Study The Regulatory Mechanism Between NLRP3 And IL-33 Based On The Effects Of Components From Yupingfengsan In Allergic Disease

Allergic disease is a chronic inflammatory disease with complex pathogenesis involving several immune pathways.Exposure to antigens leads to the susceptibility of respiratory system,digestive system and skin,etc.,which further induces the clinical symptoms such as swelling,itching,asthma,rhinorrhea.Yu-Ping-Feng-San(YPFS),composed of Radix Astragali,Rhizoma Atractylodis Macrocephalae and Radix Saposhnikoviae,is widely used to reduce allergic diseases relapse rate.Previous studies by our group showed that YPFS significantly decreased the expression of NLRP3 and IL-33 in MC903-induced atopic dermatitis(AD)mice model,suggesting that there might be a correlation between NLRP3 and IL-33.To investigate the function of NLRP3 in allergic disease,and the mechanism of components from YPFS against AD,this study mainly focuses on the following four aspects:1.Effects of components from YPFS both in vivo and in vitro.MC903-induced AD model mice were treated with calycosin,formononetin and cimifugin,and ear swelling,ear histopathology,pro-inflammatory cytokines levels,NLRP3 and IL-33 expression were detected.The results showed that formononetin had an obvious inhibitory effect on the expression of NLRP3 and IL-33.The expression of NLRP3 elevated in HaCaT cells after being stimulated with MC903,FITC,LPS and ATP.Effects of YPFS and its components on the expression of NLRP3 in HaCaT cells were detected by Western Blotting.As shown,NLRP3 increased significantly in HaCaT cells stimulated with LPS and ATP.Moreover,formononetin incubation reversed the abnormal expression of NLRP3.2.NLRP3 influences IL-33 in vitro.Changes of NLRP3 and IL-33 in HaCaT cells or human bronchial epithelial cells(16HBE)transfected with NLRP3 siRNA,IL-33 siRNA or NLRP3 plasmid were detected.We found that overexpression of NLRP3 significantly increased IL-33,and NLRP3 interfering also markedly decreased IL-33.However,siIL-33 showed negligible effects on the expression of NLRP3.Collectively,NLRP3 could positively regulate the expression of IL-33.3.IRF4 involves in the NLRP3-IL-33 pathway.The predicted transcription factors of IL-33 including p-NF-KB,p-STAT3,AP-1,IRF3,IRF4 and IRF7 were screened in HaCaT cells.Effects of IRF4 on the expression of NLRP3,IL-33 in epithelial cells were evaluated by utilizing IRF4 siRNA or IRF4 plasmid.The interaction between IRF4 protein and NLRP3 protein in epithelial cells was detected by immunoprecipitation(IP).As a result,the data suggested that the expression of IRF4 was elevated in HaCaT cells with stimulus.Furthermore,IRF4 plasmid significantly increased NLRP3 and IL-33 while siIRF4 markedly decreased NLRP3 and IL-33.And IP assay suggested that there was a interaction between IRF4 and NLRP3.Collectively,IRF4,which was synergetic with NLRP3,could regulate the expression of IL-33.4.NLRP3 protein binds with IL-33 promoter.The distribution of NLRP3 in HaCaT cells was observed.And the interaction between NLRP3 protein and IL-33 DNA in HaCaT cells was detected by Chromatin immunoprecipitation(ChIP).Besides,whether NLRP3 protein binds with IL-33 promoter was confirmed by yeast one-hybrid.Furthermore,effects of MCC950,ASC oligomeric inhibitor,and VX-765,a Caspase-1 activity inhibitor,on the MC903-induced AD model mice were evaluated.Results showed that NLRP3 primarily expressed in the nucler of epithelial cells,and NLRP3 protein binded with the promoter of IL-33.Moreover,MCC905 or VX-765 had no significant influence on the expression of IL-33 in MC903-induced AD mice model.Conclusions:1.Formononetin,as one of the active components of YPFS,significantly alleviated ear swelling and inflammatory infiltration and inhibited the expression of in MC903-induced AD model mice.The inhibitory effects of formononetin on NLRP3 and IL-33 were also confirmed in vitro.2.In epithelial cells,NLRP3 regulated IL-33 expression in an inflammasome-independent manner,and the receptor NLRP3 is a transcription regulator of IL-3 3.3.IRF4 played a pivotal role in the expression of IL-33,and the NLRP3-IRF4 interaction might regulate transcriptional functions of IRF4.