| BackgroundWith the development of society,the number of cardiovascular diseases continues to increase.The complications and sequelae caused by myocardial ischemia and myocardial infarction seriously affect human health and quality of life.Cardiomyocytes are known to be mechanically and functionally coupled at highly specialized intercellular junctions,known as intercalated discs,which serve as highly coordinated structures of the myocardium and maintain the structural and functional integrity of myocardial tissue.When the myocardium is damaged,the integrity of the myocardium is damaged,the connections between cells are damaged,and the transmission of substances and electrical signals is abnormal,resulting in adverse results such as heart failure or arrhythmia,which is a hot issue in the prevention and treatment mechanism of this disease in recent years.The clinical application of Qi-invigorating and blood-activating Chinese medicine effectively improved the patient’s symptoms and myocardial damage.Yiqi Huoxue decoction includes:Astragalus,ginseng,angelica,ligusticum Chuanxiong and panax notoginseng.Established according to the theory of qi and blood in traditional Chinese medicine,it is an empirical decoction with good curative effect for the clinical treatment of myocardial ischemia,myocardial infarction and sequelae.Its main effect is supplementing qi and nourishing heart,promoting blood circulation and regulating veins.According to our previous studies,this formula can improve many clinical symptoms of patients,and it has also been confirmed in rat experimental models that it can improve cardiac function and morphology of rats after myocardial infarction.Therefore,it is important to study the molecular mechanism of cellular connectivity after myocardial infarction to ensure myocardial integrity and improve cardiac function,and to further explain the efficacy of Yiqi Huoxue decoction.MethodsThis experiment is divided into network pharmacology,animal experiments and cell experiments.Network pharmacology research:The chemical constituents and targets of YQHXF were searched using the Online Pharmacological Database and Analysis Platform(TCMSP),CNKI,PubMed literature database and Google Scholar database.To construct the YQHXF drug-composition-target interaction network,Cytoscape software will conduct target topology analysis and Metascape online database to analyze the biological processes,molecular functions and signaling pathway enrichment of target participation.Disgenet,GeneCard,TTD,Drugbank and OMIIM were used to search the therapeutic targets of myocardial infarction(MI),and plot the targets and intersections related to MI treated by Wayne Diagram YQHXF.GO function and KEGG signal pathway enrichment were analyzed by importing Metascape.The chemical composition of YQHXF was uploaded to PharmMapper analysis platform,the target was predicted and Metascape was used for GO and KEGG analysis respectively.Animal experiment:Healthy male SD rats underwent left anterior descending coronary artery ligation to replicate the rat model of acute myocardial infarction.The rats were randomly divided into Sham group(S),Model group(Model,M),YQHX group(Y)and Perindopril group(P).Previous studies showed that myocardial ischemia of myocardial infarction used in this study was most significant at the 28-day time point,so this time point was selected for observation.After 28 days,the following tests were performed:cardiac ultrasound and HE staining were performed to observe the myocardial status in the infarct margin area of rats.The ultra-structure of myocardial tissue was observed by transmission electron microscopy.The distribution and expression of connection-related proteins Cx43,N-cadherin,α-catenin andβ-catenin were observed by immunohistochemistry.The expressions of Cx43,α-catenin,β-catenin,N-cadherin,DSG-2,DSC-2,p38 MAPK and p-p38 MAPK in myocardial infarction marginal area of rats in each group were observed by western blot assay.Cell experiment:To establish the hypoglycemic model of H9c2 cardiomyocytes.It is divided into two parts:(1)dose-effect concentration screening study.The dosage of freeze-dried powder and the observation time were determined,and the concentrations of 50μg/ml,100μg/ml,200 μg/ml and 400μg/ml were selected for the experiment.Cardiomyocyte control group(C),hypoxia and glucose deficiency model group(M),50μg/ml Yiqi Huoxue decoction(Y1),100μg/ml Yiqi Huoxue decoction(Y2),200μg/ml Yiqi Huoxue decoction(Y3),400μg/ml Yiqi Huoxue decoction(Y4),respectively.At 12 h,LDH and ROS levels in each group were determined to determine the optimal concentration of cardiomyocyte protection.(2)Study on cell connection effect and pathway mechanism.The association between cell junctions and the p38 MAPK pathway was observed using the determined optimal concentration.Observation was carried out in control group(C),model group(M),Yiqi Huoxue decoction+model group(Y),SB203580+model group(SB203580)and SB203580+Yiqi Huoxue decoction+model group(SB203580+Y).Fluo-4 AM was used to detect the change of calcium ions,and western blotting was used to observe the expression of Cx43,α-catenin,β-catenin,N-cadherin,p38 MAPK and p-p38 MAPK.Result1.Network pharmacology of YQHXF.(1)There were 993 YQHXF targets for this disease.GO and KEGG enrichment analysis showed that YQHXF had potential effects on hypoxia response,cell migration,cell death,and positive regulation of phosphorylation,as well as potential effects on MAPK signaling pathway and calcium signaling pathway.(2)The topological analysis of YQHXF and MI screening core target network obtained 136 nodes and 2526 edges.The top GO analysis functions were;positive regulation of cell migration,cell response to chemical pressure,response to oxygen content reduction,positive regulation of phosphorylation,positive regulation of cell death,etc.;KEGG analysis of the top:fluid shear stress and atherosclerosis,p53 signaling pathway,VEGF signaling pathway,etc.PharmMapper predicted the top targets of YQHXF in GO and KEGG analysis,focusing on lipid and atherosclerosis,adhesion connection and so on.It achieves the expected effect of this project and provides a common basis for the project research with strong pertinence and no detours.2.Cardiac function and structure evaluation.After 28 days,compared with group S,LVEF and LVFS in group M were significantly decreased(P<0.01),while LVIDs and LVIDd were increased(P<0.05);Compared with group M,LVEF and LVFS in group Y and group P were increased(P<0.05),while LVIDs were decreased(P<0.05),indicating that the function and structure of myocardial ischemia heart in myocardial infarction were changed,and Yiqi Huoxue decoction had the prevention and control effect.3.Tissue changes in the marginal area of myocardial infarction.In group S,the myocardial fibers were arranged neatly,the cells were arranged tightly and the structure was clear.In group M,the myocardial fibers in the infarct margin area were disorganized,the cell morphology was chaotic,and a large number of inflammatory cells could be seen in the interstitial.The fibrous arrangement of infarcted myocardium in groups Y and P was slightly irregular,and a small amount of fibrous hyperplasia was observed in the interstitial tissue,and the degree of myocardial structure destruction was significantly improved compared with that in the model group.4.Ultrastructural changes of infarct tissue.The myocardial fibers of group S were arranged neatly,with complete muscle bars,clear Z and M lines,regular myocardial cells,uniform distribution of mitochondria,clear ridge structure,and uniform distribution of matrix particles.In group M,myofilaments were damaged,Z-line and M-line were blurred,myocardial cell structure was seriously damaged,mitochondria were disordered,cristae was difficult to distinguish,and some mitochondria showed vacuolation.The arrangement of damaged myofilaments in groups Y and P tended to be linear,and the internal structure was more regular than that in group M.Near the broken myofilaments were filled with mitochondria.The overall arrangement of mitochondria was smooth,the internal structure was more regular,and the distribution of matrix particles was more uniform.5.Compared with group S,the expressions of Cx43,α-catenin,N-cadherin,DSG-2 and DSC-2 in group M were decreased(P<0.01,P<0.05),and β-catenin in group M were increased(P<0.05).The expressions of Cx43,α-catenin and N-cadherin were increased,while the expressions of β-catenin were decreased(P<0.01,P<0.05).6.Immunohistochemistry of connexin in the marginal region of myocardial infarction.Cx43,α-catenin and N-cadherin were more expressed in the S group,while the positive staining in the M group was decreased,and the positive staining in the Y and P groups was changed compared with that in the M group.β-catenin positive staining was increased in group M,and slightly improved in medication group.7.Expression of p3 8 MAPK protein in the marginal area of myocardial infarction.The P-P3 8 MAPK/p38 MAPK of the M group was increased(P<0.05),and the P-P38 MAPK/p38 MAPK of the medication group was decreased compared with that of the M group(P<0.05).8.Effect of Yiqi Huoxue decoction on survival of H9c2 cardiomyocytes.(1)The optimal concentration effect of Yiqi Huoxue decoction.After 12 h of hypoxia and glucose deficiency,cell survival rate of group M decreased(P<0.01).Yiqi Huoxue decoction had protective effect on cells at concentrations of Y2,Y3,Y4(P<0.01).LDH content and ROS in the medication group were decreased compared with that in the M group,and the comprehensive effect was better at the concentration of 200 μg/ml,so the concentration was selected to continue the experiment.(2)Effect of 200 μg/ml.After 12 hours of hypoxia,the survival rate of cells in group M was significantly lower than that in group C(P<0.01).The survival rate of Y group,SB203580 group and SB203580+Y group was increased compared with that of M group(P<0.05).9.Influence of Yiqi Huoxue decoction on cardiomyocyte connectin and p38 MAPK pathway.After 12 hours of hypoxia and glucose deficiency,the content of calcium ions in group M was increased(P<0.01),and the content of calcium ions in medication and inhibitor groups was decreased compared with that in group M.Compared with Y group,SB203580+Y group had statistical difference(P<0.05).Compared with group C,the expression of Cx43,α-catenin and N-cadherin in group M was decreased(P<0.05),and the expression of β-catenin was increased(P<0.05),and the expression of Cx43 was increased(P<0.05)and β-catenin was decreased(P<0.05)after the use of inhibitor.Conclusion1.The results of network pharmacology research provide a solid foundation for the research of this project with strong pertinence and sufficient evidence.This study revealed the possible mechanism of the relevant network action of the component-target-regulatory pathway of Yiqi Huoxue deccotion,which provided a strong support for the research of this project to explore the multi-target action,biomolecular function and signal pathway of Yiqi Huoxue deccotion in the treatment of MI,analyze and predict the correlation and effectiveness of Yiqi Huoxu deccotion on cell connection and MAPK signal pathway.2.Yiqi Huoxue decoction can improve myocardial tissue structure damage and cardiac function decline caused by ischemia and hypoxia.Yiqi Huoxue decoction can improve the expression of connexin between cardiomyocytes after myocardial infarction,improve the survival of cardiomyocytes and repair the damaged myocardia.3.The use of SB203580,a p38MAPK pathway inhibitor,can affect the survival of cardiomyocytes and calcium ion transport,as well as the expression of some connectin,indicating the correlation between p38 MAPK pathway and some connectin.4.Yiqi Huoxue decoction can inhibit the pathway of p38 MAPK and affect the connexin through this pathway,so as to reduce cell damage and contribute to cell and tissue repair.This effect may be one of the reasons for improving damaged myocardial tissue and improving cardiac function. |
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