以磷酸二酯酶为主要靶点的阿魏酸抗阿尔兹海默病作用机制研究

发布时间：2017-12-27 17:04

本文关键词：以磷酸二酯酶为主要靶点的阿魏酸抗阿尔兹海默病作用机制研究　出处：《北京工业大学》2016年博士论文　论文类型：学位论文

【摘要】：越来越多证据表明,磷酸二酯酶(Phosphodiesterase,PDE)在学习和记忆过程中发挥了很大作用,其相应的抑制剂能通过调控细胞内环磷酸腺苷(cyclic adenosine monophosphate,c AMP)含量来改善中枢神经系统退行性疾病所致的学习记忆障碍。因此寻找特异性PDE抑制剂是目前国内外各大药理实验室的研究热点。我们近期的实验结果显示,阿魏酸(ferulic acid,FA)能调节PDE的表达及活性,从而影响生物体的各种代谢。但是FA是如何对PDE进行调控以及面对PDE这样一个庞大的超家族,FA是针对其中某一种亚型发挥特异性调节作用还是发挥非特异性PDE调节作用并不明确。本项目通过现代分子生物学手段明确FA对PDE亚型表达有抑制作用,并对其下游信号通路具有调节作用,并在整体动物学实验上验证FA抗阿尔茨海默病(Alzheimer’s disease,AD)样炎症作用与其对PDE的表达抑制作用密切相关。这些工作将不仅为AD的特异性靶向治疗提供安全有效的天然药物,同时也为FA的药理作用及机制深入研究提供理论支撑。本课题展开了相关的研究工作,包括以下四个部分。第一,运用分子对接的方法模拟FA与PD4B2的相互作用。分子对接结果表明,FA可与包括Tyr233、His234、Met347、Asn395、Phe414、Gln443、和Phe446在内的氨基酸残基发生强烈的相互作用,数据还表明,FA可进入PDE4B2的结合腔,并与氨基酸残基Phe446和Phe414形成π~π相互作用。在FA与氨基酸残基Gln443和His234之间可发现有氢键。FA的构象位于疏水腔区域,表明FA与PDE4B2之间存在静电和疏水性相互作用。分子对接结果从理论上支持了FA对PDE4B酶活性及表达的潜在影响,表明FA可被用作基本结构来设计PDE4抑制剂,应进行进一步的研究来证实该相互作用。第二,体外研究FA对Aβ_(25-35)和脂多糖(lipopolysaccharide,LPS)所致PC12细胞损伤的保护作用。检测了超氧化物歧化酶(superoxide dismutase,SOD)活性,炎性因子(TNF-α和IL-1β)的表达;检测与抗AD作用密切相关c AMP和Ca~(2+)浓度;另外,检测FA对神经生长因子(nerve growth factor,NGF)对PC12细胞分化的促进作用。实验结果表明FA能显著降低Aβ_(25-35)和LPS所致TNF-α和IL-1β水平的升高,同时升高SOD的表达水平,从而验证了其强大的抗炎和抗氧化功能。FA能够抑制LPS所引起的c AMP水平的降低;通过激光共聚焦技术检测细胞内Ca~(2+)浓度的变化,发现FA能够有效降低LPS所引起的PC12细胞内Ca~(2+)浓度的升高;FA与NGF共孵育,增强NGF诱导PC12细胞的分化作用。初步推测FA有可能对PDE表达具潜在抑制作用。第三,PDE4B可调节细胞内c AMP水平和c AMP/c AMP应答元件结合蛋白(c AMPresponse element binding protein,CREB)通路。进一步研究FA对LPS诱导PC12细胞磷酸二酯酶4B(PDE4B)上调表达及活性的影响,使用激光共聚焦显微镜检测FA对LPS诱导PC12细胞骨架蛋白F-actin变化的影响。我们进一步检测FA对LPS诱导的c AMP特异性PDE活性和细胞炎性因子(TNF-α和IL-1β)及ROS产生的影响;通过实时荧光定量RT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain reaction,Q-PCR)检测LPS诱导PC12细胞PDE4B m RNA表达;通过免疫印迹对PDE4B、CREB和磷酸化CREB(p CREB)的蛋白表达进行检测。FA对LPS所致PC12细胞F肌动蛋白(F-actin)骨架的损伤具有保护作用,明显改善细胞骨架蛋白F-actin的分布和结构。显著抑制LPS诱导的TNF-α、IL-1β及活性氧(reactive oxygen species,ROS)的产生。酶活性试验表明,FA可明显减弱LPS诱导PC12细胞c AMP依赖性PDE酶活性的上调。对PC12细胞PDE4B m RNA的研究表明,FA预处理可减少由LPS诱导的PDE4B m RNA表达上调。免疫印迹表明,FA预处理后,抑制LPS诱导的PDE4B上调作用;对抗LPS诱导的CREB和p CREB下调作用。FA对LPS诱导的PDE4B表达上调的抑制作用及其对下游信号分子CREB和p CREB表达下调的对抗作用可能是其对神经炎症性疾病,比如AD样神经炎症具有治疗作用的一个机制。第四,检测FA对LPS诱导Sprague-Dawley(SD)大鼠学习和记忆缺失的保护作用;探讨FA对LPS诱导海马神经细胞损伤的保护作用及机制?在本研究中,Morris水迷宫试验检测FA对抗LPS诱导的大鼠学习和记忆缺失作用,苏木精-伊红染色(Hematoxylin-eosin staining,HE)检测FA对LPS损伤大鼠大脑皮层和海马神经元的组织病理学保护作用?免疫荧光法检测FA对LPS损伤海马神经元细胞骨架蛋白β-tubulin的保护作用?为阐明FA对LPS诱导的海马神经元的保护作用,检测SOD活性;Q-PCR检测FA抗LPS诱导的IL-1β?caspase-1?Nod样受体蛋白3(Nod like receptor protein,NLRP3)和PDE4B的m RNA表达作用;免疫印迹法检测PDE4B、NLRP3和CREB及p CREB蛋白的表达?免疫组织化学染色法检测FA对LPS诱导大鼠海马神经元CA1?CA2和CA3区PDE4B蛋白表达的影响?Morris水迷宫试验表明FA预处理对抗LPS诱导的大鼠学习记忆功能障碍;HE染色显示,预先给予不同剂量FA可以减轻腹腔注射LPS诱导的SD大鼠海马神经元损伤;与LPS组相比,FA预处理组大鼠海马神经元β-tubulin蛋白表达增加;FA预处理显著维持大鼠大脑皮层和海马神经元中被LPS抑制的SOD的活性;FA预处理显著抑制LPS诱导的IL-1β?capase-1?NLRP3和PDE4B的m RNA水平的增加?Western蛋白印迹分析表明,相较于LPS组,FA预处理组的CREB和p-CREB的表达增加,同时抑制LPS诱导的大鼠海马神经元中PDE4B和NLRP3的表达;免疫组化染色结果显示,FA预处理显著抑制了LPS诱导的海马神经元中CA1?CA2和CA3区PDE4B表达的上调?FA对LPS诱导的大鼠学习和记忆损伤以及海马神经元损伤发挥保护作用。FA的神经保护作用与其抗炎,抗氧化作用密切相关;FA抑制LPS诱导的PDE4B上调并对抗LPS对CREB和p CREB的下调作用,FA可作为AD样神经炎性疾病的治疗药物。
[Abstract]:More and more evidence that phosphodiesterase (Phosphodiesterase, PDE) play a very important role in the process of learning and memory, the inhibitor can through the regulation of intracellular cyclic adenosine monophosphate (cyclic adenosine monophosphate, C AMP) content to improve learning and memory disorder caused by the degeneration of central nervous system. Therefore, the search for specific PDE inhibitors is a hot spot in the major pharmacological laboratories at home and abroad. Our recent experimental results show that ferulic acid (ferulic acid, FA) can regulate the expression and activity of PDE, thus affecting the metabolism of the organism. But how FA regulates PDE and faces such a large superfamily of PDE, FA is not clear about whether it plays a specific regulatory role in one of the subtypes or plays a role in the regulation of non-specific PDE. This project through modern molecular biology methods clear FA on the expression of PDE isoforms are inhibited, and can regulate its downstream signaling pathways, and in the whole animal experiment to validate FA against Alzheimer's disease (Alzheimer 's disease, AD) and the role of inflammation like inhibition of PDE expression is closely related to. These works will not only provide safe and effective natural medicines for specific targeted therapy of AD, but also provide theoretical support for further research on the pharmacological actions and mechanisms of FA. This topic has carried out the relevant research work, including the following four parts. First, the interaction between FA and PD4B2 is simulated by the method of molecular docking. Molecular docking results show that FA can interact strongly with amino acid residues including Tyr233, His234, Met347, Asn395, Phe414, Gln443, and Phe446, the data also indicate that combining cavity FA can enter the PDE4B2, and the formation of PI ~ interaction with amino acid residues Phe446 and Phe414. Hydrogen bonds can be found between FA and amino acid residues Gln443 and His234. The conformation of FA is located in the hydrophobic cavity region, indicating that there is an electrostatic and hydrophobic interaction between FA and PDE4B2. The docking results support the potential effect of FA on PDE4B enzyme activity and expression in theory, indicating that FA can be used as a basic structure to design PDE4 inhibitors. Further studies should be carried out to confirm the interaction. Second, in vitro FA of A beta _ (25-35) and lipopolysaccharide (lipopolysaccharide, LPS) PC12 cell injury caused by the protective effect. Detection of superoxide dismutase (superoxide dismutase, SOD) activity, inflammatory factor (TNF- alpha and IL-1 beta) expression; and the role of anti AD detection AMP and Ca~ is closely related to C (2+) concentration; in addition, the detection of FA on nerve growth factor (nerve growth, factor, NGF) on PC12 cell differentiation role. The experimental results show that FA can significantly reduce the A beta _ (25-35) increased and LPS caused by TNF- alpha and IL-1 beta levels, and increased the expression level of SOD, which proves its strong anti-inflammatory and antioxidant function. Reduced FA can inhibit LPS induced C AMP levels; were detected by confocal laser technology in Ca~ (2+) concentration, FA can effectively reduce LPS induced PC12 cells in Ca~ (2+) concentration increased; FA incubated with NGF, enhance the differentiation of PC12 cells induced by NGF. It is preliminarily conjectured that FA may have potential inhibitory effects on PDE expression. Third, PDE4B can regulate the intracellular C AMP level and the C AMP/c AMP response element binding protein (C AMPresponse element binding protein, CREB) pathway. To further investigate the effect of FA on the up regulation and activity of phosphodiesterase 4B (PDE4B) in LPS induced PC12 cells, we used laser confocal microscopy to detect the effect of FA on LPS induced PC12 cytoskeletal protein F-actin changes. We further detection of FA on LPS induced C AMP specific activity of PDE and inflammatory cytokines (TNF- alpha and IL-1 beta) effect and ROS production; by real-time fluorescence quantitative RT-PCR (real-time fluorescent quantitative reverse transcription polymerase chain reaction Q-PCR PDE4B m RNA) expression of PC12 cells induced by Western blotting to detect LPS; PDE4B CREB, and phosphorylation of CREB (P CREB) was used to detect the expression of protein. FA has a protective effect on the damage of F actin (F-actin) skeleton in PC12 cells induced by LPS, and obviously improves the distribution and structure of cytoskeleton protein F-actin. The production of TNF- - alpha, IL-1 beta and reactive oxygen species (reactive oxygen species, ROS) induced by LPS was significantly inhibited. The enzyme activity test showed that FA significantly reduced the up regulation of LPS induced C AMP dependent PDE activity in PC12 cells. The study of PDE4B m RNA in PC12 cells showed that FA pretreatment could reduce the up-regulated RNA expression of PDE4B m induced by LPS. Immunoblotting showed that FA preconditioning inhibited the up regulation of PDE4B induced by LPS, and antagonized the down regulation of CREB and P CREB induced by LPS. The inhibitory effect of FA on LPS induced upregulation of PDE4B expression and its down-regulation effect on downstream signal molecules CREB and P CREB expression may be a mechanism for its therapeutic effect on neuroinflammatory diseases, such as AD like neuritis. Fourth, detection of FA on LPS induced Sprague-Dawley (SD) rats learning and memory deficits; to investigate the protective effect and mechanism of FA on LPS induced injury of hippocampal neural cells? In this study, learning and memory in rats of missing the Morris water maze test against FA induced by LPS and hematoxylin eosin staining (Hematoxylin-eosin staining, HE) the protective effect of histopathological detection of FA on LPS damage in rat cerebral cortical and hippocampal neurons? By immunofluorescence detection of FA protection on LPS injury of hippocampal neurons cytoskeleton protein beta -tubulin? To elucidate the protective effects on LPS induced FA in hippocampal neurons, SOD activity detection; Q-PCR detection FA induction of anti LPS beta IL-1? Caspase-1? Nod like receptor protein 3 (Nod like receptor protein, NLRP3 m) RNA and PDE4B expression; Western blot detection of PDE4B, NLRP3 and CREB and P CREB protein expression Detection of FA induced hippocampal deity induced by LPS in rats by immunohistochemical staining
【学位授予单位】：北京工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.16

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