Wu K X, Yan W G, Tian T, Wang Y F, Huang M
School of Public Health and Management, Ningxia Medical University, Yinchuan 750004, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2020 Mar 20;38(3):180-186. doi: 10.3760/cma.j.cn121094-20190924-00543.
To investigate the effects of Paraquat on autophagy level in SH-SY5Y cell and the mechanism of abnormal aggregation of α-synuclein. 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, 600 μmol/L) for 24 hours, and induced by 150 μmol/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, 600 μmol/L PQ for 24 hours, and induced by 150 μmol/L PQ for different hours to detect intracellular LDH activity. The expression levels of autophagy-related proteins(LC3I, LC3II, Beclin1 , Vps34 and p62) and α-synuclein were detected by Western blot. The gene expression level of α-synuclein was assayed by Real-time quantitative PCR. The expression level of α-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 and α-synuclein were detected by Western blot. 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 (<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 (<0.05) ; The protein and gene expression of α-synuclein were increased significantly after PQ treatment ( <0.05) ; Immunofluorescence showed the fluorescence intensity of α- synuclein in cells was significantly enhanced ( <0.05) . Compared with PQ group, the expression levels of autophagy-related proteins LC3II/LC3I and Beclin1 were significantly increased whlie α-synuclein protein level was decreased after RAPA induction (<0.05) . Similarly, the IF result showed the fluorescence signal of α- synuclein significantly decreased after RAPA induction (<0.05) . Paraquat induced autophagy dysfunction in SH-SY5Y cells, which leads to an abnormal aggregation of α-synuclein.
探讨百草枯对SH-SY5Y细胞自噬水平的影响及α-突触核蛋白异常聚集的机制。以人神经母细胞瘤细胞（SH-SY5Y细胞）作为体外多巴胺能神经元模型。将细胞用不同浓度的百草枯（0、18.75、37.5、75、150、300、600μmol/L）处理24小时，并用150μmol/L百草枯诱导0、12、24、36、48、60、72、96小时，检测细胞相对存活率并确定剂量/时间效应关系。将细胞用0、75、150、300、600μmol/L浓度的百草枯处理24小时，并用150μmol/L百草枯诱导不同时间，检测细胞内乳酸脱氢酶（LDH）活性。通过蛋白质免疫印迹法检测自噬相关蛋白（LC3I、LC3II、Beclin1、Vps34和p62）及α-突触核蛋白的表达水平。通过实时定量PCR检测α-突触核蛋白的基因表达水平。还通过免疫荧光评估α-突触核蛋白的表达水平。细胞先用100nmol/L自噬诱导剂雷帕霉素（RAPA）预处理6小时。通过蛋白质免疫印迹法检测自噬相关蛋白及α-突触核蛋白的表达水平。细胞计数试剂盒8（CCK8）检测显示百草枯诱导细胞存活率呈时间和剂量依赖性下降；与对照组相比，百草枯处理后细胞上清液中LDH活性显著升高（<0.05）；蛋白质免疫印迹分析显示，百草枯处理后自噬相关蛋白LC3II/LC3I、Beclin1和Vps34蛋白表达比例显著降低，而p62蛋白表达升高（<0.05）；百草枯处理后α-突触核蛋白的蛋白和基因表达显著增加（<0.05）；免疫荧光显示细胞中α-突触核蛋白的荧光强度显著增强（<0.05）。与百草枯组相比，雷帕霉素诱导后自噬相关蛋白LC3II/LC3I和Beclin1的表达水平显著升高，而α-突触核蛋白水平降低（<0.05）。同样，免疫荧光结果显示雷帕霉素诱导后α-突触核蛋白的荧光信号显著降低（<0.05）。百草枯诱导SH-SY5Y细胞自噬功能障碍，导致α-突触核蛋白异常聚集。
