雷诺嗪预处理对缺氧/复氧损伤H9C2心肌细胞自噬的调控及对心肌细胞的保护作用

Regulation of ranolazine preconditioning on autophagy of H9C2 cardiomyocytes injured by hypoxia/reoxygenation and its protective effect on cardiomyocytes

目的:探讨雷诺嗪预处理对缺氧/复氧损伤H9C2心肌细胞的保护作用,从自噬调控角度阐明雷诺嗪保护缺血再灌注损伤心肌的作用机制。方法:将H9C2心肌细胞随机分为正常对照组、缺氧/复氧模型组、雷诺嗪预处理组、雷诺嗪+渥曼青霉素组、雷帕霉素组。正常对照组细胞不做任何处理,其余各组H9C2细胞以相应药物预处理30 min后,行缺氧4 h、复氧3 h处理。采用CCK-8法检测各组H9C2细胞活性,采用乳酸脱氢酶(LDH)试剂盒检测各组H9C2细胞中LDH活性,免疫荧光法检测各组H9C2细胞中微管相关蛋白轻链3(LC3)阳性表达率,Western blotting法检测各组H9C2细胞中LC3、哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化mTOR(p-mTOR)蛋白表达水平,计算LC3Ⅱ/LC3Ⅰ和p-mTOR/mTOR比值。结果:与缺氧/复氧模型组比较,雷诺嗪预处理组和雷帕霉素组H9C2细胞活性升高(P<0.01),H9C2细胞中LDH活性降低(P<0.01),LC3阳性表达率升高(P<0.05)。Western blotting法检测,与正常对照组比较,缺氧/复氧模型组H9C2细胞中LC3Ⅱ/LC3Ⅰ比值升高(P<0.01),p-mTOR/mTOR比值降低(P<0.01);与缺氧/复氧模型组比较,雷诺嗪预处理组和雷帕霉素组H9C2细胞中LC3Ⅱ/LC3Ⅰ比值升高(P<0.01),p-mTOR/mTOR比值降低(P<0.01)。结论:雷诺嗪预处理对缺氧/复氧损伤H9C2心肌细胞具有保护作用,其作用机制可能与雷诺嗪抑制mTOR蛋白磷酸化从而诱导缺氧/复氧H9C2心肌细胞自噬有关联。

Objective:To explore the protective effect of ranolazine preconditioning on the H9C2 cardiomyocytes injured by hypoxia/reoxygenation,and to elucidate the mechanism of ranolazine in protecting myocardium of ischemiareperfusion injury of the point of regulation autophagy view.Methods:The H9C2 cells were randomly divided into normal control group,hypoxia/reoxygenation model group,ranolazine preconditioning group,ranolazine+Wortmannin group,and rapamycin group.The cells in normal control group didn’t receive any treatment,and the H9C2 cells in the other groups were pretreated with the corresponding drugs,and then were treated with hypoxia for 4 h and reoxygenation for 3 h.The viabilities of the H9C2 cells in various groups were detected by CCK-8 method;the levels of lactate dehydrogenase(LDH)in the H9C2 cells in various groups were detected by LDH kit;the positive expression rates of microtubule-associated protein light chain 3(LC3)in the H9C2 cells in various groups were analyzed by immunofluorescence method;Western blotting method was used to detect the expression level of LC3,mammalian target of rapamycin(mTOR)and phosphorylated mTOR(p-mTOR)in the H9C2 cells in various groups,and the ratios of LC3Ⅱ/LC3Ⅰand p-mTOR/mTOR were calculated.Results:Compared with hypoxia/reoxygenation model group,the viabilities of H9C2 cells in ranolazine precoditioning group and rapamycin group were increased significantly(P<0.01),the activities of LDH in the H9C2 cells were decreased significantly(P<0.05),and the positive expression rates of LC3 were increased significantly(P<0.05).The Western blotting results showed that compared with normal control group,the ratio of LC3Ⅱ/LC3Ⅰin the H9C2 cells in hypoxia/reoxygenation model group was increased significantly(P<0.01)and the ratio of p-mTOR/mTOR was decreased significantly(P<0.01).Compared with hypoxia/reoxygenation model group,the ratios of LC3Ⅱ/LC3Ⅰin the H9C2 cells in ranolazine preconditioning group and rapamycin group were increased significantly(P<0.01),and the ratios of p-mTOR/mTOR were decreased significantly(P<0.01).Conclusion:Ranolazine preconditioning has a protective effect on the H9C2 cells injured by hypoxia/reoxygenation,and its mechanism may be related to its inhibition in mTOR protein phosphorylation and induction of the autophagy of H9C2 cells injured by hypoxia/reoxygenation.