Study On The Protective Effect And Mechanism Of Tongguan Capsule On Ischemic Heart Failure

Heart failure is a complex clinical syndrome with a high mortality rate.Standardised treatment in Western medicine has reduced mortality in patients with heart failure.Tongguan capsule(TG)has been used in the treatment of angina pectoris,myocardial infarction,heart failure,etc.However,there are few studies on the mechanism of TG in the treatment of heart failure,so it is necessary to study the mechanism of action of TG in the treatment of heart failure.Therefore,this study was conducted to investigate two important pathological aspects of myocardial fibrosis and hypoxic injury in the process of heart failure,and to provide new ideas,new targets of drug action and theoretical basis for the application of TG to prevent and treat ischemic heart failure.Study Ⅰ Effect and Mechanism of Tongguan Capsule in Intervention of Heart Failure and Myocardial FibrosisObjective:To observe the effect of TG on ISO induced heart failure in rats,predict the possible molecular mechanism of Tongguan capsule in improving heart failure based on transcriptome,and explore the mechanism of TG intervening in ECM reconstruction to prevent myocardial fibrosis and heart failure.Methods:1.The rats were randomly divided into 4 groups:control group,ISO model group,captopril group and TG group.14 days after modeling and administration,ejection fraction(EF)and short-axis shortening(FS),left ventricular end-diastolic internal diameter(LVEDD)and left ventricular end-systolic internal diameter(LVESD)were measured by cardiac ultrasound in each group;Markers of myocardial damage(LDH)and oxidative stress(MDA,SOD)were detected;HE staining,Masson staining and Sirius red staining were used to observe myocardial fibrotic lesions;immunohistochemistry and western blot were used to observe a-SMA,Collagen Ⅰ and Collagen Ⅲ expression.2.Transcriptomics was applied to sequence the heart tissues of control group,ISO model group and TG group,and complete the screening and analysis of differential genes in each group to predict the key targets and metabolic pathways of heart failure regulated by TG,and the effect of TG on the differential protein expression of ECM was observed by western blot.3.Primary cardiac fibroblasts(CFs)were isolated and cultured,given different concentrations(0,0.01,0.05,0.1,1uM)of Ang Ⅱ for 24h,and the proliferation viability of CFs was observed by CCK-8 method to determine the appropriate concentration for AngⅡ-induced CFs pro-fibrotic model;CFs were divided into three groups:control group,AngⅡ model group,and TG group.Transwell assay and scratch assay were used to observe the migration of CFs,and immunofluorescence staining technique was used to observe the expression of a-SMA protein;Western blot was used to observe the expression of cellular ECM-related proteins a-SMA,CollagenI,CollagenⅢ,MMP2,MMP9 and TIMP-1 protein.Results:1.Compared with the ISO model group,TG could improve EF and FS(p<0.05)and reduce LVEDD and LVESD(p<0.05)after 2 weeks of treatment;compared with the ISO model group,the heart weight index,LDH and MDA levels were significantly decreased and SOD was significantly increased in the TG group(p<0.05);pathological and immunohistochemical results showed that:TG could improve the cardiac histopathological changes and reduce the collagen accumulation in ISO-induced heart failure rats compared with ISO model group.2.The mRNA expression profiles of rats in control group,ISO model group and TG administration group were analyzed by high-throughput sequencing technology;the effect of TG in treating heart failure was closely related to the ECM-receptor interaction pathway by KEGG pathway enrichment analysis.3.CFs grew well after 24 h of culture and the proliferation of CFs induced by Ang Ⅱin a dose-dependent manner,and luM stimulation for 24 h was selected as an ideal model for inducing fibrotic changes in CFs.stimulation for 24 h.Compared with Ang Ⅱ group,the fluorescence intensity of CFs a-SMA in TG group decreased,the number of cells passing through Transwell chamber decreased,and the scratch width was longer;Compared with Ang Ⅱ group,TG significantly decreased the expression levels of a-SMA,Collagen I,Collagen Ⅲ,MMP2,and MMP9 proteins in CFs after administration,and increased the expression of TIMP-1 protein.Conclusion:1.TG inhibited myocardial injury caused by ISO-induced heart failure in rats,improved cardiac function,alleviated pathological morphological and structural changes,reduced oxidative stress injury,and regulated myocardial ECM differential protein expression and deposition in rats with heart failure.2.TG inhibits the phenotypic differentiation and migration of CFs by a mechanism that may improve myocardial fibrosis by regulating the expression of α-SMA and MMPs/TIMP-1,thus achieving an anti-heart failure therapeutic effect at the cellular level.Study 2:Effect and Mechanism of Tongguan Capsule on Ischemic Heart FailureObjective:To clarify the cardiac protective effect of Tongguan Capsule on LAD induced ischemic heart failure and H9c2 cardiomyocyte hypoxia injury in rats,and to predict the key targets and metabolic pathways of TG in regulating heart failure through network pharmacology.Methods:1.LAD ligation was used to establish a model of heart failure in rats,and rats were randomly divided into 4 groups:control group(sham operation),model group(LAD ligation),positive drug group(metoprolol+LAD ligation),and TG group(TG+LAD ligation).Cardiac ultrasound was used to detect EF and FS in each group;markers such as myocardial injury(CTnT)were detected,and HE staining,Masson staining and Sirius red staining were used to observe myocardial fibrotic lesions.2.The TCMSP database was used to screen the active ingredients and their targets of TG,and the GeneCar and OMIM databases were used to screen the targets related to heart failure.The STRING online database was used to construct a protein interaction network for the targets intersected by both of them and to screen the key genes,and to construct a"drug-active ingredient-rake target-disease" visualization network in Cytoscape 3.7.1 software.Gene ontology enrichment analysis and KEGG pathway enrichment analysis of common genes were performed using DAVID tool to explore the mechanism of action of potential targets.3.After induction with different concentrations(0,0.625,1.25,2.5,5,10,20,40 mmol/L)of sodium hyposulfite(Na2S2O4),the effects of different concentrations of Na2S204 on cell morphology,cell proliferation,cell damage,and apoptosis were observed and detected.The optimal intervention concentration of Na2S204 was screened to construct a hypoxic injury model for H9c2 cardiomyocytes.4.Western blot was used to observe the expression of apoptosis-related proteins(Bax,Bcl-2,cleaved caspase-3)and ERK/STAT3.Results:1.Compared with the model group,TG improved EF and FS in rats after 2 weeks of treatment(P<0.05).The pathological results showed that TG could improve the cardiac histopathological changes and reduce collagen deposition in LAD-induced heart failure in rats compared with the model group.3.147 active ingredients were screened from 4 drugs of TG through network pharmacology,and the number of drug targets was 876;1573 targets were used for the treatment of heart failure disease,and 176 targets were intersected between the two;its core target was MAPK family,and the biological process of GO enrichment analysis mainly involved apoptosis,MAPK activation,etc.It was initially verified that TG could inhibit the expression of p-ERK and p-STAT3expression.3.Na2S204 dose-dependently inhibited H9c2 cardiomyocyte proliferation and promoted apoptosis in the range of administration concentrations 0-20 mmol/L.Finally,5 mmol/L stimulation for 2 h was selected as the ideal model for inducing hypoxia in H9c2 cardiomyocytes.4.Compared with the model group,TG was able to reduce the Bcl-2/Bax ratio,inhibit the expression of cleaved caspase-3,p-ERK and p-STAT3 and suppress apoptosis;Conclusion:1.TG can improve cardiac function,reduce myocardial infarct size and myocardial damage in LAD ligation-induced heart failure in rats,2.Network pharmacology revealed that the core targets of Tongguan capsule for prevention and treatment of ischemic heart failure were STAT3,MAPK,CASP3,etc.GO enrichment analysis showed that biological processes mainly involved in the positive regulation of apoptosis,MAPK activity and protein phosphorylation.3.Tongguan Capsule can improve the Na2S204-induced damage to H9c2 cardiomyocytes and inhibit the apoptosis of H9c2 cardiomyocytes.It has been preliminarily verified that Tongguan Capsule may play a cardiac protective role by regulating the ERK/STAT3 signaling pathway.