MFN1 ubiquitination mediates lipopolysaccharide-induced mitochondrial dysfunction and pyroptosis in Raw264.7 mouse macrophages
10.3760/cma.j.cn112309-20211006-00329
- VernacularTitle:脂多糖通过介导MFN1泛素化调控小鼠Raw264.7巨噬细胞线粒体稳态失衡和焦亡
- Author:
Jian MEI
1
;
Xiangrui ZHU
;
Langlin OU
;
Zhaosi WANG
;
Lixin ZHANG
;
Yueshan LYU
;
Xiaoying WANG
;
Siyu HE
;
Jun′e BAI
;
Hao YUAN
;
Xiaoyu GUAN
;
Cui MA
Author Information
1. 哈尔滨医科大学大庆校区医学检验与技术学院免疫教研室，大庆 163319
- Keywords:
Mitofusin 1 (MFN1);
Ubiquitination;
Raw264.7 cells;
Mitochondria;
Pyroptosis
- From:
Chinese Journal of Microbiology and Immunology
2022;42(9):705-713
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the regulatory effects of mitofusin 1 (MFN1) on lipopolysaccharide (LPS)-induced Raw264.7 mouse macrophages pyroptosis and to provide reference for further study on the prevention of inflammation and fibrosis caused by macrophage dysfunction.Methods:Raw264.7 mouse macrophages were cultured in vitro and used to construct a model of LPS-induced pyroptosis. CCK-8 staining, PI staining, LDH release assay and Western blot were used to verify the Raw264.7 pyroptosis induced by LPS. MFN1 expression was detected by Western blot. DCFH-DA probe was used to detect the synthesis of total reactive oxygen species (ROS); Mito-SOX was used to detect mitochondrial ROS; JC-1 mitochondrial membrane potential was detected by fluorescence probe to reflect mitochondrial damage. Based on Ubibrowser database, it was predicted that MFN1 could bind to a variety of E3 ubiquitin ligases. Then, immunofluorescence and co-immunoprecipitation (CO-IP) were used to analyze MFN1 ubiquitination. An overexpression plasmid for MFN1 was constructed and transfected into Raw264.7 cells to detect the changes in pyroptosis and mitochondrial function. Results:LPS could induce the pyroptosis of Raw264.7 cells and mitochondrial dysfunction. MFN1 expression was decreased after LPS stimulation. Ubiquitinated MFN1 was detected by CO-IP. Ubiquitination inhibitor MG-132 inhibited LPS-induced expression of pyroptosis-related proteins including NLRP3, Pro-caspase-1, Caspase-1, IL-1β and IL-18 and improved mitochondrial function. MFN1 overexpression relieved the mitochondrial dysfunction and pyroptosis of Raw264.7 cells induced by LPS.Conclusions:The ubiquitination of MFN1 induced by LPS was involved in mitochondrial dysfunction and macrophage pyroptosis, suggesting that MFN1 was a potential target for the treatment of macrophage-induced inflammation and related diseases.
