上调自噬抑制LPS诱导的小胶质细胞死亡

Activation of autophagy suppresses LPS-induced microglia death

目的:观察脂多糖(lipopolysaccharide,LPS)处理后,N9细胞自噬水平的变化,并探究LPS刺激下不同自噬水平对N9细胞的损伤和存活的影响。方法:体外培养N9细胞,给予24 h LPS处理,免疫荧光(IF)检测微管相关蛋白1轻链3(LC3)及溶酶体相关膜蛋白2(LAMP2)的表达,Western Blot检测LC3与Beclinl的表达。LPS处理后分别使用自噬诱导剂雷帕霉素(RAP)和自噬抑制剂3-甲基腺嘌呤(3-MA)调节N9细胞自噬水平,Western Blot检测不同处理组自噬标记蛋白LC3、Beclinl的表达;使用乳酸脱氢酶(lactic dehydrogenase)试剂盒、Cell Count Kit(CCK8)试剂盒检测细胞损伤及存活。结果:(1)LPS刺激造成N9细胞损伤,LDH释放量(113.6%±3.2%)较对照组(100.0%±3.9%)显著增加(P<0.01),存活细胞数(69.8%±2.1%)较对照组(100.0%±3.9%)显著减少(P<0.001);(2)与对照组相比,LPS显著上调N9细胞LC3以及Beclinl水平(P<0.05)并同时上调LAMP2水平;(3)RAP上调LPS处理后N9细胞的自噬水平并减少小胶质细胞损伤(100.8%±2.3%),促进细胞存活(87.4%±5.7%)(P<0.05)。3-MA则抑制LPS处理后N9细胞自噬水平并降低细胞存活(60.6%±2.9%),而细胞损伤(123.2%±3.7%)与LPS组相比未见统计学差异;(4)RAP与3-MA分别促进或抑制LPS诱导的N9细胞自噬相关蛋白的表达(P<0.05)。结论:上调自噬水平减少LPS刺激诱导的N9细胞损伤,促进细胞存活。

Objective：To observe the change of autophagy level in N9 cell line induced by lipopolysaccharide（LPS）and to explore the role of autophagy in LPS-induced N9 cell damage and survival by modulating autophagy level.Methods：N9 cells were treated with LPS for 24 h.The change of autophagy marker protein Beclinl,Microtubule-associated protein1 light chain 3（LC3） were detected with Western Blot.Moreover,Lysosomal-associated membrane protein 2（LAMP2）and LC3 were detected with immunofluorescence（IF）.Autophagy inducer rapamycin（RAP） or autophagy inhibitor3-methlyadenine（3-MA） were used to up- or down-regulate the autophagy level in N9 cells under LPS treatment,respectively.Twenty-four hours after LPS treatment,the level of Beclinl and LC3 were detected with Western Blot.Meanwhile,the lactic dehydrogenase（LDH） release and viability of N9 cells were assayed with LDH and Cell Count Kit（CCK8）.Results：（1） LPS induced N9 cells damage（113.6%±3.2%）,in which the LDH release was much more than that in control（Ctrl）（100.0%±3.9%,P0.01）,and decreased N9 cells viability（69.8%±2.1%） compared with Ctrl（100.0%± 3.9%,P〈0.001）.（2） LPS induced increase of Beclinl,LC3（P〈0.05） and LAMP2 compared with Ctrl.（3） Up-regulation of autophagy with RAP decreased LPS induced cytotoxicity（100.8%±2.3%） and alleviated LPS induced N9 cell death（87.4%±5.1%,P〈0.05）.While down-regulation of autophagy in N9 cells with 3-MA decreased cell survival（60.6%±2.9%）,however,LDH release didn＇t show difference（123.2%±3.6%） compared with LPS group.（4） 3-MA inhibited LPS-induced autophagy in N9 cells,while RAP enhanced LPS-induced autophagy in N9 cells（P0.05）.Conclusion;Activation of autophagy suppresses LPS-stimulated microglia cell death.