The Mechanism Of Xinfeng Capsule To Improve The Inflammatory Response Of Rheumatoid Arthritis Was Studied Based On MiR-486-5p/ETS-1 Signalaxis

1 Purpose The effects of Xinfang Capsule(XFC)on clinical inflammatory indexes in patients with rheumatoid arthritis(RA)were observed by using Apriori association rule and random walk model data mining method,and the long-term clinical efficacy of XFC in the treatment of RA was discussed.Clinical experiments were conducted to observe the effects of XFC on the expression levels of immune indexes,cytokines and related m RNA in RA patients.Cell viability,clonogenesis ability,apoptosis level,inflammatory factors,m RNA expression levels of mi R-486-5p and ETS-1 in fibroblastoid synoviocytes and the influence of XFC on them were studied by in vitro cell experiments,and the mechanism of XFC in treating RA was explored from the perspective of mi R-486-5p /ETS-1 signaling axis regulating inflammatory response.2 Methods2.1 Clinical data mining researchThis study collected RA patients from June 2022 to March 2023 in The First Affiliated Hospital of Anhui University of Chinese Medicine.The control group was given basic western medicine treatment,and the XFC group was given Xinfeng capsules(3 times a day,3 capsules once)in addition to basic western medicine treatment.The Apriori association rule method was used to explore the relationship between XFC and clinical inflammatory indicators erythrocyte sedimentation rate(ESR),c-reactive protein(CRP),vascular endothelial growth factor(VEGF)and serum amyloid protein A(SAA),and the long-term improvement effect of XFC on inflammatory indicators was evaluated by random walk model.2.2 Clinical experimental researchRA patients were divided into XFC group and control group by random number table method,with 50 cases in each group.Control group was given Leflunomide tablets(10mg/ time,once a day),XFC group was given XFC tablets(3 capsules/time,3 times a day)on the basis of Leflunomide tablets.The treatment course was 3 month.We observed the expression levels of RF,anti-CCP,IL-1β,IL-10,mi R-486-5p and ETS-1before and after treatment.2.3 In vitro cell experimentRA and normal joint fibroblastic synovial cells were purchased from Cybkon(Shanghai)Biotechnology Co.,LTD.The cells were cultured by passage,and the experiment began at the 3rd generation.The cells were cultured for 24 h,and the plasmid was transferred into the cells by Lipofectamine 2000 when the cells reached 0.70.The cells were divided into the following six groups: FLS group,RA-FLS group,XFC+RA-FLS group,RA-FLS+mi R-486-5p mimics-NC group,RA-FLS+mi R-486-5p mimics group,XFC+RA-FLS+mi R-486-5p mimics group.By CCK-8,plate cloning experiments,scratch experiment,cell migration,flow cytometry,RT-PCR,ELISA and dual luciferase report experiments to investigate the effects of XFC on synovial cell vitality into fiber samples,clone formation,invasion,migration,cell cycle,apoptosis levels and the expression levels of IL-1β,IL-10,mi R-486-5p,ETS-1.3 The results3.1 Results of clinical data mining studies3.1.1 Association rule for Apriori To observe the relationship between Xinfeng Capsule and immune inflammatory indexes in RA patientsThe results of the Apriori association rule model showed that the use of XFC was strongly correlated with the decrease of ESR,CRP,SAA and VEGF.3.1.2 Random walk model To observe the long-term efficacy of Xinfengcapsules on immune inflammatory indexes in RA patientsThe results of random walk model showed that CRP,ESR and VEGF in XFC group were superior to control group in terms of improvement coefficient.3.2 Results of clinical trials3.2.1 RA group and normal group RF,anti-CCP,IL-1 β,IL-10 expression level Compared with normal group,RA group RF,anti-CCP,IL-1 β level increased significantly,IL-10 level decreased obviously(P<0.01).3.2.2 Comparison of m RNA expression levels of mi R-486-5p and ETS-1 in RA group and normal groupCompared with the normal group,the m RNA expression level of mi R-486-5p in RA group was significantly increased,while the m RNA expression level of ETS-1 was significantly decreased(P<0.01).3.2.3 Mi R-486-5 p,ETS-1 m RNA expression level and IL-1β,IL-10,RF,anti-CCP the level of correlation in RA patientsMi R-486-5p in RA patients was positively correlated with RF,IL-1 β;ETS-1 was positively correlated with IL-10(P<0.01).3.2.4 Clinical efficacy of XFCThe total effective rate of observation group was higher than control group(P<0.01).3.2.5 Changes of RF and anti-CCP levels in the observation group and the control group before and after treatmentAfter treatment,the levels of RF and anti-CCP were significantly decreased in both groups.Compared with the control group,RF and anti-CCP levels in the observation group decreased significantly after treatment(P<0.01).3.2.6 Changes of IL-1β,IL-10 levels in observation group and control group before and after treatmentCompared with before treatment,two groups of IL-1 β levels significantly decreased after treatment;IL-10 level increased significantly;After treatment,the levels of IL-1βand IL-10 in the observation group were better than those in the control group(P<0.01).3.2.7 Changes of m RNA levels of mi R-486-5p and ETS-1 in the observation group and the control group before and after treatmentCompared with before treatment,m RNA expression level of mi R-486-5p was significantly decreased and ETS-1 m RNA expression level was significantly increased in both groups after treatment.After treatment,the m RNA expression levels of mi R-486-5p and ETS-1 in the observation group were better than those in the control group(P<0.05,P<0.01).3.3 Results of cell experiment3.3.1 Dual luciferase reporter gene assay verified the targeting relationship between mi R-486-5p and ETS-1Mi R-486-5p inhibited luciferase activity of ETS-1 wild-type 3’UTR reporter gene,but not ETS-1 mutant 3’UTR.3.3.2 Screening of drug-containing serumThe activity of fibroblast synoviocytes was significantly inhibited by 20% concentration of XFC medicated serum.3.3.3 Effects of XFC on the activity and proliferation of RA fibroblastic synovial cellsCompared with FLS group,the cell viability and proliferation levels in RA-FLS group were significantly increased(P<0.01).Compared with RA-FLS group,after XFC intervention,cell viability and proliferation in XFC+RA-FLS group were significantly decreased(P<0.01).Compared with the RA-FLS+mi R-486-5p mimics NC group,the cell viability and proliferation levels of RA-FLS+mi R-486-5p mimics group were significantly increased(P<0.01).After overexpression of mi R-486-5p,XFC could reduce the cell viability and proliferation level of fibroblastoid synoviocytes(P<0.01).3.3.4 Effect of XFC on migration level of RA fibroblastic synovial cells Compared with FLS group,RA-FLS constituted fibrous synovial cell migration level was significantly increased(P<0.01).After treatment with XFC,the cell migration level was significantly decreased(P<0.01).Compared with the RA-FLS+mi R-486-5p mimics NC group,the cell migration level in the RA-FLS+mi R-486-5p mimics group was significantly increased(P<0.01).In the case of overexpression of mi R-486-5p,the cell migration level was significantly decreased under the intervention of XFC(P<0.01).3.3.5 Effects of XFC on the cycle and apoptosis level of fibroblastic synovium cells in RACompared with FLS group,the proportion of S+G2 cells in RA-FLS group was significantly increased,and the apoptosis level was significantly decreased(P<0.01).After treatment with XFC,the proportion of cells in S+G2 phase was significantly decreased,and the apoptosis level was significantly increased(P<0.01).Compared with the RA-FLS+mi R-486-5p mimics NC group,the proportion of S+G2 cells in RA-FLS+mi R-486-5p mimics group was significantly increased,and the apoptosis level was significantly decreased(P<0.01).Under the overexpression of mi R-486-5p,the proportion of cells in S+G2 phase was significantly decreased and the apoptosis level was significantly increased under the intervention of XFC(P<0.01).3.3.6 Effect of XFC on the expression of IL-1β and IL-10 in fibroblast-like synovial cells of RACompared with FLS group,RA-FLS group of IL-1 β expression level increased significantly,IL-10 expression level decreased obviously(P < 0.01).After treated with XFC,IL-1 β expression level decreased obviously,IL-10 expression level increased significantly(P < 0.01);Compared with RA-FLS+mi R-486-5p mimics NC group,RA-FLS+mi R-486-5p mimics group IL-1 β expression level increased significantly,IL-10 expression level decreased obviously(P < 0.01);In overexpression of mi R-486-5p cases,XFC intervened IL-1 β expression level significantly decreased,IL-10 expression level increased significantly(P < 0.01).3.3.7 Effect of XFC on m RNA expression levels of mi R-486-5p and ETS-1 in RA fibroblast-like synovial cellsCompared with FLS group,m RNA expression level of mi R-486-5p in RA-FLS group was significantly increased,while m RNA expression level of ETS-1 was significantly decreased(P<0.01).After XFC treatment,the m RNA expression level of mi R-486-5p was significantly decreased,while the m RNA expression level of ETS-1 was significantly increased(P<0.01).Compared with the RA-FLS+mi R-486-5p mimics NC group,m RNA expression level of mi R-486-5p in RA-FLS+mi R-486-5p mimics group was significantly increased,while m RNA expression level of ETS-1 was significantly decreased(P<0.01).In the case of overexpression of mi R-486-5p,the m RNA expression level of mi R-486-5p was significantly decreased under the intervention of XFC,while the m RNA expression level of ETS-1 was significantly increased(P<0.01).4 Conclusion4.1 XFC has good long-term improvement effect on clinical inflammatory indexes in RA patients.The use of XFC was strongly correlated with the decrease of ESR,CRP,SAA and VEGF.The improvement coefficients of CRP,ESR and VEGF in XFC group were better than those in control group.4.2 RF,anti-CCP,IL-1 β,mi R-486-5p levels increased significantly,IL-10 level decreased obviously in RA patients.mi R-486-5p in RA patients was positively correlated with RF,IL-1β;ETS-1 is positively correlated with IL-10,suggesting that there is an imbalanced immunoinflammatory response and m RNA expression in RA patients,as well as a close relationship between mi R-486-5p and ETS-1 and immunoinflammatory response.4.3 Group total effective rate was higher than the control group.After treatment,the levels of RF,anti-CCP,IL-1β,IL-10,mi R-486-5p and ETS-1 in the observation group were better than those in the control group.The above results indicate the exact improvement of XFC on immune inflammation and targets in RA patients.4.4 Mechanism of action of XFC to improve inflammatory response of RA fibroblastic synovial cells:4.4.1 XFC can decrease the activity,proliferation,migration,S+G2 phase cell ratio and increase the apoptosis level of fibroblastoid synoviocytes,thus reducing the production of fibroblastoid synoviocytes.4.4.2 XFC can inhibit the expression of IL-1β,promote the expression of IL-10 and balance the inflammatory response.4.4.3 Targeting mi R-486-5p to inhibit ETS-1,XFC further enhances the expression of ETS-1 by inhibiting the expression of mi R-486-5p,and further improves the inflammatory state of fibroblastoid synovial cells.