Mechanism of Qingre Yangyin Chushi Pill in the treatment of gout based on NLRP3/GSDMD pyroptosis pathway

Background:The study was conducted with the interest of exploring the effects of Qingre Yangyin Chushi pill as anti-inflammatory agent in gout treatment based on the NLRP3/GSDMD coke death pathway.Methods:In this study,48 male SD rats were randomly divided into 6 groups,namely model group,blank control group(BC),Qingre Yangyin Chushi pill low-dose treatment group(MSD1),medium-dose treatment group(MSD2),high-dose treatment group(MSD3),and colchicine group(PC),with eight members in each group.The BC and model groups were administered with saline twice a day.The MSD1,MSD2,and MSD3 groups were administered with Qingre Yangyin Chushi pill at a dose of 1.8 g·kg^?1,3.6 g·kg^?1,and 7.2 g·kg^?1 twice a day.The colchicine group was administered with a colchicine suspension at a dose of 0.6×10^?3 g kg^?1 twice a day for 7 days.After gavage of animals in each group for 4 days,the rats'ankle joints were injected with sodium urate suspension×3 days to induce a gouty arthritis model.Then,serum and tissue samples were collected on the third and seventh day after gavage.The synovial tissue from the rat ankle joints was taken,and immunohistochemical technique was used to detect NOD-like receptor protein 3(NLRP3),inflammatory bodies,cysteine protease-1(Caspase-1),and Gasdermin-D protein(GSDMD)expression.Image-Pro Plus image analysis system was used to measure the average integrated absorbance and calculate IHS.Integral enzyme-linked immunosorbent assay was used to determine the interleukin 1β(IL-1β)and interleukin 18(IL-18)expression of tumor necrosis factor-α(TNF-α).Western blot was used to detect NLRP3,Caspase-1,GSDMD,and apoptosis-associated speck-like protein containing a CARD(ASC)expression levels.Results:After 48 hours of modeling,compared with the PC group,the arthritis indices of the MSD1,MSD2,and MSD3 groups were insignificant(P>0.05).The levels of IL-1β,IL-18,and TNF-αwere lower in the PC and MSD2 groups compared with the BC group.However,compared with the control group,MSD1,MSD2,and PC groups had lower levels of IL-1βand IL-18.Regarding TNF-αlevels(P<0.05);compared with the PC group,the levels of IL-1β,IL-18,and TNF-αin MSD2 decreased more significantly(P<0.05).The levels of NLRP3,Caspase-1,GSDMD,ASC,IHS score,and mRNA were lower in the PC and MSD2 groups(P<0.05)compared with the control group.The MSD1 and MSD2 groups had lower levels of NLRP3,Caspase-1,GSDMD,ASC protein levels,IHS points,and mRNA levels(P<0.05)compared with the PC group.Moreover,NLRP3,Caspase-1,GSDMD,ASC protein levels,IHS points,and mRNA levels were more reduced in the MSD2 group(P<0.05).Conclusion:Qingre Yangyin Chushi pill can inhibit the activation of the NLPR3 inflammatory complex,reduce the production of GSDMD protein,regulate the occurrence of pyroptosis,reduce the expression of inflammatory factors,and thus reduce arthritis.All these processes are achieved through the NLRP3/GSDMD pathway.