梓醇诱导自噬减轻高糖诱导的血管内皮细胞DNA损伤的机制研究

Study of catalpol-induced autophagy in reducing DNA damage in high glucose-induced vascular endothelial cells

目的:观察梓醇(CAT)减轻血管内皮细胞DNA损伤的作用,并从自噬角度来探讨其可能的作用机制。方法:将常规培养的EA.hy926细胞随机分为空白对照(CON)组、高糖(HG)组、梓醇高剂量(HG+CAT-H)组、梓醇低剂量(HG+CAT-L)组和自噬抑制剂3-甲基腺嘌呤(HG+CAT-H+3-MA)组。流式细胞术检测细胞活性氧(ROS)水平;ELISA法检测细胞上清中8-羟基脱氧鸟苷(8-OHdG)含量,免疫荧光检测细胞磷酸化组蛋白H2AX变异体(γH2AX)水平,Western blot方法检测各组细胞内γH2AX、beclin-1、p62和LC3-Ⅱ/LC3-Ⅰ蛋白的表达;电镜观察自噬小体的形成。结果:与HG组比较,HG+CAT-H组ROS生成明显减少,8-OHdG含量明显降低,γH2AX蛋白表达明显减少,beclin-1和LC3-Ⅱ/LC3-Ⅰ蛋白表达显著升高,p62蛋白表达则显著降低,自噬小体明显增多(P<0.05)。结论:梓醇能够有效地减轻HG诱导的EA.hy926细胞DNA损伤,其机制可能与梓醇促进细胞自噬进而增强损伤修复有关。

AIM:To observe the effect of catalpol(CAT)on DNA damage in vascular endothelial cells,and to explore its possible mechanism from autophagy.METHODS:The routine cultured EA.hy926 cells were randomly divided into blank control(CON)group,high glucose(HG)group,high-dose CAT(HG+CAT-H)group,low-dose CAT(HG+CAT-L)group,and autophagy inhibitor 3-methyladenine(HG+CAT-H+3-MA)group.The production of reactive oxygen species(ROS)was observed by flow cytometry.The content of 8-hydroxy-2’-deoxyguanosine(8-OHdG)was detected by ELISA.The production ofγH2AX was observed by immunofluorescence.The protein levels ofγH2AX,beclin-1,p62 and LC3-Ⅱ/LC3-Ⅰwere detected by Western blot.The formation of autophagosomes was observed by electron microscopy.RESULTS:Compared with HG group,ROS production,8-OHdG content and the expression of p62 andγH2AX in HG+CAT-H group were decreased,while beclin-1 and LC3-Ⅱ/LC3-Ⅰwere increased.Autophagosomes were increased in HG+CAT-H group.CONCLUSION:Catalpol reduces DNA damage in EA.hy926 cells induced by HG,and the mechanism may be related to the enhanced autophagy.