1-甲基-4-苯基吡啶离子调控线粒体自噬对线粒体氧化应激损伤的影响

Effect of 1-methyl-4-pehny1-pyridine regulated mitochondrial autophagy on mitochondrial oxidative stress injury

目的探讨1-甲基-4-苯基吡啶离子(MPP+)诱导线粒体自噬在帕金森病(PD)发病机制中的作用。方法将细胞分为MPP+(0 mmol/L)对照组、MPP+(1 mmol/L)处理组和MPP+(2 mmol/L)处理组,共同转染EGFP-LC3和RFP-MI-TO后加入MPP+处理48 h。Western blot检测细胞自噬水平的变化,甲丹磺酸尸胺(MDC)检测自噬空泡聚集,免疫荧光法检测EGFP-LC3和RFP-MITO亚细胞共定位,流式技术检测线粒体膜电位及活性氧。结果 MPP+1 mmol/L及MPP+2 mmol/L组LAMP2 A、Beclin1和LC3-Ⅱ/LC3-Ⅰ的灰度值与对照组相比均上调,差异有统计学意义(P<0.05)。与对照组相比,MPP+处理组自噬水平增加,自噬空泡增加,外源性LC3表达上调,EGFP-LC3和RFP-MITO存在亚细胞共定位。MPP+1 mmol/L及MPP+2 mmol/L组线粒体膜电位较对照组降低;MPP+1 mmol/L及MPP+2 mmol/L组线粒体活性氧较对照组增加,差异均有统计学意义(P<0.05)。结论 MPP+通过调控线粒体自噬水平致线粒体氧化应激损伤。

Objective To investigate the role of 1-methyl-4-pehny1-pyridine（MPP＋） regulated mitochondrial autophagy in the pathogenesis of Parkinson＇s disease.Methods Human neuroblastoma cell lines（SH-SY5Y） were treated with MPP＋（0,1 or 2 mmol/L） for 48 hrs and then were transfected with EGFP-LC3 and RFP-MITO.Expression of LAMP2A,Beclin1 and LC3-II/LC-I proteins in SH-SY5Y cells was detected by Western blot after MPP＋ treatment for 48 hrs.Cellular immunefluorescent chemical method was used to observe the change of autophagic vacuolization and the signals of EGFP-LC3 and RFP-MITO,and their subcellular colocalization.The flow cytometry was used to detect the mitochondrial membrane potential and reactive oxygen species content.Results The gray values of LAMP2A,Beclin1 and LC3-II/LC3-I in the 1 mmol/L and 2 mmol/L MPP＋ treatment groups were significantly increased compared with the control group（0 mmol/L MPP＋ treat-ment）（P〈0.05）.The autophagy levels increased,the number of autophagic vacuolization increased,and expression of exogenous LC3 increased significantly in the two MPP＋ treatment groups compared with the control group.EGFP-LC3 and RFP-MITO subcellular colocalization was observed in the MPP＋ treatment groups.Mitochondrial reactive oxygen increased and mitochondrial membrane potential decreased significantly in the two MPP＋ treatment groups compared with the control group.Conclusions MPP＋ can induce mitochondrial oxidative stress injury through regulating levels of mitochondrial autophagy.