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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 changes of autophagy in N9 cells treated with lipopolysaccharide (LPS) and to explore the effect of different levels of autophagy on the damage and survival of N9 cells under LPS stimulation. Methods: N9 cells were cultured in vitro and treated with LPS for 24 h. The expression of LC3 and LAMP2 was detected by immunofluorescence (IF). LC3 and Beclin1 expression. Autophagy inducer rapamycin (RAP) and autophagy inhibitor 3-methyladenine (3-MA) were used to regulate the autophagy of N9 cells after LPS treatment. Western Blot was used to detect the expression of autophagy marker protein LC3 , Beclinl expression; cell injury and survival were detected using the lactic dehydrogenase kit and the Cell Count Kit (CCK8) kit. Results: (1) N9 cells were injured by LPS stimulation, the release of LDH was significantly increased (113.6% ± 3.2% vs 100.0% ± 3.9%, P <0.01) and the number of surviving cells was 69.8% ± 2.1% (2) Compared with the control group, LPS significantly upregulated the expression of LC3 and Beclin1 in N9 cells (P <0.05) and up-regulated the expression of LAMP2 at the same time (3) RAP up-regulated the autophagy level of N9 cells and decreased microglial injury (100.8% ± 2.3%) and cell survival (87.4% ± 5.7%) (P <0.05). 3-MA inhibited the autophagy of N9 cells after LPS treatment and decreased cell survival (60.6% ± 2.9%), while cell injury (123.2% ± 3.7%) showed no significant difference compared with LPS group; (4) RAP And 3-MA promoted or inhibited LPS-induced autophagy-related protein expression in N9 cells (P <0.05). Conclusion: Upregulation of autophagy can reduce the damage of N9 cells induced by LPS stimulation and promote cell survival.