| Background and research objectives:Myocardial infarction is the primary cause of death in patients with cardiovascular diseases.Promoting the recovery of coronary blood flow is currently the main means of treating acute myocardial infarction and saving patients’ lives.The formation of new blood vessels can fundamentally improve local microcirculation,restore blood perfusion in the ischemic area,and fundamentally alleviate myocardial infarction.The preliminary research results of the research grouP found that during myocardial ischemia,intervention therapy with traditional Chinese medicine for supplementing qi and promoting blood circulation can alleviate or slow down myocardial fibrosis,maintain microvascular integrity,increase capillary density,increase blood flow perfusion,and improve local myocardial blood supply.Therefore,this study aims to investigate the mechanism of Yiqi Huoxue herbs in promoting angiogenesis after myocardial infarction,based on the changes in microvascular endothelial cells after myocardial infarction.Research methods:1.Use bioinformatics and network pharmacology data to mine differentially expressed genes in post myocardial infarction angiogenesis and targets related to Yiqi Huoxue drugs.Use GO/KEGG and GSEA enrichment analysis to analyze the signaling pathway of post myocardial infarction angiogenesis treated with Yiqi Huoxue drugs.Screen hub genes through PPI protein interaction network analysis and validate them using survival analysis and a database.Molecular docking between target components of Yiqi Huoxue drugs and Hub genes.2.After 7 days of administration of traditional Chinese medicine to SD rats via gavage,serum samples were taken at 30 minutes,1 hour,2 hours,3 hours,and 6 hours after the last gavage(on the 7th day).Analyze and detect the serum components related to Yiqi Huoxue medicine in rats after administration using liquid chromatography-mass spectrometry.3.Replicate an animal model of myocardial infarction in SD rats by ligating the left anterior descending branch of the coronary artery.It was randomly divided into a model group,a Yiqi Huoxue drug group,a Peiduopril group,and a sham operation group,totaling 4 groups.Each group was given corresponding medication by gavage starting from the second day after surgery,once a day,and the changes in relevant indicators were observed 28 days after treatment.Evaluate the success of animal model replication of myocardial infarction through small animal echocardiography,and examine the effects of Yiqi Huoxue drugs on cardiac function and structural changes in myocardial infarction rats.4.HE staining was used to observe the pathological changes of heart histiocyte in rats with myocardial infarction,and transmission electron microscopy was used to observe the changes of microvascular structure,endothelial cell morphology and quantity,to explore the effect of Yiqi Huoxue drugs on endothelial cells in the process of angiogenesis after myocardial infarction in rats.5.Immunohistochemical staining of CD31 and VEGFR2 was used to observe the effect of Yiqi Huoxue medicine on angiogenesis after myocardial infarction in rats.6.Use protein blotting and real-time fluorescence quantification methods to detect the changes in the expression levels of angiogenic related proteins PAK1,KLF2,VCAM1,VEGFR2,PI3K,pPI3K,AKT,pAKT protein and mRNA in the tissue of myocardial infarction marginal zone.Observe the changes in endothelial cell proliferation and the pathway of Yiqi Huoxue drugs in myocardial infarction rats.Results:1.Bioinformatics analysis section:Screening drugs and disease targets through network pharmacology.After intersection,GO/KEGG enrichment analysis was performed to screen the toP five pathways with the highest scores,namely PI3K/AKT pathway.Hub genes SRC,MAPK1,AKT1,STAT3,MAPK3.The ROC survival curve analysis verified the diagnostic value of the gene.The molecular docking display docking results showed that the minimum binding energy of the important components of the Qi invigorating and blood activating drugs,kynurine,isorhamnetin,ginsenoside Rh4,quercetin,panaxadiol,and gomicin B,as small molecular ligands,with the receptor protein encoded by the key genes SRC,MAPK1,AKT1,STAT3,and MAPK3,were all negative,and could form a stable hydrogen bond structure.2.High performance liquid chromatography-mass spectrometry analysis detected a total of 16 effective components in the serum of SD rats treated with gastric lavage Qi tonifying and blood activating drugs.,and 647 target genes were obtained after de duplication.The results were enriched with GOKEGG to obtain enrichment pathways,and related to angiogenesis after myocardial infarction were mainly PI3K-Akt signaling pathway.3.Evaluation of cardiac function and structural changes in myocardial infarction rats using echocardiography:Compared with the sham operated group,the left ventricular ejection fraction(LVEF)and left ventricular short axis rate(LVFS)of the model grouP rats were significantly reduced,while the left ventricular end systolic diameter(LVIDs)and left ventricular end diastolic diameter(LVIDd)were significantly increased(P<0.01).After 28 days of administration,compared with the model group,the LVEF and LVFS in the administration grouP were significantly increased,with significant differences(P<0.05).The LVIDs and LVIDd of the Yiqi Huoxue grouP were significantly reduced(P<0.05),while the LVIDd of the perindopril grouP was significantly reduced(P<0.05).The LVIDs were reduced but not statistically significant(P>0.05).The results showed that left ventricular dilation and decreased systolic and diastolic function after myocardial infarction.Using Yiqi Huoxue drugs can alleviate left ventricular dilation,improve left ventricular systolic and diastolic function,and positively affect the recovery of cardiac function after myocardial infarction.4.The HE staining results showed that the myocardial cells in the sham surgery group were arranged neatly,with normal size and morphology,clear structure,normal nuclear morphology,and no hypertrophy of myocardial cells was observed;The volume of some myocardial cells in the model grouP significantly increased,the number of normal cells significantly decreased,the myocardial arrangement was disordered,and there were inflammatory cell infiltration.The damage in each medication group was reduced compared to the model group.Under transmission electron microscopy,in the sham surgery group,cardiomyocytes showed well-organized myofibrils,with clearly visible M-lines,Z-lines,sarcomere discs,and striations.A large number of mitochondria had distinct cristae,and the capillary structure was intact,with tightly connected endothelial cells.In the model group,cardiomyocytes in the infarct border zone were severely damaged,with myofibril disruption,mitochondrial swelling and deformation,endothelial cell swelling and deformation in capillaries,sparse gap junctions,and indistinct Weibel-Palade bodies.In the Yiqi Huoxue formula group and the perindopril group,cardiomyocyte structures in the infarct border zone were partially preserved,with relatively intact capillary structures,enlarged endothelial cells showing significant proliferation,and gaP junctions tending to be tighter.The number of internal damage markers,Weibel-Palade bodies,was significantly reduced,and the overall condition was better than that of the model group.5.Immunohistochemical staining of myocardial infarction tissue showed that the number of CD31 positive endothelial cells and VEGFR2 positive endothelial cells in the model grouP increased compared to the sham operation group.After 28 days of administration,the number of CD31 positive endothelial cells and VEGFR2 positive endothelial cells in the medication grouP increased compared to the model group.The results showed that Yiqi Huoxue formula can promote the proliferation of endothelial cells in the marginal zone of myocardial infarction,promote angiogenesis,and establish effective collateral circulation to alleviate myocardial fibrosis,thereby alleviating myocardial ischemia and hypoxia.6.RT-PCR and Western Blot:Compared with sham surgery,at 28 days,the levels of PAK1,KLF2,VEGFR2,PI3K,AKT,and phosphorylation in the myocardial tissue of the model grouP rats in the infarct margin area were significantly reduced,while VCAM1 was significantly increased,with statistical significance(P<0.05);Compared with the model group,the Yiqi Huoxue grouP and Peiduopril grouP showed a significant increase in PAK1,KLF2,VEGFR2,PI3K,AKT,and phosphorylation levels in the myocardial tissue of the infarct margin area,while VCAM1 was significantly reduced(P<0.05).The results indicate that Yiqi Huoxue drugs can effectively promote angiogenesis of ischemic myocardium after myocardial infarction and improve microcirculation of ischemic hypoxic myocardium through the PI3K/AKT pathway.Conclusion:1.Yiqi Huoxue formula may prevent and treat myocardial infarction by targeting proteins such as SRC,MAPK1,AKT1,STAT3,and MAPK3,which have potential biomarker value.2.Yiqi Huoxue formula can effectively improve the structural and functional changes in the hearts of rats with myocardial infarction and enhance the angiogenesis ability of rats following myocardial infarction.3.Yiqi Huoxue formula promote angiogenesis after myocardial infarction by mediating endothelial cells through the VEGFR2/PI3K/AKT signaling pathway. |
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