siRNA沉默Drp1对鱼藤酮诱导的帕金森病模型细胞自噬和凋亡的影响

Effects of siRNA silencing of Drp1 on Autophagy and cell Apoptosis in Parkinson's disease model cells induced by rotenone

目的探究siRNA沉默动力相关蛋白1(Drp1)后对鱼藤酮诱导的帕金森病模型细胞自噬以及细胞凋亡的影响。方法体外培养人神经母细胞瘤细胞SH-SY5Y,用1μm、5μm、10μm、20μm鱼藤酮进行处理24 h、48 h,MTT法检测细胞抑制情况。将细胞分为鱼藤酮最佳浓度处理组(20μm鱼藤酮处理,损伤组)、阴性转染组(损伤组基础上转染Drp1阴性序列)、Drp1siRNA处理组(损伤组基础上转染Drp1 siRNA)、Drp1抑制剂组(损伤组基础上添加Mdivi-1试剂)、自噬促进剂组(损伤组基础上添加雷帕霉素)。观察各组线粒体自噬情况、细胞线粒体功能相关蛋白(Mfn2、Drp1、NRF1)、自噬相关蛋白(Beclin-1、LC3、P62)、凋亡蛋白(Bcl-2、Bax)表达情况和细胞凋亡情况。结果与对照组相比,鱼藤酮处理组细胞抑制率均升高,随着浓度以及培养时间的增加,细胞抑制率逐渐增加(P<0.05)。损伤组、阴性转染组、自噬促进剂组LC3II定位在细胞线粒体,且细胞线粒体结构受损,外周出现自噬双层膜。与Drp1 siRNA转染组、Drp1抑制剂组相比,损伤组、阴性转染组、自噬促进剂组Drp1、LC3II、Beclin-1、Bax蛋白表达和细胞凋亡率升高,Mfn2、NRF1、P62、Bcl-2蛋白表达降低(P<0.05)。结论 Drp1参与鱼藤酮诱导帕金森病细胞线粒体自噬,沉默Drp1后可降低鱼藤酮诱导帕金森病细胞线粒体自噬以及细胞凋亡,具有保护作用。

Objective To investigate the effects of siRNA silencing of Drp1 on autophagy and cell apoptosis in Parkinson＇s disease model cells induced by Rotenone. Methods Human neuroblastoma cell SH-SY5 Y was cultured in vitro,the 1 μm,5 μm,10 μm,20 μm Rotenone was used to treat for 24 h,48 h,and MTT assay was used to detect cell inhibition. The cells were divided into rotenone optimal concentration group（ 20 μm rotenone treatment,injury group）,the negative transfection group（ based on the injury group was transfected with Drp1 negative sequence）,the Drp1 siRNA treatment group（ based on the injury group was transfected with Drp1 siRNA）,Drp1 inhibitor group（ added Mdivi-1 reagent on the basis of injury group）,the autophagy promoter group（ adding rapamycin on the basis of the injury group）. The autophagy of mitochondria in each group was observed,mitochondrial function related proteins（ Mfn2,Drp1,NRF1）,autophagy related proteins（ Beclin-1,LC3,P62）,apoptotic protein（ Bcl-2,Bax） and apoptosis were observed. Results Compared with the control group,the cell inhibition rate of Rotenone treatment group increased,with the increases of concentration and culture time,the cell inhibition rate increased gradually（ P〈0. 05）. The damage group,the negative transfection group and the autophagy promoter group LC3 II were located in the cell mitochondria,the mitochondria structure of the cells was damaged,and the autophagic bilayer membrane appeared in the peripheral area. Compared with Drp1 siRNA transfection group,Drp1 inhibitors group,damage,feminine transfection group,autophagy promoter Drp1,LC3 II,Beclin 1,Bax protein and the rate of apoptosis were increased,the expression of Mfn2,NRF1,P62 and Bcl-2 decreased（ P〈0. 05）. Conclusion Drp1 participates in autophagy of mitochondria in Parkinson＇s disease cells induced by Rotenone,after silencing of Drp1,the mitochondria autophagy and apoptosis of Parkinson＇s disease cells induced by Rotenone were reduced,it has a protective effect.