【作者】 光红梅；

【导师】 杜冠华；

【作者基本信息】 中国协和医科大学 ， 神经药理与新药发现， 2006， 博士

【摘要】 化合物DL0108的主要成份是生松素，我们实验室首先发现其较好的神经保护作用，本研究旨在系统研究其对大鼠脑缺血损伤的药理作用，并对作用特点和作用机制进行探讨。论文评价了化合物DL0108对急、慢性脑缺血损伤模型的治疗作用；并根据实验结果，结合当前脑缺血病理生理机制研究的重点，探讨了化合物DL0108对以线粒体为中心，CREB为靶点的作用机制。 一、化合物DL0108对大鼠急性脑缺血损伤的保护作用及机制研究 1．化合物DL0108对大鼠急性局灶性脑缺血损伤的保护作用 采用大脑中动脉内线栓法建立大鼠急性局灶性脑缺血模型，观察化合物DL0108的作用。结果显示，化合物DL0108能够改善大鼠脑缺血损伤的神经症状，减小脑梗塞体积，降低乳酸脱氢酶水平，提高能量代谢指数（P＜0.05）。神经细胞死亡率降低，生存细胞的形态改善。化合物DL0108对脑组织MDA、SOD、GSH-Px水平无影响。本部分结果是化合物DL0108用于急性脑缺血治疗的肯定，且不是通过作用于氧化／抗氧化机制实现。 2．化合物DL0108对大鼠急性缺血性脑组织线粒体的保护作用 体外提取线粒体，观察化合物DL0108对缺血性脑组织线粒体的作用。结果显示，化合物DL0108（10mg／kg）处理组线粒体SOD活性提高了24.4％、MAO-B活性降低了23.7％、Na⁺-K⁺-ATP酶、Ca²⁺-ATP酶活性分别升高了24.9％、及34.0％、线粒体钙离子浓度降低了22.0％，与溶剂组相比均具有显著性差异（P＜0.05）。化合物DL0108（3mg／kg，10mg／kg）处理组线粒体超氧阴离子水平分别降低了20.2％及30.6％、线粒体肿胀程度分别降低了26.1％及46.0％，与溶剂组相比均具有显著性差异（P＜0.05）。本部分结果提示，化合物DL0108对脑组织急性缺血损伤后线粒体结构和功能的保护作用，可能与减轻线粒体钙超载损伤有关。 3．化合物DL0108对大鼠急性缺血性脑组织CREB信号通路的影响 基于分子生物学及组织化学技术，研究化合物DL0108对参与CREB通路部分因子在核酸及蛋白质水平表达的影响。结果显示，化合物DL0108能够增加BDNF、c-FOS在mRNA及蛋白水平的表达水平（P＜0.05）；化合物DL0108能够增加P-CREB／CREB表达比值、降低CaN表达（P＜0.05）。本部分结果提示，化合物DL0108对CREB通路的作用，是其发挥神经保护作用的机制之一。更多还原

【Abstract】 Background and PurposeCompound DL0108 (pinocembrin) had been found on its neuroprotective effects against glutamate damage in our previous research. In vivo, it had also been proved a potentially beneficial drug for treatment on cerebral ischemia from its neurological protection on ischemia brain in rats. The purpose of this study was to further evaluate its actions on acute and chronic cerebral ischemia in rat, and if it did so, to explore the possible mechanisms mitochondria and related CREB signal pathway involved.MethodsAcute cerebral ischemia and chronic cerebral ischemia were respectively achieved by operation of middle cerebral artery occlusion (MCAO) and bilateral carotid artery ligation (2-VO) in rats. Neurological effects of compound DL0108 were evaluated in animal behavior performances and pathological morphology. Three types of cell including PC12, SH-SY5Y, and primary cortex neuron were cultured, on which established the glutamate and energy deprivation cell model in vitro. Isolated mitochondria from rat brain were detected on structure and function changes by spectrophotometric and fluorescence assay. Immunocytochemical method was used to identify the purity of primary neuron and BDNF/c-FOS expression level. Immunohistochemical and western blotting methods were applied to estimate phosphorated level of cAMP response element binding (CREB) and expression level of its downstream transcription factors. Effects of compound DL0108 on CRE transcription activity were evaluated by luciferase and electro mobility shift assay, simultaneously.ResultsIn vivo, compound DL0108 exhibited neurological protections against both acute cerebral ischemia induced by MCAO and chronic cerebral hypoperfusion induced by 2-VO, including improvement on neurobehavior performances and neuron morphological.更多还原