ZC-13 Regulated The Myocardial Fibrosis By Mediating P2X7R-NLRP3 Signaling Pathway

Objective:Myocardial fibrosis(MF)exists in a variety of cardiovascular diseases,if not treated in time will cause heart failure,arrhythmia,sudden cardiac death and other diseases.Zhachong Shisanwei pills(ZC-13)is a traditional Mongolian medicine prescription,which has the functions of dispelling wind and opening the orifices,relaxing the muscles and promoting blood circulation,calming and tranquilizing the nerves,and has been proved to have a good curative effect on cardiovascular and cerebrovascular diseases.P2X7r-NLRP3 signaling pathway is one of the research hotspots,which is closely related to atherosclerosis,ischemic stroke,traumatic brain injury and other diseases.In this study,isoproteronol(ISO)was used to induce myocardial fibrosis in mice,and TGF-β was used to stimulate and activate human umbilical vein endothelial cells(HUVECs)in vitro.To explore the regulation of ZC-13 on myocardial fibrosis and its potential molecular mechanism,we focused on extracellular matrix extracellular matrix(ECM)deposition,epithelial-mesenchymal transformation(EMT),inflammatory response and P2X7r-NLRP3 signaling pathway.Methods:C57BL/6 male mice were randomly divided into normal group,ISO group,low,medium and high ZC-13 dose groups(0.328 g·kg-1,0.656 g·kg-1 and 1.312 g·kg-1),ZC-13 group(1.312 g·kg-1)and the positive control group of Captopril(5 mg·kg-1).The mouse model of myocardial fibrosis was established by subcutaneous injection of ISO(5 mg·kg-1)for 7 days.After 7 days of ISO stimulation,mice in the administration group were given different concentrations of ZC-13 or Captopril by intragastric administration according to their body weight,for consecutive 28 days;normal group mice were given normal saline.After the end of the experimental period,the contents of hydroxyproline(HYP)and brain natriuretic peptide(BNP)in myocardium were detected;histopathological changes of myocardium were observed by H&E,Sirius red and immunohistochemistry;the changes of fibrosis markers,EMT markers,inflammatory factors(caspase-1,IL-1β,IL1R1 and IL-6)and P2X7r-NLRP3 signaling pathway in mouse myocardium were analyzed by western blot and RT-PCR.HUVECs was used in the in vitro experiment to detect the effect of ZC-13 on its survival rate by MTT assay.After stimulated with TGF-β(10 ng·mL-1)for 2 h and incubated with different concentrations of ZC-13(7.8125 μg·mL-1,15.625 μg·mL-1,31.25 μg·mL-1 and 62.5 μg·mL-1)for 8 h,the expression of fibrosis markers,EMT markers,inflammatory factors and P2X7r-NLRP3 signaling pathway were detected by western blot assay,RT-PCR and immunofluorescence staining.HUVECs were stimulated by TGF-β and/or ATP(3 mM)respectively in vitro,and then incubated with ZC-13 or P2X7r inhibitor A438079(10 μM)to observe the effect of P2X7r on ZC-13 regulating myocardial fibrosis.Results:Compared with the normal group,the levels of HYP and BNP in myocardium tissue of ISO-induced mice were significantly increased,while ZC-13 could significantly inhibit the levels of HYP and BNP in myocardium tissue.H&E staining and Sirius Red staining showed that ZC-13 could reduce inflammatory cell infiltration,restore myocardial cell alignment,reduce cell space,and significantly reduce collagen fiber deposition.Western blot,RT-PCR and immunohistochemical staining results showed that ZC-13 could effectively inhibit the expression of fibrosis markers α-SMA,collagenI and TIMP-1,up-regulate the expression of MMP13,and significantly decrease the expression of EMT markers Vimentin and β-catenin.Increase the level of E-cadherin.In addition,ZC-13 significantly decreased the expression of caspase-1,IL-1β,IL1R1 and IL-6,and the protein and mRNA expressions of P2X7r and NLRP3 in myocardial tissue.MTT results showed that 0-62.5 μg/mL ZC-13 had no significant effect on the survival rate of HUVECs.ZC-13 significantly decreased the expression of α-SMA and collagenI in activated HUVECs,down-regulated the ratio of TIMP-1 to MMP13,significantly decreased the expression of Vimentin and β-catenin,and increased the level of E-cadherin.Inhibit the secretion of caspase-1,IL-1β,IL1R1 and IL-6 inflammatory cytokines and the activation of P2X7r-NLRP3 signaling pathway.ZC-13 significantly inhibited the expression of P2X7r,NLRP3,α-SMA,Vimentin,and IL-1β in activated HUVECs cells.In addition,ZC-13 showed similar effects to P2X7r inhibitor A438079.Conclusions:ZC-13 has a protective effect on myocardial fibrosis,and can significantly inhibit ECM deposition,EMT process and the expression of inflammatory factors.The regulatory mechanism of ZC-13 in improving myocardial fibrosis may be mediated by the P2X7r-NLRP3 signaling pathway to intervene in the changes of ECM,EMT and inflammatory factors.The results provide reference for ZC-13 in clinical treatment of myocardial fibrosis.