胰岛素对高糖引起H9c2细胞损伤的保护作用及机制研究

THE STUDY ON EFFECTS AND MECHANISMS OF INSULIN PROTECTING H9c2 AGAINST HIGH GLUCOSE INDUCING INJURY

目的:观察胰岛素对高糖诱导H9c2细胞凋亡的对抗作用,并探讨与内质网应激的相关机制。方法:培养大鼠心肌来源的H9c2细胞,以低糖(5.5 mmol/L)作为对照。H9c2细胞用高糖(35 mmol/L)培养基孵育不同时间(6h、12h、24h、48h、72h),建立心肌细胞高糖损伤模型,后用胰岛素(100nmol/L)处理细胞,采用MTT法和Hoechst染色检测细胞活力和凋亡;不同时间收集细胞mRNA,RT-PCR检测内质网应激分子标志物GRP78的表达;在高糖损伤的基础上分别用胰岛素以及LY294002(PI3K/Akt抑制剂)处理细胞,Western blotting检测各组细胞中t-Akt和p-Akt的蛋白表达水平。结果:高糖刺激H9c2细胞48h后,与对照组比较,细胞发生凋亡,细胞活力下降(P<0.05)。与对照组比较,高糖处理24h时,GRP78mRNA表达水平上调(P<0.01);与高糖组相比,胰岛素+高糖组p-Akt蛋白的表达显著增加(P<0.01),且LY294002能明显抑制胰岛素引起的p-Akt的表达上调(P<0.01)。结论:高糖可通过内质网应激引起心肌细胞凋亡,胰岛素通过PI3K/Akt信号通路的活化,保护心肌细胞因高糖刺激引起的内质网应激损伤。

Objective：To investigate the protective effect of insulin（INS）on high glucose（HG）-induced H9c2 apoptosis and the underlying mechanisms involving endoplasmic reticulum stress.Methods：H9c2cardiac myoblast cells were cultured in low glucose medium（5.5mmol/L）as control,while H9c2 cells were treated with 35 mM HG for 6,12,24,48 and 72hto establish a HG-induced damage model.MTT assay and Hoechst 33258 nuclear staining were utilized to assess the viability and apoptosis of the H9c2 cells.Total mRNA was extracted from cells at different time-points.A marker of endoplasmic reticulum stress,GRP78 mRNA expression was evaluated by RT-PCR.Bases on HG injury model,cells were also treated with INS（100nmol/L）or an PI3 Kinhibitor LY294002for 30 min.The expressions of t-Akt and p-AKT were detected by western blotting.Results：More apoptotic cells were presented in HG group compared with the control group（P〈0.05）.The expression of GRP78 mRNA was increased when cultured with HG for 24h（P〈0.01）.Compared with the HG group,the p-Akt expression was elevated significantly in INS＋HG group（P〈0.01）,and LY294002 abolished the effects of INS（P〈0.01）.Conclusion：HG could induce the apoptosis of cardiac cells by endoplasmic reticulum stress.Insulin could protect cells against HG-induced cardiac injury via the activation of the PI3K/Akt signaling pathway.