本文關(guān)鍵詞：以磷酸二酯酶為主要靶點(diǎn)的阿魏酸抗阿爾茲海默病作用機(jī)制研究　出處：《北京工業(yè)大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 阿魏酸 磷酸二酯酶 阿爾茨海默病 脂多糖

【摘要】：越來(lái)越多證據(jù)表明,磷酸二酯酶(Phosphodiesterase,PDE)在學(xué)習(xí)和記憶過(guò)程中發(fā)揮了很大作用,其相應(yīng)的抑制劑能通過(guò)調(diào)控細(xì)胞內(nèi)環(huán)磷酸腺苷(cyclic adenosine monophosphate,c AMP)含量來(lái)改善中樞神經(jīng)系統(tǒng)退行性疾病所致的學(xué)習(xí)記憶障礙。因此尋找特異性PDE抑制劑是目前國(guó)內(nèi)外各大藥理實(shí)驗(yàn)室的研究熱點(diǎn)。我們近期的實(shí)驗(yàn)結(jié)果顯示,阿魏酸(ferulic acid,FA)能調(diào)節(jié)PDE的表達(dá)及活性,從而影響生物體的各種代謝。但是FA是如何對(duì)PDE進(jìn)行調(diào)控以及面對(duì)PDE這樣一個(gè)龐大的超家族,FA是針對(duì)其中某一種亞型發(fā)揮特異性調(diào)節(jié)作用還是發(fā)揮非特異性PDE調(diào)節(jié)作用并不明確。本項(xiàng)目通過(guò)現(xiàn)代分子生物學(xué)手段明確FA對(duì)PDE亞型表達(dá)有抑制作用,并對(duì)其下游信號(hào)通路具有調(diào)節(jié)作用,并在整體動(dòng)物學(xué)實(shí)驗(yàn)上驗(yàn)證FA抗阿爾茨海默病(Alzheimer’s disease,AD)樣炎癥作用與其對(duì)PDE的表達(dá)抑制作用密切相關(guān)。這些工作將不僅為AD的特異性靶向治療提供安全有效的天然藥物,同時(shí)也為FA的藥理作用及機(jī)制深入研究提供理論支撐。本課題展開(kāi)了相關(guān)的研究工作,包括以下四個(gè)部分。第一,運(yùn)用分子對(duì)接的方法模擬FA與PD4B2的相互作用。分子對(duì)接結(jié)果表明,FA可與包括Tyr233、His234、Met347、Asn395、Phe414、Gln443、和Phe446在內(nèi)的氨基酸殘基發(fā)生強(qiáng)烈的相互作用,數(shù)據(jù)還表明,FA可進(jìn)入PDE4B2的結(jié)合腔,并與氨基酸殘基Phe446和Phe414形成π~π相互作用。在FA與氨基酸殘基Gln443和His234之間可發(fā)現(xiàn)有氫鍵。FA的構(gòu)象位于疏水腔區(qū)域,表明FA與PDE4B2之間存在靜電和疏水性相互作用。分子對(duì)接結(jié)果從理論上支持了FA對(duì)PDE4B酶活性及表達(dá)的潛在影響,表明FA可被用作基本結(jié)構(gòu)來(lái)設(shè)計(jì)PDE4抑制劑,應(yīng)進(jìn)行進(jìn)一步的研究來(lái)證實(shí)該相互作用。第二,體外研究FA對(duì)Aβ_(25-35)和脂多糖(lipopolysaccharide,LPS)所致PC12細(xì)胞損傷的保護(hù)作用。檢測(cè)了超氧化物歧化酶(superoxide dismutase,SOD)活性,炎性因子(TNF-α和IL-1β)的表達(dá);檢測(cè)與抗AD作用密切相關(guān)c AMP和Ca~(2+)濃度;另外,檢測(cè)FA對(duì)神經(jīng)生長(zhǎng)因子(nerve growth factor,NGF)對(duì)PC12細(xì)胞分化的促進(jìn)作用。實(shí)驗(yàn)結(jié)果表明FA能顯著降低Aβ_(25-35)和LPS所致TNF-α和IL-1β水平的升高,同時(shí)升高SOD的表達(dá)水平,從而驗(yàn)證了其強(qiáng)大的抗炎和抗氧化功能。FA能夠抑制LPS所引起的c AMP水平的降低;通過(guò)激光共聚焦技術(shù)檢測(cè)細(xì)胞內(nèi)Ca~(2+)濃度的變化,發(fā)現(xiàn)FA能夠有效降低LPS所引起的PC12細(xì)胞內(nèi)Ca~(2+)濃度的升高;FA與NGF共孵育,增強(qiáng)NGF誘導(dǎo)PC12細(xì)胞的分化作用。初步推測(cè)FA有可能對(duì)PDE表達(dá)具潛在抑制作用。第三,PDE4B可調(diào)節(jié)細(xì)胞內(nèi)c AMP水平和c AMP/c AMP應(yīng)答元件結(jié)合蛋白(c AMPresponse element binding protein,CREB)通路。進(jìn)一步研究FA對(duì)LPS誘導(dǎo)PC12細(xì)胞磷酸二酯酶4B(PDE4B)上調(diào)表達(dá)及活性的影響,使用激光共聚焦顯微鏡檢測(cè)FA對(duì)LPS誘導(dǎo)PC12細(xì)胞骨架蛋白F-actin變化的影響。我們進(jìn)一步檢測(cè)FA對(duì)LPS誘導(dǎo)的c AMP特異性PDE活性和細(xì)胞炎性因子(TNF-α和IL-1β)及ROS產(chǎn)生的影響;通過(guò)實(shí)時(shí)熒光定量RT-PCR(real-time fluorescent quantitative reverse transcription polymerase chain reaction,Q-PCR)檢測(cè)LPS誘導(dǎo)PC12細(xì)胞PDE4B m RNA表達(dá);通過(guò)免疫印跡對(duì)PDE4B、CREB和磷酸化CREB(p CREB)的蛋白表達(dá)進(jìn)行檢測(cè)。FA對(duì)LPS所致PC12細(xì)胞F肌動(dòng)蛋白(F-actin)骨架的損傷具有保護(hù)作用,明顯改善細(xì)胞骨架蛋白F-actin的分布和結(jié)構(gòu)。顯著抑制LPS誘導(dǎo)的TNF-α、IL-1β及活性氧(reactive oxygen species,ROS)的產(chǎn)生。酶活性試驗(yàn)表明,FA可明顯減弱LPS誘導(dǎo)PC12細(xì)胞c AMP依賴(lài)性PDE酶活性的上調(diào)。對(duì)PC12細(xì)胞PDE4B m RNA的研究表明,FA預(yù)處理可減少由LPS誘導(dǎo)的PDE4B m RNA表達(dá)上調(diào)。免疫印跡表明,FA預(yù)處理后,抑制LPS誘導(dǎo)的PDE4B上調(diào)作用;對(duì)抗LPS誘導(dǎo)的CREB和p CREB下調(diào)作用。FA對(duì)LPS誘導(dǎo)的PDE4B表達(dá)上調(diào)的抑制作用及其對(duì)下游信號(hào)分子CREB和p CREB表達(dá)下調(diào)的對(duì)抗作用可能是其對(duì)神經(jīng)炎癥性疾病,比如AD樣神經(jīng)炎癥具有治療作用的一個(gè)機(jī)制。第四,檢測(cè)FA對(duì)LPS誘導(dǎo)Sprague-Dawley(SD)大鼠學(xué)習(xí)和記憶缺失的保護(hù)作用;探討FA對(duì)LPS誘導(dǎo)海馬神經(jīng)細(xì)胞損傷的保護(hù)作用及機(jī)制?在本研究中,Morris水迷宮試驗(yàn)檢測(cè)FA對(duì)抗LPS誘導(dǎo)的大鼠學(xué)習(xí)和記憶缺失作用,蘇木精-伊紅染色(Hematoxylin-eosin staining,HE)檢測(cè)FA對(duì)LPS損傷大鼠大腦皮層和海馬神經(jīng)元的組織病理學(xué)保護(hù)作用?免疫熒光法檢測(cè)FA對(duì)LPS損傷海馬神經(jīng)元細(xì)胞骨架蛋白β-tubulin的保護(hù)作用?為闡明FA對(duì)LPS誘導(dǎo)的海馬神經(jīng)元的保護(hù)作用,檢測(cè)SOD活性;Q-PCR檢測(cè)FA抗LPS誘導(dǎo)的IL-1β?caspase-1?Nod樣受體蛋白3(Nod like receptor protein,NLRP3)和PDE4B的m RNA表達(dá)作用;免疫印跡法檢測(cè)PDE4B、NLRP3和CREB及p CREB蛋白的表達(dá)?免疫組織化學(xué)染色法檢測(cè)FA對(duì)LPS誘導(dǎo)大鼠海馬神經(jīng)元CA1?CA2和CA3區(qū)PDE4B蛋白表達(dá)的影響?Morris水迷宮試驗(yàn)表明FA預(yù)處理對(duì)抗LPS誘導(dǎo)的大鼠學(xué)習(xí)記憶功能障礙;HE染色顯示,預(yù)先給予不同劑量FA可以減輕腹腔注射LPS誘導(dǎo)的SD大鼠海馬神經(jīng)元損傷;與LPS組相比,FA預(yù)處理組大鼠海馬神經(jīng)元β-tubulin蛋白表達(dá)增加;FA預(yù)處理顯著維持大鼠大腦皮層和海馬神經(jīng)元中被LPS抑制的SOD的活性;FA預(yù)處理顯著抑制LPS誘導(dǎo)的IL-1β?capase-1?NLRP3和PDE4B的m RNA水平的增加?Western蛋白印跡分析表明,相較于LPS組,FA預(yù)處理組的CREB和p-CREB的表達(dá)增加,同時(shí)抑制LPS誘導(dǎo)的大鼠海馬神經(jīng)元中PDE4B和NLRP3的表達(dá);免疫組化染色結(jié)果顯示,FA預(yù)處理顯著抑制了LPS誘導(dǎo)的海馬神經(jīng)元中CA1?CA2和CA3區(qū)PDE4B表達(dá)的上調(diào)?FA對(duì)LPS誘導(dǎo)的大鼠學(xué)習(xí)和記憶損傷以及海馬神經(jīng)元損傷發(fā)揮保護(hù)作用。FA的神經(jīng)保護(hù)作用與其抗炎,抗氧化作用密切相關(guān);FA抑制LPS誘導(dǎo)的PDE4B上調(diào)并對(duì)抗LPS對(duì)CREB和p CREB的下調(diào)作用,FA可作為AD樣神經(jīng)炎性疾病的治療藥物。
[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
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R749.16

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