Based On TGF-β1/Smads Signaling Pathway The Mechanism Of Xinfeng Capsule In Regulating The Polarization Of Macrophages In Rheumatoid Arthritis Was Discussed

1 ObjectiveBased on clinical research and cell experiments,the effects and mechanisms of Xinfeng capsule(XFC)and si RNA on TGF-β1 / Smads signaling pathway and the polarization of rheumatoid arthritis(RA)macrophages were investigated.2 method2.1 Clinical methodsThe subjects were divided into three groups : healthy group,leflunomide(LEF)group and XFC group.After 4 weeks of treatment.Rheumatoid factor(RF),anti-cyclic citrullinated peptide antibodies(CCP-Ab),erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),immunoglobulin G(Ig G)and immunoglobulin M(Ig M)were observed.The number of joint tenderness and swelling was recorded,and the visual analogue scale(VAS),Disease Activity Score in 28 jionts(DAS28)and TCM syndrome score were calculated.Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the messenger RNA(m RNA)expression of micro RNA-145-5p(mi R145-5p),transforming growth factor β1(TGF-β1),Smad3 and Smad7.Western blot(WB)was used to detect TGF-β1,Smad3 and Smad7.2.2 Experimental methodRA synovial fibroblasts(FLS)were co-cultured with macrophages,and Smad3-specific small interfering RNA(si RNA)transfection and XFC intervention were performed respectively.RT-q PCR and WB were used to detect the transcription and protein expression of macrophage-related m RNA.The concentration of inflammatory cytokines in macrophages was determined by ELISA.The specific detection indicators were the same as clinical experiments.The proliferation of RA fibroblast-like synoviocytes(RA-FLS)was detected by CCK-8 assay.The migration of RA-FLS was determined by Transwell chamber.3 results3.1 Clinical results3.1.1 Effect of XFC on joint pain,swelling number,VAS,DAS28 scores and TCM syndrome score in RA patientsThe joint tenderness,swelling number,VAS,DAS28 scores and TCM syndrome scores of RA patients were significantly higher than those of normal(P < 0.05).After treatment with LEF and XFC,both showed a downward trend(P < 0.05).When compared between groups,XFC showed certain advantages in reducing TCM syndrome scores(P < 0.05).3.1.2 Effect of XFC on RF,CCP-Ab,Ig G and Ig M in RA patientsThe levels of RF,CCP-Ab,Ig G and Ig M in RA patients were significantly higher than those in normal controls(P < 0.05).After treatment with LEF and XFC,the levels of RF,CCP-Ab and Ig G showed a downward trend(P < 0.05),and there was no significant difference between the two groups(P > 0.05).Ig M levels were significantly decreased after LEF treatment(P < 0.05).3.1.3 Effect of XFC on ESR and CRP in RA patientsThe levels of ESR and CRP in RA patients were significantly higher than those in normal controls(P < 0.05).Both LEF and XFC showed a downward trend after treatment(P < 0.05),and there was no significant difference between the two groups(P > 0.05).3.1.4 Effect of XFC on IL-23 and IL-10 in cell supernatant in RA patientsThe levels of IL-23 in RA patients were significantly higher than those in normal controls,and the levels of IL-10 were significantly lower than those in normal controls(P < 0.05).After treatment with LEF and XFC,the level of IL-23 showed a downward trend and the level of IL-10 showed an upward trend(P < 0.05),and there was no significant difference between the two groups(P > 0.05).3.1.5 Effect of XFC on TGF-β1,Smad3 and Smad7 protein expression in RA patientsThe expression levels of TGF-β1 and Smad3 protein in RA patients were significantly higher than those in normal controls,and the expression levels of Smad7 were significantly lower than those in normal controls(P < 0.05).After treatment with LEF and XFC,the expression levels of TGF-β1 and Smad3 protein showed a downward trend,and the expression level of Smad7 showed an upward trend(P <0.05).After XFC treatment,the down-regulation of TGF-β1 and Smad3 and the up-regulation of Smad7 were more significant(P < 0.05).3.1.6 Effect of XFC on the m RNA expression of TGF,Smad3 and Smad7 in RA patientsThe expression levels of TGF-β1 m RNA and Smad3 m RNA in RA patients were significantly higher than those in normal controls,and the expression level of Smad7 m RNA was significantly lower than that in normal controls(P < 0.05).After LEF and XFC treatment,the expression levels of TGF-β1 m RNA and Smad3 m RNA protein in patients showed a downward trend,and the expression level of Smad7 m RNA showed an upward trend(P < 0.05),and there was no significant difference between the two groups(P > 0.05).3.2 Cell experiment results3.2.1 Screening of the optimal concentration of serum containing XFCThe cell viability after XFC intervention for 48 h was significantly lower than that at 0 h,24 h and 72 h(P < 0.01).After XFC intervention for 48 h,the activity value of the model group + XFC(20 %)group was significantly lower than that of the model group,the model group + XFC(5 %)group,the model group + XFC(10 %)group and the model group + XFC(30 %)group(P < 0.05).3.2.2 Effect of si RNA on Smad3 m RNA expressionThe transfection efficiency of Smad3-si RNA2 was significantly higher than that of NC-si RNA group and model control group,higher than that of Smad3-si RNA1 group and Smad3-si RNA3 group.3.2.3 Effect of XFC and si RNA on the activity in RA-FLSCompared with the RA group,the cell viability of the model group,XFC group,smad3-si RNA-NC group,smad3-si RNA group and XFC + smad3-si RNA group was significantly increased(P < 0.05).Compared with the model group,the cell viability of XFC group,smad3-si RNA group and XFC + smad3-si RNA group was significantly decreased(P < 0.05).Compared with XFC group,the cell viability of smad3-si RNA-NC group and smad3-si RNA group increased(P < 0.05),and the cell viability of XFC +smad3-si RNA group decreased(P < 0.05).3.2.4 Effect of XFC and si RNA on the migration in RA-FLSCompared with the RA group,the transmembrane cells in the model group,XFC group,smad3-si RNA-NC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly increased(P < 0.05).Compared with the model group,the number of migrated cells in XFC group,smad3-si RNA group and XFC + smad3-si RNA group decreased significantly(P <0.05).Compared with the XFC group,the number of migrated cells in the smad3-si RNA-NC group and the smad3-si RNA group increased(P < 0.05),and the number of migrated cells in the XFC + smad3-si RNA group decreased(P < 0.05).3.2.5 Effect of XFC and si RNA on IL-10 in macrophageCompared with the RA group,the secretion level of IL-10 in the supernatant of the model group,XFC group,smad3-si RNA-NC group,smad3-si RNA group and XFC +smad3-si RNA group was significantly decreased(P < 0.05).Compared with the model group,the secretion level of IL-10 in the supernatant of XFC group,smad3-si RNA-NC group,smad3-si RNA group and XFC + smad3-si RNA group was significantly increased(P < 0.05).Compared with the XFC group,the secretion level of IL-10 in the supernatant of the smad3-si RNA group and the XFC + smad3-si RNA group increased significantly(P< 0.05),while the secretion level of IL-10 in the smad3-si RNA-NC group decreased,and the difference was statistically significant(P < 0.05).3.2.6 Effect of XFC and si RNA on IL-23 and TGF-β1 in macrophageCompared with the RA group,the secretion levels of IL-23 and TGF-β1 in the supernatant of the model group,XFC group,smad3-si RNA-NC group and smad3-si RNA group were significantly increased(P < 0.05).Compared with the model group,the secretion levels of IL-23 and TGF-β1 in the supernatant of XFC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly decreased(P < 0.05).Compared with the XFC group,the secretion levels of IL-23 and TGF-β1 in the supernatant of smad3-si RNA-NC group increased(P < 0.05).3.2.7 Effects of XFC and si RNA on TGF-β1 and Smad3 protein in TGF-β1 / Smads pathway in macrophage.Compared with the RA group,the expression levels of TGF-β1 and Smad3 protein in the model group,XFC group,smad3-si RNA-NC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly increased(P < 0.05).Compared with the model group,the expression levels of TGF-β1 and Smad3 protein in XFC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly decreased(P < 0.05).Compared with the XFC group,the expression levels of TGF-β1 and Smad3 protein in the smad3-si RNA-NC group increased,and the expression levels of TGF-β1and Smad3 protein in the XFC + smad3-si RNA group decreased(P < 0.05).3.2.8 Effects of XFC and si RNA on Smad7 protein in TGF-β1 / Smads pathway in macrophage.Compared with the RA group,the expression levels of Smad7 protein in the model group,XFC group,smad3-si RNA-NC group,smad3-si RNA group and XFC +smad3-si RNA group were significantly decreased(P < 0.05).Compared with the model group,the expression levels of Smad7 protein in XFC group,smad3-si RNA group,smad3-si RNA-NC group and XFC + smad3-si RNA group were significantly increased(P < 0.05).Compared with the XFC group,the expression level of Smad7 protein in the smad3-si RNA-NC group decreased,and the expression level of Smad7 protein in the XFC + smad3-si RNA group increased(P < 0.05).3.2.9 Effect of XFC and si RNA on the expression level of mi R145-5p m RNA of TGF-β1/Smads pathway in macrophageCompared with the RA group,the expression level of mi R145-5p m RNA in macrophage in the model group,XFC group,smad3-si RNA-NC group,XFC +smad3-si RNA group and smad3-si RNA group was significantly decreased(P < 0.05).Compared with the model group,the expression levels of mi R145-5p m RNA in macrophage of XFC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly increased(P < 0.05).Compared with XFC group,the expression level of mi R145-5p m RNA in macrophage of smad3-si RNA-NC group and smad3-si RNA group decreased,and the expression level of mi R145-5p m RNA in XFC + smad3-si RNA group increased(P <0.05).3.2.10 Effect of XFC and si RNA on the expression level of TGF-β1m RNA of TGF-β1/Smads pathway in macrophageCompared with the RA group,the expression levels of TGF-β1 m RNA in macrophage of the model group,XFC group,smad3-si RNA-NC group and smad3-si RNA group were significantly increased(P < 0.05).Compared with the model group,the expression levels of TGF-β1 m RNA in macrophage of XFC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly decreased(P < 0.05).Compared with XFC group,the expression level of TGF-β 1 m RNA in macrophage of smad3-si RNA-NC group increased(P < 0.05),and the expression level of TGF-β1 m RNA in XFC + smad3-si RNA group decreased(P < 0.05).3.2.11 Effect of XFC and si RNA on the expression level of Smad3 m RNA of TGF-β1/Smads pathway in macrophageCompared with the RA group,the expression levels of Smad3 m RNA in macrophage of the model group,XFC group,smad3-si RNA-NC group and smad3-si RNA group were significantly increased(P < 0.05).Compared with the model group,the expression levels of Smad3 m RNA in macrophage of XFC group,smad3-si RNA group and XFC + smad3-si RNA group were significantly decreased(P < 0.05).Compared with XFC group,the expression level of Smad3 m RNA in macrophage of smad3-si RNA-NC group and smad3-si RNA group increased,and the expression level of Smad3 m RNA in XFC + smad3-si RNA group decreased(P < 0.05).3.2.12 Effect of XFC and si RNA on the expression level of Smad7 m RNA of TGF-β1/Smads pathway in macrophageCompared with the RA group,the expression levels of Smad7 m RNA in macrophage of the model group,XFC group,smad3-si RNA-NC group,XFC +smad3-si RNA group and smad3-si RNA group were significantly decreased(P < 0.05).Compared with the model group,the expression level of Smad7 m RNA in macrophage of XFC group and XFC + smad3-si RNA group was significantly increased(P < 0.05).Compared with XFC group,the expression level of Smad7 m RNA in macrophage of smad3-si RNA-NC group and smad3-si RNA group decreased,and the expression level of Smad7 m RNA in XFC + smad3-si RNA group increased(P < 0.05).4 conclusion4.1 clinical researchXFC and LEF can significantly reduce the secretion level of RF,CCP-Ab,Ig G,Ig M,the index of inflammatory activity ESR and CRP,the number of joint tenderness and swelling,the score of VAS,DAS28 and the score of traditional Chinese medicine syndromes,and improve the clinical symptoms of RA patients.The clinical effect of XFC on RA patients is not inferior to that of LEF,and it shows certain advantages in improving the patients’ feelings.Both XFC and LEF may regulate macrophage polarization by inhibiting TGF-β1 /Smads pathway.On the one hand,it can improve the high expression of TGF-β1,Smad3 and IL-23 in RA patients,on the other hand,it can improve the low expression of Smad7 and IL-10.4.2 Cell researchExogenous TGF-β1 stimulates macrophages to promote the polarization of M1 macrophages,thereby enhancing the proliferation and migration of RA-FLS and aggravating RA synovial inflammation.The specific regulatory mechanism is related to TGF-β1 / Smads signaling pathway.Silencing Smad3 can inhibit the activation level of TGF-β1 / Smads signaling pathway,inhibit the polarization of M1 macrophages,reduce the level of anti-inflammatory factors,promote the polarization of M2 macrophages,increase the level of pro-inflammatory factors,inhibit the proliferation and migration of RA-FLS,and improve the inflammatory state of RA.XFC has a si RNA-like effect.It can also inhibit the transduction of TGF-β1 /Smads pathway,regulate the imbalance of macrophage polarization,and reduce the proliferation and migration of RA-FLS.In addition,XFC combined with si-RNA is superior to XFC alone in inhibiting TGF-β1 / Smads pathway transduction and regulating macrophage polarization.