SQSTM1蛋白在嗜肺军团菌感染RAW264.7细胞自噬中的作用机制

SQSTM1 in Legionella pneumophila infected RAW264.7 cells mechanism of autophagy

目的以SQSTM1蛋白(P62)基因敲除及过表达的RAW264.7小鼠巨噬细胞为研究对象,建立嗜肺军团菌感染巨噬细胞模型,观察细胞内细菌增殖情况以及自噬流和自噬小体的变化,检测自噬相关因子表达水平变化,探讨P62在嗜肺军团菌感染RAW264.7巨噬细胞自噬中的作用机制。方法嗜肺军团菌以感染复数10、50和100感染RAW264.7巨噬细胞组、KO-P62细胞组、OE-P62细胞组,同时以未加嗜肺军团菌感染作为对照组;细菌增殖实验观察嗜肺军团菌在巨噬细胞内的增殖情况;透射电镜观察嗜肺军团菌与细胞共培养12 h时RAW264.7巨噬细胞组、KO-P62细胞组、OE-P62细胞组的自噬小体、自噬溶酶体及相关细胞器等超微结构;pmCherry-C1-EGFP-LC3B双荧光指示系统检测巨噬细胞自噬流的变化;采用免疫印迹试验(Western blotting)及实时荧光定量(RT-qPCR)法检测RAW264.7巨噬细胞组、KO-P62细胞组、OE-P62细胞组P62、自噬相关基因AMBRA1、溶酶体关联膜蛋白2(LAMP2)、自噬相关蛋白5(Atg5)、自噬效应蛋白1(Beclin1)、自噬微管相关蛋白轻链3(LC3B)的表达水平。结果细菌增殖实验检测在RAW264.7细胞组内嗜肺军团菌的数量随着时间的增长逐渐增长,在KO-P62细胞组及OE-P62细胞组内嗜肺军团菌的数量随着时间的增长均逐渐减少;透射电镜可观察到嗜肺军团菌感染RAW264.7细胞组后,自噬小体及自噬溶酶体减少,与RAW264.7细胞组相比,KO-P62细胞组及OE-P62细胞组自噬小体及自噬溶酶体均增多;pmCherry-C1-EGFP-LC3B双荧光指示系统检测自噬流结果显示,嗜肺军团菌感染RAW264.7细胞组,自噬流均减弱,KO-P62细胞组及OE-P62细胞组自噬流增加;Western blotting及RT-qPCR结果显示,与RAW264.7细胞组相比:KO-P62细胞组Beclin1先升高后降低,P62基本不表达,LC3Ⅱ/Ⅰ比值、AMBRA1、LAMP2及Atg5蛋白表达均明显升高,差异有统计学意义(P<0.05);OE-P62细胞组Beclin1先降低后升高,P62、AMBRA1、LAMP2、Atg5及LC3Ⅱ/Ⅰ比值均明显上调,差异有统计学意义(P<0.05)。结论敲除、过表达P62均可抑制嗜肺军团菌在RAW264.7巨噬细胞内的增殖,促进自噬,其机制可能与Atg5-P62-AMBRA1信号通路有关。

Objective To explore the role and mechanism of P62 in the autophagy of RAW264.7 macrophages infected with Legionella pneumophila.Methods RAW264.7 macrophages,KO-P62 cells and OE-P62 cells were infected with Legionella pneumophila at multiplicities of infection(MOI)of 10,50,and 100;unaffected groups were set as controls.The proliferation of Legionella pneumophila in macrophages was observed with bacterial proliferation assay.After co-culture with Legionella pneumophila for 12 h,the ultrastructure of autophagic vesicles,autophagic lysosomes and related organelles in the RAW264.7 group,KO-P62 group and OE-P62 group were observed with transmission electron microscopy.The changes in the autophagic flow of macrophages were detected with pmCherry-C1-EGFP-LC3 B dual fluorescence indicator system.The expression levels of autophagy-related factors,P62,AMBRA1,LAMP2,Atg5,Beclin1 and LC3 B in each group were detected with Western blotting and RT-qPCR.Results Bacterial proliferation assay detected that the number of Legionella pneumophila in RAW264.7 cells gradually increased over time,but decreased in KO-P62 and OE-P62 cells.Transmission electron microscopy showed that after Legionella pneumophila infected RAW264.7 cells,the autophagosomes and autolysosomes decreased;compared with the RAW264.7 group,the KO-P62 and OE-P62 groups had increased autophagosomes and autolysosomes.The results of pmCherry-C1-EGFP-LC3B dual fluorescence indicator system showed that the autophagic flux decreased in the RAW264.7 group infected with Legionella pneumophila,but increased in KO-P62 and OE-P62 groups.Western blotting and RT-qPCR showed that,compared with the RAW264.7 group,the KO-P62 group had firstly increased and then decreased Beclin1,P62 was basically not expressed,and the levels of LC3II/I,AMBRA1,LAMP2 and Atg5 were significantly increased(P<0.05);the OE-P62 group had firstly decreased and then increased Beclin1,and the levels of P62,AMBRA1,LAMP2,Atg5 and LC3Ⅱ/Ⅰwere significantly increased(P<0.05).Conclusion Knockout and overexpression of P62 can inhibit the proliferation of Legionella pneumophila in RAW264.7 macrophages and promote autophagy.The mechanism may be related to the Atg5-P62-AMBRA1 signaling pathway.