Screening Of Differentially Expressed Proteins In Acute Myocardial Infarction Based On ITRAQ Technology And Study On The Mechanism Of Serum Amyloid A1 In Myocardial Ischemic Injury

Objective:Acute myocardial infarction(AMI)is a kind of acute myocardial ischemic disease which belongs to acute coronary syndrome(ACS),served as a severe coronary heart disease with myocardial ischemia and necrosis when the coronary artery is acute blocked.It has acute onset,rapid progression,high mortality and disability rate,served as a serious threat to the health of citizens all over the world.Due to its high incidence rate,it can lead to many diseases such as heart failure,arrhythmia,myocarditis,valvulopathy,cardiac shock,etc.Therefore,it has become an urgent problem to be solved in daily clinical practice for the timely prevention and treatment of AMI.However,in addition to invasive detections such as coronary angiography currently,there still be a lack of biomarkers for early identification of the disease.Thus,the iTRAQ technology,animal experiments,cytological experiments were used to compare and identify the differential expressed proteins in AMI,so as to explore the biomarkers related to AMI,and explore its mechanism in the occurrence and development of AMI.Methods:Part ? Screening and verification of specific proteins of acute myocardial infarction based on iTRAQ technology.1)Detection of differentially expressed proteins in serum of patients with acute myocardial infarction(including STEMI and NSTEMI)and Control group using iTRAQ technology,and performing a series of bioinformation analysis such as Cluster,GO and KEGG,in order to screen differential expressed proteins associated with acute myocardial infarction.2)ELISA assay was used to verify whether SAA1 was highly expressed in the serum of patients with AMI,and to determine whether increased SAA1 levels was an independent risk factor associated with the presence of AMI.ROC curve analysis was used to evaluate the specificity and sensitivity of SAA1 for the diagnosis of AMI.Part ? Study on the changes of SAA1 levels and the effect of inflammatory injury after acute myocardial ischemia-reperfusion in rats.1)The model of acute myocardial ischemia-reperfusion in rats was established by ligating the left anterior descending branch for 30 minutes and then loosening the ligation line for 120 minutes.2)H&E staining was performed to observe the pathological changes of myocardial tissue in rats.3)TTC staining was performed to observe the myocardial infarction and calculate the percentage of infarct area;4)Serum CK-MB and LDH levels were measured to evaluate the extent of myocardial injury;5)ELISA assay was performed to measure the serum SAA1 levels;6)Western Blot technique was performed to detect the expression of NLRP3?Caspase-1 in myocardium.Part ? Study on the the role and mechanism of SAA1 molecule in cardiomyocyte pyroptosis after hypoxia/reoxygenation.1)Cell samples were divided into the following groups:(A)Control group:cell samples were cultured in complete medium under normal conditions;(B)H/R group:H9C2 cell samples cultured under normal conditions were replaced with glucose and serum-deprived DMEM medium,and then cultured in three gas mixed incubator(94%N₂,5%CO₂,1%O₂)for 4 h to complete the hypoxia process,then replaced with normal complete medium and cultured under normal conditions for2 h to complete the reoxygenation process;(C)H/R+SAA1 group:cell samples were incubated with SAA1 for 24 hours,and then subjected to hypoxia/reoxygenation;(D)H/R+SAA1+BAY group:cell samples were incubated with 5?M BAY11-7082(NLRP3 inflammasome inhibitor)for 1 hour,and then incubated with SAA1 for 24hours.2)CCK-8 assay was performed to the viability of cardiomyocytes;3)LDH levels were measured to evaluate the extent of cardiomyocytes damage;4)ELISA assay was performed to measure the serum SAA1 levels;5)The expression of NLRP3 and caspase-1 were determined by immunofluorescence staining;6)Calcein-AM/PI staining assays were performed to detect the cell pyroptosis;7)Western Blot technique was performed to detect the expression of NLRP3,ASC,caspase-1 and IL-1?in cell samples.Results:Part ? Screening and verification of specific proteins of acute myocardial infarction based on iTRAQ technology.1)In this part,we identified 95 differentially expressed proteins,of which 44 proteins were found between STEMI and NSTEMI groups,48 proteins were found between NSTEMI and Control groups,28 proteins were found between STEMI and Control groups,and 24 proteins were found among STEMI?NSTEMI?Control groups;2)The levels of serum SAA1 in STEMI and NSTEMI groups were significantly higher than Control group;3)The ROC curve analysis showed that the sensitivity,specificity and AUC of SAA1were 84.9%,90.4%and 0.927 respectively;4)Multivariate regression analysis showed that the increased SAA1 levels was an independent risk factor associated with the presence of AMI.Part ? Study on the changes of SAA1 levels and the effect of inflammatory injury after acute myocardial ischemia-reperfusion in rats.1)HE staining showed that myocardial tissue was damaged significantly in I/R group;2)TTC staining showed that the infarct size of I/R group was significantly higher than that of Sham group(P<0.01);3)The serum levels of CK-MB and LDH in I/R group were higher than Sham group(all P values were less than 0.05);4)The results of ELISA assay showed that the levels of serum SAA1 in I/R group were higher than Sham group(P<0.05);5)The expression levels of NLRP3 and Caspase-1 in myocardial tissue of I/R group were significantly higher than Sham group conducted by Western Blot technique(all P values were less than 0.05).Part ? Study on the the role and mechanism of SAA1 molecule in cardiomyocyte pyroptosis after hypoxia/reoxygenation.1)The results of CCK-8 assay showed that the viability of cardiomyocytes in H/R group were significantly lower than that in Control group(P<0.01).After the intervention of SAA1(10?g/ml),the viability of cardiomyocytes were significantly lower than H/R group(P<0.01);2)LDH level in H/R group was significantly higher than Control group(P<0.01).After the intervention of SAA1,LDH level was further increased(P<0.05).In addition,the levels of LDH were lower in the NLRP3 inflammasome inhibitor(BAY11-7082)and SAA1 co-treatment group compared with the levels in SAA1group after H/R treatment(P<0.05);3)The results of ELISA assay showed that the levels of IL-1?in H/R group were significantly higher than that in Control group(P<0.01).After the intervention of SAA1,the level was further increased(P<0.01).In addition,the levels of IL-1?were lower in the BAY11-7082 and SAA1 co-treatment group compared with the levels in SAA1 group after H/R treatment(P<0.01);4)The immunofluorescence staining showed that the expression of NLRP3 and caspase-1 in H/R group was significantly higher than Control group.After the intervention of SAA1,the expression of NLRP3 and caspase-1 in H/R group was higher than H/R group.In addition,the expression of NLRP3 and caspase-1 were lower in the BAY11-7082 and SAA1 co-treatment group compared with the levels in SAA1 group after H/R treatment;5)The Calcein-AM/PI staining showed that the extent of pyroptotic cell death in H/R group was significantly higher than Control group.After the intervention of SAA1,the extent was significantly higher than that in H/R group.In addition,the extent was significantly lower in the BAY11-7082 and SAA1 co-treatment group compared with the levels in SAA1 group after H/R treatment;6)The expression levels of NLRP3,ASC,Caspase-1 and IL-1?in H/R group were significantly higher than that in Control group(all P values were less than 0.05).After the intervention of SAA1,the expression of each index in SAA1 group was significantly higher than that in H/R group(all P values were less than 0.05).In addition,the expression levels of NLRP3,ASC,Caspase-1 and IL-1?were significantly lower in the BAY11-7082 and SAA1 co-treatment group compared with the levels in SAA1 group after H/R treatment(all P values were less than 0.05).Conclusions1)We identified 95 differentially expressed proteins by using iTRAQ technology,and24 proteins were found among STEMI?NSTEMI?Control groups.The levels of serum SAA1 were verified to be consistent with proteomic results by ELISA assay,and the increased SAA1 levels was an independent risk factor associated with the presence of AMI,which could be regarded as a biomarker for the diagnosis of AMI;2)The levels of serum SAA1 were increased and the inflammatory injury of myocardial tissue was aggravated in the occurrence of acute myocardial ischemia-reperfusion.3)SAA1 can participate cardiomyocyte pyroptosis by activating NLRP3inflammasome after hypoxia/reoxygenation treatment.