Discovery And Mechanism Study Of Metabolic Markers Of Acute Coronary Syndrome Based On Metabonomics

Part Ⅰ:Serum metabonomics study in patients with acute coronary syndromeObjective:the aim of this study was to identify potential metabolic biomarkers inpatients with ACS.Methods:the peripheral blood of 29 healthy adults and 45 patients with ACS were collected.The serum was collected by centrifugation,and the data were collected by serum metabonomics method based on ultra performance liquid chromatography(UPLC)/Orbitrap mass spectrometry(MS).Multivariate statistical analysis was used to screen the biomarkers of ACS.KEGG and HMDB databases were used to identify the differential substances.The pathway enrichment analysis of differential metabolites was carried out by bioinformatics analysis.The network construction of differential metabolites was carried out by IPA to find the pathogenesis of ACS.Results:a total of 69 biomarkers were identified as enriched in 19 metabolic pathways;43 biomarkers were significantly up-regulated,while 26 biomarkers were significantly down regulated in ACS group.The main species are lysophosphatidylcholine(SM),cinnamic acid,choline and primary amide.Receiver operating characteristic(ROC)curve analysis showed that lysoPC(20:4(8Z,11z,14z,17Z)/0:0)(area under ROC curve,AUC=0.936),SM(d18:0/16:0)(area under ROC curve,AUC=0.932)and SM(d18:1/14:0)(ROC,AUC=0.923)had higher diagnostic power for ACS.The AUC value of the diagnostic model constructed with these combined biomarkers was 0.96.Therefore,these biomarkers can improve the diagnostic effect of ACS.Conclusion:the results of this study also showed that glycerophospholipids metabolism,unsaturated fatty acid biosynthesis,linoleic acid metabolism and valine,leucine and isoleucine biosynthesis played an important role in ACS.IPA network analysis showed that these biomarkers were related to myocardial hypertrophy signaling pathway,ERK/MAPK signaling pathway,NF kappaB signaling pathway,cardiovascular nitric oxide(no)signaling pathway and TLR signaling pathway.These findings will help to improve the ability of accurate diagnosis and intervention of acute coronary syndrome.Part Ⅱ is the mechanism of functional metabolic markersObjective:To investigate the effects of the target metabolite on the function of human vascular smooth muscle cells(VSMC),human aortic vascular smooth muscle cells(THP1)and human umbilical vein endothelial cells(HUVEC).Methods:CCK8 was used to detect 8Z,11z,The effects of 14z eicosatrienoic acid(eame)on the proliferation of three kinds of cells were studied.The expression of cytokines in the supernatant of THP1 cells was detected by ELISA.The changes of proliferation and apoptosis related proteins were detected by Western blot.The expression levels of key proteins in related signaling pathways were detected by Western blotResults:the results showed that 8Z,11 z,14z eicosatrienoic acid had significant effects on the proliferation and proliferation of the above three kinds of cells,suggesting that the metabolite may play a role in promoting angiogenesis.Furthermore,we evaluated the effect of 8Z,11z,14z eicosatrienoic acid on the intracellular inflammatory factors.The results showed that the cytokines increased significantly under the intervention of 8Z,11z,14z eicosatrienoic acid.The results of Q-PCR also confirmed that the proteins related to apoptosis were significantly up-regulated.Therefore,we can preliminarily determine 8Z,11z,14z eicosatrienoic acid,14z eicosatrienoic acid should be a kind of fatty acid metabolite with regulatory function,and may play an important role in promoting cell apoptosis.Western blot and RTPCR detected the changes of PI3K/Akt/mTOR pathway related proteins.Conclusion:8Z,11z,14z eicosatrienoic acid is a kind of fatty acid metabolite with regulatory function,and it may play an important role in promoting apoptosis,and the mechanism may be through regulating PI3K/Akt/mTOR signaling pathway.Part Ⅲ is the functional evaluation of functional metabolic markers in atherosclerotic ratsObjective:To study the role of 8Z,11z,14z eicosatrienoic acid in atherosclerosis rat model.Methods:30 rats were randomly divided into control group,model group(CHD)and CHD+eame group(CHD+eame).The normal group was fed with normal diet all the time;the model group was fed with high-fat diet(cholesterol 1%,bile salt 0.1%,lard 10%,egg yolk powder and whole milk powder 5%,the rest was normal diet)for 8 weeks;eame group was fed with high-fat diet for the first 4 weeks,and eame 1 mg/kg for the next 4 weeks.The experiment lasted for 8 weeks.At the end of the experiment,rat serum,aorta and liver were taken and conveniently divided into three parts,one of which was 0.5 mm?3 tissue and fixed with 4%paraformaldehyde-0.1m phosphate buffer(pH 7.3-7.4)for transmission electron microscope examination;the second part was fixed with formalin for oil red staining;the last part was fixed with formalin for pathomorphological observation.The serum lipid levels of TG,TC,LDL-C,HDLC,TNF-α,IL-6 and IL-1 β were determined by enzyme-linked immunosorbent assay.Results:at the end of the 8th week,compared with the normal control group,the plaque area in the atherosclerotic hyperlipidemia group was significantly increased,indicating that high-fat feeding can cause the atherosclerotic hyperlipidemia model in rats;the plaque area in the drug group was decreased compared with the model group.Compared with the normal group,there was no endothelium in the atherosclerotic hyperlipidemia group,the structure of smooth muscle cells was loose,and there were a lot of vacuoles in the cytoplasm;while a small amount of vascular endothelium,a small amount of lipid droplets and vacuoles in the smooth muscle cells were found in the treatment group.Compared with the normal group,the vascular endothelial cells in the atherosclerotic hyperlipidemia group were seriously damaged,the intimal permeability increased,and the lipid invasion was visible;the above symptoms in the treatment group were improved in varying degrees.Compared with the normal control group,the levels of serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)in the atherosclerotic hyperlipidemia model group were significantly increased(P<0.05).Compared with atherosclerotic hyperlipidemia model group,serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)in eame group were significantly decreased,while high density lipoprotein cholesterol(HDL-C)was significantly increased,the difference was statistically significant(P<0.05)Conclusion:eame can improve the function of atherosclerosis rat model.