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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。
Objective To clarify the protective effect of Parkin (an E3 ubiquitin ligase) -mediated mitochondrial autophagy on primary cardiomyocyte injury induced by high glucose and high fat. Methods Primary cardiomyocytes of SD rats were transfected with LV-lac Z (Lac Z virus) or LV-Parkin (Parkin overexpression lentivirus) for 48 h, then cultured in medium containing glucose (5.5 mmol / L NG) Cardiomyocytes were cultured in high glucose and high fat medium containing palmitate (500μmol / L HF) and glucose (25 mmol / L HG) for 24 h. The experimental groups were as follows: (1) negative control group (NG-Lac Z), (2) normal Parkin overexpression group (NG-Parkin), (3) high glucose and high fat negative control group 4) High glucose and high fat Parkin overexpression group (HG-HF-Parkin). The expression of PTEN-mediated protein kinase 1 (PINK1), Parkin, P62 (an autophagy-related protein) and microtubule-associated protein 1 light chain 3 (LC3) protein were detected by Western blot. The level of mitochondrial membrane potential in living cells was detected by JC-1 staining. The number of autophagosomes was detected by immunofluorescence and the apoptosis rate was detected by TUNEL. Results Compared with the control group, the expressions of LC3-II and P62 in primary cardiomyocytes were significantly increased (P <0.05), while the expression of PINK1 was not significantly different (P < 0.05). The number of autophagosomes increased, mitochondrial membrane potential decreased, and the apoptosis rate of cardiomyocytes increased (P <0.05). However, the expression of LC3-II protein in LV-Parkin-transfected cardiomyocytes was increased (P <0.05), while the expression of P62 was significantly decreased (P <0.05). The number of autophagosomes was further increased, The level of mitochondrial membrane potential increased and the rate of cardiomyocyte apoptosis decreased (P <0.05). Conclusion High glucose and high fat can cause the autophagic flux of primary cardiomyocytes of SD rats to decrease, increase of autophagic bodies and disorder of mitochondrial autophagy. Parkin overexpression of lentivirus through activation of mitochondrial autophagy pathway in myocardial cells, improve autophagy flux, improve mitochondrial function and reduce the rate of myocardial apoptosis.