Yan W G, Wang Y F, Tian T, Wang K D, Zhang B F, Zhang B Y, Huang M
Ningxia Key Laboratory of Environmental Factors and Chronic Disease Control, School of Public Health and Management, Ningxia Medical University, Yinchuan 750004, China.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2021 Apr 20;39(4):241-247. doi: 10.3760/cma.j.cn121094-20201221-00705.
To explore the mechanism of HDAC6 mediated aggresome-autophagy-lysosome pathway in paraquat-induced autophagy in dopaminergic neurons. Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with terminal concentrations of 0, 25, 50, 100, 200 and 400μmol/L PQ for 24 hours, and the cells were induced by 100 μmol/L PQ for different time (0, 12, 24, 36, 48, 60 and 72 h) . Cell viability was detected by CCK-8 assay. The expression levels of HDAC6, α-syn, Dynein IC1/2, LC3, Beclin1, p62 and Lamp-1 were detected by Western blot. Immunofluorescence double-labeling method was used to observe the expression and localization of HDAC6, α-syn, Dynein IC1/2, LC3, Lamp-1 and γ-tubulin in cells. CCK-8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner (=-0.950、-0.960, <0.05) .Western blot showed that compared with control group, the protein levels of HDAC6, α-syn, p62 in PQ-exposed group were significantly increased (<0.05) , but there was a significant decrease in expression level of the ratio of autophagy-related protein LC3 Ⅱ/LC3 Ⅰ, Beclin1, Dynein IC1/2, Lamp-1in PQ-exposed group (<0.05) . The results of immunofluorescence double-labeling showed that compared with the control group, the fluorescence signals of HDAC6 and α-syn in the PQ-exposed group increased, and the protein expression level increased, while the fluorescence signals of Dynein IC1/2, LC3, and Lamp-1 decreased. The protein expression level is reduced. HDAC6 gradually accumulates from the diffuse shape to the nucleus; Under normal circumstances, α-syn, Dynein IC1/2, γ-tubulin, LC3, and Lamp-1 are mainly distributed in the cytoplasm. After PQ is infected, they gather in the nucleus and co-localize with HDAC6 in the area around the nucleus. PQ may induce abnormal aggregation of α-syn by inducing HDAC6-mediated aggresome-autophagy-lysosomal pathway disorder.
探讨组蛋白去乙酰化酶6（HDAC6）介导的聚集体自噬-溶酶体途径在百草枯诱导的多巴胺能神经元自噬中的作用机制。体外以人神经母细胞瘤细胞（SH-SY5Y细胞）作为多巴胺能神经元模型。细胞分别用终浓度为0、25、50、100、200和400μmol/L的百草枯处理24小时，并用100μmol/L百草枯诱导不同时间（0、12、24、36、48、60和72小时）。采用CCK-8法检测细胞活力。通过蛋白质免疫印迹法检测HDAC6、α-突触核蛋白、动力蛋白中间链1/2（Dynein IC1/2）、微管相关蛋白1轻链3（LC3）、Beclin1、p62和溶酶体相关膜蛋白1（Lamp-1）的表达水平。采用免疫荧光双标法观察HDAC6、α-突触核蛋白、Dynein IC1/2、LC3、Lamp-1和γ-微管蛋白在细胞中的表达及定位。CCK-8法检测显示，百草枯诱导细胞存活率下降呈时间和剂量依赖性（r=-0.950、-0.960，P<0.05）。蛋白质免疫印迹法显示,与对照组相比，百草枯染毒组HDAC6、α-突触核蛋白、p62蛋白水平显著升高（P<0.05），但自噬相关蛋白LC3Ⅱ/LC3Ⅰ、Beclin1、Dynein IC1/2、Lamp-1表达水平显著降低（P<0.05）。免疫荧光双标结果显示，与对照组相比，百草枯染毒组HDAC6和α-突触核蛋白荧光信号增强，蛋白表达水平升高，而Dynein IC1/2、LC3和Lamp-1荧光信号减弱，蛋白表达水平降低。HDAC6从弥散状逐渐向细胞核聚集；正常情况下，α-突触核蛋白、Dynein IC1/2、γ-微管蛋白、LC3和Lamp-1主要分布于细胞质中，百草枯染毒后，它们在细胞核内聚集，并在核周区域与HDAC6共定位。百草枯可能通过诱导HDAC6介导的聚集体自噬-溶酶体途径紊乱，诱导α-突触核蛋白异常聚集。
