Apelin-13对葡萄糖剥夺乳鼠心肌细胞自噬的影响及机制

目的观察血管紧张素1型受体相关蛋白配体（apelin-13）对损伤心肌细胞自噬的影响,探讨其可能的作用机制。方法体外培养3 d龄内的SD乳鼠心肌细胞,通过葡萄糖剥夺（glucose deprivation,GD）方法建立心肌细胞损伤模型,将细胞随机分为5组：正常对照组（Control组）、葡萄糖剥夺组（GD组）、Apelin-13预处理组（GD＋Apelin-13组）、Tricribine＋Apelin-13预处理组（GD＋Apelin-13＋Tricribine组）和阻断剂组（Tricribine组）。采用透射电镜观察细胞内自噬体的变化,免疫沉淀脂质激酶分析法检测磷脂酰肌醇3-激酶（PI3K）的活性,Western-blotting法检测细胞内自噬相关蛋白-微管相关蛋白1轻链3（LC3）的表达,以及信号通路相关蛋白Akt、p-Akt、mTOR、p-mTOR的表达。结果葡萄糖剥夺可诱导乳鼠心肌细胞自噬体数量、LC3-Ⅱ/LC3-Ⅰ比值增加（P〈0.01）;Apelin-13预处理能减轻葡萄糖剥夺引起的心肌细胞损伤,使自噬体数量、LC3-Ⅱ/LC3-Ⅰ比值下降,提高PI3K的活性,上调Akt、mTOR蛋白的磷酸化水平（P〈0.01）;Akt特异性阻断剂Tricribine可以使Apelin-13的上述保护作用减弱（P〈0.01）。结论 Apelin-13在一定程度上可以抑制GD诱导的心肌细胞自噬,机制可能与激活PI3K/Akt/mTOR信号通路有关。

Effect of apelin-13 on glucose deprivation-induced autophagy of cardiomyocytes in suckling mice and its mechanism

Objective To observe the effect of angiotensin 1 receptor-related apelin-13 on glucose deprivation（GD）–induced autophagy of injured cardiomyocytes and its mechanism.Methods Cardiomyocytes from 3 days old SD suckling mice were incubated in vitro.A cardiomyocyte injury model was induced by GD.The cardiomyocytes were divided into control group,GD group,GD＋apelin-13 group,GD＋apelin-13＋tricribine group,and tricribine group.Intracellular autophagosomes were observed under transmission electron microscope.Activity of phosphoinositide-3-kinase（PI3K） was tested by immunoprecipitation lipid kinase assay.Expressions of autophagy-related microtubule light chain protein 3（LC3） and signal pathway-related proteins（Akt,p-Akt,mTOR and p-mTOR） were detected by Western blot.Results GD increased the number of autophagosomes and the LC3-II/LC3-I ratio（P0.01）.Apelin-13 pretreatment attenuated the GD-induced injury of cardiomyocytes,reduced the number of autophagosomes and the LC3-II/LC3-I ratio,up-regulated the activity of PI3K and the phosphorylation levels of Akt and mTOR（P0.01）.However,tricribine blocked the protective effect of apelin-13（P0.01）.Conclusion Apelin-13 can inhibit GD-induced autophagy of cardiomyocytes at a certain extent by activating the signaling pathway of PI3K,Akt and mTOR.