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目的观察热休克蛋白(HSP)对1-甲基-4-苯基-吡啶离子(MPP+)引起的线粒体功能障碍和氧化应激的保护作用。方法采用免疫印迹法观察热休克诱导HSP的表达以及转染的HDJ-1基因在细胞内的过表达。通过5,5′,6,6′-四氯-1,1′,3,3′-四乙基苯丙咪唑羰花青碘化物(JC-1)和2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)流式细胞术及荧光显微镜观察MPP+对细胞线粒体膜电势和活性氧族(ROS)的影响以及HSP的保护作用。结果热休克后4h即有Hsp70(7.37±1.17)和HDJ-1(2.32±0.37)增加,并至少持续到72h;同样,转染24h后HDJ-1基因在细胞内过表达(1.26±0·06),并至少持续到72h。MPP+能引起线粒体膜电势降低(60.77±3.68),同时细胞内ROS上升(483.18±16.98)。热休克和HDJ-1基因过表达不仅能维持线粒体膜电势(热休克组68.32±3.42,转HDJ-1组66.13±3.31),而且还能抑制ROS的产生(热休克组449.45±18.80,转HDJ-1组470.56±23.53),其中热休克的作用更强。结论HSP通过保护线粒体功能、减少氧化应激来减轻MPP+毒性,从而发挥保护细胞的作用。
Objective To observe the protective effect of heat shock protein (HSP) on mitochondrial dysfunction and oxidative stress induced by 1-methyl-4-phenyl-pyridinium ion (MPP +). Methods Western blotting was used to observe the expression of HSP induced by heat shock and the overexpression of HDJ-1 in transfected cells. Tetrachloro-1,1 ’, 3,3’-tetraethylbenzimidazole carbocyanine iodide (JC-1) and 2’, 7’-dichloro fluorescence The effect of MPP + on mitochondrial membrane potential and reactive oxygen species (ROS) and the protective effect of HSP were observed by flow cytometry and fluorescence microscopy with DCFH-DA. Results Hsp70 (7.37 ± 1.17) and HDJ-1 (2.32 ± 0.37) increased 4 h after heat shock and lasted for at least 72 h. Similarly, HDJ-1 gene was overexpressed within 1 h after transfection (1.26 ± 0 · 06) for at least 72h. MPP + caused a decrease in mitochondrial membrane potential (60.77 ± 3.68) and an increase in intracellular ROS (483.18 ± 16.98). Heat shock and HDJ-1 gene overexpression not only maintained mitochondrial membrane potential (heat shock group 68.32 ± 3.42, HDJ-1 group 66.13 ± 3.31), but also inhibited ROS production (heat shock group 449.45 ± 18.80, HDJ -1 group 470.56 ± 23.53), of which the role of heat shock stronger. Conclusions HSP protects cells by protecting mitochondrial function and reducing oxidative stress to reduce MPP + toxicity.