凝集素样氧化型低密度脂蛋白受体-1对乙醇诱导的大鼠心肌细胞自噬的影响

Effect of lectin-like oxidized low-density lipoprotein receptor-1 on ethanol-induced autophagy in rat cardiomyocytes

目的探讨乙醇诱导下大鼠心肌细胞模型中凝集素样氧化型低密度脂蛋白受体-1(lectin-like oxidized low density lipoprotein receptor 1,LOX-1)的表达及其与自噬相关蛋白Beclin-1和自噬微管相关蛋白轻链3抗体Ⅱ(autophagy microtubule-associated protein light chain 3 antibodyⅡ,LC3-Ⅱ)的相关关系。方法选取对数生长期的大鼠心肌细胞随机分为以下6组:Blank组、乙醇组、乙醇+sh-NC组、乙醇+sh-LOX-1组、乙醇+OE-LOX-1组和乙醇+OE-LOX-1+si-NC组,除空白对照组外,其余各组细胞均用200 mmol/L酒精孵育1 d,体外构建乙醇诱导的大鼠心肌细胞模型。电镜观察自噬小体的数量。实时荧光定量PCR(Realtime PCR)和蛋白印迹法(Western blot)分别检测LOX-1、Beclin-1、LC3-I、LC3-Ⅱ在各组中的表达水平,并进行比较与分析。结果急性乙醇暴露后可使大鼠心肌细胞中LOX-1、Beclin-1、LC3-I和LC3-Ⅱ在基因和蛋白水平表达明显升高(P<0.01)。对LOX-1的表达抑制后,Beclin-1、LC3-I和LC3-Ⅱ表达明显下调(P<0.01),而在对LOX-1进行过表达处理后,则能够增加Beclin-1、LC3-I和LC3-Ⅱ的表达水平(P<0.01)。透射电镜下,急性乙醇摄入可造成大鼠心肌细胞中自噬小体增多;下调LOX-1后可减少自噬小体数量,而过表达LOX-1则会增加自噬小体数量(P<0.01)。结论LOX-1在急性乙醇诱导下的大鼠心肌细胞模型中表达上调,且与自噬相关因子(Beclin-1、LC3-Ⅱ)呈协同变化趋势,提示LOX-1可能通过上调Beclin-1和LC3-Ⅱ表达水平造成自噬水平的过度激活从而推动酒精致心肌损伤的病理过程。

AIM To investigate the expression of lectin-like oxidized low-density lipoprotein receptor-1(LOX-1)and its correlation with autophagy-associated protein Beclin-1 and autophagy microtubuleassociated protein light chain 3 antibody II(LC3-II)in an ethanol-induced rat cardiomyocyte model.METHODS Rat cardiomyocytes in logarithmic growth phase were randomly divided into the following 6 groups:Blank group,ethanol group,ethanol+sh-NC group,ethanol+sh-LOX-1 group,ethanol+OELOX-1 group and ethanol+OE-LOX-1+si-NC group,and the cells of all the groups except those of blank control group were incubated with 200 mmol/L alcohol for 1d.The ethanol-induced rat cardiomyocyte model was constructed in vitro and the number of autophagy bodies was observed by electron microscopy.The expression levels of LOX-1,Beclin-1,LC3-I and LC3-II in each group were detected by real-time fluorescence quantitative PCR(Realtime PCR)and protein blotting(Western blot),respectively,and their expressions were compared and analyzed.RESULTS Acute ethanol exposure resulted in significantly higher expressions of LOX-1,Beclin-1,LC3-I and LC3-II in rat cardiomyocytes at both gene and protein levels(P<0.01).After inhibition of LOX-1 expression,Beclin-1,LC3-I and LC3-II expressions were significantly down-regulated(P<0.01),whereas after over-expression treatment of LOX-1,the expression levels of Beclin-1,LC3-I and LC3-II increased(P<0.01).Under transmission electron microscopy,acute ethanol ingestion caused an increase in autophagy bodies in rat cardiomyocytes.Down-regulation of LOX-1 reduced the number of autophagy bodies,whereas over-expression of LOX-1 increased the number of autophagy bodies(P<0.01).CONCLUSION LOX-1 expression is up-regulated in an acute ethanol-induced rat cardiomyocyte model and shows a synergistic trend with autophagyrelated factors(Beclin-1 and LC3-II),suggesting that LOX-1 may contribute to the pathological process of alcohol-refined myocardial injury by up-regulating the expression levels of Beclin-1 and LC3-II,resulting in the over-activation of autophagy levels.