Establishing The Rat Model Of Acute Hypobaric Hypixia Injury And Proteomics Research Of Cerebral Cortex Mitochondria

Objective: To observe the changes of rats'cerebral cortex and lung after hypobaric hypoxia injury simulated acute high altitude exposure, and analyze the expression of mitochondria proteins from cerebral cortex by 2-DE and MALDI-TOF-MS to find any possible molecular markers and drug treatment targets associated with altitude exposure.Methods: Female Sprague-Dawley rats were randomly divided into control group and hypobaric hypoxia injury groups. The rats of injury groups were subdivided into hypobaric hypoxia 6h, 12h and 24h groups according to the duration of hypobaric hypoxia exposure. The rats were put into a specially designed decompression chamber with controlled temperature, relative humidity, PO2, barometric pressure to simulate altitude of 7000m. After hypobaric hypoxia exposure, the morphological changes of cerebral cortex and lung were studied by H.E and toluidine blue stain. Water content in brain and lung were determined by the wet-dry method, and the expression of AQP1,AQP4 were detected individually by western blot. A series of biochemical indicators like LDH, SOD, CAT and MDA in serum were assayed with biochemistry method.The protein samples extracted from cerebral cortex mitochondria of different groups were analyzed by 2-DE, the differentially expressed protein spots selected by PDQuest7.0 were identified by mass spectrometry. The types and roles of the differently expressed proteins could be ascertained preliminarily. As to whether or not these proteins were expressed in cerebral cortex mitochondria of rat, which would be checked by Western-blot in the end.Results: 1 The morphological changes in cerebral cortex of rats after hypobaric hypoxia: Different degrees of cellular injury were seen in hypobaric hypoxia injury groups. The morphology changes included cyton deflated, karyopyknosis and karyolysis, cytoplasm hyperchromatic. With increasing duration of hypoxia, the injury became serious.2 The detection of water content in brain and lung: Compared with control group, the water concents in brain and lung were remarkably increased in hypobaric hypoxia groups (P<0.01), and increased gradually with the injure duration increased.3 The detection of AQP1,AQP4 in brain and lung: The expression of AQP1 and AQP4 in brain and lung were increased after hypobaric hypoxia exposure compared with control group.4 The detection of biochemical indicators in serum: Compared to control group, the activities of LDH were remarkably increased in hypobaric hypoxia groups (P<0.01), and the activities of SOD were remarkably depressed in hypobaric hypoxia groups (P<0.01). Compared to control group, the activities of CAT were remarkably depressed in hypobaric hypoxia 6h, 12h groups (P<0.01), whereas there was a little increased in hypobaric hypoxia 24h groups than 12h group(P<0.05). Compared with control group, the contents of MDA were increased in hypobaric hypoxia groups.5 Mitochondrial isolation and electron microscopy: TEM indicates that pellets extraction were mainly mitochondria. The ultrastructure of purified mitochondria show that most of them were intact, and both outer and inner membrane ridges could be observed. The purity of mitochondria was over than 95%. 6 The results of 2-DE of mitochondrial proteins in cerebral cortex following hypobaric hypoxia injury: The immobilized gradient IPG strips (17cm, pH 3-10, Linear) were used, and most of these spots were dispersed into the scopes of PI 4.5-10 and MW 15kDa-105kDa. Compared with control group (P < 0.05), there was 12 up-regulated spots and 4 down-regulated spots in hypobaric hypoxia 6h group, 16 up-regulated spots and 5 down-regulated spots in hypobaric hypoxia 12h group, and 30 up-regulated spots and 6 down-regulated spots in hypobaric hypoxia 24h group analysised by PDQuest software. And 25 points of them represented same tendency in the three hypobaric hypoxia groups. Select the spots which had same expression tendency to be analysised by MALDI-TOF-MS.7 Identification of the different expressed protein spots via MALDI-TOF- MS: Ten protein spots were analyzed via MALDI-TOF-MS, which scores were more than 61 (p<0.05). They are dihydropyrimidinase-related protein 2, creatine kinase, isovaleryl-CoA dehydrogenase, Ts translation elongation factor, F1-ATPase beta subunit, 3-mercaptopyruvate sulfurtransferase, Electron transfer flavoprotein alpha subunit, Chain A, 2-Enoyl-Coa Hydratase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, tropomyosin beta chain.8 The result of Western-blot: F1-ATPase beta subunit and Electron transfer flavoprotein alpha subunit were validated via Western-blot. The result demonstrated that they were expressed in cerebral cortex mitochondria of rat, and has the same tendency of protein change with the 2D images which were analyzed by PDQuest7.0 software.Conclusion: 1 The morphological changes in cerebral cortex of rats after hypobaric hypoxia included cellularoedema, karyopyknosis, karyolysis and apoptosis. The injury was aggravated gradually with increased hypoxia duration. 2 The hypobaric hypoxia injury would lead to oxidative stress injury. The activities of SOD and CAT were depressed, and the contents of MDA was increased after hypobaric hypoxia injury. The activities of LDH were remarkably raised. 3 The water concent was increased after hypobaric hypoxia injury with increased expression of AQP1 and AQP4 in brain and lung. The degree of edema was aggravated with the hypobaric hypoxia injury duration increased. 4 The hypobaric hypoxia injury can cause cerebral cortex mitochondrial protein expression changes. Protein expression profiles were different in hypoxia injury groups compared with control group, indicating that many mitochondrial protein expression were changed and they might participate in the hypobaric hypoxia damage. Energy metabolism was influenced as proteins related to fatty acid oxidation and oxidative phosphorylation changed after hypobaric hypoxia injury. Hypobaric hypoxia damage could increase protein decomposing and synthetic functions to remodeling the mitochondrial structure protein damaged by oxidative stress injury. Hypobaric hypoxia injury could cause brain damage while the brain would use a series of compensatory mechanism to deal with the damage.