Molecular Mechanisms Of MicroRNA-146a Regulating Myocardial Fibrosis In Constrictive Pericarditis And Ultrasound Imaging Study

Objective: Constrictive pericarditis(CP)refers to a variety of causes of pericardial fibrosis or calcification,resulting in a series of circulatory disorders.Long-term patients can cause myocardial disuse atrophy or myocardial fibrosis(MF).Sengupta and other studies have found that patients with CP have reduced circumferential deformation,torsion and unwinding ability.Similarly,we used two-dimensional speckle-tracking imaging(STI)to observe the left ventricular myocardial rotation function of CP patients and found myocardial rotation angle and the overall rotation angle in subepicardial are significantly lower than those of normal people in apex horizon.This reduction in contractile function is related to the fibrosis of myocardium to some extent.However,the regularity and mechanism of myocardial fibrosis in CP patients are still unclear.Intercellular communication of cardiac fibroblasts(CFs)plays an important role in the pathogenesis of CP myocardial fibrosis.Several kinds of microRNAs have been reported to be related to the survival of myocardial fibroblasts and related signal transduction pathways.The purpose of this study was to investigate the expression of microRNAs in myocardial fibrosis of constrictive pericarditis and the activation of their regulatory target genes,to understand the molecular mechanism of myocardial fibrosis of constrictive pericarditis and to find new intervention targets.Method: 1.After establishing a model of constrictive pericarditis in Wistar rats,myocardial tissue was collected and the distribution of miRNA in myocardium was analyzed by high-throughput sequencing.2.Preparation of constrictive pericarditis model rats of different disease duration.Thirty rats were randomly divided into 3 groups: a 16-week model group(CP16W),an 8-week model group(CP8W),and a N group,10 in each group.The changes of left ventricular myocardial function in rats were evaluated by STI,and the correlation between myocardial function changes and myocardial fibrosis was evaluated.3.Real-time quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of microRNA-146a(miR-146a),IL-1 receptor-associated kinase 1(IRKI1)and tumor necrosis factor receptor-associated factor 6(TRAF6)downstream gene of Toll-like receptor 4(TLR4).Western blotting was used to detect the expression of IRAK1,TRAF6,nuclear factor-κB(NF-κB)and p-NF-κB in the TRL4 signaling pathway.The primary cultured rat cardiac fibroblasts were transfected into miR-146 a.After LPS treatment,RT-PCR and Western Blot were used to detect the expression of downstream effectors,and miRNA-146 a regulates cardiac fibroblast function through TLR4 signaling pathway was verified.Results: 1.Based on constrictive pericarditis rat model,microRNA analysis was performed on 3 samples of CP model group and control group by high-throughput sequencing method,17 up-regulated miRNAs and 17 down-regulated miRNAs were found.MiR-146 a showed the highest expression 2.MiR-146 a showed dynamic change in different disease term.Ultrasound evaluation of myocardial fibrosis: Compared with the control group,the circumferential strain(SC)of left ventricular free wall outer and middle layer in CP8 W group decreased,and the SC of left ventricular free wall outer,middle and inner layer in CP16 W group all decreased.Compared with the CP8 W group,the SC of left ventricular free wall outer and middle layer in the CP16 W group further decreased.Compared with the control group,longitudinal strain(SL)in the CP8 W and CP16 w group had the same trend of change.Compared with the control group,the radial strain(SR)of the CP8 W group did not change significantly,but SR of the left ventricular whole and free wall in CP16 W group decreased.The myocardial fibrosis in CP8 W group was mainly located in the left ventricular free wall subepicardial myocardium,while in CP16 W group,the myocardial fibrosis was located in all layers of the left ventricular free wall myocardium.According to pathological results,compared with CP8 W group,α-SMA,COLI and COL III mRNAs in the model group were higher than those in the control group.The α-SMA and COLI mRNA in the CP16 W model group were higher than those in the CP8 W model group.3.The expression of target genes IRAK1 and TRAF6 in CP rat model: RT-PCR and Western blot results shown IRAK1 and TRAF6 in the CP8 W group were lower than those in the control group,and there was no significant difference between the CP16 W group and the control group,which was higher than that in the CP8 W group.4.Compared with the control group,the expression of IRAK1,TRAF6,p-NF-κB and α-SMA increased in the LPS-treated group;after transfection of the miR-146 a mimics,compared with the LPS group,the expression of IRAK1,TRAF6,p-NF-κB and α-SMA decreased;after transfection of miR-146 a inhibitors,the expression of IRAK1,TRAF6,p-NF-κB and α-SMA increased compared with LPS group.Conclusion: 1.This study successfully applied a rat model of constrictive pericarditis by intrathoracic pericardial injection of LPS and talc mixture.2.Analyze the distribution of miRNAs in myocardium using high-throughput sequencing.MiR-146 a showed the highest expression.3.MiR-146 a showed dynamic change in different disease term.STI technique can be used to evaluate the left ventricular function of CP rat model from three aspects: longitudinal,radial and circumferential strain.Myocardial fibrosis in CP rats model gradually progresses from the outer layer to the inner layer of the myocardium.4.MiR-146 a is involved in the process of myocardial fibrosis in CP rats.miR-146 a can inhibit myocardial fibrosis by inhibiting the target genes TRAF6 and IRAK1 in the Toll-like receptor 4 signaling pathway.