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SIRT1表达可减轻TNF-α诱导的心肌细胞炎症损伤
作者:郑婉  林云  张光星  杨洋  颜亚妮 
单位:海南医学院第一附属医院 心血管内科, 海南 海口 570102
关键词:心肌细胞 核转录因子κB 沉默信息调节因子1 炎症损伤 
分类号:R364.5;R-33
出版年·卷·期(页码):2019·38·第四期(579-585)
摘要:

目的:研究沉默信息调节因子1(SIRT1)在肿瘤坏死因子-α(TNF-α)诱导的心肌细胞炎症损伤中的作用。方法:用20μg·L-1的TNF-α处理大鼠心肌细胞H9c2,分别用real-time PCR和Western blotting方法测定细胞中SIRT1表达水平。用含有SIRT1的慢病毒感染H9c2细胞,筛选稳定感染的细胞株,给予TNF-α刺激以后,采用real-time PCR和Western blotting方法测定感染效果。用四甲基偶氮唑蓝(MTT)法测定细胞增殖,流式细胞术测定细胞凋亡,同时用Western blotting方法检测细胞中p-p65蛋白水平。用核转录因子κB(NF-κB)激活剂PMA处理过表达SIRT1的H9c2细胞,再用上述方法分析NF-κB激活剂对TNF-α环境下SIRT1对H9c2细胞增殖凋亡的逆转作用。结果:TNF-α处理后细胞中SIRT1表达水平降低,稳定感染SIRT1慢病毒上调TNF-α条件下心肌细胞中SIRT1的表达水平。TNF-α处理后H9c2细胞增殖活性降低,细胞凋亡率升高。与只经过TNF-α诱导的细胞比较,过表达SIRT1的H9c2细胞经TNF-α诱导后,细胞增殖活性升高,细胞凋亡率降低,细胞中p-p65蛋白水平降低,差异均有统计学意义(P<0.05)。NF-κB激活剂PMA可逆转过表达SIRT1对H9c2细胞增殖活性和凋亡的作用。结论:SIRT1通过抑制NF-κB减少TNF-α诱导的心肌细胞凋亡,提高细胞增殖活性,减少炎症损伤。

Objective: To study the myocardial cell inflammatory injury in SIRT1 induced by TNF-α. Methods: Rat cardiomyocytes H9c2 were treated with 20 μg·L-1 TNF-α. The expression of SIRT1 in rat cardiomyocytes was measured by real-time PCR and Western blotting, respectively. H9c2 cells were infected with lentivirus containing SIRT1, screening stable infected cell lines, after stimulation with TNF-α, real-time PCR and Western blotting were used to determine the infecting effect. Cell proliferation was measured by MTT assay. Cell apoptosis was measured by flow cytometry. At the same time, the level of p-p65 protein was detected by Western blotting. H9c2 cells overexpressing SIRT1 were treated with PMA, an activator of NF-κ B. The above methods were used to analyze the reversal effect of NF-κ B activator on proliferation and apoptosis of H9c2 cells induced by SIRT1 in TNF-α environment. Results: The expression of SIRT1 in TNF-α treated cells decreased. Stable infection of SIRT1 lentivirus up-regulated the expression of SIRT1 in myocardial cells under TNF-α condition,the proliferation activity of H9c2 cells treated with TNF-α decreased, the apoptotic rate increased. H9c2 cells overexpressing SIRT1 were induced by TNF-α, cell proliferation activity increased, the apoptotic rate decreased, the level of p-p65 protein in cells decreased, compared with the cells induced only by TNF-α, the difference was significant (P<0.05). PMA treatment with NF-κ B activator reversed the effect of SIRT1 overexpression on proliferation and apoptosis of H9c2 cells. Conclusion: SIRT1 reduces TNF-α-induced cardiomyocyte apoptosis by inhibiting NF-κB.

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