Parkin介导的线粒体自噬在高糖高脂导致的心肌细胞损伤中的保护作用

Protective role of Parkin-mediated mitophagy in cardiomyocyte injury induced by high glucose and high fat

目的明确Parkin(一种E3泛素化连接酶)介导的线粒体自噬对高糖高脂导致的原代心肌细胞损伤的保护作用。方法以LV-lac Z(Lac Z空病毒)或LV-Parkin(Parkin过表达慢病毒)转染SD大鼠原代心肌细胞48 h,再用含葡萄糖(5.5 mmol/L NG)的培养基或含棕榈酸盐(500μmol/L HF)和葡萄糖(25 mmol/L HG)的高糖高脂培养基培养心肌细胞24 h。实验分组:(1)阴性对照组(NG-Lac Z),(2)正常Parkin过表达组(NG-Parkin),(3)高糖高脂阴性对照组(HG-HF-Lac Z),(4)高糖高脂Parkin过表达组(HG-HF-Parkin)。用Western blot法检测PTEN介导的假定激酶蛋白1(PINK1)、Parkin、P62(一种自噬相关蛋白)、微管相关蛋白1轻链3(LC3)蛋白表达水平。采用JC-1染色法检测活细胞内线粒体膜电位水平。免疫荧光法检测自噬体数量,TUNEL法检测细胞凋亡率。结果与对照组相比,高糖高脂处理的原代心肌细胞自噬相关蛋白LC3-II,P62表达水平上调(P<0.05),PINK1表达未发生统计学差异,Parkin表达水平下调(P<0.05),自噬体数量增多,线粒体膜电位下降功能损伤,心肌细胞凋亡率升高(P<0.05)。而用高糖高脂处理LV-Parkin转染的心肌细胞,LC3-II蛋白表达水平进一步升高(P<0.05),而P62表达水平显著下降(P<0.05),自噬体数量进一步增多,细胞内线粒体膜电位水平上升,心肌细胞凋亡率下降(P<0.05)。结论高糖高脂可引起SD大鼠原代心肌细胞自噬流量降低,自噬小体增多,线粒体自噬发生障碍。Parkin过表达慢病毒通过激活心肌细胞内线粒体自噬途径,提高自噬流量,改善线粒体功能,降低心肌细胞凋亡率。

AIM To determine the role of Parkin-mediated mitophagy in cardiomyocytes exposed to high glucose and saturated fatty acid stimulation. METHODS Neonatal mouse ventricular myocytes were separated and cultured in DMEM with normal （ 5.5 mmol/L） dose of glucose. LV-LacZ or LV-Parkin was transfected into cardiomyocytes. After transfected for 48 hrs, eardiomyocytes were cultured in DMEM with normal （5.5 retool/L） or high dose of glucose （25 mmol/L HG） and palmitate （16：0; 500mmol/L HF） for 24hrs. So we divided the experiment into four groups： NG-LacZ; NG-Parkin; HG-HF- LacZ; HG-HF-Parkin. The expression of proteins was analyzed by Western blot and the number of autophagosomes was counted by immunofluoreseence. The mitoehondrial membrane potential was measured by JC-1 staining and the apoptotic index was evaluated by TUNEL. RESULTS Compared with that inLC3II was significantly increased （P 〈 0. 05）, indicating the accumulation of autophagosomes. Up-regu- lated P62 （P 〈 O. 05 ） suggested that HG and HF resulted in impaired clearance of autophagosomes. Compared with those in HG-HF group,, LV-Parkin up-regulated the expression of Parkin and LC3II （P 〈 O. 05） and decreased the level of P62 significantly （ P 〈 0. 05 ） following HG-HF treatment, indicating that Parkin over-expression enhanced mitophagy and autophagy flux. Parkin reduced the apoptosis （P 〈 0. 05 ） caused by high glucose and high plamitate in cardiomyocytes. CONCLUSION Parkin-mediated mitophagy plays a protective role in cardiomvocvte injury induced by high glucose and high palmitate.