| BackgroundS100A8/A9protein is a newly discovered inflammation-related protein, which plays a key role in the process of inflammatory response, especially in the regulation of innate immune response. S100A8/A9also known as myeloid-related protein-8/14(MRP8/14), calprotectin,27E10antigen or leukocyte protein L1. S100A8/A9is a heterodimer composed of the two proteins, S100A8and S100A9. S100A8and S100A9are first to be found as immunogenicity proteins in the neutrophils, both of which belongs to the calcium binding protein S100family members. The EF-hand structure, which can combine with Ca2+, is the most important functional domains for the protein. Once the protein combined with Ca2+, the protein will undergo conformational change, thus exposing the interaction with target proteins, which play the corresponding biological functions. Eexpression of S100A8/A9is tissue and cell depended, and is mainly expressed in myeloid cells-in neutrophils, accounting for50%of the soluble components of the neutrophil cytoplasm, and in monocytes, also take a constitutive expression. Platelets, endothelial cells, fibroblasts, epithelial cells, myocardial cells and malignant cells can also express S100A8/A9in certain circumstances. However, it can not be induced in T cells, suggesting that it mainly involved in congenital immune response.Recently, S100A8/A9as an important molecular of DAMPs, which broadly participates in the pathological process of many human inflammatory diseases, has become the hot spot of research. According to researches, serum S100A8/A9increased significantly in unstable angina, myocardial infarction; atherosclerosis of S100A9expression in platelets of acute plaque rupture and ST-segment elevation myocardial infarction patients is much higher than the amount of patients with stable and non-thromboembolic coronary heart disease; transcriptome studies found that plasma S100A8/A9can be used as a new predictor of myocardial infarction, forecast the risk of onset and recurrence of cardiovascular events.These findings suggest that S100A8/A9may be associated with coronary heart disease, especially in the inflammatory mechanism. Coronary heart disease has become the number one killer of threat to human health, and showing a rising trend in China. Myocardial reperfusion therapy is the key to the treatment of acute myocardial infarction. Clinical practice has fully proved that coronary reperfusion therapy is of great significance to save or improve left ventricular function in myocardial infarction patients, for it can save the dying myocardial, limit or reduce the size of infarction, reduce or limit the occurrence of left ventricular remodeling, and significantly improve in the fatality rate of acute myocardial infarction. However, reperfusion can cause a series of changes, such as generation of too much oxygen free radicals, excessive inflammation, calcium deposition, ect., which may lead to irreversible mitochondrial damage, causing myocardial cell death. Therefore, how to reduce myocardial ischemia-reperfusion injury has become an important topic of prevention of ischemic heart disease and clinical interventional treatment for heart disease.The pathophysiology of ischemia-reperfusion injury has not been fully elucidated, and the inflammation process during ischemia-reperfusion injury is considered to be a factor that can not be ignored. It has been reported that during the first six hours of acute myocardial infarction after reperfusion, neutrophils chemoattracted by tissue damage molecular immersed in the infarct area, and within24hours, the infiltrated neutrophils, in the promotion of cell adhesion molecules, migrated into the myocardial tissue. The one hand, the chemotaxis of neutrophils can lead to blood clots, and further increase the tissue ischemia and hypoxia. On the other hand, neutrophils activated within the tissue, and release a large number of inflammatory mediators, reactive oxygen species, collagenase and elastase, leading to further damage and destruction of the tissue, expand the area of infarction. Simple anti-neutrophil adhesion antibody and complement activation suppression therapy can not effectively inhibit the inflammatory response in reperfusion injury. S100A8/A9as an important inflammation starting and expanding factor, whether involved in ischemia-reperfusion injury, whether as a new target to inhibit tissue damage brought about by the inflammatory response in reperfusion injury are problems we are concerned to treat perfusion disorders. In addition, the injury is also an important factor in myocardial ischemia-reperfusion injury mediated by the apoptosis. A higher incidence of apoptosis in late phase reperfusion, which appeared in the necrotic myocardium surrounding area, indicates that apoptosis may play a certain role in the expansion of the scope of myocardial infarction after ischemia-reperfusion. As high concentration of S100A8/A9has a pro-apoptotic role, whether it can be take as the new target against cardiomyocyte apoptosis ischemia-reperfusion injury is a question to be answer.The S100A8/A9is the beginning of the inflammation and promoting factor, while high concentrations of S100A8/A9also has a pro-apoptotic role, can be used as a more upstream role of targets in the process of ischemia-reperfusion injury, and inducing apoptosis, blocking excessive inflammatory response, thereby reducing myocardial cell death has important significance for improving the effect of reperfusion therapy.Research purposesThis project is primarily concerned with the law in the natural course of the study of myocardial infarction model, S100A8/A9expression in myocardial tissue, and of S100A8/A9in MI tissue sources, evolution, and participation in the course of the disease stage; second study myocardial lack blood-reperfusion in the expression S100A8/A9situation of the S100A8/A9is involved in ischemia-reperfusion injury; final study S100A8/A9direct effect on myocardial cells, mainly in its withered induced myocardial S100A8/A9when the death of the role of reperfusion injury is involved in the induction of myocardial apoptosis.Research programs1.S100A8/A9expression in acute myocardial infarction modelBuild classic myocardial infarction in vivo experimental model of mouse left coronary artery ligation, and the separation of primary cardiac myocyte hypoxia as an in vitro model, detected by fluorescence quantitative PCR, Western blot and immunohistochemical methods after myocardial infarction organizations S100A8/A9spatial and temporal distribution characteristics, and expression of S100A8/A9primary myocardial hypoxia, as well as the part of the signaling pathways associated with the model activation.2. S100A8/A9expression of the myocardial cells under hypoxia-reoxygen and oxidative stressSeparation of primary cardiac cells with hypoxia-reoxygenation and hydrogen peroxide oxidative stress to deal with as ischemia-reperfusion in vitro model using the immunoblotting method of S100A8/A9in primary cardiac myocyte hypoxia- reoxygenation and oxidation should be stimulated expression and part of the kinase activation state.3. Exogenous S100A8/A9inducing apoptosis of myocardial cellsSeparation of primary cardiomyocytes, exogenous S100A8/A9protein stimulation, MTT assay and immunofluorescence techniques preliminary study on the effects of S100A8/A9on myocardial cell survival and apoptosis.Result1. S100A8/A9expression in acute myocardial infarction model1.1Successly in mice construct the left coronary artery ligation model, detection mRNA and protein expression of S100A8/A9in sham group and30min,2h,6h,12h,24h,3d,7d after myocardial infarction. The results show that the amount of S100A8/A9expression in2h-24h and7d ligation gtoup are higher than the sham group with significant difference (P<0.05). S100A8/A9in6h-12h group is80-90times y higher than sham group, and in24h group reach the peaked,130times than the sham group, after myocardial infarction3to7days there is still a certain degree of increases,5times over sham group. Protein level show S100A8/A9began to increased significantly in6h group, and in6-12h reach the peak, continue to24h, and in the third day began to decline significantly, consistent with the change of the mRNA level.1.2Select sham group and24h after myocardial infarction group for immunohistochemical and HE dyeing, the results showed that, MI team infarcted tissue parts have the obvious S100A8/A9distribution (brown particle), and sham group without dyeing positive particles. And at the same time we do the sham and MI negative control group (that is, only join second antibody to incubation and show color) to let out the S100A8/A9antibody nonspecific binding. Immunohistochemical negative antibody group in the control group and observed when, S100A8/A9 antibody show color in area overlap negative control group on inflammatory cells infiltrating area, but not in the distribution in the heart of the infarction. HE dyed shows that compared with the sham group, MI team myocardial infarction occurred parts coagulative necrosis, myocardial cell swelling, boundary is not clear, muscle fiber disorder, eosinophilic cytoplasm increased, nuclear dissolved or disappear, fusion is red dye homogeneous thing. Infarcts seen edge hyperemia and neutrophils infiltrating.1.3Immuno blot detection NF-kB, JAK1, JNK signal pathway activation time. NF-kB show activation30min after myocardial infarction, and in6to12h the most obvious, the activation is more lasting; And JAK1activation time is earlier,30min after myocardial infarction is enabled, and in2-6h reach a peak;12to24h after myocardial infarction, JNK is obviously increased, but in contrast of NF-kB and JAK1, the activation of the phase is late and relatively weak.1.4The stimulate to myocardial for4h oxygen separation, then measure the mRNA level and protein level of S100A8/A9vs control group are of no difference (P>0.05). Immune imprinting detection p38signaling pathways, show p38signal pathway in a low4h had been apparent activation.2. Influence of anoxia-reoxygenation and oxidation stress on the myocardial expression of S100A8/A92.1Set control, reoxygenation10min,2h,4h group, detection S100A8/A9mRNA and protein level changes. The results showed that anoxia for2h then reoxygenation2-4h, the mRNA all have increased, about three times as much as control group, though there was no statistically difference (P>0.05), but the protein level can be observed obviously increase, especially after oxygen4h group.2.2Set0.2mM,2mM two kind of concentration and10min,2h two time for hydrogen peroxide stimulation, detection mRNA and protein level S100A8/A9 changes. Results compared with the control, mRNA in0.2mM hydrogen peroxide treatment2h group rise60-90times, and for2mM hydrogen peroxide processing2h group the20times. Protein level of change consistent with the mRNA level changes.2.3Myocardial cells in anoxia-reoxygenation and hydrogen peroxide oxidation process of stimulation all accompanied with p38signal pathway activation. In anoxia4h and reoxygenation for2h and4h has obvious activation; Different concentration hydrogen peroxide stimulation also have different degree of activation.3. Exogenous S100A8/A9of myocardial cell apoptosis of promoting role3.1Build S100A8, S100A9mice original nuclear expression vector, then use His affinity resin chromatography target protein purification to get a pure recombination fusion protein His-S100A8and His-S100A9, protein size11.11KD and13.8KD respectively, consistent with the theoretical value and the concentration to meet the requirements.3.2The purified S100A8/A9protein undergo response in vitro to form dimers, then use50ug/ml and100ug/ml concentration to stimulate the myocardial cells48h, whereas the control with PBS. The results show that, compared with the control, S100A8/A9treatment group of myocardial survival rate drop about8-10%(**P<0.001), and as S100A8/A9stimulate concentration increases, the survival rate of myocardial cells gradually reduced.3.3The purified S100A8/A9protein undergo response in vitro to form dimers, then use50ug/ml concentration to stimulate myocyte for48h, observe under visible and fluorescence, S100A8/A9treatment group in light microscopy found myocardial cell size decreased and variable is long and narrow, fluorescence microscopic found myocardial nuclei empty bubble produced, solid condensed chromatin reach. Conclusion1. Source of high expression of S100A8/A9in parts of the myocardial infarction may be from the viable myocardium, and inflammatory cell infiltration and activation, rather than infarction of the myocardium itself.2. S100A8/A9in the process of myocardial ischemia-reperfusion injury may have an important role in survival after which reperfusion myocardial expression of S100A8/A9chemotaxis of neutrophils to the excessive accumulation and activation of the starting.3. Exogenous S100A8/A9can reduce myocardial cell survival and induced cardiomyocyte apoptosis. |
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