Studies On Inhibitory Effect And Mechanism Of Babaodan And Its Bioactive Compounds Against NLRP3 Inflammasome Activation

The NLRP3 inflammasome is a multi-protein complex in the cytoplasm that promotes inflammation and pyroptosis by inducing the maturation and secretion of inflammatory cytokines such as IL-1β and IL-18.Recognized as a key influencing factor in the pathological process of various diseases including infection,many studies have proposed inhibiting the activation of the NLRP3 inflammasome as a potential therapeutic target for related diseases.Babaodan(BBD)is a traditional Chinese medicine with a long history of clinical application,which is made of Bovis Calculus,Fel Serpentis,Cornu Saigae Tataricae,Margarita,Moschus,Notogiseng Radix et Rhizoma.Referring to its“clearing and humid heat,activating blood and removing toxicity,curing jaundice and relieving pain” effects,BBD has been widely used in clinic to treat viral hepatitis,cholecystitis,angiocholitis and urinary tract infection.In addition,we and others have verified the anti-inflammatory effects of BBD.Till now,whether BBD can inhibit the activation of NLRP3 inflammasome,what are the active substances in BBD that can be used as anti-inflammatory lead compounds as well as their molecular modes of action are still unclear.In summary,in this study:1.With the LPS-induced sepsis mouse model,it was confirmed that BBD suppressed sepsis by inhibiting NLRP3-mediated inflammasome activation.BBD improved the survival of sepsis mice accomplished with diminished inflammatory cytokines production,multiple organ injury and the expression of caspase-1 in tissues.With application of RNA sequencing(RNA-seq)technologies,we found that the NOD-like receptor signaling pathway in macrophages is a key pathway for BBD in its treatment of sepsis.Besides,while peritoneal macrophages(PMs)were stimulated with LPS plus ATP for NLRP3 inflammasome activation,BBD inhibited both the expression of NLRP3 and pro-IL-1βregulated by the NF-κB signaling pathway and the assembly of inflammasome complexes.Simultaneously,the extractions of BBD were applied for UPLC-QTOF-MS analyses,and identified compounds were mainly bile acids and saponins.The inhibitory effect of 23 compounds in BBD against NLRP3 inflammasome activation was evaluated,and the results suggested that muscone,bilirubin,lithocholic acid,taurolithocholic acid,and ginsenoside Rg3 were bioactive compounds of BBD.2.With the LPS-induced sepsis mouse model and PMs stimulated by LPS plus ATP,it was confirmed that muscone inhibited the activation of NLRP3 inflammasome.With application of RNA-seq technologies,western blotting and transmission electron microscopy,we found that Muscone promoted mitophagy in macrophages by regulating the PINK1/Parkin signaling pathway.Furthermore,muscone inhibited NLRP3 inflammasome activation by promoting mitophagy to rapidly clear damaged mitochondria and stimuli such as mt ROS released by them.In addition,macrophages isolated from muscone-treated sepsis mouse also had enhanced mitophagy.3.With the LPS-induced sepsis mouse model and PMs stimulated by LPS plus ATP,it was confirmed that bilirubin inhibited the activation of NLRP3 inflammasome.Mechanistically,with respect to the antioxidant nature of bilirubin,we evaluated the effect of bilirubin on the releasing of mt ROS from mitochondria.In addition,mitochondrial morphofunction mainly including morphology and membrane potential in contact living macrophages was analyzed by applying a newly developed multiplexed high-content mitochondrial imaging analysis system using live-cell microscopy.With the application of multiplexed high-content mitochondrial imaging analysis system,it was found that mitochondria were damaged in the process of inflammasome activation,accompanied by regular changes in mitochondrial morphofunction;bilirubin targets and stabilizes mitochondrial membrane during NLRP3 inflammasome activation,defined doses of bilirubin could be considered as a mitochondria targeted medication against inflammasome-related diseases.Taken together,BBD has pronounced bioactivity of inhibiting NLRP3 inflammasome activation,which supports its novel application value for the treatment of related diseases.As the two bioactive compounds in BBD,the mechanism of muscone and bilirubin is related to the promotion of mitophagy and the stabilization of mitochondrial membrane,respectively,which reflects the nature of traditional Chinese medicine characterized by integrated effect of "multi-component and multi-target".In addition,the live cell imaging based high-content mitochondrial morphology analysis is expected to become a new technology applicable for evaluating the activation of NLRP3 inflammasome,which is beneficial for rapid high-content screening of inflammasome inhibitors.