TBK1对NLRC4炎症小体的作用及其机制研究OA北大核心CSTPCD

Objective:To investigate the mechanism by which TANK binding kinase 1(TBK1)regulates the activation of nucleotide-binding oligomerization domain-like receptor 4(NLRC4)inflammasome.Methods:Western blot was used to detect the activation of NLRC4 inflammasome and their downstream molecules cysteine aspartic acid-specific protease 1(Caspase-1)and Gasdermin D(GSDMD)in immortalized bone marrow-derived macrophages(IBMDM)infected with Salmonella typhimurium(S.T).A lactate dehydrogenase detection kit was used to detect the content of lactate dehydrogenase in the supernatant of cell culture medium.The interaction between TBK1 and NLRC4 and their specific interaction domain was determined through protein co-immunoprecipitation experiments.Cellular immunofluorescence assay was used to determine the spatial localization of TBK1 and NLRC4.The GST pull-down experiment confirmed the direct interaction between TBK1 and NLRC4.The assembly of NLRC4 inflammasome was verified using apoptosis-associated speck-like protein containing a CARD(ASC)oligomerization detection experiments.The S.T infected animal model of C57BL/6 mice was built and the survival of mice was observed.The bacterial load of lung tissues and peritoneal cavity-flushed fluid was analyzed through smear analysis.ELISA was used to detect the content of tunor necrosis factor(TNF)-α and interleukin(IL)-1β in peritoneal cavity-flushed fluid and serum.Flow cytometry was used to detect the proportion of neutrophils in peritoneal cavity-flushed fluid.Results:In S.T infected IBMDM,inhibiting TBK1 led to a weakened activation of NLRC4 inflammasomes,decreased phosphorylation levels of NLRC4,and reduced cleavage of Caspase-1 and GSDMD.There was an interaction between TBK1 and NLRC4,and the N-terminal of TBK1 interaced with the NACHT domain of NLRC4.TBK1 and NLRC4 had spatial co-localization.TBK1 phosphorylated the NLRC4 Ser533 site.S.T animal model experiments showed that inhibiting TBK1 activity significantly improved the survival rate of mice,weakened the bacterial load in the peritoneal cavity-flushed fluid and lung tissues of mice,reduced the content of IL-1β and TNF-α in serum and peritoneal cavity-flushed fluid,and reduced the proportion of neutrophils in peritoneal cavity-flushed fluid.Conclusion:TBK1 interacts with NLRC4,phosphorylates the NLRC4 Ser533 site,and promotes the activation of NLRC4 inflammasome,which providing a theoretical basis and new potential targets for treating related diseases.