Mechanism Of Small Molecule Compound C646 In Inhibiting NLRP3 Inflammasome Activation

Objective: Aberrant activation of NLRP3 inflammasome is associated with the occurrence and development of multiple inflammatory disorders.Our previous study showed that NLRP3 acetylation modification is an important event in the activation of NLRP3 inflammasome,and by screening the acetyltransferase inhibitors,we found a small molecule inhibitor,C646 significantly inhibited the release of LDH and IL-1β.However,the mechanism of C646 on NLPP3 inflammasome has not been elucidated yet.This study aims to explore the effect of C646 on NLRP3 inflammasome activation in certain diseases,hence to provide therapeutic strategies for NLRP3 inflammasome related disorders.Methods: 1.In vitro: mice and human macrophages were pretreated with the small molecule inhibitor C646 30 minutes before administration of the stimuli of NLRP3 inflammasome,Caspase-11,AIM2 inflammasome and NLRC4 inflammasome.WB detection was performed to detect the IL-1β and Caspase-1 cleavage and other expressed proteins.Secretion of inflammatory factors IL-1β,TNF-α and IL-6 were detected by ELISA,and pyroptotic cells were estimated by LDH;ASC oligomerization was detected by DSS cross-linking and immunefluorescence evaluated Speck formation;co-immunoprecipitation was used to detect the interaction between NLRP3 and ASC or NEK7;SDD-AGE was applied for NLRP3 aggregation.2.In vivo: LPS-induced mice endotoxemia model and DSS-induced mice acute colitis model were established,then the release of inflammatory factors,expression of NLRP3 inflammasome components(methods)and multi-organ injury were evaluated by HE staining.Results: Treated before the second signal of NLRP3 inflammasome activation,C646 specifically inhibited the release of LDH,the secretion of related inflammatory factors and the cleavage of IL-1β and caspase-1in mouse peritoneal macrophages.Mechanistically,C646 decreased the expression of NLRP3 by inhibiting the activation of NF-κB.It also diminished the assembly of NLRP3 inflammasome by inhibiting the interaction between NLRP3 and ASC.In vivo,C646 was protective in inflammatory responses in LPS-induced endotoxemia and DSS-induced acute colitis,it attenuated organ inflammatory damage as well.Conclusion: C646 inhibits the activation of NF-κB signal,results in reduced NLRP3 expression.In addition,C646 interferes the interaction between NLRP3 and ASC,thus blocks the assembly of the NLRP3 inflammasome.C646 significantly alleviates LPS-induced endotoxemia inflammatory response、lung injury and DSS-induced acute colitis injury.