subunit B8(NDUFB8;complex Ⅰ),succinate dehydrogenase B(SDHB;complex Ⅱ),ubiquinol-cytochrome C reductase core protein 2(UQCRC2;complex Ⅲ),mitochondrially encoded cytochrome C oxidase Ⅰ(MTCO1;complex Ⅳ)and ATP synthase F1 subunit alpha(ATP5A;complex Ⅴ),mitochondrial dynamic fission proteins phosphorylated dynamin-related protein 1(p-Drp1)and mitochondrial fission protein 1(Fis1),fusion proteins mitofusin 1(Mfn1),Mfn2 and optic atrophy protein 1(OPA1),and apoptosis-related proteins cleaved caspase-3,Bcl-2 and Bax were detected by Western blot. The expression of NDUFB8 and MTCO1 was detected by immunofluorescence staining. TUNEL staining was applied to observe SH-SY5Y cell apoptosis.RESULTS:The SHSY5Y cells were treated with H_(2)O_(2)at the dose of 200 μmol/L to establish the oxidative stress cell model. In oxidative stress model,mitochondrial membrane potential(P<0. 01)and ATP level(P<0. 05)were down-regulated significantly,and the expression of NDUFB8,SDHB,ATP5A and MTCO1 were significantly decreased(P<0. 05 or P<0. 01). Mitochondrial fission proteins Fis1(P<0. 05)and p-Drp1(P<0. 01)were significantly increased,fusion proteins Mfn1,Mfn2 and OPA1 were significantly decreased,the expression levels of pro-apoptotic proteins Bax and cleaved caspase-3 were significantly increased(P<0. 01),and Bcl-2 was significantly decreased(P<0. 01)after treatment with H_(2)O_(2). Treatment with AST Ⅳ significantly increased mitochondrial membrane potential(P<0. 01)and ATP level(P<0. 01),significantly upregulated the complex proteins NDUFB8,SDHB,MTCO1,ATP5A and UQCRC2(P<0. 05 or P<0. 01),significantly down-regulated the expression of mitochondrial fission proteins p-DRP1 and Fis1(P<0. 01),and remarkably up-regulated the expression of mitochondrial fusion proteins OPA1,Mfn1 and Mfn2(P<0. 05 or P<0. 01). Meanwhile,AST Ⅳ significantly increased the expression of Bcl-2(P<0. 05),significantly decreased the expression of Bax and cleaved caspase-3(P<0. 01),and inhibited SH-SY5Y cell apoptosis.CONCLUSION:Astragaloside Ⅳ attenuates the apoptosis of SHSY5Y cells induced by H_(2)O_(2)through regulating mitochondrial function.