乙酰胆碱抑制自噬保护高糖诱导的H9c2细胞损伤

目的 研究乙酰胆碱(ACh)对高糖诱导的心肌细胞损伤的影响及机制。方法 应用高糖处理H9c2细胞72 h,建立心肌细胞损伤模型。实验分为:①对照组(control);②高糖组(HG);③HG+ACh(10 -4 nmol·L^-1 )组;④HG+ACh+AICAR(0.5 nmol·L^-1 )组;⑤HG+ACh+雷帕霉素(rapamycin, Rap)组(Rap 50 nmol·L^-1 );⑥HG+3-MA(2 mmol·L^-1 )组。MTT及ATP含量检测,评估各组细胞活性;Western blot观察各组自噬、凋亡及AMPK/mTOR信号通路相关蛋白表达变化;透射电镜观察各组细胞自噬小体的数量变化。结果与对照组相比,HG组LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK比值降低( P <0.05),p-mTOR/mTOR、Bax/Bcl-2比值升高( P <0.05);与HG组相比,HG+3-MA组Bax/Bcl-2比值降低( P <0.05),HG+ACh组心肌细胞活性增加( P <0.05),LC3-Ⅱ/LC3-Ⅰ、p-AMPK/AMPK、Bax/Bcl-2比值降低( P <0.05),p-mTOR/mTOR比值升高( P <0.05);与HG+ACh组相比,HG+ACh+AICAR组p-AMPK/AMPK比值升高( P <0.05),p-mTOR/mTOR比值降低( P <0.05),而LC3-Ⅱ/LC3-Ⅰ比值升高( P <0.05),HG+ACh+Rap组Bax/Bcl-2比值升高( P <0.05)。结论 ACh通过AMPK信号通路抑制自噬,保护高糖诱导的心肌细胞损伤。

Acetylcholine protects high glucose-induced H9c2 cell injury by inhibiting autophagy

Aim To study the effects of acetylcholine (ACh) on high glucose(HG)-induced cardiomyocytes injury.Methods H9c2 cells were treated with HG for 72 h to establish a model of HG-induced cardiomyocyte injury.The experiment was divided into control, HG group, HG+ACh(10 -4 nmol·L^-1 ) group, HG+ACh+AICAR (0.5 nmol·L^-1 ) group, HG+ACh+Rap(50 nmol·L^-1 ) group, and HG+3-MA (2 mmol·L^-1 ) group.Cell viability was evaluated by MTT and ATP assay.The expressions of autophagy, apoptosis and AMPK-mTOR signaling pathway were detected by Western blot.The number of autophagosomes in each group was observed by transmission electron microscopy.Results Compared with control group, the ratio of LC3-Ⅱ/LC3-Ⅰ, p-AMPK/AMPK decreased ( P <0.05), and the ratio of p-mTOR/mTOR and Bax/Bcl-2 increased in HG group ( P <0.05).Compared with HG group, the activity of cardiomyocytes in HG+ACh group increased, the ratio of LC3-Ⅱ/LC3-Ⅰ, p-AMPK/AMPK, Bax/Bcl-2 decreased ( P <0.05), the ratio of p- mTOR/mTOR increased ( P <0.05), and the ratio of Bax/Bcl-2 decreased in HG+3-MA group.Compared with HG+ACh group, the p-AMPK/AMPK and p- mTOR/mTOR increased in HG+ACh+AICAR group ( P <0.05), LC3-Ⅱ/LC3-Ⅰ was elevated ( P <0.05), and the ratio of Bax/Bcl-2 increased in HG +ACh+Rap group.Conclusions ACh inhibits autophagy through the AMPK signaling pathway to protect HG -induced cardiomyocytes injury.