动力相关蛋白1通过诱导线粒体自噬对高糖导致心肌细胞损伤的影响

Drp1 acts as a safeguard mechanism against cardiomyocyte dysfunction induced by high glucose via mitophagy

目的明确动力相关蛋白(Drp)1通过诱导线粒体自噬对高糖条件下心肌细胞起保护作用。方法以Lac Z空病毒(LV-Lac Z)或Drp1过表达腺病毒(LV-Drp1)转染大鼠原代心肌细胞24 h,再用含葡萄糖(5.5 mmol/L)的正常培养基(NG)或含葡萄糖(33 mmol/L)的高糖培养基(HG)处理大鼠心肌细胞72 h。实验分组:1:(1)阴性对照(NG+Lac Z)组;(2)正常Drp1过表达(NG+Drp1)组;(3)高糖阴性对照(HG+Lac Z)组;(4)高糖Drp1过表达(HG+Drp1)组。采用免疫荧光法检测自噬体数量,用JC-1染色法检测线粒体膜电位水平,采用Western blot法检测Drp1、Parkin(一种E3泛素化连接酶),微管相关蛋白1轻链3(LC3)、p62蛋白表达水平,TUNEL法检测细胞凋亡率。结果与对照组相比,高糖处理组自噬体数量明显减少(P<0.05),线粒体膜电位显著下降(P<0.05),线粒体自噬相关蛋白LC3-Ⅱ、Parkin水平下调(P<0.05),Drp1,P62表达水平显著增高(P<0.05),心肌细胞凋亡率升高(P<0.05);与高糖组比较,Drp1过表达组可显著增加自噬体数量(P<0.05),线粒体膜电位上升(P<0.05),Drp1,Parkin,LC3-Ⅱ表达水平显著上升(P<0.05),P62水平显著下降(P<0.05),心肌细胞凋亡率下降(P<0.05)。结论高糖可引起SD大鼠原代心肌细胞自噬水平降低和线粒体功能障碍,是糖尿病心肌病发病机制之一。Drp1通过提高线粒体自噬水平改善线粒体功能,降低高糖条件下心肌细胞凋亡率。

AIM To investigate the role of Drp1-related mitophagy in cardiomyocytes exposed to high glucose （HG）. METHODS Neonatal mouse ventricular myocytes were separated and cultured in DMEM with normal （5.5 mmol/L） doses of glucose for 48 hrs. Drp1 over-expression adenovirus was transfected into cardiomyocytes, After 24 hrs transfection, cardiomyocytes were cultured in DMEM with normal （5.5 mmol/L） or high doses （33 mmol/L） of glucose for 72 hrs. So we divided the experiment into four groups： ①NG＋lacZ group; ②NG＋Drp1 group; ③HG＋LacZ group; ④HG＋Drp group. Expressions of Drp1, Parkin, LC3 and P62 were analyzed by Western blot. The number of autophagosomes was measured by immunofluorescence, the mitochondrial membrane potential was measured by JC-1 staining and the apoptotic index was examined by TUNEL. RESULTS Compared with those in the control group, expressions of Parkin, LC3 and the number of autophagosomes were obviously decreased. The expressions of Drp1 and P62 were elevated significantly （P〈0.05）, the mitochondrial membrane potential was degraded （P〈0.05） and the apoptosis index was increased significantly （P〈0.05） when cardiomyocytes were exposed to HG. Compared with those in the HG group, LV-Drp1 up-regulated the expression of Parkin, LC3 and autophagosomes （P〈0.05）, enhanced the mitochondrial membrane potential（P〈0.05） and reduced the apoptosis index （P〈0.05） when cardiomyocytes were cultured with high glucose. CONCLUSION Drp1 improves the mitochondrial membrane potential and down-regulates the apoptosis index in cardiomyocytes exposed to high glucose via mitophagy.