目的:观察艾地苯醌对过氧化氢(H2O2)诱导的视网膜神经节细胞-5(RGC-5细胞)氧化损伤的保护作用。
方法:RGC-5细胞用各种浓度的艾地苯醌(5、10和20µM)预处理12小时,然后再接受300µMH2O212小时。通过流式细胞术测量RGC-5中的凋亡。JC-1染色检测线粒体膜电位(MMP)的变化。透射电镜观察RGC-5细胞的自噬。蛋白质印迹法检测自噬相关蛋白轻链3(LC3)的表达,Beclin-1和细胞色素c(Cyt-c)的释放。
结果:流式细胞仪显示正常对照组细胞凋亡率,H2O2组,艾地苯醌组为6.48±0.55%,27.3±0.51%,22.8±0.52%,15.45±0.81%,和12.59±0.58%,F=559.7,P<0.0001。与H2O2孵育后,自噬体数量显著增加,而艾地苯醌组下降。用H2O2孵育RGC-5细胞后,MMP水平显著降低,而艾地苯醌可以防止MMP水平的降低。与正常对照组相比,LC3II/I,H2O2组Beclin-1和Cyt-c的表达水平显著升高(P<0.05)。与H2O2组相比,LC3II/I,艾地苯醌组Beclin-1和Cyt-c表达显著降低(P<0.05)。
结论:艾地苯醌通过减少线粒体损伤和自噬活性,保护RGC-5细胞免受H2O2诱导的氧化损伤。
OBJECTIVE: To investigate the neuroprotective effect of
idebenone against hydrogen peroxide (H2O2)-induced oxidative damage in retinal ganglion cells-5 (RGC-5 cells).
METHODS: RGC-5 cells were pre-treated with various
idebenone concentrations (5, 10, and 20 µM) for 12 h and were then subjected to 300 µM H2O2 for a further 12 h. Apoptosis in RGC-5 was measured by flow cytometry. The changes of mitochondrial membrane potential (MMP) were detected by JC-1 staining. Autophagy in RGC-5 cells was observed by transmission electron microscopy. Western blots were used to measure the expression of autophagy-related protein light chain 3 (LC3), Beclin-1, and the release of Cytochrome c (Cyt-c).
RESULTS: Flow cytometry showed that the apoptosis rates in the normal control group, H2O2 group, and idebenone groups were 6.48 ± 0.55%, 27.3 ± 0.51%, 22.8 ± 0.52%, 15.45 ± 0.81%, and 12.59 ± 0.58%, respectively (F = 559.7, P < 0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, whereas it was decreased in the idebenone groups. After incubation of RGC-5 cells with H2O2, MMP levels were significantly decreased, while
idebenone could prevent the decrease in MMP levels. Compared with that in the normal control group, LC3 II/I, the expression levels of Beclin-1 and Cyt-c were increased significantly in the H2O2 group (P < 0.05). Compared with that in the H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c was significantly decreased in
idebenone groups (P < 0.05).
CONCLUSIONS: Idebenone protects RGC-5 cells against H2O2-induced oxidative damage by reducing mitochondrial damage and autophagic activity.