Study On The Mechanism Of Huoxue Yiqi Recipe Intervening Exosomal MiRNA To Promote Angiogenesis After Myocardial Infarction

Objective:To explore the mechanism of Huoxue Yiqi formula on the therapeutic angiogenesis of ischemic heart disease from the perspective of exosomes miRNAMethods:The rat model of acute myocardial infarction was established and treated with Huoxue Yiqi recipe for 4 weeks.The expression of endothelial specific CD31 and ?-SMA was detected by immunohistochemical staining,and the proliferation of microvascular endothelial cells in the marginal zone of infarction was observed.The blood of abdominal aorta was taken to extract serum exosomes,the morphological changes of exocrine body were observed by transmission electron microscope,and the particle size of exocrine body was detected by NanoSight.Flow cytometry was used to detect the expression of exosome marker proteins CD63 and CD81.Agilent2100 was used to detect the characteristics of exosome RNA,single-terminal sequencing based on illumina sequencing platform to construct miRNA sequencing library.combined with bioinformatics analysis method,differential exosome miRNA,was screened for cluster analysis of differential miRNA,and heat map was used to show the expression pattern of differential miRNA among different samples.The target genes of differential miRNA were predicted by miRand database,and the target genes were analyzed by GO and KEGG enrichment analysis to determine the biological functions or pathways mainly affected by differential miRNA.The results of high-throughput miRNA sequencing were verified by real-time fluorescence quantitative RT-PCR and the effect of Huoxue Yiqi recipe on the differentially expressed miRNA was detected.After the target genes were predicted by miRand database,the target genes enriched in specific pathways were analyzed by GO and KEGG,and the protein interactions of target genes enriched in specific pathways were analyzed to determine the biological functions and pathways mainly affected by differential miRNAResult:1.MVD was measured by CD31 in the infarct margin area.Compared with the sham group,MVD in model group increased(P<0.01),the difference was statistically significant;compared with the model group,MVD in Huoxue Yiqi formula group increased(P<0.05),the difference was statistically significant2.The microvessel density(MVD)of myocardial tissue in the infarct margin was measured by ?-SMA.Compared with the sham group,the number of MVD in model group increased(P<0.01),the difference was statistically significant;compared with the model group,the MVD in Huoxue Yiqi formula group increased(P<0.05),the difference was statistically significant.3.Under the transmission electron microscope,the exosomes were cup-shaped or saucer shaped,with a diameter of about 100nm;NTA detection showed that the peak size of the exosomes was 134.2nm,with sharp peak shape and no obvious heteropeak;flow cytometry detection showed that the exosomes marker proteins CD63 and CD81 were expressed in the sham group,model group and Huoxue Yiqi formula group.4.Compared with the sham group,there were 22 different miRNAs in the serum exosomes of the model rats.novel1007_mature?novel10_star?novel1147_mature>novel1170_mature?novel208 mature?novel238_ature?novel335_mature?novel407_mature?novel539 mature?novel65_mature?novel898_mature>novel1221_mature?novel921 mature?novel951_mature?mo-miR-484?no-miR-615?rno-miR-676 were down-expression;novel1073 mature?novel130_mature?novel311_mature?novel738_mature?nove1867 mature>nove1928_mature?nove1903_mature?mo-miR-181d-5p were up-regulated.Target genes of different miRNAs were predicted by Miranda database,and a total of 7002 target genes were predicted.Go function analysis and KEGG pathway analysis of miRNA target gene were carried out by using David database There are 3681 enrichment pathways of go,including 742 molecular functions,2577 biological processes and 362 cell components,and 113 pathways of KEGG enrichment5.22 different miRNAs were screened,and the screening conditions(P<0.01;FC(abs)>2;priority was given to the known miRNA for verification).There were 11 different miRNAs for real-time fluorescent quantitative PCR verification,and 6 miRNAs for real-time fluorescent quantitative PCR verification.Compared with the sham group,the expression of novel539_mature?novel921_mature?novel951_mature?rno-miR-484 were decreased in model group,while novel1073_mature?rno-miR-181d-5p were increased.The trend of differential gene expression between sham group and model group was consistent with that of high-throughput sequencing.6.Compared with the model group,the expression of novel539_mature?novel921_mature?novel951_mature?rno-miR-484 were increased in Huoxue Yiqi formula group,while the expression of novel1073_mature?rno-miR-181d-5p decreased.The target genes of miR-484 were predicted by Miranda database,and a total of 357 target genes were predicted.The target gene of miR-484 was analyzed by GO function analysis and KEGG pathway analysis by using David database.There are 60 enrichment of go function,including 19 molecular functions,27 biological processes and 14 cell components;the results of KEGG enrichment showed that there are 4 pathways,in which calcium signaling pathway is closely related to angiogenesis;RyR2 and CACNA1D are most closely related to other proteins in protein interaction analysis of target genes enriched in calcium signaling pathway by using String database.Conclusion:Huoxue Yiqi formula can promote angiogenesis after myocardial infarction,which may be involved in VEGF induced calcium signal transduction of endothelial cells by regulating exosomes miR-484 targeting RyR2,so as to promote angiogenesis after myocardial infarction.