右美托咪定神经保护作用的自噬机制研究

Research of Autophagy for Neuroprotection of Dexmedetomidine

目的:探讨右美托咪定发挥神经保护作用的细胞自噬和线粒体自噬机制。方法:通过对SH-SY5Y细胞进行氧糖剥夺再灌注模拟全脑的缺血再灌注损伤,将细胞随机分为7组:(1)C组:对照组;(2)OGD/R组:氧糖剥夺再灌注损伤组;(3)DEX组:右美托咪定组;(4)3MA组:3-甲基腺嘌呤组;(5)D+3MA组;(6)RAPA组:雷帕霉素组;(7)D+RAPA组。结果:与OGD/R组相比,DEX组、3MA组、D+3MA组的细胞活性、电镜下完整线粒体的数量、自噬体数量明显好于OGD/R组(P<0.05);RAPA组与OGD/R组相比上述指标无明显差异(P>0.05);而RAPA中加入右美托咪定以后,可以部分逆转RAPA的作用,细胞活性增加,完整线粒体数量增加,自噬体数量减少(P<0.05)。免疫印迹结果显示,与OGD/R组相比,DEX组、3MA组、D+3MA组LC3II/LC3I、Beclin 1表达减少,BCL-2、P62、TOM20的表达增加,RAPA组各种自噬蛋白的表达与OGD/R组相比没有统计学意义,当应用右美托咪定之后逆转了各种蛋白的表达(P<0.05)。结论:右美托咪定通过减少过度的细胞自噬和线粒体自噬发挥神经保护作用。

Objective: To evaluate the mechanism of autophagy and mitophagy in which dexmedetomidine exerts neuroprotective effects. Methods: The SH-SY5 Y cells suffering oxygen and glucose deprivation/reperfusion were used to simulate ischemia-reperfusion injury of the whole brain, and the cells were randomly divided into 7 groups:(1) Group C: control group;(2) OGD/R group: oxygen and glucose deprivation/reperfusion injury group;(3) DEX group: dexmedetomidine group;(4) 3 MA group: 3-methyladenine group;(5)D+3 MA group;(6) RAPA group: rapamycin group;(7) D+RAPA group. Results: Compared with the OGD/R group, the activity of cells,the number of intact mitochondria and autophagosomes of the DEX group, 3 MA group, and D+3 MA group were significantly better than those of OGD/R group(P<0.05);There is no significant difference between RAPA group and OGD/R group in the above indicators(P>0.05);While dexmedetomidine was added to RAPA, the effect of RAPA could be partially reversed including the addition of cell activity and the number of intact mitochondria, the decrease of the number of autophagosomes(P<0.05). Immunoblotting results showed that compared with the OGD/R group, the expression of LC3 II/LC3 I and Beclin 1 in the DEX group, 3 MA group, DEX+3 MA group reduced,the expression of BCL-2, P62, TOM20 increased. There was no statistical significance in the expression of various autophagy proteins in the RAPA group Compared with the OGD/R group. When dexmedetomidine was applied, the expression of various proteins was reversed(P<0.05). Conclusion: Dexmedetomidine exerts neuroprotective effects by reducing excessive autophagy and mitophagy.