| Objective:(1)To investigate the neuroprotection of17β-estradiol (E2) on cerebral cortical neurons subjected to oxygen-glucose deprivation/reoxygenation.(2) To observe the neuroprotection of17β-estradiol (E2) during focal cerebral ischemia in ovariectomized rats.Methods:Primary cultured cerebral cortical neurons were prepared from Sprague-Dawley rats, and a cell damage model in vitro was induced by oxygen-glucose deprivation/reoxygenation (OGD/R).To investigate the neuroprotection of E2, Cell injury was evaluated by lactate dehydrogenase (LDH) release rate, cell viability was assessed by MTT assay, and the detection of apoptotic cells was determined by the flow cytometry and Hoechst33342staining. We studied the effects of17β-estradiol on cerebral cortical neurons by the model. Then we detected the concentration of intracellular Ca2+by fluorescence duel wavelength spectrophotometer. The level of GRP78and caspase12mRNA were evaluated by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). Western bloting was used to observe the expression of145kD SBDP and procaspase12protein. The activation of caspase12 was evaluated by caspase12fluorescence activity assay. A model of cerebral ischemia/reperfusion was made by occlusion of middle cerebral artery used an intraluminal filament method in ovariectomized rats. We estimated the model by TTC staining of brain sections, infarct volum and neurological deficit scores. TUNEL staining, immunohistostaining and fluorescent quantitation were used to observe the effect of17β-estradiol on brain ischemia/reperfusion and the expression of procaspase12.Results:1. Effects of17β-estradiol on cortical neurons subjected to OGD/R(1)Estrogen receptor protein was present in both the cultured cortical neurons at10days in vitro and the cerebral cortical tissue from post-natal day10rat detected by western blotting.(2)Effects of17β-estradiol on cortical neurons subjected to OGD/R17β-estradiol on cortical neurons subjected to OGD2h/R12h:The decrease of LDH release rate by17β-estradiol was observed at1nM, and the maximum effect was at100nM; The increase of cell viability by17β-estradiol was observed at1nM, and the maximum effect was at100nM.(2)The administration of17β-estradiol at6h before OGD,12h before OGD,24h befor OGD all increased the cell viability, decreased the LDH release rate. And the most potent effect of17β-estradiol mediated cell survival was observed at24h. (3)Estrogen inhibiter ICI182,780(1μM) significantly abolished the protection mediated by17(3-estradiol in cortical neurons subjected to OGD/R.2. Relationship between the influence of17β-estradiol on neurons subjected to OGD/R with Ca2+/calpain/caspase12pathway(1)The concentration of intracellular Ca2+was increased by OGD/R, and the pretreatment of17β-estradiol can decrease it.(2)As detected by RT-PCR analysis, the level of caspase12and GRP78mRNA increased by OGD/R. and the pretreatment of17β-estradiol can inhibit these expression.(3)Western blotting analysis demonstrated that the expression of procaspase12decreased during OGD/R, and the pretreatment of17β-estradiol can increase the expression. The activity of calpain was evaluation by the level of145kD SBDP, the result indicated that the activity of calpain elevated during OGD/R, and the pretreatment of17β-estradiol decreased the activity of calpain, which can be inhibitted by ER inhibiter ICI182,780.(4)As detected by fluorescent activity assay, the activity of caspase12increased during OGD/R. and the pretreatment of17β-estradiol or calpain inhibiter ALLN(12.5μM)can inhibit the activation of caspase12.(5)Calpain inhibiter ALLN decreased the LDH release rate and apoptotic percentage, and increased cell viability. 3. The neuroprotection of17β-estradiol on brain ischemia/reperfusion in ovariectomized rats(1) The effect of17β-estradiol on brain ischemia/reperfusion in rats:The infarct volum and neurological deficit scores increased after MCAO, which can be improved by17β-estradiol. Blurry, malalignment even death cells were observed in rat cerebral cortical and hippocampus at2h ischemia following22h reperfusion when stained with HE and Nissle, while pretreatment with17β-estradiol reduced cortical and hippocampus neurons loss after ischemia/reperfusion.(2)The apoptotic cells in peripheral zone of ischemia:Rats in the sham group had very few or no TUNEL positive cell. Rats in MCAO group had positive cell located in ischemic side, Significant expression was in peripheral zone of ischemia, and the number of positive cell increased significantly compared to sham group; The number of TUNEL positive cell pretreatment with17β-estradiol were lower than those in MCAO group.(3) The expression of procaspase12in ischemic side:Rats in MCAO group had few procaspase12positive cells, compared with sham group; The number of procaspase12positive cell increased significantly in the group pretreatment with17β-estradiol. The activity of caspase12increased in MCAO group compared with sham group; pretreatment with17β-estradiol reduced the activation of caspase12(compared to MCAO group, p<0.05).Conclusion:1.17β-estradiol has a direct protection on cultured cerebral cortical neurons subjected to OGD/R in a dose-and time-dependent manner. ER inhibiter ICI182,780abolished the effect of17β-estradiol completely.2. ERS participated in the injury of OGD/R;17p-estradiol inhibited the ERS induced by OGD/R.3. Ca2+/calpain/caspase12pathway participated in the cerebral cortical neuronal apoptosis induced by OGD/R.17β-estradiol attenuated neuronal apoptosis during OGD/R by Ca2+/calpain/caspase12pathway.4.17β-estradiol protected rat from brain ischemia/reperfusion, The protection of17β-estradiol on brain ischemia/reperfusion may be related to inhibiting the activation of caspase-12.
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