文冠果壳苷对β淀粉样蛋白致痴呆小鼠学习记忆障碍的改善作用及线粒体相关机制研究

【作者】 纪雪飞；

【导师】 邹莉波；

【作者基本信息】 沈阳药科大学 ， 药理学， 2014， 博士

【摘要】 目的：文冠果壳苷是一种从文冠果果壳中提取获得的单体化合物,前期研究证实其对多种痴呆模型动物的学习记忆障碍具有改善作用。为进一步探讨文冠果壳苷对阿尔茨海默病(AD)的防治作用及作用机制,本文考察了文冠果壳苷对一次性侧脑室注射Aβ1-42致痴呆模型小鼠学习记忆障碍的改善作用及线粒体相关机制。方法：一次性侧脑室注射Aβ1-42制备痴呆小鼠模型,通过水迷宫、Y迷宫、新物体辨别及避暗实验考察文冠果壳苷对模型小鼠学习记忆障碍的影响；通过透射电镜观察小鼠大脑皮层及海马CA1区神经细胞线粒体的超微结构,并利用生物化学发光法考察大脑皮层及海马ATP的水平；利用Percoll密度梯度离心的方法分离小鼠大脑皮层线粒体,透射电镜观察获得线粒体的超微结构,并通过测定乳酸脱氢酶(LDH)的含量及线粒体呼吸链复合体Ⅳ(即细胞色素c氧化酶,COX)的活性验证分离线粒体的纯度及活性。利用分离获得的线粒体测定COX、丙酮酸脱氢酶复合体(PDHC)及a-酮戊二酸脱氢酶复合体(KGDHC)的活性。利用荧光分光光度法考察小鼠大脑皮层及海马活性氧(ROS)的水平。通过免疫组织化学方法考察小鼠大脑皮层及海马内DNA氧化损伤生物标志物8-羟基鸟苷(8-OH-dG)的表达,并考察线粒体细胞色素c的释放情况。结果：文冠果壳苷对一次性侧脑室注射Aβ1-42致痴呆模型小鼠学习记忆障碍具有显著的改善作用。与模型组比较,文冠果壳苷(0.16～0.32mg/kg)显著缩短水迷宫实验中小鼠到达安全台的逃避潜伏期及游泳路程,并显著增加小鼠在原安全台所在象限游泳时间和游泳路程百分比；显著提高Y迷宫实验中小鼠自发交替反应率；显著提高新物体辨别实验中小鼠对新物体的优先指数和辨别系数；显著提高避暗实验中小鼠进入暗室的潜伏期。文冠果壳苷可改善神经细胞线粒体的超微结构并显著增加模型小鼠大脑皮层及海马ATP的水平,改善线粒体的功能。文冠果壳苷显著对抗模型小鼠线粒体COX、PDHC及KGDHC活性的下降;显著逆转模型小鼠大脑皮层及海马ROS水平的异常升高。免疫组化结果显示,文冠果壳苷能够显著降低模型小鼠大脑皮层及海马8-OH-dG的表达；显著减少模型小鼠线粒体细胞色素c的释放。结论：文冠果壳苷对一次性侧脑室注射Aβ1-42致痴呆模型小鼠的学习记忆障碍具有显著的改善作用,并且能够改善线粒体功能,减少ROS的产生及对抗氧化应激；其对线粒体功能的改善作用可能与对抗Ap引起的线粒体呼吸链及三羧酸循环的损伤,减少线粒体细胞色素c的释放,从而对抗Ap诱导的神经细胞死亡有关。因此,本研究及前期试验结果提示,文冠果壳苷具有显著的改善学习记忆障碍及神经保护作用,可能成为治疗AD新药研发中的优良候选化合物。更多还原

【Abstract】 Aim: Xanthoceraside is a monomer extracted from the shell of the fruit of Xanthoceras sorbifolia Bunge. Our previous study showed that xanthoceraside could significantly improve learning and memory impairment in several AD animal models. In this study, we further investigated the effects of xanthoceraside on learning and memory impairment and the possible mechanism associated with the protection of mitochondria in Alzheimer’s disease (AD) model mice induced by Aβ1-42, which aims to explore the anti-AD effects and possible related mechanisms of xanthoceraside. Methods:We acquired learning and memory impairment mice model by intracerebroventricular (i.c.v.) injection of the aggregated Aβ1-42. Learning and memory ability was examined using Morris water-maze test, Y-maze test, novel object recognition test and step-through test in mice. Transmission electron microscope was used to observe the ultra-structure of mitochondria of neurons in cerebral cortex and hippocampus. The level of ATP in cerebral cortex and hippocampus was detected by bioluminescence assay. Percoll density gradient centrifugation method was applied to isolate free mitochondria from mice cerebral cortex. Transmission electron microscope observation, lactate dehydrogenase(LDH) level and cytochrome c oxidase (COX) activity assay were applied to confirm whether the mitochondria are intact and pure with good metabolic activity. The activities of COX, pyruvate dehydrogenase complex (PDHC) and a-ketoglutarate dehydrogenase complex (KGDHC) were detected using assay kits respectively with isolated free mitochondria. The ROS level in cerebral cortex and hippocampus was detected using fluorescence spectrophotometry. Immunohistochemistry was used to investigate the expression of8-OH-dG, the biomarker of DNA oxidative damage, and the release of cytochrome c from mitochondria. Results:Xanthoceraside significantly improved learning and memory impairment in mice induced by i.c.v. injection of Aβ1-42. Compared with model group, xanthoceraside (0.16～0.32mg/kg) significantly decreased the escape latency and swimming distance, increased the swimming time and percentage of the swimming distance in the fourth quadrant where the platform had been located in Morris-water maze test; increased the percentage of alternation behaviors in Y-maze test; increased the preferential index and discrimination index for the novel object in novel objective recognition test; and decreased the step-through latency in step-through test. Xanthoceraside improved the ultra-structure of mitochondria and increased the ATP level in cerebra cortex and hippocampus of model mice. Xanthoceraside significantly reversed the decrease of the activity of COX, PDHC and KGDHC and inhibited the abnormal increase of ROS induced by Aβ. Moreover, the results of immunohistochemistry showed that xanthoceraside significantly decreased the expression of8-OH-dG and reduced the release of cytochrome c from mitochondria in model mice. Conclusion:Xanthoceraside could improve learning and memory impairment, promote the function of mitochondria, decrease the production of ROS and inhibit oxidative stress. The improvement effects to mitochondria may be through withstanding the damage of Aβ to mitochondrial respiratory chain and the key enzymes in Kreb’s circle, decreasing the release of cytochrome c, and finally reducing the death of neurons. Therefore, the results from present study and previous study indicate that xanthoceraside could be a competitive candidate for the treatment of AD.更多还原