| Coronary Microembolizaiton(CME)is caused by spontaneous or iatrogenic causes of rupture of atherosclerotic plaques,small plaque debris,and secondary thrombosis that follow the blood flow to the microcirculation system(arterioles,capillaries,and venule),which leads to coronary microcirculation disorder.The widespread coronary microcirculation disorder significantly weakens the benefits of acute myocardium infarction(AMI)patients after emergency interventional therapy(PCI),which is the result of early complications,long-term heart failure and death important reason.No reflow is a common clinical manifestation of CME.The changes of creatine kinase and troponin in the blood can indirectly indicate the damage of CME to the myocardium.Doppler intracoronary artery can directly observe microembolism,while cardiac MRI Further clarify the location of the embolism and the area of infarction.Antiplatelet drugs,statins,thrombus aspiration devices and other measures can reduce the occurrence of CME,but clinical practice has not yet fully established a cardiac protection strategy to avoid coronary microembolism and rescue myocardium from microvascular occlusion.The maximum no-reflow phenomenon of AMI patients can still reach 30%.How to effectively prevent and treat CME is a problem faced by cardiovascular doctors.In-depth research on its injury mechanism and subsequent medical conversion have been the issues that researchers have paid attention to for many years.Initially,the myocardial tissue of CME animal model was detected by TUNEL staining and cleared caspase3 immunohistochemistry,and it was found that there was cardiomyocyte apoptosis in CME.Later,in-depth studies by multiple research groups including ours showed that apoptosis is one of the most important mechanisms of CME-induced myocardial injury.Mitochondrial apoptosis pathway is an internal pathway of apoptosis mechanism.In recent years,HtrA2 has attracted much attention in various fields as a newly discovered key molecule in mitochondrial apoptosis pathway.HtrA2 is a serine protease in the mitochondrial membrane space,plays an important role in mitochondrial quality control,and maintains mitochondrial homeostasis,and can promote apoptosis through a dual pathway.Recent studies have shown that the increase of serum HtrA2 in patients with acute myocardial infarction PCI is related to ischemia-reperfusion injury,but it is related to the type of reperfusion injury results,so it is not further elucidated.Clinical studies have shown that the mitochondrial ATP-dependent potassium channel activator nicorandil improves the phenomenon of no backflow.Combined with our previous research,it shows that it can inhibit apoptosis in the upstream pathway of mitochondria in cardiomyocytes and whether nicorandil inhibits HtrA2 There are no relevant reports on the downstream channels of regulation.Based on this,this subject aims to observe the correlation between the changes of peripheral blood HtrA2 and acute myocardial injury in patients with PCI with acute myocardial infarction,and explore its clinical value.Establish a CME rat model,observe the changes of HtrA2 after CME,and explore the role of HtrA2 in regulating apoptosis in CME-induced myocardial injury.To explore the role of HtrA2 / XIAP / PARP pathway in the improvement of CME myocardial injury by Nicorandil.Part 1 Expression and clinical significance of HtrA2 in peripheral blood of patients with acute myocardial infarctionObjective:1.To observe the changes of HtrA2 in peripheral blood of patients with acute myocardial infarction after PCI2.To explore the clinical significance of peripheral blood HtrA2 changes of patients with acute myocardial infarction after PCI.Then,explore the relationship between peripheral blood HtrA2 and no-reflow.Method:Patients with acute myocardial infarction(AMI group),stable coronary heart disease(Stable-CAD group),and healthy volunteers(Health-Control group)were admitted continuously.If the patient's coronary blood flow does not recover during PCI,and there is no reflow(TIMI 0-2),the patient is enrolled into the NF subgroup,and the AMI with TIMI level 3 coronary blood flow is the NNF subgroup.The blood samples of patients with acute myocardial were collected within 24 hours,72 hours,and 7 days,and stable coronary heart disease patients were within 24 hours;volunteers were after asking about no previous medical history and current disease status.A 3ml blood sample of each subject was collected with an EDTA anticoagulation tube,centrifuged at 1200 x g for 15 minutes at 4 ° C,and then the plasma was stored in a refrigerator at-80 ° C.The plasma HtrA2 concentration was detected by ELISA.SPSS 23 software was used to analyze the data,compare the concentration differences between the groups,and observe the concentration changes related to myocardial injury markers in the acute myocardial infarction group.Logistic regression analysis of the correlation between HtrA2 expression level and AMI,NF,ROC curve is used to indicate the HtrA2 diagnosis of AMI,NF cutoff point,sensitivity,specificity,area under the ROC curve.Results:1.A total of 37 subjects were selected.A total of 20 patients with acute myocardial infarction,except for one non-ST-increased myocardial infarction,the rest are myocardial infarctions with elevated ST,and all patients received emergency PCI.Seven patients with acute myocardial infarction in NF group.7 cases in the Stable-CAD group and 10 cases in the healthy control group.There was no statistical difference between the three groups(AMI?Stable-CAD?Health-Control)in terms of gender,history of diabetes,dyslipidemia,family history of coronary heart disease,blood pressure,body mass index,serum creatinine,total cholesterol and low-density lipoprotein cholesterol(P ? 0.05).Compared with the coronary heart disease group,the age,history of hypertension and medication of coronary heart disease were not statistically different(P?0.05),and LVEF was lower(P <0.05).Compared with other groups,the total number of leukocytes,absolute neutrophils,and high-sensitivity C-reactive protein in AMI patients within 24 hours of admission was higher(P <0.05),while lymphocytes and high-density lipoprotein cholesterol decreased(P <0.05).2.Plasma HtrA2-24 h in AMI group,Stable CAD group and Health-Control group were 2018.13(702.41,2781.57)pg / m,622.19(359.54,882.182)pg / m,722.87(343.16,895.49)pg / ml,compared with other two groups,AMI group Increased(P <0.05),but there was no significant difference in HtrA2 concentration between Stable CAD group and Health-Control group(P?0.05).After PCI in AMI patients,HtrA2 shows a gradual downward trend.3.The plasma HtrA2-24 h concentrations in the AMI subgroup NF and NNF were 2781.57(2203.66,4600.02)pg / ml and 1808.15(620.124,2016.52)pg / ml,respectively,and there was a significant difference between the two(P <0.05).After PCI in AMI patients,plasma HtrA2 and various markers of myocardial injury showed a gradual downward trend.The NF group and NNF group also showed a downward trend,but the plasma HtrA2 concentration in the NF group at 24 h and 72 h was higher than that in the NNF group(P <0.05).4.The plasma HtrA2 concentration within 24 hours after admission was positively correlated with CK-MB,LDH,CTn T,CTn I(P <0.001),but not significantly correlated with Pro BNP(P?0.05)5.According to the diagnosis group,logistic regression was performed with AMI as the dependent variable and HtrA2 as the independent variable.HtrA2 was significantly positively correlated with AMI(P <0.05).In the coronary heart disease patient group,HtrA2 was used as a test variable,AMI was used as a state variable,and a ROC curve was made.Plasma HtrA2 is of great significance in the diagnosis of AMI(P <0.05).The area under the curve is 0.895.When HtrA2 was 1374.43 pg / ml,the sensitivity of acute myocardial infarction diagnosed in patients with coronary heart disease was 73.7%,and the specificity was 100%.In all populations,using HtrA2 as the test variable and acute myocardial infarction as the state variable,a ROC curve was prepared with an area under the curve of 0.864(P <0.05).When HtrA2 was 1425.03 pg / ml,the sensitivity for diagnosis of acute myocardial infarction was 73.7% and the specificity was 100%.6.According to the median 2018.13 pg / ml of 24 h plasma HtrA2 concentration in AMI patients,the patients were divided into two groups.There was no statistical difference in the baseline data.Taking NF as the dependent variable and HtrA2 within 24 h as the independent variable of logarithmic regression,HtrA2 was significantly positively correlated with NF(P <0.05).In AMI,24-hour plasma HtrA2 is used as a test variable,while NF is a state variable.The area under the ROC curve of plasma HtrA2 diagnosis NF was 0.912(P <0.05).When HtrA2 was 1988.37 pg / ml,the diagnosis was NF with a sensitivity of 100.0% and a specificity of 76.9%.Conclusion:1.Peripheral blood HtrA2 level of AMI emergency PCI patients increased significantly,and gradually decreased after 24 hours.Plasma HtrA2 in NF patients were higher than NNF patients at 24 h and 72 h.2.Plasma HtrA2 is positively correlated with myocardial injury markers,and has the highest correlation with CK-MB.3.Plasma HtrA2 is positively correlated with NFPart 2 The mechanism of HtrA2/XIAP/PARP mitochondrial apoptotic pathway mediating myocardial injury in rats with coronary microemboliztionObjective:1.To explore the expression level of HtrA2 in CME;2.To explore the mechanism of HtrA2/XIAP/PARP regulating apoptosis in CME rats.Methods:1.Observation on changes of myocardial injury in CME: Sixty rats were randomly divided into Sham group(n=10)and CME group(n=50),CME group was randomly divided into 3h,6h,9h,12 h,24h subgroups(n=10 per subgroup)according to different observation time points.Cardiac ultrasound was used to measure cardiac function,HE staining and HBFP staining were used to observe myocardial microinfarction and measure infarct size.2.Exploring the role and mechanism of HtrA2 in CME: Forty rats were divided into Sham group(n=10),CME group(n=10),CME + DMSO group(n=10),CME + UCF10 group(n=10).Combining the results of the previous section,the time point of the most severe cardiac function impairment after CME modeling was used as a measure of the cardiac function and sampling time of all groups in this section.Compare cardiac function,myocardial infarction area,TUNEL staining,apoptosis and pathway-related protein changes.Results:1.Subgroup cardiac ultrasound prompts at different times: Compared with the Sham group,the cardiac function of the CME group began to decline from 3h,and the CME 9h was the lowest value.It showed that left ventricular systolic diameter(LVEDs),left ventricular end-diastolic diameter(LVEDd)increased,left ventricular ejection fraction(LVEF),and left ventricular short axis shortening rate(LVFS).,Cardiac output(CO)decreased,P <0.052.HE staining and HBFP staining display: In the CME operation groups,HE staining showed that the microvessels were blocked by the transparent polyethylene microspheres,and the microembolic balls caused the surrounding microinfarction.Myocardial cell nucleus dissolved or disappeard in microinfarction,accompanied by a large number of inflammatory cell infiltration.HBFP stained myocardial red in the micro-infarct area,yellow stained normal myocardium,and multiple micro-infarct foci were seen in the slices.3.Western blot technique shows :9h after modeling,compared with the Sham group,the CME group myocardial cytoplasmic HtrA2 expression was significantly increased,and the mitochondrial HtrA2 expression was reduced(P <0.05)4.Echocardiogram 9h after modeling :Compared with the CME group,the cardiac function of the CME + UCF101 group was improved,showing increased LVEF,LVFS,and CO,but decreased LVIDd and LVIDs(P <0.05),but the heart of the CME + DMSO group had no significant changes in function.5.HBFP staining display 9h after modeling: The areas of myocardial microinfarction in the CME group,CME + DMSO group,and CME + UCF101 group were(18.21 ± 3.41)%,(19.08 ± 3.14)%,and(9.74 ± 2.36)%.Compared with the CME group,the area of myocardial microinfarction of CME + UCF101 group was significantly reduced(P <0.05).There was no significant difference in the area of myocardial microinfarction between CME + DMSO group and CME group(P?0.05)6.Serum biochemical detection 9h after modeling: Compared with the Sham group,the serum CK-MB and serum LDH of the CME group increased,P <0.001;compared with the CME group,the serum CK-MB and serum LDH of the CME + UCF101 group were lower,P <0.05,and no difference in serum CK-MB and LDH between CME + DMSO group and CME group.7.Apoptosis staining of myocardial TUNEL 9h after modeling: Myocardial tissue section TUNEL apoptosis staining showed that the myocardial cell apoptosis index(AI)of microinfarction area in Sham group,CME group,CME + DMSO group,CME9 h + UCF101 group were(1.67 ± 0.68)%,(26.54 ± 1.87)%,(27.56 ± 2.46)%,(19.30 ± 1.95)%;compared with the Sham group,the myocardial cell apoptosis index in the CME group increased significantly(P <0.05);compared with the CME group,the AI of CME + UCF101 group was significantly reduced(P <0.05).There was no significant difference in AI between CME + DMSO group and CME group.8.Expression of apoptosis-related proteins in each group 9h after modeling: Compared with the Sham group,the CME group C-caspase3,C-caspase9,Cytochrome C protein expression levels were significantly increased(P <0.05).Compared with the CME group,the expression levels of protein C-caspase3,C-caspase9,and Cytochrome C in the CME + UCF101 group decreased(P <0.05),and there was no significant difference in up expression between CME + DMSO group and CME group.9.Expression of HtrA2 / XIAP / PARP pathway protein in each group 9h after modeling: Compared with the Sham group,the relative expression levels of cytoplasmic HtrA2 and C-PARP in the CME group increased significantly,while the relative expression of XIPA decreased(P <0.05).Compared with the CME group,the expression of HtrA2 cytoplasmic protein and C-PARPprotein in the CME + UCF101 group decreased,and the expression of XIPAprotein increased(P <0.05).Compared with the CME group,the expressionsof CME + DMSO group HtrA2 cytoplasmic protein,C-PARP and XIPA werenot significantly different.Conclusion:1.Rats with CME has a progressive decline in cardiac function after 3h,with the lowest CME 9h2.In CME,HtrA2 translocates from mitochondria to cytoplasm,and HtrA2 expression in CME cardiomyocytes is increased3.Apoptosis regulated by HtrA2 causes CME myocardial damage.Inhibiting HtrA2 activity can reduce apoptosis and improve CME-induced changes in cardiac structure and function4.HtrA2 promotes CME myocardial injury through HtrA2 / XIAP / PARP pathway-mediated apoptosis.Part 3: The role of HtrA2/XIAP/PARP pathway in Mito KATP activator in improving myocardial injury in coronary microembolizationObjective:1.To explore whether mitochondrial KATP channel activator-nicorandil has a protective effect on CME2.To explore the role and mechanism of HtrA2/XIAP/PARP pathway in the improvement of coronary microemboliztion-induced myocardial injury by KATP channel activator.Methods:Forty SD rats were randomly divided into four groups: Sham,CME,CME+NIC,CME+UCF,10 rats in each group.The CME + NIC group(nicorandil group)was injected intraperitoneally with nicorandil(5 mg / kg)10 minutes before CME modeling.The CME + UCF group received intraperitoneal injection of UCF101(1.5umol / Kg)10 minutes before CME modeling.Nine hours after modeling,echocardiography was measured,blood was taken from the abdominal aorta,and heart tissue was taken for HE staining,TUNEL staining,Western-blot protein detection,and transmission electron microscope scanning for detection.Results:1.Compared with the CME group,the echocardiography showed that the LVEDs and LVIDd decreased in the CME + NIC group,and the LVEF,LVFS,and CO increased(P <0.05).The CME + UCF group showed the same trend,and the LVEDs and LVIDd decreased,while LVEF,LVFS,CO rised(P <0.001)2.Serum biochemical test indication :Compared with the Sham group,the serum CK-MB and LDH of the CME group increased(P <0.001).Compared with the CME group,the CK-MB and LDH of the CME + NIC group,CME + UCF decreased significantly(P <0.05).There was no significant difference in CK-MB and LDH between CME + NIC group and CME + UCF group(P> 0.05).3.Transmission electron microscopy showed that the myocardial fibrillar structure of the Sham group was clear,and the mitochondrial membrane was basically intact;the CME group showed obvious mitochondrial swelling and mitochondrial ridge fracture;the mitochondrial structure of the CME + NIC and CME + UCF groups was still clear,and the mitochondria were slightly swollen without obvious mitochondrial ridge break.4.TUNEL apoptosis staining showed that the AI of Sham group,CME group,CME + NIC group,CME + UCF group were(1.56±0.78)% ?(25.34±1.45)%,(17.55 ± 1.96)%,(19.32 ± 1.94)%.Compared with the CME group,the AI in CME + NIC group and CME + UCF decreased(P <0.001);there was no statistical difference in AI between CME + NIC group and CME + UCF group(P? 0.05)5.Western Blot showed that the cytoplasmic HtrA2 expression increased in the CME group compared with the sham group(P<0.05).Compared with the CME group,both the CME + NIC group and the CME + UCF group decreased(P<0.05)6.Western-blot detection of apoptosis-related protein expression: Compared with the CME group,the expressions of protein C-caspase3,C-caspase9 and Bax in CME + NIC group and CME + UCF group decreased(P <0.05),and protein Bcl and expression increased(P <0.05).There was no significant difference in the expression of protein C-caspase3,C-caspase9,Bax,Bcl-2,between CME + NIC group and CME + UCF group.7.Western-blot detection of HtrA2 / XIAP / PARP pathway protein expression: Compared with the CME group,the expressions of protein C-PARP in CME + NIC group and CME + UCF group decreased(P <0.05),and protein XIAP expression increased(P <0.05).There was no significant difference in the expression of up proteins between CME + NIC group and CME + UCF group.Conclusion:1.Nicorandil reduces myocardial damage caused by CME and improves cardiac function;2.HtrA2 / XIAP / PARP signaling pathway may mediate nicorandil to improve the apoptosis of CME. |
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