【摘要】：阿爾茨海默�。ˋD）是一種以記憶和認知功能障礙為主要癥狀多發(fā)于老年群體的神經(jīng)退行性疾病，發(fā)病后較難治愈，AD主要的病理學特征包括腦神經(jīng)元外出現(xiàn)的β淀粉樣蛋白（Aβ）的沉積和其進一步形成的老年斑（SP），細胞內(nèi)過度磷酸化的Tau蛋白導致的神經(jīng)纖維纏結(jié)和神經(jīng)元的營養(yǎng)壞死等。很多證據(jù)表明Aβ42可溶性的寡聚體的神經(jīng)毒性最強，，很可能是AD發(fā)病的關鍵因素。如何安全而有效地阻止甚至清除Aβ42毒性肽被認為是預防和治療AD的有效方法。本論文以Aβ42的寡聚體作為AD的靶點，旨在尋找能夠抑制Aβ42的寡聚體神經(jīng)毒性并可以真正通過血腦屏障在體內(nèi)產(chǎn)生治療AD效果的天然藥物，并嘗試對其作用機制進行初步的研究和探討。 本論文首先總結(jié)分析并篩選了中醫(yī)用于治療AD的中藥，綜合近年來對AD致病機理的研究，篩選出肉蓯蓉、淫羊藿、石菖蒲、茯苓、黃芪、何首烏、紅景天、遠志、絞股藍、天麻、當歸、苦參、熟地黃、蛇床子、益智仁、女貞子共16味中藥材作為具有潛在治療阿爾茨海默病的天然藥物，通過高通量篩選，確定了十種具有抗Aβ42寡聚體神經(jīng)毒性的提取物，并鎖定了10種可能對Aβ42的細胞毒性有抑制作用的化合物，再根據(jù)所選化合物與AD治療相關的文獻報道，基于提取物中有效成分的濃度，透過血腦屏障所需的化合物的物理化學性質(zhì)的考慮，最終確定了兩種單體化合物：阿魏酸（樣品A）和苦參堿（樣品B）作為潛在具有抗Aβ42細胞毒性的目標化合物，在體外通過構建具有神經(jīng)細胞特性的SH-SY5Y細胞的AD細胞模型，進一步在細胞水平上和分子水平上檢測這兩種單體化合物對Aβ42聚集的影響和變化，同時在分子水平上對這兩種單體化合物抑制Aβ42毒性的作用機理進行初步的探討。 本論文在體外檢測了樣品A和B對Aβ42誘導的細胞毒性的抑制作用，結(jié)果顯示：以SH-SY5Y細胞為靶細胞，樣品A表現(xiàn)出抑制Aβ42單體及寡聚體誘導的細胞毒性的作用；PC12細胞和SH-SY5Y細胞為靶細胞，樣品B均表現(xiàn)出抑制Aβ42單體及寡聚體誘導的細胞毒性的作用，結(jié)果表明樣品A和B均具有抗Aβ42的神經(jīng)保護作用。進一步通過CD色譜的手段研究樣品A與Aβ42的相互作用我們發(fā)現(xiàn)樣品A可以抑制Aβ42單體中β折疊結(jié)構的形成，但當Aβ42形成寡聚體后，樣品A卻反而促進了Aβ42寡聚體中β折疊結(jié)構的形成，而樣品B則均可抑制Aβ42單體和寡聚體聚集形成β折疊。隨后通過透射電鏡的觀察樣品A和B對Aβ42聚集形態(tài)的影響結(jié)果和Th-T熒光染色檢測樣品A和B對Aβ42聚集程度的影響的結(jié)果均與CD色譜結(jié)果相符合，證實了樣品A可以抑制Aβ42單體的聚集，但是卻可以促進Aβ42寡聚體的聚集的特性。而樣品B則可以抑制Aβ42單體和寡聚體的聚集。接下來通過樣品A和B對Aβ42不同聚集狀態(tài)的特異性研究我們發(fā)現(xiàn)樣品A和樣品B對Aβ42單體，寡聚體和纖維三種聚集狀態(tài)均有特異性，樣品A對Aβ42寡聚體的特異性最低，而樣品B對Aβ42單體的特異性最低。 由于AD的病灶在腦內(nèi)，所以藥物是否可以有效的通過血腦屏障發(fā)揮作用是我們所必須優(yōu)先考慮的重要因素。因此在接下來的實驗我們檢測了樣品A和B對SH-SH5Y細胞膜的透過效率，結(jié)果顯示樣品A對SH-SH5Y細胞的細胞膜具有一定的透過性。而在對樣品B進行小鼠灌胃后的初步研究中我們在灌胃小鼠的腦勻漿提取物中檢測到了樣品B的存在，確定了樣品B可以通過血腦屏障而進入腦內(nèi)；而通過MTT實驗我們在體外證實了在體內(nèi)代謝后的樣品B仍然可以對Aβ42寡聚體誘導的細胞毒性產(chǎn)生抑制作用。 通過分析樣品A和樣品B對Aβ42的二級結(jié)構變化，聚集形態(tài)變化和特異性等多方面的影響后我們最終確定了樣品A和樣品B對Aβ42是有相互作用的，而且通過這種相互作用樣品A和樣品B可以改變Aβ42的聚集狀態(tài)，最終抑制Aβ42的誘導的神經(jīng)毒性，那這種相互作用的分子機制是什么？我們運用分子對接的方法對樣品A和B與Aβ42單體的相互作用進行了模擬，分子對接的結(jié)果顯示樣品A和樣品B分別都能夠與Aβ42單體產(chǎn)生相互作用，作用位點相似，而作用機制不同，樣品A主要通過氫鍵與Aβ42相互作用，而樣品B則依靠靜電相互作用與Aβ42相結(jié)合。 綜上所述，本論文通過對篩選后得到的兩種天然單體化合物阿魏酸（樣品A）和苦參堿（樣品B）與AD的靶點Aβ42寡聚體的相互作用的研究，確定了樣品A和樣品B均可以通過抑制或促進Aβ42的聚集使其遠離最具神經(jīng)毒性的Aβ42寡聚體結(jié)構形式，起到抑制Aβ42的神經(jīng)毒性，保護神經(jīng)細胞的作用；此外我們還發(fā)現(xiàn)，樣品A可以抑制Aβ42單體的聚集，但卻促進Aβ42寡聚體的聚集，而動物實驗證實了樣品B可以通過血腦屏障進入腦內(nèi)并在體內(nèi)代謝后仍然可以對Aβ42寡聚體誘導的細胞毒性產(chǎn)生抑制作用。初步的分子對接模擬結(jié)果也展示了樣品A和B針對Aβ42單體不同的作用機制和相似的作用位點，這些實驗結(jié)果都為進一步的研發(fā)治療AD的藥物提供了新的實驗依據(jù)和參考。
[Abstract]:Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory and cognitive dysfunction mainly occurring in the elderly. It is difficult to cure after the onset of Alzheimer's disease. The main pathological features of AD include the deposition of beta amyloid (A beta) outside the brain neurons and its progressive formation of the senile plaque (SP), and the excessive phosphorylation of Tau in the cells. A lot of evidence suggests that the neurotoxicity of A beta 42 soluble oligomers is the strongest and may be the key factor in the pathogenesis of AD. How to prevent and even remove A beta 42 toxicities safely and effectively is considered as an effective method to prevent and treat AD. In this paper, the oligomer of A beta 42 is used as an oligomer. The target of AD is to find the oligomeric neurotoxicity of A beta 42 and to produce the natural drugs that can actually produce the effect of AD through the blood brain barrier in the body, and try to make a preliminary study and Discussion on the mechanism of its action.
This paper first summarizes and analyzes the traditional Chinese medicine used in the treatment of AD, and the study of the pathogenesis of AD in recent years, and screening out the 16 herbs of Cistanche, epimedium, calamus, Poria, Radix Polygoni multiflorum, Radix Polygoni multiflorum, Radix Polygala, Gynostemma pentaphyllum, Gastrodia elata, Radix Angelicae, radix rehmanniae, Fructus Cnidii, yizhi kernel and Ligustrum lucidum. The natural drugs for Alzheimer's disease, through high throughput screening, identified ten extracts with anti A beta 42 oligomeric neurotoxicity, and locked 10 compounds that could inhibit the cytotoxicity of A beta 42, and then based on the related literature of the selected compounds and AD treatment, based on the concentration of the effective components in the extract. In consideration of the physical and chemical properties of the compounds required for the blood brain barrier, two monomers are determined: ferulic acid (sample A) and matrine (sample B) as a potential target compound that has the potential to resist A beta 42 cytotoxicity. In vitro, the AD cell model of SH-SY5Y cells with neural cell characteristics is constructed in vitro, and further at the cell level The effects and changes of these two monomers on the aggregation of A beta 42 were measured at the upper and the molecular levels, and the mechanism of the inhibition of the toxicity of the two monomers on the toxicity of A beta 42 was preliminarily discussed at the molecular level.
In this paper, the inhibitory effects of A and B on cytotoxicity induced by A beta 42 were detected in this paper. The results showed that SH-SY5Y cells were the target cells and the sample A inhibited the cytotoxicity of A beta 42 monomers and oligomers; PC12 cells and SH-SY5Y cells were target cells and sample B showed inhibition of A beta 42 monomers and oligomers. The cytotoxic effect of the sample A and B showed that both A and B had the protective effect of anti A beta 42. We further studied the interaction between sample and A beta 42 by CD chromatography. We found that the sample A could inhibit the formation of beta fold structure in A beta 42 monomer, but when A beta 42 formed oligomers, the sample A promoted beta 42 oligomer beta. The formation of the folding structure, while the sample B can inhibit the aggregation of A beta 42 monomers and oligomers to form beta folding. Subsequently, the effects of A and B on the aggregation morphology of A beta 42 by the transmission electron microscopy and the effect of A and B on the aggregation degree of A beta 42 by the Th-T fluorescence staining samples are all in accordance with the CD chromatographic results, which confirms that the sample A can be suppressed. The aggregation of A beta 42 monomers can promote the aggregation of A beta 42 oligomers, while sample B can inhibit the aggregation of A beta 42 monomers and oligomers. Next, we found that the samples A and sample B are specific to A beta 42 monomers, and three aggregation states of oligomers and fibers are specific by the specificity of samples A and B on the different aggregation states of A beta 42. The specificity of A for A beta 42 oligomers was the lowest, while the specificity of B for A A 42 was the lowest.
As the focus of AD is in the brain, it is an important factor that we have to consider whether the drug can play an effective role in the blood brain barrier. So in the next experiment, we detected the transmission efficiency of the sample A and B on the SH-SH5Y cell membrane. The results showed that the sample A had a certain transmittance to the cell membrane of the SH-SH5Y cells. In the preliminary study of mouse gastric perfusion with sample B, we detected the presence of sample B in the brain homogenate extract of the gavage mice, and determined that the sample B could enter the brain through the blood brain barrier; and through the MTT experiment, we confirmed in vitro that the sample B after metabolism in the body can still be induced by the A beta 42 oligomer. Toxicity has an inhibitory effect.
By analyzing the effects of sample A and sample B on the two stage structure changes of A beta 42, aggregation morphological changes and specificity, we finally determined that the sample A and sample B have interaction with A beta 42, and that A and sample B can change the aggregation state of A beta 42 through this interaction sample, and eventually inhibit the induced neurotoxicity of A beta 42. What are the molecular mechanisms of this interaction? We use molecular docking method to simulate the interaction of samples A and B and A beta 42 monomers. The results of molecular docking show that both sample A and sample B can interact with A beta 42 monomers respectively. The action loci are similar, and the mechanism of action is different, and the sample A is mainly through hydrogen. The bond is interacted with A beta 42, while the sample B is combined with A beta 42 by electrostatic interaction.
To sum up, through the study of the interaction between ferulic acid (sample A) and matrine (sample B) and A beta 42 oligomer of AD, the two kinds of natural monomer compounds obtained after screening have been studied. It is determined that the sample A and sample B can keep them away from the most neurotoxic A beta 42 oligomer by inhibiting or promoting the aggregation of A beta 42. It can inhibit the neurotoxicity of A beta 42 and protect the role of neural cells. In addition, we also found that sample A can inhibit the aggregation of A beta 42 monomers, but promote the aggregation of A beta 42 oligomers, and animal experiments confirm that the sample B can enter the brain through the blood brain barrier and can still be induced by the A beta 42 oligomer after metabolism in the body. The preliminary molecular docking simulation results also show the different mechanisms of action and similar action sites for the sample A and B against A beta 42 monomers. These results provide a new experimental basis and reference for further research and development of drugs for the treatment of AD.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R749.16

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