Effect Of Sivelestat Sodium Hydrate On Cerebral Ischemia-Reperfusion Injury And Proteome Study

STUDY ON THE EFFECTS OF ELASTATINAL SIVELESTAT SODIUM HYDRATE ON CEREBRAL ISCHEMIA–REPERFUSION INJURYBackground and objective:Ischemic cerebrovascular disease is a common disease for older persons, and it is also a major disease which leads to death and disability for the elderly. Many animal studies and human clinical trials reported that inflammation induced by cerebral ischemia-reperfusion injury related with neutrophilic leukocyte (polymorphonuclear leukocyte, PMN)[1]. When activated by cerebral ischemia, neutrophil release elastase(neutrophil elastase, NE) which take participate in the process of the degradation of extracellular matrix components such as elastin, fibronectin and proteoglycan; and promote the movement of PMN to endodermis and the adhesion of PMN to the vascular endothelial cell[2], induce the damage of vascular endothelial cells and tissues.On clinically, ONO-5046 was used to treat acute lung injury induced by different reason and respiratory distress syndrome[3] [4]. Recent studies have shown that ONO-5046 could significantly attenuated ischemia-reperfusion injury of hepatic, myocardium, and spinal by modulating neutrophils priming, inhibiting NE activity, decreasing inflammatory mediators and improving energy after ischemia-reperfusion[5-10]. So far, there haven't been reports about the function of ONO-5046 in total cerebral ischemia–reperfusion(CIR) injury. As there are many similar things among brain, liver and myocardial ischemia, such as inflammation, oedema and the change regional blood flow, so we can infer that ONO-5046 have protective effect for brain ischemia.Methods:SD rats were randomly divided into sham operation, model and ONO-5046 groups, The latter group were further subdivided into low, middle and high dosage subgroups. The CIR was induced by occlusion both sides of arteria carotis communis accompany with hypotension, ischemia 20 min, reperfusion 24h. ONO-5046 at doses of 3, 10 or 30 mg/kg·d with 24h continuous infusion by femoral vein, sham operation and model groups administer physiological saline. ELISA was employed to measure the content of NE. Measuring brain water content. The histopathology change was examined by HE staining. The content of malondialdehyde(MDA), activities of superoxide dismutase (SOD) and myeloperoxidase(MPO) in brain were measured by spectrophotometer. Immunohistochemistry was employed to assess the expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κB(NF-κB).The topic is aim to study whether ONO-5046 can protect the neurons against CIR and the possible mechanism of action, so as to find a new potent agent for ischemic cerebrovascular disease and other neurodegeneration diseases.Results and conclusion:(1) Sivelestat sodium hydrate inhibition can inhibit NE release, lessen the cerebral edema and pathologic change and the phenomenon in a dose-dependent manner. It is suggested that ONO-5046 have protection on the cerebral ischemia-reperfusion injury in rats.(2) Sivelestat sodium hydrate could markedly reduced the content of MDA, increased SOD activity, decreased MPO activity, and reduced the levels of inflammatory mediators(TNF-α, NF-κB) to their normal 1evel. It is suggested that the protection of ONO-5046 from cerebral ischemia-reperfusion injury maybe related to those factors. EFFECT OF SIVELESTAT SODIUM HYDRATE ON PROTEOMICS IN RATS WITH GLOBAL CEREBRAL ISCHEMIAObjective:(1)To Compare the differences of proteins between the animal model of global brain ischemia–reperfusion and give neutrophil elastase inhibitors - sivelestat sodium hydrate(ONO-5046) control rat by proteomics. Analysis of two-dimensional gel-image was performed by PDQuest7.4.0 analysis software and obtained target proteins.(2) The MS/MS of the peptides from the differential proteins digested by tripsin were analyzed by HPLC-Chip-MS/MS and the original data were pre-processed by Spectrum Mill then searched in NCBInr database identify these proteins with HPLC-CHIP-MS/MS. Expected to find out the target and effective proteins related with cerebral ischemia and then explore the pathologic mechanism and the ONO-5046 effect on proteomics in rats with global cerebral ischemia.Methods:Twelve Sprague-Dawley rats (weighting 250-300g, 10-12 weeks) were randomly divided into 3 groups (sham-operation group, model group and ONO-5046 group)with 12 rats in each. The global brain ischemia model in rats was induced by occlusion both sides of arteria carotis communis accompany with hypotension for 20 min, followed by 24h reperfusion. Twenty minute after the occlusion, ONO-5046, dissolved in saline, at doses of 30mg/kg with 24h continuous infusion by femoral vein just after the reperfusion, sham operation and model groups administer physiological saline. Rats were decapitated immediately after reperfusion and the whole brain of each animal was rapidly dissected out. Brain were cut into two halves along fissura interhemisphaerica, then cut behind half of the right cerebral hemisphere, weighing, extracting total protein. Using bradford method to measure the protein concentration, then stored in-80°C. Used the total protein to run immobilized pH gradient isoelecticfocusing (IPG-IEF), and then vertical flat sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) to obtain the 2-DE gel maps. Then gels were stained with silver and differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by HPLC-CHIP-MS/MS.Results:(1)Use pH3-10 nonlinearity IPG to analysis protein, well resolution and repetitiveness 2-DE gel images of rat brain proteome were obtained.(2)Through the analysis of 2-DE images protein spots with PDQuest7.4.0 analysis software,there were 989±22 protein spots in sham groups, 1043±34 protein spots in model groups and 1027±29 protein spots in ONO-5046 groups. Compared with sham group, there were 21 differentially expressed proteins in model group, include 2 new proteins; group; 8 proteins were significantly up-regulated and 11 down-regulated. Compared with model group, ONO-5046 group have 9 proteins were significantly up-regulated and 6 down-regulated.(3)We identified 8 proteins by MS and moscot were, Serine protease 1, Heat shock cognate 71 kDa protein, Malate dehydrogenase, Fructose-bisphosphate aldolase A, Dihydrolipoamide acetyltransferase, Thioredoxin peroxidase 1, Adenylate kinase isoenzyme 1 and Phosphatidylethanolamine-binding protein 1.Conclusion:The ideal brain protein 2-DE images could be obtained with pH3-10 IPG DryStrip, running IPG-IEF and then vertical flat SDS-PAGE. Sivelestat sodium hydrate may promote the survival of injured neurons in rats by virtue of up or down-regulating the expression levels of proteins which concerned with cerebral ischemia injury.