The Mechanism Of Huoxue Yiqi Recipe In Promoting Angiogenesis After Myocardial Infarction In Rats Was Explored From The Perspective Of Exosomes

As one of the compensatory strategies for revascularization in ischemic heart disease,therapeutic angiogenesis plays an important role in restoring blood supply in ischemic area and saving damaged myocardium in time.As a new conduction medium of intercellular communication,exosome provides a new idea for the"acellular" strategy of angiogenesis.The previous study of our group found that Blood Activation and Qi Supplement prescription,a clinical empirical prescription based on the method of activating blood circulation and replenishing qi,could promote the angiogenesis of myocardial tissue in the marginal zone of infarction and the survival,proliferation and migration of HUVECs in rats.Therefore,on the basis of the previous experimental research of the research group,this study is expected to further explore the possible mechanism of Blood Activation and Qi Supplement prescription in regulating and promoting angiogenesis based on the exosomes body.Objective:To observe the effect of serum exosomes of Blood Activation and Qi Supplement prescription on angiogenesis after myocardial infarction in rats,and its effect on the proliferation,migration,tubulation ability and angiogenesis-related signal pathway of hypoxic HUVECs,and then reveal the possible mechanism of Blood Activation and Qi Supplement prescription in promoting angiogenesis from the point of view of exosomes regulation.Methods:(1)The rat deionized water serum and the drug-containing serum of Blood Activation and Qi Supplement prescription were prepared,and the exosome was extracted by ultracentrifugation.The morphological changes of exosome were observed by transmission electron microscope,the particle size of exosome was detected by NanoSight,and the expression of exosome marker proteins CD9,CD63 and TSG101 was detected by Western blot.(2)The rat model of acute myocardial infarction was established by ligation of the anterior descending branch of the left coronary artery.Four weeks after myocardial injection of exosomes,the pathomorphological changes of myocardial tissue in the marginal zone of infarction were observed by HE staining;the expression of endothelial specific CD31 and ?-SMA was detected by immunohistochemical staining to observe the proliferation of myocardial microvascular endothelial cells in the marginal zone of infarction;the improvement of cardiac function in rats was detected by small animal ultrasound imaging system.The expressions of HIF-1 ?,VEGF and mRNA in myocardial tissue of infarct marginal zone were detected by real-time fluorescence quantitative PCR and Western blot,respectively.(3)HUVECs were cultured in vitro to establish the anoxic cell model of HUVECs.The fusion of HUVECs and exosomes was detected by immunofluorescence.The effects of serum exosomes on the proliferation,migration and tube formation of hypoxic HUVECs cells were detected by CCK-8 method,cell scratch test,Transwell migration test and Matrigel matrix gel test,respectively.Results:(1)Under the transmission electron microscope,the characteristic morphology of the exosomes was saucer-shaped,and the size of the exosomes was 30-150nm.The expression of exosomes marker proteins CD9,CD63 and TSG101 was detected by Western blot,which proved that the exosomes extracted and purified in this experiment was exosomes.(2)Under light microscope,the shape of myocardial muscle bundle in sham operation group was complete,the arrangement of cardiomyocytes was dense and the outline of nucleus was clear,while in model group and deionized water serum exosomes group,myocardial transverse striation disappeared,myocardial muscle bundles were broken and arranged in disorder,necrotic cardiomyocytes were replaced by collagen tissue,and fibrous connective tissue proliferated.In the Blood Activation and Qi Supplement prescription serum exosomes group,the myocardial muscle bundles were broken and disordered,the nucleus and cytoplasm of the cells were not clear and the necrotic cardiomyocytes were replaced by collagen tissue,but the scope and degree were less than those in the model group and deionized water serum exosomes group.(3)Compared with the sham operation group,the LVEF and LVFS of the model group decreased,while the LVIDd and LVIDs increased,and the difference was statistically significant(P<0.01).Compared with the model group,the LVEF and LVFS of the serum exosomes group of Blood Activation and Qi Supplement prescription increased,the LVIDd and LVIDs of the serum exosomes group of Blood Activation and Qi Supplement prescription tended to decrease,but there was no statistical significance,and the LVEF and LVFS of the serum exosomes group of deionized water showed an upward trend,but there was no statistical significance.In the serum exosomes group of deionized water,the levels of LVIDd and LVIDs increased,but there was no statistical significance.(4)Compared with the sham operation group,the CK,CK-MB and LDH of the model group increased,but there was no statistical difference.Compared with the model group,the CK,CK-MB and LDH in the serum exosomes group of Blood Activation and Qi Supplement prescription decreased significantly,and the CK,CK-MB and LDH in the deionized water serum exosomes group tended to decrease,but the difference was not statistically significant.(5)Compared with the sham operation group,the expression of CD31 and ?-SMA in the myocardial tissue of the infarct marginal zone in the model group was significantly higher than that in the sham operation group(P<0.01).Compared with the model group,the expression of CD31 and ?-SMA in the myocardial tissue of the infarct marginal zone in the Blood Activation and Qi Supplement prescription serum exosomes group was significantly higher than that in the model group,while the expression of CD31 and ?-SMA in the deionized water serum exosomes group had no significant difference(P>0.05).(6)Compared with the sham operation group,the expression levels of HIF-1?,VEGF and mRNA in the model group were significantly higher than those in the sham operation group(P<0.01 or P<0.05).Compared with the model group,the expression levels of HIF-1 ?,VEGF and mRNA in the deionized water serum exosomes group decreased,but there was no statistical significance,while in the Blood Activation and Qi Supplement prescription serum exosomes group,the VEGF mRNA expression level increased and the HIF-1a expression level decreased,but the difference was not statistically significant.The expression levels of HIF-1 and VEGF in the serum exosomes group of Blood Activation and Qi Supplement prescription were up-regulated,and the difference was statistically significant(P<0.01 or P<0.05).Compared with the serum exosomes group of deionized water,the expression of HIF-1 a and VEGF mRNA in the serum exosomes group of Blood Activation and Qi Supplement prescription increased,but the difference was not statistically significant(P>0.05),and the expression of HIF-1 a and VEGF in the serum exosomes group of Blood Activation and Qi Supplement prescription was up-regulated.(7)Compared with the normoxic group,the cell proliferation in the hypoxic group was significantly lower than that in the normoxic group(P<0.01).Compared with the hypoxia group,the cell proliferation in the deionized water serum exosomes group was significantly increased(P<0.05),and that in the Blood Activation and Qi Supplement prescription serum exosomes group was significantly higher than that in the hypoxia group(P<0.01).Compared with the serum exosomes group of deionized water,the cells proliferated in the serum exosomes group of Blood Activation and Qi Supplement prescription,but there was no statistical difference(P>0.05).(8)Compared with the normoxic group,the percentage of cell migration area and the migration area in the hypoxia group were significantly lower than those in the normoxic group(P<0.01).Compared with the hypoxia group,the percentage of cell migration in the deionized water serum exosomes group and Blood Activation and Qi Supplement prescription serum exosomes group tended to increase,but there was no statistical difference(P>0.05).Compared with the hypoxia group,the migration area of the serum exosomes group of deionized water increased,but there was no statistical difference(P>0.05),and the cell migration area of the serum exosomes group of Blood Activation and Qi Supplement prescription increased with statistical significance(P<0.05).Compared with the deionized water serum exosomes group,the percentage of cell migration in the Blood Activation and Qi Supplement prescription serum exosomes group tended to increase,but there was no significant difference(P>0.05),and the cell migration area increased significantly(P<0.01).(9)Compared with the normoxic group,the length of neovascularization in the hypoxia group was significantly shorter than that in the normoxic group(P<0.05).Compared with the hypoxia group,the length of neovascularization in the deionized water serum exosomes group and Blood Activation and Qi Supplement prescription serum exosomes group was significantly longer than that in the hypoxia group(P<0.05).Compared with the serum exosomes group of deionized water,the length of neovascularization in the serum exosomes group of Blood Activation and Qi Supplement prescription increased,but there was no statistical difference(P>0.05).(10)Compared with normoxic group,the expression of HIF-1 a and VEGF mRNA in hypoxia group was up-regulated,and the expression of HIF-1 a was down-regulated,but there was no significant difference between hypoxia group and normoxic group(P>0.05);the expression of VEGF was down-regulated in hypoxia group,but there was no significant difference between hypoxia group and normoxic group.Compared with the hypoxia group,the expression levels of HIF-1? and VEGF mRNA were up-regulated in the deionized water serum exosomes group,while the expression levels of HIF-1 a and VEGF tended to decrease,but there was no statistical difference.Compared with the hypoxia group,the expression levels of HIF-1?,VEGF and mRNA in the serum exosomes group of Blood Activation and Qi Supplement prescription were up-regulated,and the difference was statistically significant(P<0.01 or P<0.05).Compared with the serum exosomes group of deionized water,the expression level of HIF-1a in the serum exosomes group of Blood Activation and Qi Supplement prescription group was down-regulated,and the difference was statistically significant(P<0.01).The expression level of VEGF mRNA was up-regulated,and the expression levels of HIF-1 a and VEGF were up-regulated,the difference was statistically significant.(11)Compared with the normoxic group,the expression levels of FAK,p38 and MAPKAPK mRNA were all up-regulated in the hypoxia group,and the expression of HSP27 mRNA tended to be up-regulated,but there was no significant difference(P>0.05).The expression of FAK,p38,MAPKAPK and HSP27 protein tended to be down-regulated,but there was no statistical difference(P>0.05).Compared with the hypoxic group,the expression of FAK,p38,MAPKAPK and HSP27 mRNA in the deionized water serum exocrine group was up-regulated,and the difference was statistically significant(P<0.01 or P<0.05).The expression of FAK and p38 protein tended to be down-regulated,but there was no significant difference in the expression of MAPKAPK and HSP27 protein(P>0.05).The expressions of FAK,p38,MAPKAPK and HSP27mRNA were all up-regulated in the serum exocrine group of Blood Activation and Qi Supplement prescription,and the difference was statistically significant(P<0.01).The expression of FAK,p38 and HSP27 protein was up-regulated,and the difference was statistically significant(P<0.05 or P<0.01).The expression of MAPKAPK protein was up-regulated,but there was no statistical difference.Compared with the serum exocrine group of deionized water,the expression of FAK,p38,MAPKAPK,HSP27 mRNA was up-regulated in the serum exocrine group of Blood Activation and Qi Supplement prescription,and the difference was statistically significant(P<0.01 or P<0.05).