JNK/MAPK通路在结核杆菌感染的巨噬细胞凋亡、自噬中的作用机制研究

Mechanism of JNK/MAPK pathway in apoptosis and autophagy of macrophages infected by Mycobacterium tuberculosis

目的探讨c-Jun N端激酶(JNK)/丝裂原活化蛋白激酶(MAPK)通路在结核杆菌感染的巨噬细胞凋亡、自噬中的作用机制研究。方法体外培养小鼠巨噬细胞RAW264.7细胞建立卡介苗结核分枝杆菌(BCG)细胞感染模型,将BCG感染的RAW264.7细胞使用JNK抑制剂SP600125处理,将细胞随机分为对照组、BCG组、对照+SP600125组、BCG+SP600125组;PCR法检测Bax、Bcl-2、caspase-3表达情况;吖啶橙/溴乙锭(AO/EB)染色检测各组细胞自噬情况;噻唑蓝法(MTT法)检测各组细胞存活率变化情况;流式细胞术检测各组细胞凋亡情况;蛋白印迹分析法检测各组细胞JNK、p-JNK、LC3、Beclin-1、P62蛋白表达变化。结果与对照组相比,BCG组RAW264.7细胞中JNK蛋白及磷酸化表达水平、细胞自噬水平、凋亡率、Bax、caspase-3 mRNA表达水平、LC3、Beclin-1蛋白表达水平显著升高,细胞存活率、Bcl-2 mRNA表达水平、P62蛋白表达水平显著降低,差异有统计学意义(P<0.05);与BCG组相比,BCG+SP600125组JNK蛋白及磷酸化表达水平、细胞自噬水平、凋亡率、Bax、caspase-3 mRNA表达水平及LC3、Beclin-1蛋白表达水平显著降低,细胞存活率、Bcl-2 mRNA表达水平、P62蛋白表达水平显著升高,差异有统计学意义(P<0.05)。结论结核杆菌感染可抑制巨噬细胞增殖、诱导巨噬细胞凋亡与自噬,可能与激活JNK/MAPK通路有关。

Objective To investigate the mechanism of c-Jun N-terminal kinase(JNK)/mitogen-activated protein kinase(MAPK)pathway in apoptosis and autophagy of macrophages infected by Mycobacterium tuberculosis.Methods Mouse macrophage RAW264.7 cells were cultured in vitro to establish Bacille Calmette-Guerin vaccine(BCG)Mycobacterium tuberculosis cells infection model,and RAW264.7 cells infected with BCG were treated with JNK inhibitor SP600125 and randomly divided into the control group,the BCG group,the control+SP600125 group and the BCG+SP600125 group.The expression of Bax,Bcl-2 and caspase-3 was detected by PCR.The autophagy was detected by acridine orange/ethidium bromide(AO/EB)staining.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the cell survival rate.Cell apoptosis was detected by flow cytometry.Western blot analysis was used to detect the expression of JNK,p-JNK,LC3,Beclin-1 and P62.Results Compared with those in the control group,JNK protein and phosphorylation levels,autophagy level,apoptosis rate,Bax and caspase-3 mRNA expression levels,LC3 and Beclin-1 protein expression levels were significantly higher in the BCG group,the cell survival rate,Bcl-2 mRNA expression level and P62 protein expression level were significantly lower(P<0.05).Compared with those in the BCG group,JNK protein and phosphorylation levels,autophagy level,apoptosis rate,Bax and caspase-3 mRNA expression levels,LC3 and Beclin-1 protein expression levels were significantly lower in the BCG+SP600125 group,the cell survival rate,Bcl-2 mRNA expression level and P62 protein expression level were significantly higher(P<0.05).Conclusion Mycobacterium tuberculosis infection can inhibit macrophage proliferation,induce macrophage apoptosis and autophagy,which may be related to the activation of JNK/MAPK pathway.