The Molecular Mechanism Of Serum Exosomes MiR-223-5p Mediating Macrophage Apoptosis In Patients With Phlegm Turbidity Stagnation Syndrome Of Coronary Heart Disease

Purpose:To investigate the interrelationship between coagulation index,echocardiography index,serum exosomal miR-223-5p expression and patients who have coronary heart disease with phlegm and stasis.To explore the possible pathogenesis of CHD from views of miR-223-5p-mediated macrophage apoptosis.To investigate the difference between coagulation index and echocardiography in these three common syndromes: Qi deficiency and blood stasis syndrome,Qi stagnation and blood stasis syndrome and phlegm turbidity stasis syndrome in CHD patients and to provide experimental evidence for clinical TCM classification.Using miRNA-seq method to compare serum exosomes micro RNA distribution in patients with coronary heart disease of different syndromes and normal people and to screen the meaningful micro RNAs(ie miR-223-5p).To verify the expression of miR-223-5p in exosomes of patients who have coronary heart disease with phlegm and stasis,as well as its downstream target genes in mononuclear cells.Material and method:To compare the difference and importance between coagulation index and color Doppler flow imaging in patients who have coronary heart disease with three syndromes: qi deficiency and blood stasis,qi stagnation and blood stasis and phlegm turbid and blood stasis,and to explore the relationship between them and syndrome types.To extract serum exosomes from patients with phlegm and blood stasis,then to identify and quantify results.To culture THP-1 cells in vitro and to induce it to be macrophages with 100ng/μl PMA.To label exosomes with fluorescent probe and to observe the uptake of serum exosomes by macrophages with living cell imaging technology.To use RT-q PCR to detect miR-223-5p downstream target genes ALDH2 and apoptotic genes caspase-3 or bcl-2 after the uptake of serum exosomes by macrophages in ox-LDL(50μg / ml)environment.To compare them with normal control group.Results:1.Statistical results of coagulation indexes showed that PT activity significantly differed among three groups(p<0.05).PT in phlegm turbidity stasis syndrome group was higherthan Qi deficiency and blood stasis syndrome group and Qi stagnation and blood stasis syndrome group.There was no significant difference in other test indexes(P > 0.05).The trends of coagulation indexes were showed here: PT,TT and INR value of phlegm turbidity stasis syndrome group < Qi stagnation and blood stasis group < Qi deficiency and blood stasis group;APTT and D-DI value of Qi stagnation and blood stasis group < phlegm turbidity stasis syndrome group < Qi deficiency and blood stasis group;FBG value of Qi stagnation and blood stasis group < Qi deficiency and blood stasis group <phlegm turbidity stasis syndrome group;PT value of Qi deficiency and blood stasis group < Qi stagnation and blood stasis group <phlegm turbidity stasis syndrome group.2.Results of color Doppler echocardiography showed that there was of a significant difference in LA among Qi deficiency and blood stasis syndrome,Qi stagnation and blood stasis syndrome and phlegm turbidity stasis syndrome(P < 0.05).The difference in other indexes between these groups was not statistically obvious.(P > 0.05).3.Micro RNA sequencing of exosomes showed that mir-6503-3p,mir-6763-5p,mir-33a-5p,mir-223-5p,mir-3928-3p and mir-605-3p were highly expressed in both Qi deficiency group and normal group,and mir-223-5p was closely related to coronary heart disease.Blood vessel development,cell response to organic nitrogen compound,growth factor,heart development,cholesterol metabolism,fluid shear stress and atherosclerosis,FOXO signaling pathway,HIF-1 signaling Pathway and adipocytokine signaling pathway are involved in the regulation of coronary heart disease.4.Results of PCR showed that the levels of mir-223-5p,caspase-3 and Bcl-2 in the patients with phlegm stasis syndrome were higher than those in the normal people(P < 0.05).5.After 10 minutes incubation of macrophages and serum exosomes from patients with phlegm and stasis obstruction,it was found that PKH67 labeled exosomes was absorbed by macrophages.Compared with control groups,the expression of ALDH2 and Caspase-3 in ox LDL group,exo group and ox LDL + exo group increased(P < 0.01),while the expression of Bcl-2 decreased(P < 0.01).Compared with ox LDL group,the expression of ALDH2 and caspase-3 decreased and the expression of Bcl-2 increased in ox LDL + exo + mir-223 inhibitor group(P < 0.01).Conclusion:1.There were differences in coagulation index and color Doppler echocardiography between patients with coronary heart disease and different TCM syndromes.As the diagnostic basis,the combined detection of Pt activity and left atrium diameter can be used to identify different TCM Syndromes of coronary heart disease.2.There were differences in the distribution of serum micro RNA in patients with different types of coronary heart disease and normal people.As reference indexes,those differences can be used to diagnose coronary heart disease.Exo-mir-223-5p was highly expressed in the serum exosomes of patients who have coronary heart disease with phlegm and stasis.3.Exo-mir-223-5p in the serum of patients who have coronary heart disease with phlegm and stasis can reduce the expression of ALDH2 in macrophages and induce apoptosis of macrophages.