上调自噬抑制LPS诱导的小胶质细胞死亡

发布时间：2019-01-28 19:13

【摘要】：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(P0.01),存活细胞数(69.8%±2.1%)较对照组(100.0%±3.9%)显著减少(P0.001);(2)与对照组相比,LPS显著上调N9细胞LC3以及Beclinl水平(P0.05)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%)(P0.05)。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(P0.05)。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。
[Abstract]:Aim: to observe the changes of autophagy level in N9 cells treated with lipopolysaccharide (lipopolysaccharide,LPS) and to explore the effects of different autophagy levels stimulated by LPS on the injury and survival of N9 cells. Methods: N9 cells were cultured in vitro and treated with LPS for 24 h. The expression of light chain 3 (LC3) and lysosomal associated membrane protein 2 (LAMP2) of microtubule associated protein 1 (LC3) and lysosomal associated membrane protein 2 (LAMP2) were detected by immunofluorescence (IF). Western Blot was used to detect the expression of LC3 and Beclinl. After LPS treatment, autophagy markers LC3, were detected with autophagy inducer rapamycin (RAP) and autophagy inhibitor 3-methyladenine (3-MA) respectively., Western Blot was used to detect autophagy level of N9 cells. Beclinl expression; Lactic dehydrogenase (lactic dehydrogenase) kit (, Cell Count Kit (CCK8) was used to detect cell damage and survival. Results: (1) N9 cells were injured by LPS, and the release of LDH (113.6% 卤3.2%) was significantly higher than that of control group (100.0% 卤3.9%) (P0.01). The number of surviving cells (69.8% 卤2.1%) was significantly lower than that of control group (100.0% 卤3.9%) (P0.001). (2) compared with the control group, LPS upregulated the levels of LC3 and Beclinl in N9 cells (P0.05) and increased the level of LAMP2 at the same time. (3) RAP upregulated the autophagy level of N9 cells treated with LPS and reduced the damage of microglia (100.8% 卤2.3%). 3-MA inhibited the autophagy level of N9 cells treated with LPS and decreased the cell survival (60.6% 卤2.9%), which promoted cell survival (87.4% 卤5.7%). The cell injury (123.2% 卤3.7%) was not significantly different from that in LPS group. (4) RAP and 3-MA promoted or inhibited the expression of autophagy associated protein in N9 cells induced by LPS (P0.05). Conclusion: upregulation of autophagy can reduce the injury of N9 cells induced by LPS and promote the survival of N9 cells.
【作者单位】：第四军医大学西京医院麻醉及围术期医学科;广西医科大学第一附属医院麻醉科;
【基金】：国家自然科学基金-国际合作与交流项目(81420108013)
【分类号】：R741

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