线粒体电压依赖性阴离子通道在心肌细胞缺氧/复氧损伤中的作用

Role of voltage-dependent anion channel in cardiomyocytes subjected to anoxia/reoxygenation

目的:探讨线粒体电压依赖性阴离子通道(VDAC)在H9c2心肌细胞缺氧/复氧(A/R)损伤中的作用及机制。方法:以pSUPER质粒为载体,构建shRNA VDAC1质粒。H9c2细胞随机分为5组:control组、缺氧/复氧(A/R)组、缺氧预处理(APC)组、pSUPER-VDAC1-A/R组和pSUPER-A/R组。Western blotting法测定VDAC1的蛋白表达,MTT法检测细胞存活率,生化自动分析仪测定乳酸脱氢酶(LDH)和肌酸磷酸激酶(CPK)活性,流式细胞仪法检测线粒体膜电位。结果:A/R处理后,VDAC1表达上调,细胞存活率下降,LDH和CPK活性增高,线粒体膜电位崩溃;而APC则能抑制VDAC1的上调,并对抗A/R损伤;与APC相似,通过RNA干扰下调VDAC1后,亦能有效保护心肌细胞,维持线粒体膜电位,提高细胞存活率。结论:下调VDAC1可有效对抗A/R损伤引起的线粒体通透性转换孔道的开放,维持线粒体膜电位,保护心肌细胞。

AIM：To investigate the role of voltage-dependent anion channel（VDAC） in H9c2 cardiomyocytes subjected to anoxia/reoxygenation（A/R）.METHODS： The shRNAs targeting VDAC1 mRNA were inserted into pSUPER plasmid.H9c2 cells were randomly divided into 5 groups as follows： control group,A/R group,anoxia preconditioning（APC） group,pSUPER-VDAC1-A/R group and pSUPER-A/R group.The expression of VDAC1 was detected by Western blotting.Cellular injury was evaluated by measuring the cell viability and the release of lactate dehydrogenase（LDH） and creatine phosphokinase（CPK）.The mitochondria membrane potential was determined by flow cytometry.RESULTS： VDAC1 expression was up-regulated in A/R group and was inhibited in APC group.Similarly,down-regulation of VDAC1 expression by shRNA protected H9c2 cells from A/R injury.Moreover,we found that,with silencing VDAC1 expression,mitochondrial membrane potential was well preserved in H9c2 cells subjected to A/R.CONCLUSION： Down-regulation of VDAC1 protects H9c2 cells against A/R injury and its possible mechanism appears to be related to the regulation of mitochondial permeability transition pore opening.