| Background: Acute myocardial infarction(AMI) is a serious cardiovascular emergency and critical disease that harm to human health. Clinical treatment of AMI is mainly thrombolysis(urokinase,streptokinase, etc.) and direct percutaneous transluminal coronary angioplasty(PCI) in the shortest time after infarction, to reduce the infarct size, save the myocardial cells lack of oxygen and energy supply which were on the verge of death. It is often accompanied by myocardial ischemia reperfusion injury in the treatment and natural turnover course of AMI, which causes a large range of myocardial cell death, mainly because the process of reperfusion accelerates the death of the severely injured cells, accompanied by a strong inflammatory response whose characteristic is inflammatory cell infiltration and inflammatory cytokine production, may eventually lead to myocardial infarction, fibrosis, cardiac hypertrophy and heart failure.Recent studies found that the level of inflammatory cytokines is much directly related with the damage of cardiac function and necrosis of cells post ischemic,indicates inflammatory response played a very important role in myocardial ischemia- reperfusion injury(MI/RI).It is exciting that many of the basic experimental results confirmed that the occurrence and development of inflammatory responses through drug antagonism can effectively down regulate the inflammatory response, thereby improving the ischemia-reperfusion injury. In the myocardial ischemia-reperfusion model of rats, Fucoidan can inhibit activation of inflammatory pathways, and reduce the expression of inflammatory mediators and inflammatory cell infiltration, reduce the damage caused by ischemia-reperfusion, thus protects rat liver.Dongdong Sun's research also proved that luteolin can inhibit the activity of MPO and expressions of inflammatory cytokines including IL-6, IL-1? and TNF-a, which are beneficial to reduce the myocardial infarction area and improve cardiac function after myocardial ischemia-reperfusion in diabetic rats.After more than ten years of exploration research, our group found that TRAIL/DR5 closely associated with myocardial infarction, the early use of human sDR5 derived fromyeastthrough blocking TRAIL/DR5 pathway significantly reduced myocardial infarct size in AMI model rats, reduced the extent of myocardial infarction, relevant achievements have been granted national invention patent(patent number:ZL 201210442685.6). Additionally, TRAIL binding to its receptor can mediate inflammatory responses. Inorder to extend the half-life, increase the efficacyin vivo, facilitate the production and purification of sDR5,and better conversion to drug development, We further integration the sDR5 and human IgG1 Fc fragment formed sDR5-Fc antibody fusion proteinsand build stably transfected cell line of CHO for large-scale expression of sDR5-Fc fusion protein, obtained high yield and good activity of theantibody fusion protein.And hypothesized that sDR5-Fc protein can also block TRAIL, inhibit inflammation, improve myocardial ischemia reperfusion induced inflammatory damage, limit the expansion of myocardial infarction area to protect the myocardial cells.On the basis of reports and our previous lab study, to explore the effect of inflammatory response and sDR5-Fc in myocardial ischemia-reperfusion, we detect the effect of sDR5-Fc on myocardial infarct size and expression of inflammatory cytokines, inflammatory cells infiltrationand myocardial fibrosis in rats,evaluate the feasibility of human sDR5-Fc protein as a new therapeutic drug for myocardial infarction. The results will provide experimental basis for further revealing the mechanism of myocardial ischemia reperfusion injury, and to provide new ideas for the effective prevention and treatment of MI/R.Purpose:Establish MI/R of rats and sought to present the mechanism of sDR5-Fc in antagonism to myocardial ischemia and reperfusion and related inflammatory reaction from morphological structure, the level of protein and RNA and histology of heart tissue in experimental rats.Method:1. Models and materials: we choose 8-week old Wistar male rats, mice were divided into three groups according to the random number table method. Through the ligation the left descending coronary artery for the myocardial ischemia and 1h after releasing the ligature for reperfusion, the model of rat acute myocardial ischemia-reperfusion was successfully established. and give corresponding treatment: drug group(sDR5-Fc, 16mg/kg), phosphate buffered saline solution group(phosphate buffer solution, PBS,) and sham operation group(Sham, only thoracotomy without ligation, the else were treated same to PBS group).3h, 24 h and 2w after reperfusion, heart tissue were isolated below the ligature of the heart. Sham group set3 h, 24 h, 2w 3 time-point as the control;2. Identification of the model and the effect of drug: by Evans blue-2,3,5-chloride triphenyl tetrazolium(Evans blue-2,3,5-Triphenyltetrzolium chloride, Evans blue-TTC) double staining method to detect myocardial infarction area; using hematoxylin-eosin staining(Hematoxylin-Eosin, HE) to detectmorphology and the structure of tissue slice;3. Detection the expression of inflammatory factors in the model: Determination of the expression of inflammatory factors: TNF-?, IL-1? and IL-6 in myocardial tissue by real-time quantitative polymerase chain reaction(RT-PCR) and Western blot(Western blot, WB);Analysis the results of WB by Quantity One.4. Inflammatory cell infiltration was detected in the model: the expression of inflammatory cells such as macrophages and T lymphocytes was detected by immunohistochemical(IHC);5. Heart functiondetection: the cardiac functionof the rats with 2 weeks reperfusion were tested by echocardiography;6. Myocardial fibrosis detection: detect the myocardial fibrosis of the rats with 2 weeks' reperfusion by Masson three color staining.Result:1. The AMI/R model of rat was successfully established.2. Morphological changes were detected in the rat myocardial tissue by HE staining. It is visible that myocardial cells arranged in disorder at infarction site of PBS group, some cells showed nuclear fragmentation and myocardial striations disappear;Compared with PBS group, in the infarcted zone of myocardial cells were spindle shaped arrangement is neat and orderly, and myocardial striations were obvious of the I/R+sDR5-Fc group.3. RT-PCR detect the expression of inflammatory cytokines in myocardial tissue. Results show that,compared with sham group, the expression of TNF-?, IL-1? and IL-6 increased in PBS group; compared with PBS group, the expression of TNF-?, IL-1? and IL-6 was down regulated in rats with sDR5-Fc injection.4. Western blotting detect the expression of inflammatory cytokines in myocardial tissue. Results show that, compared with sham group, the expression of TNF-?, IL-1? and IL-6 increased in PBS group;compared with PBS group, the expression of TNF-?, IL-1? and IL-6 was down regulated in rats with sDR5-Fc injection.5. Immunohistochemical detection found that macrophages, T lymphocytes increased in in the PBS group compared with Sham group; while after treated with sDR5-Fc, the infiltration of macrophages, Tlymphocytes decreased than PBS group.6. In the rats of myocardial ischemia for 1h and reperfusion for 2 weeks, Echocardiography showed that, compared with the Sham group, there was obvious myocardial movement, ST segment elevation in the PBS group. After treated with sDR5-Fc, the ST segment recovered smoothly, cardiac function was significantly improved.7. In the rats of myocardial ischemia for 1h and reperfusion for 2 weeks, compared with the Sham group, the myocardial fibrosis was obvious in PBS group, and the myocardial fibrosis size of sDR5-Fc group was significantly decreased than that of PBS group.Conclusion: sDR5-Fc can reduce the infarction size, the expression of inflammatory cytokines secretion and inflammatory cell infiltration of ischemia-reperfusion myocardial, and reduce the fibrosis size and improve cardiac function in rats. Thus proves that sDR5-Fc plays a protective role of myocardial cells in rats with ischemia-reperfusion injury. |
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