针对阿尔茨海默病中Aβ42靶点的天然药物及其分子机制的研究

发布时间：2018-07-16 23:39

【摘要】：阿尔茨海默病（AD）是一种以记忆和认知功能障碍为主要症状多发于老年群体的神经退行性疾病，发病后较难治愈，AD主要的病理学特征包括脑神经元外出现的β淀粉样蛋白（Aβ）的沉积和其进一步形成的老年斑（SP），细胞内过度磷酸化的Tau蛋白导致的神经纤维缠结和神经元的营养坏死等。很多证据表明Aβ42可溶性的寡聚体的神经毒性最强，，很可能是AD发病的关键因素。如何安全而有效地阻止甚至清除Aβ42毒性肽被认为是预防和治疗AD的有效方法。本论文以Aβ42的寡聚体作为AD的靶点，旨在寻找能够抑制Aβ42的寡聚体神经毒性并可以真正通过血脑屏障在体内产生治疗AD效果的天然药物，并尝试对其作用机制进行初步的研究和探讨。本论文首先总结分析并筛选了中医用于治疗AD的中药，综合近年来对AD致病机理的研究，筛选出肉苁蓉、淫羊藿、石菖蒲、茯苓、黄芪、何首乌、红景天、远志、绞股蓝、天麻、当归、苦参、熟地黄、蛇床子、益智仁、女贞子共16味中药材作为具有潜在治疗阿尔茨海默病的天然药物，通过高通量筛选，确定了十种具有抗Aβ42寡聚体神经毒性的提取物，并锁定了10种可能对Aβ42的细胞毒性有抑制作用的化合物，再根据所选化合物与AD治疗相关的文献报道，基于提取物中有效成分的浓度，透过血脑屏障所需的化合物的物理化学性质的考虑，最终确定了两种单体化合物：阿魏酸（样品A）和苦参碱（样品B）作为潜在具有抗Aβ42细胞毒性的目标化合物，在体外通过构建具有神经细胞特性的SH-SY5Y细胞的AD细胞模型，进一步在细胞水平上和分子水平上检测这两种单体化合物对Aβ42聚集的影响和变化，同时在分子水平上对这两种单体化合物抑制Aβ42毒性的作用机理进行初步的探讨。本论文在体外检测了样品A和B对Aβ42诱导的细胞毒性的抑制作用，结果显示：以SH-SY5Y细胞为靶细胞，样品A表现出抑制Aβ42单体及寡聚体诱导的细胞毒性的作用；PC12细胞和SH-SY5Y细胞为靶细胞，样品B均表现出抑制Aβ42单体及寡聚体诱导的细胞毒性的作用，结果表明样品A和B均具有抗Aβ42的神经保护作用。进一步通过CD色谱的手段研究样品A与Aβ42的相互作用我们发现样品A可以抑制Aβ42单体中β折叠结构的形成，但当Aβ42形成寡聚体后，样品A却反而促进了Aβ42寡聚体中β折叠结构的形成，而样品B则均可抑制Aβ42单体和寡聚体聚集形成β折叠。随后通过透射电镜的观察样品A和B对Aβ42聚集形态的影响结果和Th-T荧光染色检测样品A和B对Aβ42聚集程度的影响的结果均与CD色谱结果相符合，证实了样品A可以抑制Aβ42单体的聚集，但是却可以促进Aβ42寡聚体的聚集的特性。而样品B则可以抑制Aβ42单体和寡聚体的聚集。接下来通过样品A和B对Aβ42不同聚集状态的特异性研究我们发现样品A和样品B对Aβ42单体，寡聚体和纤维三种聚集状态均有特异性，样品A对Aβ42寡聚体的特异性最低，而样品B对Aβ42单体的特异性最低。由于AD的病灶在脑内，所以药物是否可以有效的通过血脑屏障发挥作用是我们所必须优先考虑的重要因素。因此在接下来的实验我们检测了样品A和B对SH-SH5Y细胞膜的透过效率，结果显示样品A对SH-SH5Y细胞的细胞膜具有一定的透过性。而在对样品B进行小鼠灌胃后的初步研究中我们在灌胃小鼠的脑匀浆提取物中检测到了样品B的存在，确定了样品B可以通过血脑屏障而进入脑内；而通过MTT实验我们在体外证实了在体内代谢后的样品B仍然可以对Aβ42寡聚体诱导的细胞毒性产生抑制作用。通过分析样品A和样品B对Aβ42的二级结构变化，聚集形态变化和特异性等多方面的影响后我们最终确定了样品A和样品B对Aβ42是有相互作用的，而且通过这种相互作用样品A和样品B可以改变Aβ42的聚集状态，最终抑制Aβ42的诱导的神经毒性，那这种相互作用的分子机制是什么？我们运用分子对接的方法对样品A和B与Aβ42单体的相互作用进行了模拟，分子对接的结果显示样品A和样品B分别都能够与Aβ42单体产生相互作用，作用位点相似，而作用机制不同，样品A主要通过氢键与Aβ42相互作用，而样品B则依靠静电相互作用与Aβ42相结合。综上所述，本论文通过对筛选后得到的两种天然单体化合物阿魏酸（样品A）和苦参碱（样品B）与AD的靶点Aβ42寡聚体的相互作用的研究，确定了样品A和样品B均可以通过抑制或促进Aβ42的聚集使其远离最具神经毒性的Aβ42寡聚体结构形式，起到抑制Aβ42的神经毒性，保护神经细胞的作用；此外我们还发现，样品A可以抑制Aβ42单体的聚集，但却促进Aβ42寡聚体的聚集，而动物实验证实了样品B可以通过血脑屏障进入脑内并在体内代谢后仍然可以对Aβ42寡聚体诱导的细胞毒性产生抑制作用。初步的分子对接模拟结果也展示了样品A和B针对Aβ42单体不同的作用机制和相似的作用位点，这些实验结果都为进一步的研发治疗AD的药物提供了新的实验依据和参考。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

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