利福平通过促进自噬保护帕金森病模型细胞损伤

Rifampin protects Parkinson's disease model cell by promoting autophagy

目的:探讨利福平对α-突触核蛋白(α-synuclein,α-syn)多聚体水平的影响及其与细胞自噬的关系。方法:用不同浓度鱼藤酮、利福平、自噬抑制剂氯喹和自噬促进剂雷帕霉素处理SH-SY5Y细胞,MTT法检测细胞存活率,筛选最佳实验浓度;蛋白质印迹法检测氯喹和雷帕霉素处理后不同时间点细胞内Beclin-1表达水平,确定最佳作用时间;蛋白质印迹法检测鱼藤酮、氯喹和雷帕霉素处理后Beclin-1和α-syn多聚体的表达;将SH-SY5Y细胞分为对照组、鱼藤酮组、利福平+鱼藤酮组和利福平组,流式细胞术检测4组细胞凋亡率,蛋白质印迹法检测α-syn多聚体及自噬标志蛋白Beclin-1、LC3-Ⅱ/Ⅰ、p62的表达;在上述分组基础上,蛋白质印迹法检测氯喹和雷帕霉素预处理后各组细胞α-syn多聚体的表达。结果:利福平浓度为150μmol/L时细胞存活率最高;氯喹、雷帕霉素调节自噬的最佳处理条件分别为10μmol/L(1 h)、10 nmol/L(2 h);与对照组相比,鱼藤酮、氯喹均能显著增加细胞内α-syn多聚体表达(P均<0. 01),降低Beclin-1表达(P均<0. 05),雷帕霉素能显著提高Beclin-1表达(P <0. 01);与对照组比较,鱼藤酮组细胞凋亡率明显上升(P <0. 05),p62和α-syn多聚体表达增加,Beclin-1和LC3-Ⅱ/Ⅰ表达明显降低(P均<0. 01);与鱼藤酮组相比,利福平+鱼藤酮组细胞凋亡率明显降低(P <0. 05),p62和α-syn多聚体表达明显减少,Beclin-1和LC3-Ⅱ/Ⅰ表达明显升高(P均<0. 01);与利福平+鱼藤酮组比较,氯喹预处理后α-syn多聚体表达明显增加(P <0. 01)。结论:利福平能减少鱼藤酮诱导的多巴胺能神经元凋亡,可能通过促进细胞自噬降低α-syn多聚体表达。

Objective:To investigate the effect of rifampicin on the level of a-synuclein(a-syn)mul-timer and its relationship with autophagy.Methods:Cells were treated with different concentrations of rotenone,rifampicin,autophagy inhibitor chloroquine and autophagy promoter rapamycin,MTT assay was used to detect the cell viability,and to screen the best experimental concentration.After cells were treated with chloroquine and rapamycin at different time points,Western blotting was used to detect the expression of Beclin-1 and a-syn multimer.SH-SY5Y cells were divided into four groups:control group,rotenone group,rifampicin+rotenone group and rifampicin group.The apoptosis rate of each group was detected by flow cytometry.Expression of the a-syn multimer and autophagy marker proteins Beclin-1,LC3-II/I and p62 were detected by Western blotting.On the basis of the above-mentioned grouping,Western blotting was used to detect the a-syn multimer expression of each group after pretreatment with chloroquine or rapamycin.Results:The cell survival rate was the highest when the concentration of rif-ampicin was 150(xmol/L.The optimal treatment conditions for chloroquine and rapamycin to regulate au-tophagy were 10(xmol/L(1 h)and 10 nmol/L(2 h),respectively.Compared with the control group,ro-tenone and chloroquine significantly increased a-syn multimer expression(P<0.01)and decreased Bec-lin-1 expression level(P<0.05).Rapamycin increased Beclin-1 expression level(P<0.01).Compared with the control group,the rotenone group showed higher apoptosis rate(P<0.05)and higher expression of p62 and a-syn multimer,while with lower levels of Beclin-1 and LC3-II/I(P<0.01).Compared with the rotenone group,the rifampicin group showed lower apoptosis rate(P<0.05)and lower expression of p62 and a-syn multimer,Beclin-1 level and LC3-II/I(P<0.01).The expression of a-syn multimer was increased after chloroquine treatment compared with rifampicin treatment group(P<0.01).Conclusion:Rifampicin reduces rotenone-induced apoptosis in dopaminergic neurons and may reduce a-syn multimer levels by promoting autophagy.