| BackgroundCoronary heart disease is one of the major reasons of human death, because myocardial ischemic injury and ischemia-reperfusion Injury can lead to death or very high incidence of heart failure. For patients with acute myocardial infarction, the using of thrombolysis or percutaneous coronary intervention in the early treatment can reduce myocardial infarct size after the myocardial reperfusion and can be an effective way to improve clinical outcome. However, it is very difficult how to predict and reduce damage after myocardial ischemia and ischemia-reperfusion injury of cardiac function and sudden cardiac events. By studying the mechanism about myocardial ischemia and reperfusion injury, we found that the main reasons are neutrophil accumulation, calcium overload or calcium redistribution, of mitochondrial energy synthesis barriers,the generation of oxygen free radicals. But can we achieve the desired effect by studying these changes with regulators detection and intervention? Recently it is the hotspot research to study the role of microRNA in the cardiovascular system.Ischemia is a blood disorder caused by insufficient blood supply to tissue pathology. Tissue ischemia caused by the disease known as ischemic disease, it can happen in a variety of tissues and organs of the common diseases. Reperfusion after ischemia refers to the organization of the process to restore the blood supply.Ischemia-reperfusion actually includes two aspects.One is ischemia and another is reperfusion. The cause of the disease is not only caused by ischemia, but also the conditions of reperfusion. Timely reperfusion therapy, such as thrombolysis, percutaneous coronary intervention (PCI), etc. can be reconstructed coronary revascularization, myocardial reperfusion obtained. Thereby.reducing the area of myocardial necrosi is the key way of the treatment about acute myocardial infarction (AMI). However, recent studies have found that a sudden opening of the coronary arteries and restore blood flow might cause endothelial cell dysfunction, activation of inflammatory factors. But increasing ischemic myocardial injury could result in reperfusion injury. Animal with AM study results I show that40%to50%of myocardial necrosis is caused by reperfusion injury and this injury weakens the active presence of reperfusion therapy benefits obtained. Therefore, it is to reduce reperfusion injury that become an important part of prevention and treatment of AMI.Injury after myocardial ischemia and ischemia-reperfusion injury is currently mainly the following points:1, free radical damage theory. When the body produces free radicals damage exceeding the body’s ability to clear, the body will produce injury. Study confirmed during ischemia-reperfusion, ischemic area there are plenty of free radicals, such as reduced ability to scavenge oxygen free radicals or reactive oxygen species (ROI). ROI can produce too much will lead to ROI accumulation caused by oxidative stress. Oxidative stress reperfusion of ischemic tissue is one of the characteristics. And the application of free radical scavengers Coenzyme Q10can reduce ischemic cell damage irrigation.2, Calcium overload caused by myocardial injury is an important mechanism of ischemic hypoxia.The myocardial intracellular acidosis, intracellular H+to increase and restore reperfusion intracellular formation of pH gradient difference, stimulating Na+-H+exchange, so that Na+a large influx. Since the energy supply after reperfusion and pH recovery, promote Na+-Ca2+exchange, a large number of extracellular Ca2+influx, resulting in Ca2+overload. So that the cell membrane and organelle membrane damage caused by oxygen free radicals promote myocardial injury. Ischemic vascular smooth muscle and because a significant increase in Ca2+influx can cause vasoconstriction spasm, vascular resistance increased circulating adverse ischemic area, expand the infarct. Study found that the changes of the concentration of intracellular calcium are caused by reperfusion injury.It is one of the important reasons. Calcium overload is a variety of causes cell damage and death common pathway.3,Myocardial energy is metabolism, ATP is the main energy source of cell metabolism, cell in which the life of the state and all life activities ultimately depend AT P levels. If mild or transient hypoxia reoxygenation after severe hypoxia, cells can still generate some ATP and the energy required for apoptosis to provide protection to promote apoptosis. Conversely, if a longer time severe hypoxia,cell apoptosis due to AT P plummeted lead to insufficient energy needed necrosis; When ATP production was completely inhibited cell necrosis, Study suggests that hypoxia-induced ATP in the cell during cell death manner of death of the decisive factors. Energy metabolism is the basis of free radicals, free radicals can also increase energy metabolism, they are mutually reinforcing relationship. Therefore, mitochondrial damage and energy metabolism of myocardial ischemia-reperfusion injury is a major cause of intracellular AT P. It is to determine the level of apoptosis or necrosis of the main factors.4,Anti-oxidation system damage endothelial cells. Studies have shown that the antioxidant activity of endothelial cells and their sensitivity to oxidation activity relationship between certain. When myocardial ischemia and reperfusion, the endothelial cells not only produce large amounts of reactive oxygen species, and also greatly reduce its antioxidant activity.Exogenous reactive oxygen generation system has high sensitivity. It can cause a large number of reactive oxygen species, far endothelial cells than defense system, and ultimately lead to myocardial injury. Other studies have shown that ischemia-reperfusion endothelial cell dysfunction occurs and the reduction of NO synthesis,. Endothelin (ET) ET increase release of myocardial ischemia. Reperfusion ET release further increased causing intense vasoconstriction.It can be directly cause myocardial ischemia, increased vascular dysfunction, cell irreversible damage. Tissue hypoxia and increased secretion of adrenaline can lead to the ET mRNA and ET releasing. Myocardial ischemia and reperfusion can cause myocardial cell ET receptors, and makes the ET coronary vessels increased sensitivity to small branches. And thus coronary Easy spasms that can lead to myocardial no-reflow phenomenon. Endothelial cells and neutrophil adhesion ischemia, reperfusion induced neutrophil adhesion to endothelial cells increased neutrophils and endothelial contact is activated, the release of OFR and other toxic products and destructive proteases, vascular permeability changes. At the same time an increasing in reactive oxygen species will damage to the endothelial cells and other cells, and its role in endothelial cells or granulocyte cell surface adhesion molecules, promote endothelial cell adhesion and endothelial function hypoxic injury itself. Changing its adhesion eventually leading to endothelial cell damage, edema and dysfunction. Capillary chamber is blocked, leading to the great vessels although the area of ischemia-reperfusion, but still no reflow phenomenon.5, the related cytokines. Bing stimulation of the hypoxia when hindered gas exchange, coronary vascular will injury swelling of endothelial cells. And endothelial cells and smooth muscle cell damage the blood vessels are not well expanding the endovascular neutrophil and platelet aggregation through incarceration, clogging the capillaries, so that more reduced coronary blood flow;Followed by vascular endothelium and neutrophils produce cytokines, adhesion molecules, the neutrophils to vascular endothelial cells stick attached, and the release of particulate elastin, reactive oxygen species, lysosomal enzymes, cytokines and other inflammatory mediators. These substances can damage the endothelium, vascular smooth muscle cells and cardiac myocytes.6.Involving in the process of cell apoptosis. The apoptosis is ischemia-reperfusion tissue injury in important cause loss of function. It is a very complicated process. Cell apoptosis is Inducted by many factors. And in myocardial ischemia and reperfusion inducing cell apoptosis was the main mechanism.Intracellular free radicals increased Ca2+levels. Because ischemic hypoxia endogenous antioxidants such as SOD inactivation or depletion, AT P metabolites accumulate in hypoxic-ischemic myocardial tissu eand result in a large number of oxygen free radicals. Unstable nature of oxygen free radicals once formed to intracellular DNA strand breaks quickly make sarcolemma glucose and lipid peroxidation, protein denaturation and enzyme inactivation, and can induce apoptosis.MicroRNAs is a recently discovered small endogenous non-coding single-stranded RNA(about22nucleotides) and the control of gene expression. It is considered to be a negative regulator of gene expression, via its target mRNA molecule3’end complementary to the non-coding region match by inhibiting translation of the mRNA molecule (1-3). Currently, there is about one-third of the genes regulated by MicroRNAs. Recent research suggests that some MicroRNAs are highly expressed in the cardiovascular and they are of great significance in the regulation of cardiovascular development and disease, which will bring hope to the treatment of cardiovascular diseases. The study found that the persistent disorders of miR-320expression in model of the rats away from the body and in vivo myocardial ischemia/reperfusion.They identify that heat shock protein20(a known myocardial protection protein) is the target body of miR-320. After knockdown of endogenous miR-320, the heat shock protein20will lose the protection myocardial ischemia-reperfusion-induced myocardial cell death/apoptosis. Another study found that the miR-21Expression of the ischemic area of the heart is decreased after six hours of ischemia in rats, while the expression is increased in the peripheral edge area. Transfected with miR-21in vitro through the implementation of its target gene activator protein1pathway to reduce cell apoptosis in the edge of the area and the ischemic area. In the vivo experiments of MiR-1and miR-133show that the effect of apoptosis is counterproductive. MiR-1is linked to cell death by inhibiting the transporter of insulin-like growth factor1. Recently study found that miRNAs can be detected in the blood of the patient’s peripheral circulation. Therefore, we can assume that the peripheral blood miRNAs reflect damage of tissue. So the detected miRNAs can be serum biomarkers as acute myocardial infarction,.As a member of the miRNAs family, miR-126can regulated the cell apoptosis in mouse artery atherosclerosis model. The role is down regulator for the formation of blood vessels by the inhibition of cell-derived factor1expression in human endothelial cells. Regulation of vascular integrity of the vascular endothelial cell proliferation and angiogenesis. Sun X, and other studies suggest that the downward and upward role of miR-126was not no significant correlation with coronary heart disease. It had only significant correlation with cholesterol metabolism and the expression levels in the low-density cholesterol (LDL) in patients with high coronary heart disease was reduced. Guangwen Long thought that miR-126expression was correlation with acute myocardial infarction and the expression of miR-126in patients with acute myocardial infarction is reduced. He found the up and down regulation was no correlation with time. In different expression of time curve curve is consistent with the expression and serum troponin I, but no significant correlation with each other.MiR-92a as a another member of the miRNAs family.The study found miR-92a was highly expressed in the control of angiogenesis in human endothelial cells. Overexpression of miR-92a in endothelial cells in vivo and in vitro would damaged angiogenesis. In vitro studies have shown that inducible ischemia miR-92a expression was significantly up-regulated in the organization. Inhibition of miR-92a expression in acute myocardial infarction model could promote the recovery of heart function. Experimental studies have shown that miR-92a was expressed not only in the endothelial cells and also in cardiac fibroblasts, cardiac myocytes. Antagonizing miR-92a could be significantly reduced apoptosis. It did not affect cell survival. The study also found that miR-92a dependent regulation of NO synthase to control vascular tone. Studies have shown that miR-92a was high expressed in patients with coronary heart disease. Statins could reduce miR-92a expression to reduce blood lipids. Inhibition of miR-92a might be reduced myocardial infarct size and improved myocardial infarction remodeling and neovascularization. Studies have shown that miR-92a by promoting cardioprotective protein synthesis played a positive role in myocardial ischemia/reperfusion.Therefore, this study from the clinical and basic aspects of miR-92a and miR-126in myocardial ischemia and reperfusion in the role of research, methods and measures for the diagnosis and treatment of coronary heart disease in the future.Objective:1. Could the detection of miR-92a and miR-126be the early diagnosis of acute myocardial infarction by the miR-92a and miR-126expression studies in the peripheral blood of patients with acute myocardial infarction? 2. To elaborate the possible role of the miRNAs in reperfusion of acute myocardial infarction in patients by detecting the miR-92a and miR-126expression in peripheral blood before and after treatment of reperfusion3. To explain the possible role of miR-92a and miR-126in the no-reflow phenomenon after reperfusion therapy for acute myocardial infarction through studying the expression of miR-92a and miR-126.4.To infer the possible mechanism of miR-92a and miR-126in myocardial ischemia reperfusion injury regulatory through the establishment of the mice with myocardial ischemia and reperfusion and the experimental studies.Methods:1.The collection and testing of specimens for clinical peripheral blood in miRNAs1.136patients with coronary heart disease and3health s which age to match the patients were selected into the study of clinical.Clinical studies. Groups:patients with unstable angina, patients with unstable angina and non-ST-segment elevation acute myocardial infarction, ST-segment elevation myocardial infarction group and the healthy control group. All patients with coronary heart disease were treated with Percutaneous coronary intervention (PCI) treatment and patients with AMI are undergoing emergency PCI treatment.1.2The venous blood in the patients with coronary heart disease was taken before PCI,1and5days after PCI. So did the healthy control group. Their blood were collected to separate vials for good transferr. Putting it to-70℃freezer in short-term storage and liquid nitrogen for long-term preservation.1.3Using real-time quantitative fluorescent PCR (qRT-PCR) to measure the expression of miR-92a, miR-126. All detection processing was carried out in accordance with the kit instructions on kangcheng company in shanghai city. At the same time we detected indicators in the blood of patients,such as.:the serum troponin I (cTNI), N-terminal pro-brain natriuretic peptide (NT-proBNP), ultra-sensitivity C-reactive protein (hs-CRP), the DD dimer (D-D). we analysised the patients with a complex flow phenomena after PCI surgery.1.4Statistical analysis:SPSS13.0statistical software. Using the t test or analysis of variance, the parameter is x±s. The correlation analysis is the Pearson correlation method, P<0.05was considered statistically significant.2. The collection and testing of specimens for animal experiments peripheral blood in miRNAs2.1Mice myocardial ischemia-reperfusion model and grouping:The left coronary descending artery of Kunming mice was ligation. The successfully mark of model is the ECG ST-segment elevation and mouse myocardial white colors. Thirty minutes later, removing of the ligature. The mouse myocardial colors recover to red and ST segment depression as a sign of success of reperfusion model of myocardial recovery. This animal model is mature. Groups:sham group (9cases), model group (9cases), the the intervention model group (9cases), with three in each group were made to histological sections. The intervention model group was given Atorvastatin (2mg/kg) and the other two groups were given saline, a total of seven days.2.2Specimen collection and completion of relevant indicatorsSix mouse in each group were designed to coronary artery ligation for30minutes before reperfusion12h. Some portion of the serum blood was used to detect the related indicators,the other whole blood (about400ul) were collected good transferred to separate vials putting to-70℃refrigerator for short-term storage and liquid nitrogen for long-term preservation. After killed mice different specimens (ischemia area, marginal zone) organization were deal with by fixed-dehydration-embedded in paraffin-slices.2.3To detect relevant indicators The specimens was stained by Evans blue. The apoptotic index was detected by the TUNEL method. Whole blood was used by quantitative real-time fluorescence PCR (qRT-PCR) to determine the expression of miR-92a, miR-126. CK-MB with immune chemiluminescence, a chemical test of superoxide dismutase (SOD), malonic aldehyde (MDA).2.4statistical analysisSPSS13.0statistical software. Using the t test or analysis of variance, the parameter is x±s. The correlation analysis is the PERSON correlation method, P<0.05was considered statistically significant.Results:Results of clinical trials1.The results of qRT-PCR showed that the expression level of miR-92a and the healthy control group of patients with coronary heart disease is increased between the two group.There is a significant difference (P<0.05); In the different types of patients with coronary heart disease the miR-92a expression increased with the severity of increased (P<0.05); After PCI miR-92a expression levels reduced.The miR-92a expression levels between before and after PCI was significant differences (P<0.05); same type of patients with coronary heart disease the expression level of miR-92a reduced over the time change (P<0.05).2. The expression level of miR-126and the healthy control group of patients with acute myocardial infarction (non-ST-segment elevation myocardial infarction and ST-segment elevation myocardial infarction) reduced, a significant difference between the two group (P<0.05); MiR-126expression levels of other types of patients with coronary heart disease and the healthy control group had no significant difference (P>0.05); Acute myocardial infarction after PCI miR-126expression levels increased. The miR-126expression between before and after PCI was significant differences (P<0.05);The same type of patients miR-126expression levels with coronary heart disease after PCI increased and changed over time (P <0.05).3we found that the cTNI,hs-CRP and DD.level in the blood of patients with acute myocardial infarction were not correlated with the expression level of miR-92a was (r=0.162,0.424,0.361,P>0.05). NT-proBNP levels was significantly correlated with miR-92a expression levels (r=0.583, P<0.05). NT-proBNP, hs-CRP, DD levels were significantly correlated with miR-126expression levels (r were-0.605,-0.666,-0.616, P<0.05). cTNI were not significant correlation with the expression level of miR-126(r=-0.368, P>0.05).4. The study about no-reflow phenomenon in patients with acute myocardial infarction and reperfusion treatment of the emergence PCI:(1) miR-126expression levels, miR-92a expression levels, hs-CRP levels and D-D levels between the two groups was significant difference (P<0.05).(2) miR-126expression levels was significantly correlated with hs-CRP, D-D levels (r1=-0.666, r2=-0.616, P<0.05), there are significant relationship between the expression level of miR-92a and hs-CRP levels, D-D level (r1=0.638, r2=0.623, P<0.05).The results of animal experiments1.The miR-92a, miR-126relative expression level (2-△△CT)were detected by using qRT-PCR method in the mice myocardial ischemia-reperfusion group (sham group, ischemia-reperfusion group, drug intervention group).We found that the expression levels both in the sham group and ischemia-reperfusion group were significant difference in the drug intervention group (P<0.05)2. The relationship between serum markers of myocardial cell apoptosis index and miR-92a and miR-126in three groups:Sham group, ischemia-reperfusion group, drug intervention reperfusion group.(1) miR-92a and CK-MB, MDA changes are consistent with and the change of SOD is the opposite.(2) The change of miR-126expression is opposite to the changes of CK-MB and MDA and are consistent with SOD.(3) The changes miR-92a expression is consistent apoptosis index trend. The trend of changes in the amount of miR-126expression is opposite to apoptosis index.Conclusions:1. MiR-92a and miR-126were involved in the process of myocardial ischemia and reperfusion. It is Possible for early diagnosis and prediction of no-reflow phenomenon and may provide ideas for early intervention.2.We fond that the expression of miR-92a and miR-126was associated with oxygen free radicals and myocardial apoptosis in myocardial ischemia-reperfusion injury,3.The intervention of statins can alter the expression of miR-92a and miR-126. |
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