| 1 ObjectiveThe purpose of this study is to examine the impact of Xinfeng capsules(XFC)on immune-inflammatory markers,cytokines,DAS-28,SAS,SDS,TCM syndrome score,HAQ score,and VAS score in patients with rheumatoid arthritis(RA)through clinical research.The study also aims to observe the changes in toe swelling,arthritis index,joint pathological morphology,macrophage polarization markers,mi R145-5p/Smads signaling pathway,and the effect of XFC on them in adjuvant arthritis(AA)rats.Furthermore,this study explores the mechanism by which XFC improves RA inflammation from the perspective of mi R145-5p/Smads signaling pathway regulation of macrophage polarization.2 Methods2.1 Changes and correlation analysis of clinical indicators such as IL-8 and IL-13 in RA patients and the effect of XFC on themFrom May 2021 to May 2022,60 hospitalized RA patients were gathered from the Anhui Provincial Hospital of TCM.30 persons in each of the observation and control groups,assigned at random.Also,20 healthy people were chosen as the usual group from the health assessment center.We observed the effects of Xinfeng Capsule(XFC)on immune inflammatory indicators(RF,anti-CCP,ESR,CRP,Ig A,Ig G,Ig M,C3,C4),cytokines(IL-8,IL-13),DAS-28,SAS,SDS,Chinese medicine syndrome score,HAQ and VAS in RA patients.2.2 Effect of XFC on mi R145-5p/Smads signal pathway and macrophage polarization in AA ratsSix rats from each of the four groups(MC,MTX,XFC and NC group),were randomly selected from a total of 24 SD rats.With the exception of the NC group,each rat in the other three groups received an injection of Freund’s complete adjuvant to cause inflammation for modeling.Injections of 0.1 m L of FCA induced inflammation were administered to the back 3 groups of mice to model.19 days after the successful replication of the model,each group began to administer the drug and lasted 4 weeks.(1)NC group and MC group 1 times / day,physiological salt water 1ml / 100 g irrigation of the stomach;(2)XFC group: prepared to contain XFC(0.034 g / ml)mixed liquid,1 time/ day for 1 ml / 100 g irrigations;(3)MTX group: manufactured to include MTX(0.095 mg / ml),1 time per week,1 ml/100 g irigation.Under a microscope,the histological alterations of the synovial membrane and the swelling and arthritis index of the four groups of rats were examined.IL-8 and IL-13 levels were discovered using an ELISA.The polarization markers CD80 and CD206 of synovial macrophages,as well as the protein expression of TGF-β1/Smads signaling pathway,were detected by WB.The relative expression of mi R145-5p/Smads signaling pathway genes in synovial tissue was detected by RT-q PCR.3 Results3.1 Changes and correlation analysis of clinical indicators such as IL-8 and IL-13 in RA patients and the impact of XFC on them3.1.1 Comparison of IL-8 and IL-13 levels between RA group and normal groupCompared to the normal group,the RA group showed a significant increase in IL-8levels and a significant decrease in IL-13 levels(P<0.01).3.1.2 Comparison of immune-inflammatory markers between RA group and normal groupThe expression levels of markers of inflammation were considerably higher in the RA group in comparison with the control group(P<0.01),whereas the level of Ig G、RF、anti-CCP was substantially higher among the immune markers(P<0.01),and there were no meaningful variations in the expression levels of the other immune markers(P>0.05).3.1.3 Correlation analysis between IL-8,IL-13 and laboratory indicators,DAS-28 in the RA groupIn the RA group,IL-8 was favorably linked with Ig G,RF,anti-CCP levels and markers of inflammation(P<0.01);In the RA group,IL-13 was inversely linked with Ig G、RF、anti-CCP levels and markers of inflammation(P<0.01).3.1.4 Correlation analysis of IL-8 and IL-13 with SAS and SDS in RA groupSAS and SDS were positively connected with IL-8 in the RA group(P<0.01),whereas SAS and SDS were negatively correlated with IL-13(P<0.01).3.1.5 Correlation analysis of IL-8 and IL-13 with TCM syndrome score in RA groupJoint indications,diarrhea,and joint heaviness were all favorably connected with IL-8 in the RA group and negatively correlated with IL-13(both P<0.01).3.1.6 Correlation analysis of IL-8 and IL-13 with VAS and HAQ scores in RA groupIL-8 in the RA group was positively correlated with VAS and HAQ scores(P<0.01);IL-13 was negatively correlated with VAS and HAQ scores(P<0.01).3.1.7 Comparison of clinical efficacy between the observation group and the control group There was no discernible distinction among the two groups’ total effective rates,which were 86.7% in the observation group and 83.3% in the control group(P>0.05).3.1.8 Changes in IL-8 and IL-13 levels before and after treatment in the observation group and control groupCompared with before treatment,IL-8 levels significantly decreased(P<0.01)and IL-13 levels significantly increased(P<0.01)after treatment in both groups,but the changes in IL-8 and IL-13 levels in the observation group were better than those in the control group(P<0.01).3.1.9 Changes in inflammatory immune levels and DAS-28 before and after treatment in the observation group and control groupFollowing therapy,the expression of other immune markers did not change considerably(P>0.05).but the levels of inflammatory markers reduced significantly in both groups(P<0.01).as well as the level of Ig G、RF、anti-CCP.After therapy,the observation group’s CRP level significantly decreased when compared to the control group(P<0.01).3.1.10 Changes in TCM Syndrome Scores before and after Treatment in the Observation and Control GroupsBoth groups had a substantial reduction in joint complaints following therapy compared to before(P<0.01).Joint heaviness,a marker of spleen shortage,significantly decreased following treatment in both groups(P<0.01),with the observation group seeing a more dramatic improvement(P>0.05).Following therapy,the observation group’s diarrhea significantly improved(P<0.01),but the control group’s diarrhea did not improve(P>0.05).Other spleen insufficiency symptoms did not significantly better in either group following therapy(P>0.05).3.1.11 Changes in SAS and SDS Scores before and after Treatment in the Observation and Control GroupsWhen compared to before treatment,both groups had a substantial drop in SAS and SDS levels(P<0.01).After treatment,there was no significant variation in the decrease in SAS and SDS scores between the observation and control groups(P>0.05).3.1.12 Changes in HAQ and VAS Scores before and after Treatment in the Observation and Control GroupsAs comparison to before treatment,both groups had a substantial decline in HAQ and VAS ratings after therapy(P<0.01).After treatment,there was no substantial distinction in the changes in HAQ and VAS levels among the 2 groups(P>0.05).3.2 Effects of XFC on mi R145-5p/Smads signaling pathway and macrophage polarization in AA rats3.2.1 Changes in toe swelling and arthritis index in AA rats and the effect of XFCThe toe swelling and arthritis index of the MC,MTX,and XFC groups were considerably higher the day before treatment compared to the NC group(P<0.01),although there was no substantial difference between the MC group and the MTX or XFC groups(P>0.05).Following 4 weeks of therapy,the MC group’s arthritis index and toe swelling index were substantially higher than in the NC group(P<0.01)compared to the NC group.The arthritis index and toe edema in the MTX group and XFC group were significantly reduced(P<0.01)as compared to the MC group.Between the MTX group and the XFC group,there was no discernible change in the arthritis index or toe edema(P>0.05).3.2.2 Pathological morphological changes of AA rat joints and the effect of XFC on themThe synovial tissue of the NC group rats seems to be clean under a microscope,with neatly distributed cells and no inflammatory cell infiltration.The synovial tissue of the MC group rats exhibits proliferation with many inflammatory cell infiltrations as compared to the NC group.Rats in the MTX and XFC groups significantly reduced synovial proliferation and inflammatory cell infiltration compared to the MC group,and the XFC group improved synovial proliferation and inflammatory cell infiltration more than the MTX group did.3.2.3 Correlation analysis of peripheral blood cytokines and macrophage polarization markers in synovial tissue of AA ratsThrough correlation analysis,it was discovered that CD80 and CD206 had opposite relationships to IL-8 and IL-13,with CD80 having a positive correlation with IL-8 and a negative relationship with IL-13(P<0.01).3.2.4 Correlation analysis of TGF-β1/Smads pathway protein expression and macrophage polarization markers CD80 and CD206 in synovial tissue of AA ratsThrough correlation analysis,it was found that M1 macrophage marker CD80 was positively correlated with Smad3 and TGF-β1(P<0.05 or P<0.01)and negatively correlated with Smad7(P<0.01);M2 macrophage marker CD206 was negatively correlated with Smad3 and TGF-β1(P<0.05 or P<0.01)and positively correlated with Smad7(P<0.05).3.2.5 Correlation analysis of TGF-β1/Smads pathway protein expression and mi R145-5p in synovial tissue of AA ratsThrough correlation analysis,it was found that mi R145-5p was negatively correlated with Smad3 and TGF-β1(P<0.05 or P<0.01)and positively correlated with Smad7(P<0.01).3.2.6 Changes in expression of IL-8 and IL-13 in peripheral blood of AA rats and the effect of XFCWith statistical significance(P<0.01),the MC group’s expression of IL-8 was statistically substantially higher than that of the NC group and statistically considerably lower than that of IL-13.In the MTX and XFC groups,IL-8 expression was statistically considerably reduced compared to the MC group,but IL-13 expression was dramatically elevated(P<0.01).The changes in IL-8 and IL-13 levels were superior in the XFC group as compared to the MTX group(P<0.05 or P<0.01).3.2.7 Changes in relative expression of macrophage polarization markers in AA rat synovial tissue and the effect of XFCIn the synovial tissue of MC rats,CD80 expression was statistically substantially higher than that of CD206 and significantly lower than that of the NC group(P<0.05 or P<0.01).In the MTX and XFC groups,compared to the MC group,there was a statistically significant(P<0.05 or P<0.01)drop in CD80 expression and an increase in CD206 expression.The level of CD80 was higher in the XFC group compared to the MTX group(P<0.05),while there was no discernible change in the level of CD206(P>0.05).3.2.8 Changes in relative expression levels of TGF-β1/Smads pathway factors in synovial tissue of AA rats and the effect of XFC on themIn the synovial membrane of rats in the MC group,Smad7 expression was dramatically downregulated compared to the NC group,but Smad3 and TGF-1 levels were markedly upregulated,with statistical significance(P<0.05 or P<0.01).In comparison to the MC group,both the MTX and XFC groups showed significant decreases in TGF-β1 expression and significant increases in Smad7 expression(P<0.05 or P<0.01).While no differ significantly in Smad3 level was observed in the MTX group(P>0.05),the XFC group showed a significant decrease in Smad3 expression with statistical significance(P<0.01).Furthermore,the XFC group showed a significant decrease in Smad3 expression compared to the MTX group,with statistical significance(P<0.05),while no significant difference was observed in the expressions of TGF-β1and Smad7 between the two groups(P>0.05).3.2.9 Changes in relative expression levels of mi R145-5p/Smads pathway genes in synovial tissue of AA rats and the effect of XFC on themRats in the MC group had considerably higher levels of Smad3 in their synovial tissue compared to the NC group,whereas their levels of Smad7 and mi R145-5p were much lower.These changes were statistically meaningful(P<0.01).Smad3 expression was substantially lower in the MTX and XFC groups compared to the MC group,whereas Smad7 and mi R145-5p expression was significantly higher,with significant statistical distinctions(P<0.01 or P<0.05).Compared to the MTX group,the XFC group showed statistically significant differences in the expression of Smad3 and mi R145-5p(P<0.05 or P<0.01),while the expression of Smad7 showed no significant differences(P>0.05).4 Conclusion4.1 XFC can improve DAS-28,SAS,SDS,traditional Chinese medicine symptom score,HAQ score,and VAS score in RA patients,while reducing the expression of IL-8 and immune inflammatory markers(RF,ESR,CRP,anti-CCP,Ig G)and increasing the expression of IL-13.Correlation analysis suggests that there is a correlation between IL-8 secreted by M1 polarized macrophages and IL-13 secreted by M2 polarized macrophages and immune inflammatory markers(RF,ESR,CRP,anti-CCP,Ig G)in RA patients.Imbalanced macrophage polarization leads to excessive secretion of pro-inflammatory cytokines,resulting in immune inflammation in RA.This study indicates that XFC can reduce the level of IL-8 and increase the level of IL-13 in RA patients,possibly by regulating macrophage polarization,improving immune inflammation response,alleviating symptoms,and improving patients’ physical and mental quality of life.4.2 The arthritis index,degree of toe edema,and synovial histology of AA rats can all be improved by XFC,thereby reducing the inflammatory response of arthritis.4.3 Intervention mechanism of XFC on improving immune inflammation response in AA rats:4.3.1 XFC can inhibit the expression of pro-inflammatory cytokine IL-8,while upregulating the expression of anti-inflammatory cytokine IL-13,inhibiting inflammation response in AA rats.4.3.2 XFC has the ability to upregulate CD206 cell expression while downregulating CD80 cell expression.XFC can regulate macrophage polarization,restore the M1/M2 dynamic balance,inhibit the proliferation of synovial cells induced by pro-inflammatory cytokines,reduce inflammatory cell infiltration,improve the degree of cartilage destruction in joints,and alleviate immune inflammatory response.4.3.3 After XFC treatment,TGF in AA rats-β 1.The expression of Smad3,CD80,and IL-8 decreased,while the expression of mi R145-5p,Smad7,CD206,and IL-13 increased,indicating that the mi R145-5p/Smads signaling pathway axis was activated.At the same time,macrophage polarization towards M1 was also inhibited,indicating that XFC can regulate macrophage polarization through the mi R145-5p/Smads signaling pathway axis,suppress the immune inflammatory response of AA rats,and improve RA immune inflammatory indicators. |
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