Xinfeng Capsule Improves The Cardiac Function Of RA Patients Through Regulating The MiRNA-21/TLR4/MAPK/NF-?B Signaling Pathway

ObjectiveTo study the effects and the mechanism of Xinfeng capsule on cardiac function in RA using miRNA-21/TLR4/MAPK/NF-?B signaling pathway as a candidate target.Methods1.Animal experimentsMale Wistar rats were used in this study.each rat was not treated with any chemical in the control group?NC?.The other rats were injected with 0.1 ml of Freund's complete adjuvant?Freund's complete adjuvant,FCA,Sigma products?to the right foot to induce adjuvant-induced arthritis?AA?rat model.After nineteenth days of the Freund's complete adjuvant injection,there rats were randomly divided into 5 groups:the model group?Model?,the XFC group,the triptolide group?TPL?,the Huangqi glycoprotein group?HQGP?,and the methotrexate?MTX?group.Rats in these groups were treated with respective drugs for 30 days.After medication,the animals were sacrificed.Hematoxylin-eosin?HE?staining was used to examine the myocardial morphologic changes.The ultrastructure of myocardial cells was observed by transmission electron microscopy.TUNEL staining was used to detect the myocardial cell apoptosis.Serum levels of tumor necrosis factor-alpha?TNFa?,interleukin-6?IL-6?,interleukin 17?IL-17?were determined by enzyme-linked immunosorbent assay?ELISA?.Western blotting was used to detect TLR4,p-p38,and p-p65 levels.2.In vitro experimentThe suspensions of Xinfeng capsule?XFC?were given to rats by 1 ml/100 g,1 times a day for 3 days.The control group was given the same amount of normal saline.After the last administration of 1 h,the rats were anaesthetized with the abdo minal aorta to collect the whole blood and separate the serum.The H9C2 myocardial cells treated with LPS were used as the model of RA cardiac function change at cellular level.The cells were divided into the bcontrol group?vehicle treatment?,the TPL group?TPL treatment?,the LPS stimulation group?LPS treatment?,the XFC containing serum group?XFC+LPS treatment?,the miRNA-21 inhibitor group?XFC+ miRNA-21+LPS treatment?.The cell viability of cardiac myocyte was determined by MTT.MiRNA-21 was determined by qPCR.The effect of drugs on myocardial cell proliferation activity was determined by MTT method.The levels of TLR4,p-p38 and p-p65 were determined by Western blotting and immunofluorescence staining.The levels of TNF-a,IL-6,and IL-17 in the supernatant of myocardial cell were examined by ELISA.Flow cytometry was used to determine the apoptosis of H9C2 cells.Results1.In vivo experimentsCompared with the control group,the myocardium ultrastructure of the rat AA model group was obviously damaged according to HE staining or electron microscopy,and the TUNEL staining positive cells in the cardiomyocytes were significantly higher than that of the normal control group.Compared with the normal control group,the serum inflammatory factors TNF-alpha,IL-6,IL-17,miRNA-21 and TLR4 in the myocardium were highly expressed in the AA model group,and the level of MAPK p3 8 pathway and NF kappa B-p65 phosphorylation level was significantly increased.The positive staining of myocardial cells in rats treated with XFC,astragalus polysaccharides and TPL was significantly less than that in model rats.The expression level of serum inflammatory factors,miRNA-21 and TLR4 in the groups of XFC and its components were decreased,and the activation level of MAPK p38 and NF kappa B p65 pathway protein in the downstream of TLR4 was reduced.The magnitude of changes induced by XFC were significantly higher than those by other intervention drugs.2.In vitro experimentLow concentration of TPL?1,10 ng/ml?had no significant effect on the growth of rat H9C2,while high concentration of TPL?20 ng/ml?could significantly reduce the viability of rat H9C2 cardiomyocytes.Triptolide can inhibit the expression of miR-21,inhibit the expression of TLR4,p-p38 and p-p65,and reduce the expression level of TNF-,IL-6 and IL-17 without affecting the viability and apoptosis rate of cardiac myocytes.XFC containing serum significantly inhibited LPS-induced miRNA-21 expression?P<0.01?,and the level of miRNA-21 in the combined treatment group of XFC containing serum and miRNA-21 inhibitor was significantly lower than that of the XFC containing serum group.Western blotting Tests revealed that XFC containing serum inhibited the expression levels of TLR4,p-p38 and p-p65 induced by LPS,and miRNA-21 inhibitor reverses this inhibitory effect.The results detected by immunofluorescence were consistent with Western blotting results.The XFC containing serum inhibited the expression levels of TNF-alpha,IL-6 and IL-17 induced by LPS.Compared with the group of XFC containing serum,the levels of TNF-alpha,IL-6 and IL-17 in the Combined Xinfeng Capsule serum and miRNA-21 inhibitor group were further increased.The XFC containing serum group inhibited LPS-induced cardiomyocytes apoptosis,and.Compared with the serum group of Xinfeng capsule,the apoptosis rate of cardiac muscle cells in the combined Xinfeng Capsule and miRNA₂1 inhibitor treatment group was increased.Conclusion1.Xinfeng capsule,astragalus polysaccharide and Tripterygium wilfordii can improve the changes of RA toe swelling and arthritis index,myocardial histological structure and ultrastructure.2.Xinfeng capsule,astragalus polysaccharide and triptolide can inhibit the apoptosis of RA cardiomyocytes.3.Xinfeng capsule,astragalus polysaccharide,and Tripterygium wilfordii can inhibit the levels of RA serum inflammatory factors.The inhibitory effect of Xinfeng capsule is better than those of Astragalus polysaccharides,Tripterygium Wilfordii,and methotrexate.4.The miRNA-21 was highly expressed in RA cardiac tissue.Xifeng capsule,astragalus polysaccharide,and triptolide can inhibit the high expression of miRNA-21 in RA heart tissue.The TLR4/MAPK p38/NF kappa B pathway was activated in the cardiac tissue of RA,and this was inhibited by Xinfeng capsule,astragalus polysaccharide,and triptolide.The inhibitory effect of Xinfeng capsule is better than those of Astragalus polysaccharide,Tripterygium Wilfordii,and methotrexate.5.MiR-21 inhibitor antagonizes the inhibitory effect of Xinfeng capsule containing serum.Xifeng capsule containing serum may inhibit the miR-21 and TLR4/MAPK/p38-NF kappa B pathway to inhibit the inflammation and apoptosis induced by LPS and protect the viability of cardiac myocytes.Therefore,Xinfeng capsule may inhibit RA myocarditis and apoptosis,and protect myocardial viability though inhibiting miR-21 and TLR4/MAPK/p38-NF kappa B pathway.