| PrefaceIt is considered that oxygen free radicals and their reactive lipid peroxidation is one of the mechanisms of ischemic-reperfusion damage. In process of ischemic-reperfusion damage, Oxygen free radicals can damage proteins, lipids, nucleic acids, can directly act on the biofilm of unsaturated fatty acids and injury biofilm, following with the body oxidation system being activated, oxygen free radical generation, triggering lipid peroxidation, protein Denaturation, membrane barrier function severe impairment, which leads to cell death. As an important mediator of the damage of nerve cell in ischemic brain tissue and blood-brain barrier, oxygen free radicals may exacerbate the occurrence and development of brain edema, post-ischemic hemorrhage and tissue necrosis in the pathological process after ischemic stroke.During the pathological process of brain ischemia reperfusion, the source of oxygen free radical has many kinds of ways. NADPH oxidase (reduced coenzyme?, the nicotinamide adenine dinucleotide phosphate) is one of major factors in Peroxide produce and closely related to the Pathological effects of oxygen free radicals in many cells. Large number of experimental evidences show the expression of NADPH oxidase increases in ischemic brain tissue, which is involved in death of neurons after ischemic stroke. Recent studies have shown that NADPH oxidase catalytic subunit gp91 phox is closely related to neurological disease like ischemic stroke. NADPH oxidase plays an important role of oxygen free radical generation in cardiovascular system. Overproduction of oxygen free radicals causes lipid peroxidation and destroys the integrity of blood-brain barrier. The ischemic blood-brain barrier damage of gp91phox gene knock-out mice is more significantly relieved than that of the wild-type mice's, which indicates that the NADPH oxidase plays an important role in ischemic blood-brain barrier damage.Studies have shown that in ischemic stroke, excessive production of oxygen free radicals is considered to be an important reason for induced expression of MMP-9 (matrix metalloproteinase-9) and blood-brain barrier damage, in addition to direct oxidative damage of blood-brain barrier, oxygen free radicals in ischemic brain tissue is the important triggering factor for the induced expression of MMP-9 which mediates proteolysis and degradation of structural components of blood-brain barrier. A large number of experimental data shows that proteolysis, mediated by MMP-9 in ischemia reperfusion brain damage, led to the degradation of structural components of blood-brain barrier, oxygen free radicals induced MMP-9 expression in cerebral ischemia. In the SOD1 transgenic mice (SOD over-expression) of the ischemic brain tissue, MMP-9 expression and blood-brain barrier damage were significantly reduced. Studies also have shown that in vascular system, NADPH oxidase-mediated oxygen free radicals involved in the activities of MMP-2 and MMP-9. However, there is no information involved in NADPH oxidase regulating the MMP-9 expression in the ischemic brain tissue.E-selectin is an inducible adhesion molecule, which can mediate adhesion between the activation of endothelial cells and the neutrophil, and directly involved in aggregation and infiltration of inflammatory cells. In the period of cerebral ischemia-reperfusion injury, leukocyte adhesion molecule-mediated pavement, adhesion and metastasis, so that white blood cells travel through the blood vessel wall endothelial cells and release of many organizations inflammatory factors. In the process of close adhesion of leukocytes and endothelial cells and Transfer of leukocytes across endothelium, local hypoxia can activate endothelial cells to produce oxygen free radicals, causing damaging of vascular endothelial cells, while oxygen free radicals also enhance the adhesion of endothelial and leukocytes, and release much more flammatory factors. In brain Ischemic tissue, the level of adhesion molecule mRNA significantly increased, a large number of adhesion molecule synthesis, there is a mutual promotion existence between the E selectin and the oxygen free radicals. Early in the ischemic, blocking the expression of P-selectin, neutrophil accumulation and adhesion in the ischemic area reduced which can reduce death of neurons and infarct volume, improve stroke patients' Prognosis. In a variety of animal models of cerebral ischemia, the animal survival rate increases after the use of anti E-selectin antibody treatment. NADPH oxidase and the generation of oxygen free radical damage is closely related to cerebral ischemia, the mutual influence between the NADPH oxidase and E-selectin has not been reported. ObjectiveThis research establishes the focal cerebral ischemic-reperfusion rat model (model of middle cerebral artery occlusion), detects the changes of the gp91phox NADPH oxidase subunit mRNA and protein expression in the ischemia-reperfusion brain injury, discusses the role of NADPH oxidase in ischemia-reperfusion; Meanwhile, preprocessing with NADPH oxidase inhibitors before the production of MCAO model, further observating the change of MMP-9 expression and E-selectin expression in ischemia-reperfusion brain, the correlation between NADPH oxidase and MMP-9 induced expression; discussing the role and mechanism of NADPH oxidase in the ischemia-reperfusion brain injury and the correlation between NADPH oxidase and E-selectin. Make clear whether MMP NADPH oxidase plays its role by adjusting the expression of MMP-9 and E-select element in ischemia-reperfusion injury, and to provide the new information and ideas for new treatment exploration of ischemia-reperfusion injury. Experimental Methods(1) The establishment of experimental rat model of focal cerebral ischemia18 healthy Sprague-Dawley (SD) male rats weighing 290 to 320 g. Rats were randomly divided into control and experimental groups,6 rats in each group. Normal control rats without any treatment, other processes including feeding the same with the experimental group. Experimental groups of 12 rats were divided into two subgroups, a group of six used for MCAO TTC staining to observe the success of MACO another group of 6 used for MACO modeling manufacture,18 SD male rats, weight 290-320g, using mixture gas isoflurane containing NO2:O2 (70%:30%) for anesthesia (surgical induction of isoflurane concentration was was 4%; maintainance anesthesia of isoflurane concentration was 1.75%.). In the whole experimental stage (before, during the ischemic, and ischemia reperfusion), the rats were in anesthesia.Middle cerebral artery occlusion (MCAO) was founded by Koizumi J, in 1986, bolt inside the cavity model (12). (2)TTC stained brain slicesTTC (2,3,5-Triphenyl-2 H-tetrazolium chloride,2,3,5-triphenyl tetrazolium chloride) is a fat-soluble light-sensitive compound, originally used to test the viability of seeds, from 1958, It began to dyeing to detect ischemic infarction of Mammalian tissue. It is the proton acceptor of the pyridine-nucleoside structure enzyme system in the respiratory chain. And normal tissue dehydrogenase responses in the red, but ischemic tissue cannot respond due to the dehydrogenase activity decline, which will not produce changes and present Pale color. 2g 2,3,5-triphenyltetrazolium chloride (TTC) was dissolved in 100ml phosphate buffer, dark, prepared before being used. Coronal brain slices for 1mm thick staining by 2,3,5-triphenyltetrazolium chloride (TTC) confirmed that the success of rat MCAO model. (3) Ischemic tissue after treatmentThe rats were decapitated at 2% of sodium pentobarbital over-anesthesia after reperfusion, being sacrificed by decapitation the brain, being taken back of the head to collect the brain tissue both of non-ischemic and ischemic hemisphere, then being used to analyze gp91phox protein levels and mRNA expression, NADPH oxidase activity, MMP-9 protein expression, or embedded in paraffin, sliced, E-selectin immunohistochemical stained.(4) SYBER (?) Green gp91phoxmRNA quantitative PCR analysis brain tissue, collected by the above method, placed in glass homogenizer, press 100g:3ml trizol reagent ratio of added to homogenate treatment. Then based on DNA-Free kit technical manuals to separate and purify total RNA. Strict accordance with the Trizol RNA extraction flow, after the reverse transcription reaction, the specific method in accordance with the TaqMan (?) reverse transcription kit instructions. Then 1 RT products were amplified under 7900HT Fast Real-time PCR system the expression level of mRNA were detected.(5) Gp91phox protein imprinting analysisTaking the the brain of the ischemia hemisphere and the non-ischemic hemisphere to the RIPA buffer fluid which contained a mixture of protease inhibitors to homogenate. Centrifugal and take the supernatant fluid, take 10%sds-page acrylamide gel electrophoresis, transfer to the nitration fiber membrane, join monoclonal anti-gp91phox to anti-incubation, join the HRP combined antibody of anti-rats to incubate, and then, use chemiluminescence kit to develope the fiber membrane in order to detect protein, the specific steps is according to the operation instructions, with Kodakb 4,000 digital imaging system of the image workstation to enhancement observe and quantitative calculate strip.(6) NADPH oxidase activity determination Use peroxide-induced lucigenin photoemission determination of NADPH oxidase activity, as previously papers mentioned, take the brain homogenate, then join the Krebs bufferSolution contained lucigenin, analysis NADPH oxidase activity through the photometric.(7) MMP-9 gelatin zymography analysisTake the brain tissue homogenates, both the ischemic hemisphere and non-ischemic hemisphere. Mensurating concentration of homogenate protein. MMP-9 was analyzed by gelatin zymography, separated under non-reducing conditions using 1mg/ml gelatin containing 10%SDS-polyacrylamide gel. Rinse gel with Triton X-100, then incubate in developing buffer under 37oC for 72 hours, followed by dyeing, bleaching, until the gelatin digestion with clearly displayed on dark blue background. Analyzing Gelatin digestion bands image intensity with the Kodak 4000 workstation.(8) E-selectin immunohistochemistryBrain tissue paraffin dewaxing, hydration, antigen retrieval, endogenous peroxidase blocking, adding rabbit anti-mouse E-selectin monoclonal antibody, incubated goat anti-mouse 1gG plusing. With biotin-labeled, incubated in PBS solution Adding an avidin peroxidase, adding freshly prepared AEC solution, observed under the microscope, the positive brown or brown color. Results1. TTC staining confirmed the success of MACO modeling:MCAO TTC staining is one of the most convenient way to prove the success. 1mm thick brain tissue biopsies TTC staining 90 minutes of MACO, and 22.5 hours reperfusion produced ischemic hemisphere infarction. The major areas involving the cortex, subcortical white matter, striatum, etc. shows that the MCAO model making success.2. Ischemia-reperfusion brain tissue NADPH oxidase subunit gp91phox mRNA up-regulation and protein expression increased:Quantitative RT-PCR revealed that Compared to the control group and the non-ischemic side, NADPH oxidase subunit gp91phox mRNA expression in the the experimental Group (brain ischemic-reperfusion) was significantly increased. In the experimental group rats, the NADPH oxidase subunit expression in ischemic brain tissue than the non-ischemic brain tissue is significantly increased?Western blot analysis showed that gp91phox protein in non-ischemic hemisphere was low, but the levels were significantly increased in the ischemic hemisphere in the MCAO brain.3. The changes of NADPH oxidase activity in ischemia-reperfusion brain tissue:Experiments using lucigenin enhanced chemiluminescence to detect the activity of NADPH oxidase, showed NADPH oxidase activity of the ischemic hemisphere brain in the experimental group were to be significantly reduced than the non-ischemic brain hemisphere and the control group.4. NADPH oxidase inhibitors inhibit gp91phox protein expression and enzyme activity:Western blot analysis showed that, in the menstruum control group treated rats, compared with non-ischemic hemisphere in the organization, gp91phox protein levels in ischemic hemisphere brain increased significantly (P<0.05, and non-ischemic hemisphere compared brain tissue), indicating ischemia-reperfusion have a feature to make the gp91phox protein expression significantly increase.in the ischemic hemisphere. Meanwhile, compared with menstruum control group, in the NADPH oxidase inhibitors group, the gp91phox protein expression significantly reduced, which showed NADPH oxidase inhibitors inhibit gp91phox protein expression in the ischemic hemisphere. But gp91phox protein expression did not change significantly in two groups of non-ischemic hemisphere, indicating that the NADPH oxidase inhibitors can inhibit gp91phox protein expression in ischemic brain tissue (P <0.05). At the same time, in the NADPH oxidase inhibitor group of ischemic brain tissue, NADPH oxidase activity significantly reduced than the menstruum control group', indicating that the NADPH oxidase inhibitors can inhibit the NADPH oxidase, gp91phox protein expression and enzyme activity.5. NADPH oxidase inhibitor inhibit MMP-9 expression in ischemia-reperfusion brain tissue:The results showed MMP-9 protein expression, in two brain ischemia hemisphere groups, were significantly increased than that in non-ischemic hemisphere (P< 0.05), which indicated that ischemia-reperfusion significantly induce MMP-9 brain protein expression. On the other hand, compared to the menstruum control group, the expression of MMP-9 was significantly inhibited in the NADPH oxidase inhibitors group (P< 0.05), indicating that NADPH oxidase inhibitor not only Inhibit NADPH oxidase expression and activity but also inhibit MMP-9 expression of ischemia-reperfusion brain tissue.6. NADPH oxidase inhibitors inhibit the expression of gp91phox protein and E-selectinWestern blot analysis showed that, in the menstruum control group treated rats, compared with non-ischemic hemisphere in the organization, gp91phox protein levels in ischemic hemisphere brain increased significantly, and E-selectin level also increased; however, in the NADPH oxidase inhibitors group, the gp91phox protein expression significantly reduced, and E-selectin expression also significantly reduced, which indicated that there was somewhat relation between NADPH oxidase expression and E-selectin expression.E-selectin positive cells increased significantly both in the CA-1 area of Hippocampus and in cortex in rats with ischemic/perfusion by immunohistochemistry method, but the results are much different in the NADPH oxidase inhibitors group, E-selectin reduced significantly in the same parts compared with the menstruum control group. The phenomena is obviously in the cortex area surrounded the ischemia. Conclusions1. NADPH oxidase gp91phoxmRNA, protein expression and NADPH oxidase activity were significantly increased in the ischemia-reperfusion brain tissue.2. NADPH oxidase inhibitor can inhibit NADPH oxidase activity and the induction of MMP-9 expression in the ischemic brain tissue, The expression of MMP-9 correlated NADPH oxidase gp91phox in the ischemia-reperfusion brain tissue, NADPH oxidase gp91phox is a major precipitating factor for the expression of MMP-9 induced in the ischemic brain tissue.3. The expression of E-selectin correlated NADPH oxidase in the ischemia-reperfusion brain tissue, the expression of E-selectin was suppressed followed NADPH oxidase was inhibited, which indicated that NADPH oxidase mediated E-selectin expression through some way after ischemia-reperfusion. |
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