Study On The Mechanism Of Xinfeng Capsule On Immuno-inflammation In RA Based On Pyroptosis Mediated By NLRP3/GSDMD Pathway

1 Objective Through clinical research,observe the changes of laboratory indicators,cytokines(interleukin-1β,interleukin-18),pyroptosis pathway-related indicators(NLRP3,caspase-1,GSDMD),patient experience-related indicators and Xinfeng Capsules in RA patients.(Xinfeng Capsule,XFC)on it;Based on in vitro cell experiments,explore the mechanism of XFC improving RA immune inflammation by regulating pyroptosis mediated by NLRP3/GSDMD pathway.2 Methods2.1 Clinical researchSixty patients with rheumatoid arthritis hospitalized in the Department of Rheumatology and Immunology of Anhui Provincial Hospital of Traditional Chinese Medicine from May 2021 to May 2022 were selected and divided into two groups according to the random number table method,one group was the XFC group(observation group)and one group was the LEF group(control group),in addition,30 healthy persons were selected from the Health Examination Centre of Anhui Provincial Hospital of Traditional Chinese Medicine as the NC group(healthy control group).Blood sedimentation(ESR)was measured by blood sedimentation meter,C-reactive protein(CRP),rheumatoid factor(RF),IgA,IgG,IgM,C3 and C4 were measured by automatic biochemical analyzer,anti-cyclic melon amino acid peptide antibody(CCP)was measured by chemiluminescence method,IL-1β and IL-18 levels were measured by ELISA method,and protein immunoblotting(westernblot,WB)was used.The expression of NLRP3,caspase-1 and GSDMD was measured by westernblot,WB.The questionnaires were used to record the feeling-related indexes of RA patients to observe the effect of XFC on the efficacy of RA patients and the NLRP3/GSDMD pathway.2.2 Experimental researchSynovial fibroblasts(RA-FLS)of patients with RA stimulated by lipopolysaccharide(LPS)were used as research objects,and were divided into normal control group(RA-FLS),model group(RA-FLS+LPS),XFC group(RA-FLS)FLS+LPS+XFC drug-containing serum),MCC950 group(RA-FLS+LPS+MCC950),given corresponding drugs and reagents for 48 hours of intervention;CCK-8 method was used to detect cell viability,the morphology of the pyrogenic cells was observed by electron microscopy and the viability of the cells was measured by ELISA.The concentrations of IL-1β and IL-18 were measured and the protein and mRNA expression of NLRP3,caspase-1 and GSDMD were detected by WB method and quantitative real-time polymerase chain reaction(q RT-PCR).3 Results3.1 Results of clinical studies3.1.1 The changes of laboratory indicators,cytokines and pyroptosis pathway-related indicators in RAIn 60 RA patients,patients compared with NC group what ESR,CRP,RF,CCP,IL-1β,IL-18,NLRP3,caspase-1 and GSDMD protein levels were significantly increased(P<0.01).3.1.2 The clinical curative effect of XFC in treating RAPatients was compared with LEF group,the effective rate of XFC group was 83.3%,and the effective rate of LEF group was 76.7%.The efficiency was higher than that of LEF group(P<0.05).3.1.3 The effect of XFC on the laboratory indicators and cytokines of RA Patients Compared with before treatment,ESR,CRP,RF,and CCP in both groups decreased after treatment,at the same time,in the XFC group,the reduction of ESR,CRP,and RF was more visible,and with C3,C4 increasing(P<0.05,P<0.01).Compared with before treatment,IL-1β and IL-18 in both groups decreased significantly after treatment(P<0.01).3.1.4 The effect of XFC on pyroptotic pathway-related indicators in RA Patients Compared with before treatment,NLRP3,caspase-1 and GSDMD in both groups decreased after treatment,and the reduction of GSDMD and NLRP3 in the XFC group was more obvious(P<0.05,P <0.01).3.1.5 The influence of XFC on the related indicators of RAPatients compared with before and after treatment,in the two groups,the DAS28 scores and sign scores,TCM syndrome scores,SDS scores,RA symptom,VAS scores,and SAS scores were significantly reduced,and the XFC group was significantly lower in The reduction of RA symptom and sign scores and TCM syndrome scores was more obvious(P<0.05,P<0.01).3.2 Experimental research results3.2.1 The optimal intervention concentration and time of XFC drug-containing serum acting on RA-FLSCompared with the normal group,cell viability of XFC-containing serum was significantly reduced at each concentration gradient(P<0.01,P<0.05);in the same concentration group Compared with 24 h,the cell viability of 20% and 40% of XFC drug-containing serum at 12 h was significantly higher than that of 24h(P<0.01),and the cell viability of 40% of XFC drug-containing serum at 72 h was significantly lower than that of 24h(P<0.01).Therefore,20% and 24 h were selected as the optimal intervention concentration and time of XFC drug-containing serum on RA-FLS.3.2.2 Effect of XFC drug-containing serum on RA-FLS cell viability and cell pyroptosis Compared with the normal control group,tthe survival rate of RA-FLS cells was significantly higher in the model group(P<0.05);compared with the model group,the MCC950 group and the viability of RA-FLS cells in XFC group was significantly decreased(P<0.01).Thermalization was observed by transmission electron microscopy.The cell membrane of the model group was ruptured compared to the normal control group,the cell membrane of the model group ruptured,and the cells formed a large number of vesicles(pyroptotic bodies),and the cell contents flowed out;compared with the model group,the cells of the XFC group and MCC950 group The pyroptosis situation has improved compared to before.3.2.3 Effect of XFC drug-containing serum on the expression of IL-1β,IL-18,pyroptosis-related mRNA and proteinCompared with the normal control group,IL-1β,IL-18 concentration,NLRP3,Caspase-1,GSDMD protein and mRNA expression were significantly higher in the model group(P<0.01,P<0.05);compared with the model group,IL-1β,IL-18 concentration,NLRP3,Caspase-1,GSDMD protein and mRNA expression were significantly decreased(P<0.05,P<0.01).4 Conclusions4.1 XFC can effectively reduce the antibody levels of ESR,CRP,RF,and CCP in RA patients,inhibit the secretion of cytokines IL-1β and IL-18 and the expression of Caspase-1,GSDMD and NLRP3 proteins,improve patients’ relevant sensory indicators and suppress the inflammatory response.Improve the quality of life of patients.4.2 The mechanism of XFC improving immune inflammation in RA patients may be through regulating NLRP3/GSDMD pathway,inhibiting the activation of NLRP3 inflammasome,reducing NLRP3,GSDMD,Caspase-1 mRNA and protein levels,and inhibiting cytokines IL-1β,IL-18 Secreted to inhibit pyroptosis,so as to improve the immune inflammation of RA patients.