论文部分内容阅读
目的:探讨组蛋白去乙酰化酶6(HDAC6)介导蛋白包涵体-自噬-溶酶体途径(AALP)在百草枯(PQ)诱导多巴胺能神经元自噬障碍中的作用。方法:以人神经母细胞瘤细胞(SH-SY5Y)作为多巴胺能神经元的体外模型,将细胞分为对照组和PQ染毒组(不同浓度PQ处理)。用不同浓度PQ (0、25、50、100、200和400 μmol/L)处理细胞24 h,100 μmol/L PQ处理细胞不同时间(0、12、24、36、48、60和72 h),CCK-8法检测细胞增殖活性;蛋白免疫印迹法(Western blot)检测细胞HDAC6、α-突触核蛋白(α-syn)、动力蛋白中链(Dynein IC1/2)、微管相关蛋白轻链3(LC3)、自噬相关蛋白(Beclin1)、泛素结合蛋白(p62)和溶酶体相关膜蛋白-1(Lamp-1)的表达水平;免疫荧光双标法观察细胞HDAC6、α-syn、Dynein IC1/2、LC3、Lamp-1及γ-微管蛋白(γ-tubulin)在细胞中的表达及定位。结果:PQ呈剂量和时间依赖性抑制SH-SY5Y细胞增殖活性(n R=-0.950、-0.960,n P<0.05);与对照组比较,PQ染毒组HDAC6、α-syn、p62蛋白表达水平升高,差异有统计学意义(n P<0.05),而自噬标志蛋白(LC3 Ⅱ/LC3 Ⅰ)、Dynein IC1/2、Beclin1、Lamp-1表达水平降低(n P<0.05);与对照组比较,PQ染毒组HDAC6与α-syn荧光信号增强,蛋白表达水平增高,而Dynein IC1/2、LC3、Lamp-1荧光信号减少,蛋白表达水平降低(n P<0.05);PQ染毒诱导上述蛋白表达位置也发生变化,细胞质中的HDAC6由弥散状逐渐向细胞核聚集;α-syn、Dynein IC1/2、γ-tubulin、LC3、Lamp-1也逐渐由细胞质向细胞核聚集,并与HDAC6在细胞核共定位于核周区域。n 结论:PQ可能通过诱导HDAC6介导蛋白包涵体-自噬-溶酶体途径障碍引起α-syn异常聚集。“,”Objective:To explore the mechanism of HDAC6 mediated aggresome-autophagy-lysosome pathway in paraquat-induced autophagy in dopaminergic neurons.Methods: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.Results:CCK-8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner (n R=-0.950、-0.960, n P<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 (n P<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 (n P<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.n Conclusion:PQ may induce abnormal aggregation of α-syn by inducing HDAC6-mediated aggresome-autophagy-lysosomal pathway disorder.