2021, 31(04): 307-313.
Objective: To investigate the potential mechanism of Listeria monocytogenes （Lm）infectionon pyroptosis in mice and macrophages.Methods: In vivo experiments, C57B/6 mice were divided into Lm group and negative control group（NC group）, then the mice were injected with Lm 5×104 CFU/mouse and sterile PBS via tail vein, respectively. The spleen, liver and brain tissues of mice were collected 24 hours later. The pyroptosis and NLRP3 inflammasome related protein expression were detected by Western blotting. In vitro experiments were divided into Lm group, NC group, LPS+ATP group, Lm+MCC950 group and LPS+ATP+MCC950 group. Multiplicity of infection （MOI）= 10 Lm was used to infect THP-1 derived macrophages, ELISA was used to detect IL-1β from cell supernatant, quantitative real-time PCR （qRT-RCR）and Western blotting were used to detect expression of mRNA and protein about pyroptosis and NLRP3 inflammation. Immunofluorescence （IF）was used to detect ASC-speck and co-localization with NLRP3. Results: After Lm infection of mice and macrophages, the expression level of gasdermin D （GSDMD）protein was significantly increased, and gasdermin D N-terminal （GSDMD-NT）was appeared. The levels of NLRP3, caspase-1, IL-1β, and IL-18 mRNA were significantly increased after Lm infected macrophages at 1 h, 3 h, and 6 h （P<0.05）, and the increase was the most obvious at 3 h. The protein expression of NLRP3, pro-caspase-1, pro-IL-1β and cleavage fragment caspase-1 p20, IL-1β-17 in Lminfected mouse spleen, liver, brain tissue were all significantly increased （P<0.05）.In macrophages by Lm infection, the secretion of IL-1β was increased, the secretion of caspase-1 p20 and IL-1β-17 was detected in the cell supernatant, and the expression of NLRP3, pro-caspase-1, pro-IL-1β protein in the cell lysate was increased （P<0.05）, then ASC-speck was formed and co-localized with NLRP3 protein was observed by fluorescence microscope. After pretreatment with specific NLRP3 inflammasome inhibitor MCC950, GSDMD-NT of macrophages was significantly reduced （P<0.05）. Conclusion: Pyroptosis caused by Lm infection is mainly related to the activation of NLRP3 inflammasome in mice and macrophages.
