百草枯诱导多巴胺能神经元α-突触核蛋白的自噬性降解障碍机制  

Autophagic dysfunction contributes toα-synuclein accumulation in dopaminergic neurons induced by paraquat

作　　者：武坷鑫 闫伟广 田甜 王一帆 黄敏 Wu Kexin;Yan Weiguang;Tian Tian;Wang Yifan;Huang Min(School of Public Health and Management,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院,职业卫生与环境卫生学系,银川750004

出　　处：《中华劳动卫生职业病杂志》2020年第3期180-186,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金项目(81560538);教育部"春晖计划"合作科研项目(Z2016059)。

摘　　要：目的探讨百草枯(PQ)对人神经母细胞瘤细胞(SH-SY5Y)自噬水平的影响及诱导α-突触核蛋白(α-syn)异常聚集的机制.方法以SH-SY5Y细胞作为多巴胺能神经元的体外模型,不同浓度PQ(0、18.75、37.5、75、150、300、600μmol/L)处理细胞24 h,150μmol/L PQ处理细胞不同时间(0、12、24、36、48、60、72、96 h),每组设5个复孔.CCK8法检测细胞相对存活率,确定剂量/时间-效应关系;不同浓度PQ(0、75、150、300、600μmol/L)处理细胞24h,150μmol/L PQ处理细胞不同时间,检测细胞LDH活力;蛋白质印迹法(Western blot)检测细胞内自噬微管相关蛋白轻链3(LC3)、自噬相关蛋白(Beclin1)、Ⅲ型磷脂酰肌醇-3-激酶(Vps34)、泛素结合蛋白(p62)及α-syn表达水平;实时荧光定量反转录聚合酶链反应(qRT-PCR)检测α-syn基因表达水平;免疫荧光法(Immunofluorescencetechnique,IF)检测α-syn表达水平;用自噬诱导剂雷帕霉素(Rapamycin,RAPA)100 nmol/L预处理细胞6h,Western blot检测处理前后细胞内自噬相关蛋白及α-syn的蛋白表达水平.结果PQ染毒细胞24h后,细胞相对存活率明显降低,差异有统计学意义(P<0.05).与正常对照组比较,PQ呈剂量依赖性和时间依赖性抑制SH-SY5Y细胞的增殖活性(P<0.05);与正常对照组比较,染毒组细胞上清中LDH活力明显升高(P<0.05);Western blot结果显示,与正常对照组比较,染毒组细胞内自噬相关蛋白比值(LC3II/LC3I)、Beclin1和Vps34蛋白表达水平明显下降(P<0.05),p62蛋白表达水平升高(P<0.05);细胞内α-syn蛋白和基因表达水平均明显升高(P<0.05);IF结果显示,经PQ处理后,细胞内α-syn荧光信号明显聚集于胞质,荧光强度增强(P<0.05);Western blot结果显示,与染毒组比较,RAPA干预组的LC3II/LC3I、Beclin1和Vps34蛋白表达水平明显升高(P<0.05),α-syn蛋白表达水平明显下降(P<0.05).结论PQ可能通过诱导SH-SY5Y细胞自噬功能障碍,引起细胞内α-syn的异常聚集.Objective To investigate the effects of Paraquat on autophagy level in SH-SY5Y cell and the mechanism of abnormal aggregation ofα-synuclein.Methods Human neuroblastoma cell(SH-SY5Y cell)was used as model of dopaminergic neurons in vitro.The cells were treated with different concentrations of PQ(0,18.75,37.5,75,150,300,600μmol/L)for 24 hours,and induced by 150μmol/L PQ for 0,12,24,36,48,60,72,96 hours to detect the relative survival rate of cells and determine dose/time-effect relationship.The cells were treated with concentration of 0,75,150,300,600μmol/L PQ for 24 hours,and induced by 150μmol/L PQ for different hours to detect intracellular LDH activity.The expression levels of autophagy-related proteins(LC3I,LC3II,Beclin1,Vps34 and p62)andα-synuclein were detected by Western blot.The gene expression level ofα-synuclein was assayed by Real-time quantitative PCR.The expression level ofα-synuclein was also evaluated by immunofluorescence.The cells were pretreated with 100 nmol/L autophagy inducer rapamycin(RAPA)for 6 hours.The expression levels of autophagy-related proteins andα-synuclein were detected by Western blot.Results CCK8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner;Compared with control group,the activity of LDH in the cell supernatant increased significantly after PQ exposure(P<0.05);Western blot analysis showed the ratio of autophagy-related protein LC3II/LC3I,Beclin1 and Vps34 protein expression were significantly lower after PQ treatment while the expression of p62 protein was higher(P<0.05);The protein and gene expression ofα-synuclein were increased significantly after PQ treatment(P<0.05);Immunofluorescence showed the fluorescence intensity ofα-synuclein in cells was significantly enhanced(P<0.05).Compared with PQ group,the expression levels of autophagy-related proteins LC3II/LC3I and Beclin1 were significantly increased whlieα-synuclein protein level was decreased after RAPA induction(P<0.05).Similarly,the IF result showed the fluorescence s

关 键 词：百草枯 SH-SY5Y细胞 自噬 Α-突触核蛋白 

分 类 号：R74[医药卫生—神经病学与精神病学]