The Molecular Mechanism Of Serum Exosomal-derived MiR-451 In Patients With Coronary Heart Disease With Phlegm Turbidity And Blood Stasis Syndrome Regulating The Expression Of Macrophage Inflammatory Factors

Purpose:To investigate the differential effect of serum exosomal miR-451 as a novel specific biomarker on phlegm turbid blood stasis syndrome of coronary heart disease by observing the relationship between blood lipid level,serum exosomal miR-451 and other inflammatory related indexes and patients with phlegm turbid blood stasis in coronary heart diseaseMaterial and method:Patients with 31coronary heart disease phlegm turbid blood stasis syndrome,33 coronary heart disease non-phlegm turbid blood stasis syndrome patients,26 healthy normal people were selected from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,and theirTC?TG?LDL-C?HDL-C?Apo-A1?Apo-B was measured after the separation of serum and fasting blood collection in the morning.The sera of 3 patients with coronary heart disease phlegm turbid blood stasis syndrome and 3 healthy normal patients was selected for excomes microRNAs sequencing to find out the differential expression.To review the literature and to determine microRNAs and its corresponding inflammation-related target genes through prediction websites and analysis softwareThe relative expression levels of patients with coronary heart disease phlegm turbid blood stasis syndrome and healthy normal people's serum exosomal miR-451 and their corresponding inflammatory-related target genes MIF,MMP-9,TNF-? were verified by RT-PCR techniques.The macrophage/exsome co-culture system was established.Particle size distribution of exosomes was detected by NTA,and the exocrine marker protein was detected by WB method.The uptake of exocrine by macrophages was observed by fluorescent live cell imaging.The relative expression level of macrophage MIF,MMP-9,TNF-? mRNAs is regulated by RT-PCR detection of exosomes,thus verifying the targeted regulatory relationship between serum exosomal miR-451 and inflammatory factorsResults:1.Patients with coronary heart disease phlegm turbid blood stasis syndrome compared with coronary heart disease non-phlegm turbid blood stasis syndrome patients and healthy normal people,the level of TC,TG,LDL-L increased significantly(P<0.05),while the HDL-L level showed a downward trend(P<0.05),with no significant difference in Apo-Al and Apo-B(P>0.05)2.Sequencing analysis of serum exosomal miRNAs found that there were differences in miRNAs expression in serum exocrine of patients with coronary heart disease phlegm turbid blood stasis syndrome compared with healthy normal people,and miR-451,miR-6881-3p,miR-1292-5p,miR-6786-3p,miR-3661 decreased significantly in the phlegm turbid blood stasis syndrome of coronary heart disease,among which miR-451 was closely related to the disease.It is mainly through the signal pathway such as Cholesterol metabolism,Regulation of lipolysis in adipocytes,Cytokine-cytokine receptor interaction,Chemokine signaling pathway,Leukocyte transendothelial migration to regulate phlegm turbid blood stasis type of coronary heart disease3.Compared with healthy normal people,the expression level of miR-451 in serum exosome of patients with coronary heart disease phlegm turbid blood stasis syndrome was significantly reduced(P<0.01),while the expression level of MIF,TNF-?,MMP-9 mRNAs,target genes related to inflammation in macrophages,were significantly increased(P<0.01)4.Macrophages were co-cultured with serum exosomes of healthy normal people or patients with coronary heart disease phlegm turbid blood stasis syndrome,respectively.The results showed that serum exosomes of patients with coronary heart disease phlegm turbid blood stasis syndrome could increase the expression level of MIF,MMP-9,TNF-? mRNAs in macrophages(P<0.05),which verified the regulatory relationship of miR-451 to inflammatory factors MIF,MMP-9 and TNF-? mRNAsConclusion:1.To a certain extent,the level of blood lipid has a certain role in the diagnosis of phlegm turbid blood stasis syndrome of coronary heart disease,and it is closely related to the inflammatory response in the process of atherosclerosis2.The decreased expression of miR-451 in exosomes of patients with coronary heart disease phlegm turbid blood stasis syndrome cannot effectively inhibit the release of macrophage inflammatory factors MIF,MMP-9,TNF-?,thus promoting the development of phlegm turbid blood stasis syndrome of coronary heart disease.