| OBJECTIVE:Hypertension is one of the most common cardiovascular diseases with high incidence (18%). As the major complication of hypertension, stroke makes a considerable contribution to morbidity and mortality. Given the disease burden of stroke, prevention is an important public health concern to curb the stroke pandemicly. Drugs used for blood pressure, lipid control and anticoagulation are the tradition measures for stroke prevention. But today, there are still lots of people suffering from stroke especially for sapling. Many other new drugs are needed to be researched and used for the prevention and therapy of ischemic stroke.Rapamycin (RAP) is a kind of macrolide antibiotics. It was first found in the culture medium of actinomycetes more than 30 years ago in Canada. Recent studies show that RAP has anti-inflammation, immunosuppression, anti-neoplasms, neuroprotective effects. Although it has neuroprotective effect in nerval cells, the protective effect of RAP on ischemic cerebral injury is unknown. This study was designed to investigate the protective effect of RAP on stroke.RESEARCH DESIGN AND METHODS:Experiment one:The effect of RAP on ischemic cerebral injury in stroke-prone spontaneously hypertensive rat (SHR-SP):50 male SHR-SPs, aged 3 months, were randomly divided into 5 groups and received solvent (DMSO), or RAP 0.3,1.0,3.0,10μg/kg (intraperitoneal injection, i.p.) respectively. After 3 weeks drug administration, animal model of acute ischemic cerebral injury by electric coagulation of middle cerebral artery occlusion (MCAO) were made. After 24 h, the infarct and hemisphere areas of each section (both sides) were traced and quantified by an image analysis system.Experiment two:The effect of RAP on ischemic cerebral injury in C57 mouse:70 male C57 mice, weight 18-22 g. were also randomly divided into 7 groups and received DMSO, or RAP 0.2,2,20.200μg/kg and 2,20 mg/kg (i.p.) respectively. After 3 weeks drug administration, animal model of acute ischemic cerebral injury by MCAO were made. After 24 h. the infarct and hemisphere areas of each section (both sides) were traced and quantified by an image analysis system.Experiment three:The effect of microdose RAP on longevity in SHR-SPs:30 male animals, aged 3 months, were randomly divided into 2 groups and received normal rat chow and RAP 10μg/kg respectively. All of them were carefully observed every day, the survive time were record.Experiment four:The effect of microdose RAP on apoptosis of neuron cell and mitochondrial function of cerebral cortex in SHR-SPs. (1) The effect of microdose RAP on apoptosis of neuron cell:RAP was given to the culturing neurons for 3 h, and then H2O2 or 1-methyl-4-phenylpyridiniumion (MPP+) induced oxidative stress or aging cell model. Apoptosis rate of neuron cell was detected through Hoechst or MTT method. (2) The effect of microdose RAP on mitochondrial function of cerebral cortex:9 male SHR-SP, aged 3 months, were randomly divided into 3 groups and received RAP 1μg/kg,lmg/kg and DMSO respectively. After 1w, mitochondrial mass, mitochondrial membrane potential and ROS level of cerebral cortex was measured through FCAS analysis after stained with Mitotracker Green FM,JC-1 and Dihydrorhodamine123 respectively.Experiment five:The protective mechanism of microdose RAP on molecule level. (1) protein chips:2 male SHR-SP, aged 3 months, were randomly divided into 2 groups and received DMSO and RAP 1μg/kg (i.p.) respectively. After 5 h, Cerebral cortex was detected, and the effect of microdose RAP on protein phosphorylation level was examined by protein chips technology.(2) Weastern Blot:9 male SHR-SP, aged 3 months, were randomly divided into 3 groups and received RAP 1μg/kg, lmg/kg and DMSO (i.p.) respectively. After 5 h, the level of phosphorylation in the ser-2448 site of mammalian target of rapamycin (mTOR) protein was measured through weastern-blot.(3) gene chips, immunoprecipitation and mass chromatographic analysis:9 male SHR-SP, aged 3 months, were randomly divided into 3 groups and received RAP 1μg/kg,1 mg/kg and DMSO (i.p.) respectively. After 1 w, the cerebral cortex was dissected and the change of mRAN gene expression level was measured via gene chips. The specificity protein binding with mTOR was detected using immunoprecipitation and mass chromatographic analysis. RESULTS:Experiment one:Microdose RAP (1-10μg/kg) has significantly decreased the infarct mass in SHR-SP.Experiment two:Microdose RAP (0.2-20μg/kg) has significantly decreased the infarct mass in C57 mouse. High-dose RAP (200μg/kg,2mg/kg,20mg/kg) has not decreased the infarct mass in C57 mouse and RAP (2mg/kg,20mg/kg) decreased the body weight of C57 mice and caused the mice in a state of feeble.Experiment three:The survival time is expressed by Kaplan-Meier survival curves. The lifespan was significantly increase by RAP (3μg/kg) in SHR-SPs.Experiment four:RAP (0.2,2 pM) significantly decreased the neurons apoptosis induced by H2O2, while RAP (0.02,20,200 pM) did not decreased the neurons apoptosis. The neurons apoptosis induced by MPP+ was also decreased by RAP (2 pM). In the other experiment, RAP (1μg/kg) significantly increased mitochondrial mass and membrane potential in SHR-SP. The production of ROS was significantly decreased in RAP (1μg/kg) group.Experiment five:The level of protein phosphorylation have been significantly changed in mTOR signaling pathway such as AMPK, PDK1, S6K1 and 4EBP1. The mammalian target of rapamycin (mTOR) protein was activated by the phosphorylation in the ser-2448 site. The gene chip data showed that the pathways of P53, cyclin E, Gadd45, etc. were changed.CONCLUSIONS:Microdose RAP decreased the cerebral infarct mass and prolonged the lifespan of SHR-SP. The protective effect of microdose RAP may be related with antiapoptosis, antioxidative stress and regulating mitochondrial function. The mTOR protein might be the target of microdose RAP. |
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