冬凌草甲素改善阿尔茨海默病模型小鼠认知功能及机制研究

发布时间：2018-04-28 16:37

本文选题：冬凌草甲素 + 阿尔茨海默病　；参考：《南京中医药大学》2016年博士论文

【摘要】：研究背景：阿尔茨海默病(Alzheimer's disease, AD)是一种致死性的中枢神经系统变性疾病,是导致老年痴呆的最主要原因,其主要临床表现为进行性学习记忆损伤和认知功能障碍,典型病理特征包括细胞外p-淀粉样蛋白(Amyloid-β, Aβ)沉积成老年斑以及细胞内过度磷酸化的Tau蛋白形成神经纤维缠结(Neurofibrillary Tangles, NFTs)。目前认为Aβ级联假说是AD最重要的发病机制,而越来越多的研究发现Aβ诱导的突触丢失和神经炎症在AD的进程中发挥重要作用。目的：(1)观察冬凌草甲素(Oridonin, Ori)对AD模型小鼠认知功能障碍的影响；(2)体内外研究Ori对Aβ1-42诱导的突触丢失的影响及其机制；(3)体内研究Ori对Aβ1-42诱导的神经炎症的影响及其机制。方法：(1)以立体定位法将Aβ1-42注射至C57BL/6 (B6)小鼠双侧海马内以制备AD小鼠模型,Aβ1-42处理原代培养的小鼠皮层神经元为体外AD模型,体内外均随机建立AD模型组及Ori治疗组。利用水迷宫检测Ori能否改善AD模型小鼠认知功能障碍。(2)第一部分利用Western blot及免疫荧光分析Ori对体内及体外AD模型中突触标记物PSD-95和synaptophysin的影响,MTT检测Ori处理AD模型小鼠突触小体活性的变化,Golgi染色观察小鼠海马内树突形态的变化,Western blot及免疫荧光检测Ori能否激活海马内BDNF/TrkB/CREB信号通路；(3)第二部分利用RT-PCR和Western blot检测Ori处理的AD模型小鼠海马内炎症因子mRNA和蛋白表达水平,免疫荧光分析小胶质细胞和星型胶质细胞的激活状况,Western blot检测线粒体的损伤情况以及Ori能否抑制NF-κB通路的激活。结果：(1)行为学结果显示Ori显著降低AD模型小鼠逃避潜伏期和上台前搜索距离,并增加其穿越平台的次数；Western blot及免疫荧光结果显示Ori上调体内外AD模型中PSD-95和synaptophysin的表达；Golgi染色结果显示Ori增加AD小鼠海马内树突长度及分叉,并增加树突棘的数量；此外,Ori上调AD小鼠突触小体内PSD-95和synaptophysin的表达,MTT结果显示Ori可提高突触小体活性；Western blot及免疫荧光结果显示Ori可激活海马内BDNF/TrkB/CREB信号通路。(2)RT-PCR及Western blot结果显示Ori可抑制AD模型小鼠海马内炎症因子的表达；免疫荧光显示Ori可抑制海马内小胶质细胞和星型胶质细胞的激活；Western blot结果显示Ori抑制AD小鼠海马内NF-κB通路的激活同时改善线粒体损伤。结论：Ori明显改善AD小鼠认知功能障碍,其作用机制可能为Ori改善AD小鼠海马内的突触丢失和缓解AD小鼠海马内的神经炎症反应,提示Ori可作为治疗AD的潜在药物。
[Abstract]:Background: Alzheimer's disease (ADD) is a fatal degeneration of the central nervous system (CNS) and is the main cause of Alzheimer's disease (AD). The main clinical manifestations of AD are progressive learning and memory impairment and cognitive impairment. Typical pathological features include the deposition of extracellular pamyloid- 尾 (A 尾) into senile plaques and the formation of neurofibrillary tangles (NFTs) by hyperphosphorylation of Tau proteins in cells. The A 尾 cascade hypothesis is considered to be the most important pathogenesis of AD, and more and more studies have found that A 尾 -induced synaptic loss and neuroinflammation play an important role in the progression of AD. Objective: to observe the effect of Oridonin (Ori2) on cognitive dysfunction in AD model mice in vivo and in vitro to study the effect of Ori on synaptic loss induced by A 尾 1-42 and its mechanism in vivo) to study the effect of Ori on neuroinflammation induced by A 尾 1-42 and its mechanism in vivo. Methods A 尾 1-42 was injected into the hippocampus of C57BL/6 B6 mice by stereotaxic method. The primary cultured cortical neurons of AD mice were treated with A 尾 1-42 as AD model in vitro. The AD model group and Ori treatment group were randomly established in vivo and in vitro. Using water maze to detect whether Ori can improve cognitive dysfunction in AD model mice. Part I: using Western blot and immunofluorescence to analyze the effect of Ori on PSD-95 and synaptophysin in AD model in vivo and in vitro. Ori was used to detect the treatment of AD model with Ori. Changes of synaptosome activity in mice by Golgi staining the morphological changes of dendrites in the hippocampus of mice were observed. Western blot and immunofluorescence were used to detect whether Ori could activate the BDNF/TrkB/CREB signaling pathway in the hippocampus. Part 2: the AD model treated with Ori was detected by RT-PCR and Western blot. The expression of inflammatory factor mRNA and protein in hippocampus of mice, The activation of microglia and astrocytes was analyzed by immunofluorescence. The damage of mitochondria was detected by Western blot and whether Ori inhibited the activation of NF- 魏 B pathway. Results (1) Behavioral results showed that Ori significantly decreased the escape latency and search distance before entering the stage in AD mice. The results showed that Ori up-regulated the expression of PSD-95 and synaptophysin in AD model in vivo and in vitro. The results showed that Ori increased the length and bifurcation of dendrites in hippocampus and increased the number of dendritic spine in AD mice. In addition, Ori upregulated the expression of PSD-95 and synaptophysin in synaptosomes of AD mice. The results showed that Ori could increase the activity of synaptosomes. Western blot and immunofluorescence showed that Ori could activate BDNF/TrkB/CREB signaling pathway in hippocampus. RT-PCR and Western blot showed that Ori could inhibit AD. The expression of inflammatory factors in hippocampus of model mice; Immunofluorescence showed that Ori could inhibit the activation of microglia and astrocytes in hippocampus. Western blot showed that Ori inhibited the activation of NF- 魏 B pathway in hippocampus of AD mice and improved mitochondrial damage. Conclusion Ori can improve cognitive dysfunction in AD mice, and the mechanism may be that Ori can ameliorate synaptic loss in hippocampus and relieve neuritis in AD mice, suggesting that Ori may be a potential drug for AD.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5;R-332

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