SIRT6-shRNA预处理对缺氧/复氧诱导的H9C2心肌细胞损伤的影响及其机制

Effect of pretreatment of SIRT-shRNA on H9C2 cardiomyocytes injure induced by anoxia/reoxygenation and its mechanism

目的探讨通过RNA干扰技术抑制H9C2心肌细胞内的SIRT6基因表达对缺氧/复氧(A/R)诱导的细胞损伤的影响和机制。方法将H9C2心肌细胞随机分为正常对照组(Con组)、A/R组、阴性对照SIRT6-shRNA质粒处理组(NC组)和SIRT6-shRNA质粒处理组(shRNA组)。检测4组H9C2心肌细胞的存活率、凋亡率、Caspase-3活性及SIRT6、核因子(NF)-κBp65、I-κBα表达水平及细胞培养液中白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α水平并进行比较。结果与Con组相比,A/R组H9C2心肌细胞存活率和细胞浆中I-κBα蛋白表达水平明显降低,凋亡率、细胞SIRT6mRNA和蛋白、细胞核中NF-κBp65蛋白表达水平、细胞培养液中IL-6和TNF-α水平及Caspase-3活性明显升高(P<0.05)。与A/R组比较,shRNA组H9C2心肌细胞存活率、细胞SIRT6 mRNA和蛋白及细胞浆中I-κBα蛋白表达水平明显降低,细胞凋亡率、细胞核中NF-κBp65蛋白、细胞培养液中IL-6和TNF-α水平及Caspase-3活性明显升高(P<0.05)。结论抑制H9C2心肌细胞内SIRT6基因表达能促进炎症因子的分泌,激活NF-κB信号通路,诱导细胞凋亡,加重A/R诱导的心肌细胞损伤。

Objective To explore the effect of inhibiting intracellular SIRT6 gene expression in H9C2 cardiomyocytes by RNA interference technology on cell injure induced by anoxia/reoxygenation(A/R)and its mechanism.Methods H9C2 cardiomyocytes were randomly divided into control(Con)group,A/R group,negative control(NC)group and SIRT6-shRNA plasmid-treated group(shRNA group).Survival rate,apoptotic rate,Caspase-3 activity,expression levels of SIRT6,nuclear factor(NF)-KBp65,I-kBα and in terleukin(IL)-6 and tumor necrosis factor(TNF)-α in culture medium of H9C2 cardiomyocytes in four groups were assessed and compared.Results Compared with Con group,survival rate of H9C2 cardiomyocytes and the expression level of I-kBα protein in cytoplasm decreased significantly in A/R group,apoptotic rate of H9C2 cardiomyocytes,expression levels of SIRT6 mRNA and protein,NF-KBp65 protein in nucleus,IL-6,TNF-a in culture medium and Caspase-3 activity increased significantly in A/R group(P<0.05).Compared with A/R group,survival rate of H9C2 cardiomyocytes,expression levels of SIRT6 mRNA and protein and I-kBa protein in cytoplasm decreased significantly in shRNA group,while apoptotic rate of H9C2 cardiomyocytes,expression levels of NF-KBp65 protein in nucleus,IL-6,TNF-α in culture medium and Caspase-3 activity increased significantly in shRNA group(P<0.05).Conclusion Inhibition of intracellular SIRT6 gene expression in H9C2 cardiomyocytes can promote the secretion of inflammatory cytokines,activate NF-kB signaling pathway,induce cell apoptosis and aggravate cardiomyocytes injury induced by A/R.