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Research Objective: The incidence of hypertension in adults up to 18%, China has almost 200 million patients with hypertension. The stroke is one of the most important complications of hypertension, in the event the patient dead residues. Prevention and treatment of stroke has become an urgent need to solve the problems in the scientific research. The prevention and treatment of stroke, antihypertensive drugs control blood lipids and anticoagulation measures. But the incidence of stroke in recent years continued to show a rising trend, the global hair stroke 15 million per year is still new, increasingly younger. Pathophysiological mechanisms of stroke is very complex, it is necessary to carry out further in-depth research to find out more prevention and treatment of drug. Rapamycin (Rapamycin, RAP) is the beginning of the 1970s by the Canadian Institute for Ayerst from actinomycetes culture the liquid separated macrolide antibiotics as immunosuppressive agents have been widely used in clinical. · As a hot research at home and abroad, the current study found a variety of biologically active anti-aging and neurotrophic RAP. Since neuroprotective activity the RAP on ischemic brain damage no protective effect? ??RAP protection of ischemic brain injury, then what is the exact mechanism? Therefore, the subject of research focused on these two issues, in order to find the RAP protective effect of ischemic brain injury, and preliminary exploration of its mechanism. Experimental design and methods: experimental, RAP acute ischemic brain damage in stroke-prone spontaneously hypertensive rats (SHR-SP): 50-month-old male SHR-SPs were randomly divided into five groups, were given the solvent dimethyl sulfoxide (DMSO) and RAP0.3 1.0,3.0,10 μg / kg intraperitoneal injection (ip) administered three weeks after electric coagulation brain artery occlusion (MCAO) surgery, 24 hours after brain stained Analysis. Second experiment, RAP C57 mice with acute ischemic brain injury: 70-month-old male C57 mice were randomly divided into seven groups were given the solvent DMSO and RAP 0.2,20,200 μg / kg, 2 mg / kg , 20 mg / kg, continuous ip administration three weeks after the weigh in, and MCAO surgery, 24 hours after brain staining analysis. The experiment three trace RAP long-term administration of SHR-SP life: 30-month-old male SHR-SPs were randomly divided into 2 groups, respectively to feeding to trace the RAP Drug feed (3μg/kg/d) and normal diet, record survival time. Experiments, RAP neuronal apoptosis and cerebral cortex mitochondrial function. 1, RAP primary neuronal apoptosis: cultured primary neurons SHR-SP fetal rats (20-23 days old) and RAP administered 3 hours after hydrogen peroxide (H2O2) or 1 - methyl - 4 - phenylpyridinium ion (MPP) model, primary neuronal cell apoptosis induced by Hoechst law or MTT (MTT) assay of apoptosis. 2, RAP cortex mitochondrial function: a 9-month-old male SHR-SPs were randomly divided into three groups, were given RAP fish was 1μg/kg. 1 mg / kg, and the solvent DMSO, continuous ip administration to take the cerebral cortex cells, respectively the Mitotracker GreenFM, JC-1, and Dihydrorhodamine123 staining, flow cytometry, to observe the RAP number of mitochondria in the cerebral cortex of SHR-SP cells, the level of the membrane potential and reactive oxygen species (ROS). Experiment 5: study trace RAP protection molecular mechanisms of ischemic brain injury: a protein phosphorylation chips: two male SHR-SPs were divided into two groups were given the RAP 1μg/kg and the solvent DMSO, 5 hours later to take the cerebral cortex , to do protein phosphorylation chips, to find larger changes in the level of phosphorylation of the administration of the RAP trace protein. Trace RAP cortex of SHR-SP the mTOR protein phosphorylation levels: nine male SHR-SPs were randomly divided into three groups, were given RAP fish was 1μg/kg. 1 mg / kg and the solvent DMSO5 hours after cerebral cortex, Western-blot method to detect changes in the level of phosphorylation of the mTOR protein Ser2448 sites. 3, gene chip and co-immunoprecipitation Mass Spectrometry: 9 male SHR-SPs were randomly divided into three groups, were given RAP 1μg, 1 mg / kg, and the solvent DMSO, administered continuously for one week, take the cerebral cortex do GeneChip and immunoprecipitation mass spectrometry to detect protein mRNA gene expression in the cerebral cortex cell apoptosis and oxidative stress pathway, looking downstream trace RAP administration when combined with mTOR specific protein. Experimental results: one, prophylactic administration of trace RAP (1-10μg/kg) significantly reduced the area of ??cerebral infarction of SHR-SP, a good protective effect on acute ischemic brain injury. Trace RAP (0.2-20μg/kg) Prophylactic administration significantly reduced the C57 mice infarction area, when the dose was increased to 200μg/kg acute ischemic brain damage, RAP disappeared, but the C57 mice weight loss, apathetic state. , Micro-RAP (3μg/kg) long-term administration can be significant in delaying the occurrence of stroke in SHR-SP, to prolong its life. IV, RAP within a certain range of concentrations can significantly reduce primary neuronal apoptosis caused by oxidative stress modeling and MPP modeling. In addition, trace RAP (1μg/kg) significantly increase the number of mitochondria of the cerebral cortex of the SHR-SP cells, mitochondrial membrane potential, reducing the generation of ROS doses under 1mg/1kg of not obvious. Trace RAP protection of the molecular mechanisms of ischemic brain injury, protein chip results show: compared with the control group the trace the RAP (1μg/kg) group, the mTOR signaling pathway the mTOR upstream protein AMPK, AKT, PDK1 downstream protein S6K1 and 4EBP1 phosphorylation levels changed significantly. 2, Western-blot detection: Compared with the control group, the micro-RAP (1μg/kg) group were significantly increased mTOR phosphorylation levels of the protein the Ser2448-bit point, the role of RAP (1 mg / kg) group is not obvious. 3, the results of the gene chip: trace RAP administration a week after the nerve cells in the cerebral cortex of the SHR-SP P53 signaling pathway, cyclinE gene expression raised Gadd45 genes were downregulated. Conclusion: 1, trace RAP acute ischemic brain damage, very good protection, long-term administration can significantly delay the incidence of stroke in SHR-SP, to prolong its life. Trace RAP protective effect of ischemic brain injury may be related to anti-oxidative stress, anti-apoptosis, enhanced mitochondrial function-related. 3, trace the RAP protective effect may be related with the mTOR protein.
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