To Study The Mechanism Of XFC Capsule On RA In Ragulating M1/M2 Inflammatory Polarization Based On The MiR-486-5p/ETS-1/Notch1 Signaling Pathway

1.ObjectiveIn this project,rheumatoid arthritis(RA)patients were studied and Xinfeng capsule(XFC)was used as an intervention to explore the mechanism of XFC’s action in the treatment of RA in terms of spleen deficiency and dampness symptom and sign scores,health evaluation index quantitative criteria,disease activity,pain score,inflammatory immune index,and inflammatory polarized surface markers.We observed the degree of toe swelling,arthritis index(AI),pathological morphology,inflammatory factors,CD86 and CD206 in adjuvant arthritis(AA)rats through animal experiments,and explored the mechanism of action of the Chinese herbal compound XFC against RA from the perspective of miR-486-5p/ETS-1/Notch1 signaling axis regulating macrophage inflammatory polarization.2.Methods2.1 Clinical studyIn this study,,batch number:Anhui Pharmaceutical Preparation Z20050062,3 capsules/time,3 times/day;the control group was given methotrexate(MTX),Shanghai ShangPharma Xinyi Pharmaceutical Co.(LEF),Metro Pharmaceuticals Co.,Ltd.,Guohua Zhuanzi:H20080047,10mg/dose,1 time/day,oral treatment,both groups were treated continuously for 4 weeks.The changes in systemic symptom scores,quantitative health assessment indexes,disease activity,pain score,inflammatory immune indexes,inflammatory polarized surface markers and correlations between the indexes were analyzed according to the experimental data of RA patients.2.2 Experimental studyThirty SD female rats were randomly divided into normal group,model group,MTX group,XFC group,Notchl inhibitor group and miR-486-5p inhibitor group,5 rats in each group.Except for the normal group,0.3 ml of FCA was injected subcutaneously into the right toe of the rats in each group to induce an inflammatory response and replicate the AA rat model.The drug was administered 14 days after the successful replication,as described in the animal experiments.3 Results3.1 Clinical study results3.1.1 Changes in the systemic symptoms and signs of RA patients in the two groups before and after treatmentThere was no statistical difference between the two groups before treatment(P>0.05).The observation group showed significant improvement in systemic symptoms and signs compared with those before treatment(P<0.05 or P<0.01),while the control group also showed greater improvement in all symptoms and signs after treatment(P<0.05),except for bloating after eating(P>0.05);in comparison,the observation group showed more significant improvement in tiredness and loss of appetite than the control group(P<0.05).3.1.2 Changes in HAQ,DAS-28 and VAS of RA patients in the two groups before and after treatmentThere was no statistical difference between the two groups before treatment(P>0.05).HAQ,DAS-28 and VAS scores in both groups were significantly better than before treatment(P<0.01),and there was no statistically significant difference between the two groups after treatment(P>0.05).3.1.3 Changes in ESR,hs-CRP,RF and anti-CCP of RA patients in the two groups before and after treatmentThere was no statistical difference between the two groups before treatment(P>0.05).The ESR,hs-CRP,RF and anti-CCP were significantly lower in the observation and control groups than before treatment(P<0.01 or P<0.05);compared with the two groups,the observation group was more significant in reducing RF and anti-CCP(P<0.01).3.1.4 Changes in IgG,IgM,C3 and C4 of RA patients in the two groups before and after treatmentThere was no statistical difference between the two groups before treatment(P>0.05).The observation group had a substantial decrease in IgG,IgM,C3 and C4 after treatment(P<0.01),while the control group had a significant decrease in IgG,C3 and C4(P<0.01 or P<0.05),and the difference in IgM before and after treatment was not significant(P>0.05);compared with the two groups,the observation group was better than the control group in improving C3 and C4(P<0.01).3.1.5 Clinical efficacy of XFC in the treatment of RAThere was no statistically significant difference in the total effective rate between the two groups(P>0.05).3.1.6 Changes in inflammatory polarisation surface markers in RA patients in the two groups before and after treatmentThere was no statistically significant difference between the two groups before treatment(P>0.05).After treatment,CD86 decreased and CD206 increased in both groups(P<0.01);compared with the two groups,the decrease in CD86 and the increase in CD206 in the observation group were more significant than those in the control group(P<0.01).3.1.7 Correlation analysis of inflammatory polarization surface markers with systemic joint symptoms and sign scores in RA patientsCD86 was significantly positively correlated with joint swelling,joint pain and morning stiffness in RA(P<0.01 or P<0.05),while CD206 levels were significantly negatively correlated with joint swelling,pain and morning stiffness(P<0.01).CD206 was negatively correlated with loss of appetite and joint regain(P<0.01).3.1.8 Correlation of inflammatory polarisation markers with HAQ,DAS-28 and VAS in RA patientsThe level of CD86 in RA patients was positively correlated with HAQ,DAS-28 and VAS(P<0.01 or P<0.05);the level of CD206 was negatively correlated with HAQ,DAS-28 and VAS(P<0.01 or P<0.05).3.1.9 Correlation analysis of CD86,CD206 and inflammatory immune indexes in RA patientsThere was a significant positive correlation between RF,anti-CCP,hs-CRP,C3,C4 and CD86 in RA patients(P<0.01 or P<0.05);there was a significant negative correlation between RF,anti-CCP,hs-CRP,C3,C4 and CD206(P<0.01 or P<0.05).3.2 Results of the experimental study3.2.1 Changes in toe swelling and arthritis index of AA rats in each groupBefore drug administration,toe swelling was significantly higher(P<0.01)and arthritis index was higher(P<0.05)in the model group,XFC group,MTX group,Notch 1 inhibitor group and miR-486-5p inhibitor group compared with the normal group;except for the normal group,toe swelling and arthritis index in the model group were not significantly different compared with the other groups(P>0.05).After 30 days of treatment,the toe swelling and arthritis index in the model group were significantly greater compared with the other groups(P<0.05 or P<0.01);the toe swelling and arthritis index in the other groups,except the normal group,were significantly lower compared with the model group(P<0.05 or P<0.01);the differences between the four groups and the XFC group were not significant except for the model group(P>0.05).3.2.2 Morphological changes of joint pathology in AA rats in each groupIn the normal rat group,the joint and cartilage structures were intact and clear,the fibrous synovial membrane and synovial epithelium were well organized without hyperplasia and swelling,and there was no obvious inflammatory cell infiltration.In the model group,the synovial tissue on the joint surface was hyperplastic and extruded into the joint cavity,with a large number of inflammatory cells infiltrating the joint,the distribution of synovial vessels was blurred and the synovial epithelial tissue was disorganized.The synovial cells were still normally arranged and a small number of inflammatory cells infiltrated the synovial membrane and blood vessel wall,and the overall structure of the joint surface was more regular.In the methotrexate group,the synovial cells were more regularly arranged,the joint surface was not clear,there was mild damage to the synovial tissue and mild vascular blurring in the joint wall.miR-486-5p inhibitor group,mild inflammatory cell infiltration in the synovial tissue and vascular wall was observed.3.2.3 Changes in serum cytokines in the joints of AA rats in each groupSerum levels of IL-23 and TNF-α were significantly higher in the model group compared to the normal group,while the levels of IL-4 and IL-13 were significantly lower(P<0.01).With the intervention of XFC,MTX,Notchl inhibitor and miR-486-5p inhibitor,the expression of IL-23 and TNF-α was significantly reduced,while the expression of IL-4 and IL-13 was significantly increased(P<0.05 and P<0.01).Compared to the XFC group,IL-23 and TNF-α were increased in the MTX,Notch1 and miR-486-5p inhibitor groups,while IL-4 and IL-13 were decreased in the MTX group(P<0.05).3.2.4 Changes in CD86 and CD206 in peripheral blood of AA rats in each groupCompared with the normal group,CD86 expression was significantly higher in the model group,while CD206 expression was significantly lower(P<0.05).Compared with the model group,CD86 expression was decreased and CD206 expression was increased in the XFC,MTX,Notchl inhibitor and miR-486-5p inhibitor groups(P<0.05).Except for the model group,the XFC group showed significantly more increased CD86 expression and decreased CD206 expression than the other four groups(P<0.05).3.2.5 Correlation analysis of peripheral blood CD86 and CD206 with serum cytokines in AA ratsThere was a significant positive correlation between CD86 and IL-23 and TNF-α(P<0.05 or P<0.01),and a significant negative correlation between CD86 and IL-4 and IL-13(P<0.05);there was a significant negative correlation between CD206 and IL-23(P<0.01),and a significant positive correlation between CD206 and IL-4 and IL-13(P<0.01 or P<0.05).There was a significant positive correlation between CD206 and IL-4 and IL-13(P<0.01 or P<0.05).3.2.6 Expression of genes related to miR-486-5p/ETS-1/Notch1 signalling pathway in synovial tissue of AA rats in each groupAfter 30 days of administration,the expression levels of miR-486-5p and Notchl in the model group were significantly higher(P<0.05),while the expression level of ETS-1 was significantly lower(P<0.05)compared with the normal group.Except for the normal group,the expression levels of miR-486-5p and Notchl were decreased(P<0.05)and ETS-1 expression levels were increased(P<0.05)in the other four groups compared with the model group.miR-486-5p and Notchl expression levels were increased in the MTX group,Notchl inhibitor group and miR-486-5p inhibitor group compared with the XFC group(P<0.05)and ETS-1 expression level was decreased(P<0.05).3.2.7 Expression of miR-486-5p,ETS-1 and Notchl in the synovial tissue of AA rats in each groupAfter 30 days of administration,the expression of miR-486-5p and Notchl protein increased significantly in the model group,XFC group,MTX group,Notchl inhibitor group and miR-486-5p inhibitor group compared with the normal group(P<0.05),and the expression of ETS-1 protein decreased(P<0.05).,miR-486-5p inhibitor group compared with the model group,Notchl and miR-486-5p protein expression were all decreased(P<0.05)and ETS-1 protein expression was increased(P<0.05).miR-486-5p protein expression was decreased in the XFC group compared with the MTX group,Notchl inhibitor group and miR-486-5p inhibitor group(P<0.05)and ETS-1 protein expression was increased(P<0.05),while Notchl protein expression was not significantly different in the Notch1 inhibitor group(P>0.05).3.2.8 Correlation analysis of CD86 and CD206 in peripheral blood and miR-486-5p/ETS-1/Notch signaling pathway protein expression in synovial tissue of AA ratsThe correlation analysis of miR-486-5p/ETS-1/Notch1 signaling axis-related proteins revealed that CD86 was positively correlated with miR-486-5p and Notchl(P<0.01 or P<0.05),while it was negatively correlated with ETS-1(P<0.01);CD206 was significantly positively correlated with the expression level of ETS-1(P<0.01),while significantly negatively correlated with the expression levels of miR-486-5p and Notchl(P<0.05).4 Conclusions4.1 XFC can effectively improve joint symptoms and signs score values,improve related scale score values,reduce inflammatory immune laboratory indexes,and repulsively adjust inflammatory polarized surface markers in RA patients,thus reducing the inflammatory immune response in the RA organism.4.2 XFC significantly reduced toe swelling and arthritis index in AA rats and attenuated synovial inflammatory responses.4.3 Mechanisms underlying the regulation of inflammatory immune indexes by XFC in AA rats4.3.1 XFC reduced the immune inflammatory response in AA rats by decreasing IL-23 and TNF-α and increasing IL-4 and IL-13.4.3.2 XFC inhibits inflammatory CD86 expression and promotes inflammatory CD206 expression,exerting a beneficial effect in balancing the repair of the immune inflammatory response in AA rats.4.3.3 XFC down-regulated miR-486-5p and Notchl expression,leaving ETS-1 expression uninhibited,indicating that XFC could regulate the miR-486-5p/ETS-1/Notch1 signaling pathway and modulate macrophage inflammatory polarization,thereby reducing the inflammatory response in AA rats at an overall level and improving inflammatory immune indexes in RA.