Screening And Application Of NLRP3 Inflammasome Inhibitors

The innate immunity is the body’s first line of defense,eliminating pathogens and autoantigen.The target signals of innate immune response are pathogen-related exogenous danger signals termed-pathogen-associated molecular patterns(PAMPs)and the endogenous danger signals named-damage-associated molecular patterns(DAMPs).The recognition of PAMPs and DAMPs require the pattern recognition receptors(PRRs).NLRP3(NOD-like receptor(NLR)family,pyrin domain-containing-3)is one of the important PRRs in the cytoplasm,and it is also the most widely studied PRRs.NLRP3responds to stress signals via forming inflammasome complex with the adaptor protein ASC(apoptosis-associated speck-like protein containing a CARD)and the active protein Caspase1.When the inflammasome complex was assembling,caspase1 were cleaved into activated form termed cleaved-Caspase1.The cleaved-Caspase1 could cleave Pro-IL-1βand Pro-IL-18 into the form of activated IL-1βand activated IL-18.At the same time,the a cleaved-Caspase1 cleave the pore-punching protein GSDMD(Gasdermin D)into C-GSDMD and N-GSDMD.N-GSDMD anchors and punches pores in the cell membrane,activated IL-1βand IL-18 was released outside the cell through this pore and cause inflammatory.It was reported that NLRP3 inflammasome was widely involved in inflammatory diseases,such as infectious diseases,cardiovascular diseases,autoimmune diseases,neurodegenerative diseases and psychiatric disorders.Therefore,development and utilization of effective NLRP3 inflammasome activation inhibitors would have very important clinical significance.Essential oils(EOs)are liquid mixtures of volatile and low molecular weight organics extracted from aromatic plants.EOs have long been used for medical and commercial purposes with their various biological activities,including antibacterial,antifungal,antiviral and antioxidant properties.Importantly,growing number of studies revealed the anti-inflammatory effects of EOs in different models of inflammation in vitro and in vivo.However,the mechanism by which EOs exert the anti-inflammatory activity remains incompletely understood.In addition,owing to their complex components,the active compound(s)of EOs that display anti-inflammatory property need to be identified.In this study,we established a drug screening system that targeted NRLP3inflammasome,aiming to pick out the active compound(s)from different constituents of EOs that directly and specifically inhibit NRLP3 inflammasome activity.We expected to clarify the mechanisms of the active component(s)inhibiting NLRP3 inflammasome and verify the effects of the active component(s)in neurological diseases through different neurological disease animal models.We hope this study can provide available theoretical and practical basis for subsequent clinical applications.First of all,we used immortalized bone marrow-derived macrophages(i BMDM)as model cells,LPS as the first stimulus signal and nigericin as the second stimulus signal,established a stable NLRP3 inflammasome activation and evaluation system.Using this system,we successfully screened out 1,2,4-trimethoxybenzene(1,2,4-TTB)andα-terpineol could effectively inhibit the cleavage of IL-1βand caspase1.Cell viability assay shown that 1,2,4-TTB has less cytotoxicity and better inhibitory activity.In order to systematically evaluate the inhibitory effect of 1,2,4-TTB on NLRP3 inflammasome,various mononuclear macrophages(primary peritoneal macrophages and primary microglia)and inflammasome activators(ATP,Imiquimod,Pam CSK4)were used for this research.We found that 1,2,4-TTB has a good inhibitory effect on the activation of NLRP3 inflammasome.In the aspect of mechanism,we found that 1,2,4-TTB could inhibit the K~+efflux of mononuclear macrophages via K~+fluorescent probe labeling.The results of ASC/NLRP3cross-linking oligomerization experiments and cellular immunofluorescence experiments shown that 1,2,4-TTB could effectively inhibit the formation of NLRP3 and ASC oligomer.Co-IP experiments revealed that 1,2,4-TTB could inhibit the NLRP3-ASC interaction.Molecular docking predicted that 1,2,4-TTB and NLRP3 have a high binding probability at Gln225 sites,and 1,2,4-TTB also have a high binding probability with ASC protein at Ser195 site.These results proved that 1,2,4-TTB could either inhibit the K~+efflux or attenuate the formation of NLRP3 oligomers.In terms of in vivo research,we found that two times injections of low-dose LPS could induce the activation of NLRP3 inflammasome in the microglia after the second injection without causing strong systemic inflammation.This model might be as a new model to evaluate NLRP3 inflammasome activity in central nervous system in vivo.Using the new model,we found that 1,2,4-TTB could effectively inhibit the NLRP3inflammasome activation and the cleavage of IL-1βand caspase1 in mouse brain.EAE mouse model was also used to evaluate the 1,2,4-TTB inhibitory activity.Results shown that 1,2,4-TTB could attenuate the clinical score and reduce the weight loss of mice.Spinal cord slices staining and biochemical analysis presented that the ASC expression in microglia were reduce and the activation of NLRP3 inflammasome was significantly inhibited compared with control.The area of spinal demyelination was also decrease in1,2,4-TTB administration EAE model mice.Aiming to verify the effect of 1,2,4-TTB in post-traumatic stress disorder(PTSD)mouse model was also be used,and the results showed that 1,2,4-TTB could accelerate the elimination of fear memory causing by electric shock and reduce the anxiety-like and depression-like phenotypes of mice.In summary:We screened out a direct and specific NLRP3 inflammasome inhibitor,1,2,4-TTB,from essential oil components.We also revealed that 1,2,4-TTB could either inhibit the K~+efflux and attenuated the formation of NLRP3 oligomers.In vivo research:A new animal model for evaluating neuroinflammation caused by NLRP3 inflammasome had been established.This animal model was used to verify that 1,2,4-TTB could effectively inhibit the neuroinflammation induced by NLRP3 inflammasome.The EAE and PTSD mouse model were used to verify that 1,2,4-TTB could treat various central nervous system diseases with NLRP3 inflammasome involved in.Overall,1,2,4-TTB is a specific NLRP3 inflammasome activation inhibitor and may be used as a new choice for the diseases that NLRP3 inflammasome involved in.