The Effect And Mechanism Of Oridonin In Inhibition Of NLRP3 Inflammasome Activation And Alleviation Of NLRP3-driven Diseases

NLRP3 inflammasome is a protein complex formed by innate immune sensor NLRP3,adaptor protein ASC and pro-caspase-1.The assembly of this complex results in the activation of caspase-1,which then cleaves pro-IL-1? and pro-IL-18 to produce mature and functional IL-1? and IL-18,so it plays a central role in innate immunity and inflammation.NLRP3 can sense not only pathogen-associated molecular patterns(PAMPs),such as fungal zymosan,but also damage associated molecular patterns(DAMPs),including monosodium urate crystals(MSU),cholesterol crystals,amyloid-? aggregates,unsaturated fatty acids,high glucose and ceramide.Since the activators of the NLRP3 inflammasome are diverse,the specific mechanism of the NLRP3 inflammasome is still unclear.There are three hypotheses:potassium efflux,ROS generation and lysosomal damage.The dysregulation of NLRP3 inflammasomes can lead to several human diseases,such as gout,atherosclerosis,neurodegenerative diseases,and type 2 diabetes.Several NLRP3 inflammasome inhibitors have shown preventive or therapeutic activity on the mouse models of NLRP3-driven diseases.However,most of them work by suppressing the upstream events of NLRP3 inflammasome activation.So,they may have many side effects.Target the components of NLRP3 inflammasome is the best choice.But some components of NLRP3 inflammasome,such as ASC and pro-caspase-1,have function in other inflammasomes and have other physiological functions besides as components of NLRP3 inflammasome.Targeting NLRP3 is the best and direct way to specifically inhibit the NLRP3 inflammasome.A small molecule inhibitor CY-09 can directly bind to NLRP3 and inhibit activation of NLRP3 by suppressing NLRP3 ATPase activity.CY-09 has shown preventive or therapeutic activity on the mouse models of NLRP3-driven diseases,but as an artificially synthesized molecule,its application prospects needs to be further determined.Rubescens is a traditional Chinese medicine widely used in the treatment of various inflammatory diseases.Modern studies have found that it has a certain therapeutic effect on cancer.Oridonin(Ori)is a major component of Rubescens,and it can perform anti-cancer functions by inducing apoptosis and cell cycle arrest.Ori has been reported to inhibit NF-?B or MAPK activation so as to suppress the production of proinflammatory cytokines,such as TNF-a and IL-6.Moreover,Ori has also exhibited anti-inflammatory activity and protective role in colitis,sepsis and neuroinflammation.Although the anti-inflammatory activity of Ori is emerging,the underlying mechanisms and direct target are unknown.Here we show that Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain via carbon-carbon double-bond and then blocks the interaction between NLRP3 and NEK7,which results in the inhibition of NLRP3 inflammasome assembly and activation.In vivo experiments reveal that Ori exhibits remarkable preventive or therapeutic effects on the mouse models of peritonitis,gouty arthritis and type 2 diabetes by inhibition of NLRP3 activation.Taken together,our results identify NLRP3 as the direct target of Ori and elucidate the detailed mechanism of Ori leading to the anti-inflammatory activity.Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.