Experimental Study On Zhenwu Decoction Regulating Structural Remodeling-electrical Remodeling Of Hypertrophic Cardiomyocytes Through P38-MAPK Signal Pathway

When chronic heart failure（Chronic heart failure,CHF）is complicated with arrhythmias,the treatment of CHF with antiarrhythmic drugs alone has potential risks.On the one hand,the potential risk may hinder compensatory hemodynamic changes,on the other hand,it may also lead to other types of arrhythmias,further worsening CHF.Studies have found that Zhenwu decoction can effectively improve arrhythmia in the treatment of chronic heart failure,but its antiarrhythmic mechanism is not clear.Objective:The purpose of this study is to explore the mechanism of Zhenwu decoction in the treatment of heart failure-related arrhythmias from the point of view of p38-MAPK pathway regulation of hypertrophic myocardial structural remodeling and electrical remodeling,to provide reliable experimental basis for Zhenwu decoction in the treatment of heart failure,to provide theoretical reference for further study of its mechanism,and to provide reliable evidence for clinical application.Methods:Normal rats were given Zhenwu decoction granule(7.23 g kg^-1·d^-1,crude drug dose)for 7 days.Blood samples were collected from abdominal aorta,and drug-containing serum of inactivated Zhenwu decoction was separated.H9c2 cardiomyocytes were induced by AngⅡto establish cardiomyocyte hypertrophy model.The proliferation of H9c2 cardiomyocytes was measured by CCK-8 method and the expression of key protein Cx43 was determined by Westernblot method to set the optimal concentration of AngⅡand the volume fraction of low,middle and high dose serum containing drugs.According to the pre-experimental results,the best modeling concentration was set to 10^-7 mol/L,and the volume fractions of low,middle and high doses of Zhenwu decoction were set to 2%,4%and 8%,respectively.The experiment was divided into six groups:normal group（N）,model group（M）,Zhenwu decoction low dose group（D）,middle dose group（Z）,high dose group（G）and p38 pathway inhibitor group（S）.The normal group was cultured in high glucose medium containing fetal bovine serum,the model group was treated with AngⅡmedium containing 10^-6mol/L,the low,middle and high dose groups were treated with 2%,4%and 8%volume fraction of Zhenwu decoction containing serum and AngⅡmedium,respectively,and the inhibitor group was treated with medium containing p38MAPK inhibitor SB203580.After 48 hours of treatment,the growth of cardiomyocytes in each group was observed under microscope,the content of myocardial hypertrophy marker ANP was measured by Elisa method,cell senescence was detected byβ-galactosidase staining,the expression of Cx43,p38-MAPK,MMP2,MMP9,TIMP1,p53 protein in cardiomyocytes was detected by Westernblot,and the m RNA transcription levels of ANP,Cx43,p-Cx43,p38-MAPK,MMP2,MMP9 and TIMP1 in cardiomyocytes were quantitatively analyzed by q PCR.The expression and distribution of Cx43 protein were detected by immunofluorescence.Results:High dose of Zhenwu decoction could reduce the surface area of hypertrophic cardiomyocytes,but the middle and low doses had no obvious effect.Zhenwu decoction could reduce the level of ANP and m RNA,which is a marker of cardiac hypertrophy,and its effect was positively correlated with the dose.Zhenwu decoction can improve the senescence of mast cells,and the effect is similar to that of inhibitors.Zhenwu decoction can reduce the expression level of Cx43,p38-MAPK,MMP2,MMP9,p53,p-Cx43 protein and m RNA transcription level of Cx43,p38-MAPK,MMP2 and MMP9 in hypertrophic cardiomyocytes,and increase the expression level of TIMP1 and m RNA transcription level in dose-dependent manner.Zhenwu decoction can alleviate the disorder of Cx43 distribution on the surface of mast cells and reduce the lateral-lateral translocation distribution of this protein.Conclusions:1.Zhenwu decoction can slow down the senescence and fibrosis of myocardial mast cells by regulating p38-MAPK signal pathway,regulate the balance of MMPs and TIMPs,maintain the metabolic balance of extracellular matrix,and improve the structural remodeling of CHF.2.Zhenwu decoction may regulate the quantitative expression,phosphorylation level and distribution location of Cx43 in hypertrophic cardiomyocytes by regulating p38-MAPK signal pathway,improve gap junction remodeling and electrical conduction.3.Zhenwu decoction may achieve the unified regulation of structural remodeling and electrical remodeling of hypertrophic myocardium by regulating p38-MAPK signal pathway,so as to achieve the purpose of treating heart failure and related arrhythmias.