SQSTM1蛋白在嗜肺军团菌感染RAW264.7细胞自噬中的作用机制

doi:10.6040/j.issn.1671-7554.0.2023.0167

摘要/Abstract

摘要： 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。

关键词: 嗜肺军团菌, 感染, 巨噬细胞, SQSTM1, 自噬

Abstract: 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/Ⅰ were 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.

Key words: Legionella pneumophila, Infection, Macrophage, Sequestosome1, Autophagy

中图分类号:

R574

郑荣慧,李攀,曹秀琴,贺瑞霞,陈民佳,陈海霞,杨志伟. SQSTM1蛋白在嗜肺军团菌感染RAW264.7细胞自噬中的作用机制[J]. 山东大学学报 (医学版), 2023, 61(6): 10-21.

ZHENG Ronghui, Li Pan, CAO Xiuqin, HE Ruixia, CHEN Minjia, CHEN Haixia, YANG Zhiwei. SQSTM1 in Legionella pneumophila infected RAW264.7 cells mechanism of autophagy[J]. Journal of Shandong University (Health Sciences), 2023, 61(6): 10-21.

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