TRAF6基因对缺氧复氧肝细胞凋亡、Caspase-3、Caspase-9表达及线粒体膜电位的影响

Effects of TRAF6 gene on apoptosis, expressions of Caspase-3 and Caspase-9 and mitochondrial membrane potential in hypoxia-reoxygenation hepatocytes

目的探讨肿瘤坏死因子受体相关因子6(TRAF6)基因对缺氧复氧(H/R)肝细胞凋亡、Caspase-3和Caspase-9表达及线粒体膜电位的影响。方法人正常肝L02细胞分为对照组(不进行转染和H/R处理)、H/R组(细胞缺氧12 h,复氧12 h)、H/R+NC组(转染NC后进行H/R处理)和H/R+si-TRAF6组(转染si-TRAF6后进行H/R处理)。Western blotting检测TRAF6、Caspase-3和Caspase-9蛋白表达。流式细胞仪检测细胞凋亡率及膜电位变化。结果 si-TRAF6转染L02细胞后,细胞中TRAF6蛋白表达明显降低,与对照组比较,差异有统计学意义(P<0.05)。H/R处理可明显上调L02细胞TRAF6、Caspase-3和Caspase-9蛋白表达,促进细胞凋亡,降低线粒体膜电位,而抑制TRAF6蛋白表达可降低TRAF6、Caspase-3和Caspase-9表达,抑制细胞凋亡,提高线粒体膜电位。结论抑制TRAF6基因表达可降低H/R诱导的L02细胞凋亡,提高线粒体膜电位,下调Caspase-3和Caspase-9表达。

Objective To investigate the effect of TRAF6 gene on apoptosis, expressions of Caspase-3 and Caspase-9 and mitochondrial membrane potential in hypoxia-reoxygenation(H/R) hepatocytes. Methods Human normal liver L02 cells were divided into control group(without transfection and H/R treatment), H/R group(hypoxia for 12 hours, reoxygenation for 12 hours), H/R+NC group(after transfection for H/R treatment) and H/R+si-TRAF6 group(after transfection for H/R treatment). Western blotting was used to detect the expressions of TRAF6, Caspase-3 and Caspase-9 proteins. Cell apoptosis rate and membrane potential were detected by flow cytometry. Results The expression of TRAF6 protein in L02 cells transfected with si-TRAF6 was significantly lower than that in control group(P<0.05). H/R treatment can significantly up-regulate the expressions of TRAF6, Caspase-3 and Caspase-9 proteins in L02 cells, promote cell apoptosis, reduce mitochondrial membrane potential. Inhibition of TRAF6 expression could inhibit the expressions of TRAF6, Caspase-3 and Caspase-9, inhibit cell apoptosis and increase mitochondrial membrane potential. Conclusion Inhibition of TRAF6 gene expression can decrease H/R-induced apoptosis of L02 cells, increase mitochondrial membrane potential, and down-regulate the expressions of Caspase-3 and Caspase-9.