Metabonomics Of Eicosanoids Acid In Myocardial Ischemia-reperfusion Injury

Objectives Acute myocardial infarction(AMI)is the most serious type of coronary heart disease.ST-Segment Elevation Myocardial Infarction(STEMI)occurs when thrombus formation results in complete occlusion of a major epicardial coronary vessel.In some severe cases,malignant arrhythmias(ventricular tachycardia,ventricular fibrillation)can occur and even lead to sudden death.Therefore,for patients suffering acute STEMI,blood flow restoration is strongly recommended to save the ischemic myocardium as soon as possible.Percutaneous Coronary Intervention(PCI)is often the first-choice strategy for acute STEMI according to current guidelines,which can improve long-term survival in patients with acute STEMI.However,many clinical issues related to PCI need to be addressed,among which,MIRI is one of the critical problems that may be associated with symptoms such as increased MI area,arrhythmia,and heart failure.In-depth understanding of the pathophysiologic changes prior and post PCI surgery can further improve the prognosis of STEMI.Though,the exact pathological mechanisms of IRI in STEMI patients are yet to be identified..Eicosanoids are metabolites of hundreds of active lipid molecules generated by Polyunsaturated Fatty Acids(PUFAs)such as Arachidonic Acid(ARA)under the action of three major metabolic pathways: Cyclooxygenase(COX),Lipoxygenase(LOX)and Cytochrome P450(CYP450).Many of these molecules are involved in biological processes related to cardiovascular disease.Eicosanoids play either a harmful or protective role in processes such as inflammation,thrombosis,atherosclerosis,myocardial hypertrophy,arrhythmia,and heart failure.Yet,the overall metabolic pathways of eicosanoids in patients prior and post PCI is still not clear.The purpose of this study is to use eicosanoid metabolomics to: · explain the overall changes of the eicosanoid profile during myocardial ischemia-reperfusion in the mouse myocardial ischemia-reperfusion injury model and plasma samples from STEMI patients;· discover key metabolites related to myocardial ischemia-reperfusion injury;and to study the function and mechanism of key metabolites in cell models.Methods The mice model of myocardial ischemia-reperfusion injury was established by coronary artery ligation / reperfusion surgery and verified by ultrasound cardiography and TTC staining.The plasma samples of STEMI patients within a time frame consisting of 7 time points(30 minutes before surgery,surgery 6 hours,surgery 12 hours,surgery 24 hours,surgery72 hours,1 day before discharge,and 28 days after discharge)were collected,and the patient's clinical information were fully recorded(including demographic characteristics,vital characteristics,medical history,pre-hospital medication,echocardiography,myocardial infarction area,CK-MB,c Tn I and other cardiac injury marker levels,etc.).Liquid chromatography-triple quadrupole mass spectrometry was used to analyze heart tissue of mouse MIRI model and plasma of eicosanoid metabolites(including cyclooxygenase,lipoxygenase,and more than 84 metabolites under the cytochrome P450 oxidase pathway).Mann-Whitney U test,ANOVA analysis of variance,partial least squares discriminant analysis and other statistical methods were used to screen out key metabolites related to myocardial ischemia-reperfusion injury.Establish a model of hypoxia-reoxygenationinduced apoptosis of primary cardiomyocytes in neonatal rats(12 hours of hypoxia and 6 hours of normoxic conditions).In this model,the effects of key metabolites on cardiomyocyte apoptosis were studied.The profiles of eicosanoids in the heart tissue of mice MIRI model and plasma of STEMI patients were analyzed using a targeted metabolomics strategy based on liquid chromatography-triple quadrupole mass spectrometry.Through the screening principles composed of Mann-Whitney U test,ANOVA analysis of variance,partial least squares discriminant analysis and other statistical methods,key metabolites related to myocardial ischemia-reperfusion injury were screened out.A cell I/R model was established using primary cardiomyocytes from neonatal rats induced by hypoxiareoxygenation treatment(12 hours of hypoxia and 6 hours of normoxic conditions),and the effects of key metabolites on cardiomyocyte apoptosis were studied using this model.Results We identified 68 eicosanoids in mouse heart tissue.Partial least squares-discrimination analysis showed that compared with the control group,the IR model established by coronary artery ligation / recanalization in mice showed a significant change profile of eicosanoids,and 15-HETE(elevated I/R group)contributed the most to the overall differentiation between the two group;7 eicosanoids with significant changes were screened out by MannWhitney U test,including 16-HETE(decreased to below the detection limit in the I/R group),12-HETE(increased by approximately 2.08 times in the I/R group,p = 0.011),15-HETE(increased about 2.11 times in the I / R group,p = 0.014),18-HETE(decreased about 70%,p = 0.001),TXB2(increased about 1.46 times,p = 0.048).A total of 140 plasma samples collected from 20 STEMI patients at 7 time points were analyzed for eicosanoid metabolomics.A total of 68 eicosanoids were identified in these samples.Partial Least Squares Discriminatory Analysis(PLS-DA)showed that STEMI patients had significant changes in the eicosanoid metabolic profile prior and post PCI operation,of which 6-k-PGF1? contributed the most to the overall change.The time course curves of different metabolites shows different characteristics.In the early stage,which is 6-12 hours after I/R,the level of 16-HETE increased;in the middle stage,which is 12-72 hours after I/R,the level of DHETs increased,while the level of TXB2 and LXA4 decreased;In the late stage,which is 72 hours after I/R,the level of 6-k-PGF1? and LTB4 increased.In addition,PGD2 and PGE2 continue to decrease throughout the entire process.Conclusions Based on the data derived from comprehensive analysis using Mann–Whitney U test and PLS-DA multivariate statistics toghether with known knowledge of eicosanoid metabolites,we identified that 5 arachidonic acid metabolitess,i.e 12-HETE,15-HETE,16-HETE,18-HETE,and TXB2,which showed important pathophysiological functions during myocardial ischemia-reperfusion injury in mice.Based on the data analysis through ANOVA,Mann–Whitney U test and PLS-DA multivariate statistics,combined with known functions of eicosanoid metabolites,we have identified 9 arachidonic acid metabolites,i.e 6-k-PGF1??TXB2?PGD2?PGE2?LXA4?LTB4?16-HETE?20-HETE?5,6-DHET,that played important roles in the process of ischemia-reperfusion injury in STMEI patients.TXB2 and 16-HETE showed significance in both animal models and STEMI patients.In this study,we have systematically explored the overall changes of metabolic profiles of eicosanoid in myocardial ischemia-reperfusion injury,which helped to fully understand the overall characteristics of the eicosanoid system during myocardial ischemia-reperfusion injury.We have proposed an interventional strategy that targeted to analyze eicosanoid profile involved in myocardial ischemia-reperfusion injury according to the metabolomics data.