【作者】 杨冰；

【导师】 张应玖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2021， 博士

【摘要】 β-淀粉样蛋白(1-42)(β-amyloid protein 1-42,Aβ42)聚集体的形成和Aβ42寡聚体的细胞毒性是阿尔茨海默病(Alzheimer’s disease,AD)病理学的两个重要特征,在AD的发病机理中起着关键的作用。选择性抑制Aβ42聚集体的形成和Aβ42寡聚体的细胞毒性为AD治疗提供了最佳靶点。来自中草药(CHM)的某些天然化合物具有抗Aβ42聚集和抑制Aβ42神经细胞毒性的功效,能够保护神经细胞免受具有细胞毒性的Aβ42聚集体的损伤。我们在早期的研究中已发现,而且也有文献报道证明了,苦参碱(Matrine,Mat)能够抑制Aβ42寡聚体对人神经母细胞瘤细胞(SH-SY5Y)的毒性。然而,我们在前期的研究中还进一步发现,苦参碱对Aβ42寡聚体的细胞毒性的抑制功效并不完全与苦参碱的浓度成正比,这提示苦参碱对Aβ42寡聚体的抑制作用模式可能不是单一的,而可能存在着多样性,亦或也不是单方向的,但是关于这些方面的内容在此之前还没有进一步的揭示和文献报道。在本论文研究中,基于我们在前期研究中发现的苦参碱对Aβ42寡聚体毒性的抑制功效存在浓度不平行的现象,以进一步揭示苦参碱对Aβ42寡聚体靶点的(多重)作用机理,并以揭示其量效关系为目的,对苦参碱低量高效地抑制Aβ42寡聚体细胞毒性进行进一步深入细致的研究和探索,并采用纳米技术,利用具有安全性的、可生物降解的高分子化合物研究并确定苦参碱纳米粒子的最优化制备体系,借助苦参碱纳米粒子的缓释特性,使用APP/PS1转基因小鼠(阿尔茨海默病模型鼠),确定苦参碱纳米粒子在体内的持续功效。通过本论文的研究,获得如下结果:一、首次发现苦参碱通过抑制Aβ42单体的聚集,并与Aβ42单体协同作用来维持甚至增强Aβ42单体的细胞营养功效。1.苦参碱(Mat)可在远低于其IC50值的浓度下(如10μM)选择性地与Aβ42寡聚体和Aβ42单体而非与Aβ42纤维相结合。2.苦参碱与Aβ42单体或由Aβ42单体新形成的Aβ42寡聚体直接结合后,苦参碱不仅能够降低,甚至能够消除Aβ42寡聚体的细胞毒性,表现出促进体外SH-SY5Y细胞的增殖或提高其存活率。同时首次发现,苦参碱对SH-SY5Y细胞的这些保护作用与所用苦参碱的浓度没有绝对的线性相关性,只有在一定浓度下(如1.0-50μM)才能发挥更好的功效。借助苦参碱与Aβ42单体或新形成的Aβ42寡聚体的分子对接结果,首次揭示,苦参碱能够增强Aβ42单体的细胞营养作用,这可能是苦参碱通过与Aβ42单体产生了正协同作用来实现的,并推测在人脑中有可能存在苦参碱样的代谢产物,它(们)可作为Aβ42单体的分子伴侣,起到稳定Aβ42单体的作用。苦参碱对Aβ42的这种功能特性或者对Aβ42的量效关系在此之前还没有其它报道披露,我们分析这应该与Aβ42分子的两性特性和整合构象有关。二、首次发现,苦参碱对Aβ42寡聚体的细胞毒性也表现出浓度区域相关性的特点。中等浓度的苦参碱(1.0-50μM)与Aβ42寡聚体相互作用不仅能抑制后者的细胞毒性,而且还能积极促进Aβ42寡聚体解聚成Aβ42单体,进而对SH-SY5Y细胞发挥细胞营养和细胞保护作用。但是,较高浓度的苦参碱(如100μM)仅表现出抑制Aβ42寡聚体细胞毒性的作用。根据苦参碱与Aβ42寡聚体分子对接结果,通过构象分析首次提出,苦参碱在功效上的这些差异是由于苦参碱和Aβ42寡聚体在不同的分子比例下相互作用的结果,并同样指向相同的推测,即人脑中可能存在类苦参碱的代谢物,它(们)可作为Aβ42单体的分子伴侣,当缺乏这种分子伴侣时会导致Aβ42单体逐渐聚集,形成具有细胞毒性的Aβ42寡聚物。三、苦参碱能有效实现脑转运,其纳米粒子形式有利于维持其脑内的有效浓度。动物实验结果表明,苦参碱口服给药后,虽然能被迅速吸收并进入脑内,但随着时间的延长,苦参碱在血液和脑组织中的水平持续下降。为了使苦参碱持续性地发挥抑制Aβ42寡聚体的细胞毒性的功效,以及协同Aβ42单体发挥细胞营养的功效,本论文采用纳米技术,利用具有生物安全性的、可降解的高分子化合物聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]和聚乙二醇修饰的聚乳酸-羟基乙酸共聚物[Polyethylene glycol-poly(lactic-co-glycolic acid),PEG-PLGA]分别制备了性能良好的载苦参碱的纳米粒子(Mat-NPs):Mat-PLGA-NPs与Mat-PEG-PLGA-NPs,并实现了苦参碱的有效脑转运与脑内持续释放的效果。1.使用乳化溶剂挥发法,基于单因素分析,正交试验分析,经高效液相色谱(HPLC)、动态激光分析技术(DLS)以及扫描电镜检测(SEM),分别确定了两类苦参碱纳米粒子的优选制备条件,即制备Mat-PLGA-Nps的最适条件为:PLGA用量为50mg、聚乙烯醇(Polyvinylalcohol,PVA)浓度为2%、搅拌挥发时间为3小时、超声时间为8分钟以及苦参碱溶液的体积为200μL;制备Mat-PLGA-NPs的最适条件为:PEG-PLGA用量为50 mg、聚乙烯醇(Polyvinylalcohol,PVA)浓度为1%、搅拌挥发时间为3小时、超声时间为8分钟以及苦参碱溶液的体积为200μL。两类Mat-NPs的制备条件很相近,但各因素影响的权重不同,优化出的条件对相应的Mat-PLGA-NPs或Mat-PEG-PLGA-NPs来说分别是最适宜的,而且条件工艺均较简便、操作性强、可行性高。2.分别在各自最适条件下制备了十批次Mat-PLGA-NPs或Mat-PEG-PLGA-NPs,并分别分析了它们的重要性能指标:Mat-PLGA-NPs的包封率为80.67±2.5%、载药量为3.24±0.06%,平均粒径是190.5±2.43 nm;Mat-PEG-PLGA-NPs的包封率为81.75±3.96%、载药量为3.27±0.16%、平均粒径是121.2±3.15 nm。通过扫描电镜(50.00 KX)观察到所制备的纳米粒子Mat--PLGA-NPs与Mat-PEG-PLGA-NPs,它们的颗粒分散均匀,平均粒径均控制在200 nm以内(Mat-PLGA-NPs的平均粒径为121.2±3.15 nm,而Mat-PLGA-NPs的平均粒径为190.5±2.43 nm)。外观形态均基本呈球形,表面光滑,大小也比较均匀。以上结果表明,PEG的加入不但引入了它的功效,同时还可降低了纳米粒子的颗粒尺寸,这不仅有利于纳米粒子在体内的吸收与运输,更有利于提高纳米粒子的脑转运效率,而且同时也提高了纳米粒子的载药量。3.缓释是纳米粒子作为载药系统最重要的目的之一。载药纳米粒子的体外释放药物的性能也是评价所制备的纳米粒子质量的重要指标之一。通过体外释放实验检测证明,Mat-PLGA-NPs有效释放持续9天,累积释放率达到93.2±0.26%;Mat-PEG-PLGA-NPs有效释放持续10天,累积释放率达到92.4±0.69%。根据释放结果进行数学模型拟合,Mat-PLGA-NPs拟合的释放方程为Q=33.956t（？）-4.849(R2=0.9958);Mat-PEG-PLGA-NPs拟合的释放方程为Q=31.795t（？）-5.166(R2=0.9978)。因此,我们制备的两种苦参碱纳米粒子均符合Higuchi方程,属于缓释释药系统,符合制备缓释苦参碱纳米粒子的预期。Mat-PLGA-NPs和Mat-PEG-PLGA-NPs的体外释放过程大致都可以分为前2天的突释过程和3-9/10天的缓释过程。前2天的突释推测主要是由于所制备的纳米粒子表面携带的苦参碱直接释放到介质中所导致的。之后的3-9/10天的缓慢释放是因为位于纳米粒子内部的苦参碱在多聚物(PLGA或PEG-PLGA)骨架逐渐降解后暴露出来,并释放到介质中的结果。这样符合对载药纳米粒子药物释放模式的基本要求。此外,对比分析显示,Mat-PEG-PLGA-NPs较Mat-PLGA-NPs缓释苦参碱的效果更好、释放持续时间更长、对提高苦参碱的生物利用度更有利。4.两种苦参碱纳米粒子(Mat-PLGA-NPs和Mat-PEG-PLGA-NPs)总体上与游离苦参碱相似,在一定水平范围能够阻抑Aβ42的细胞毒性作用。然而,与游离苦参碱相比,两种苦参碱纳米粒子释放出的苦参碱所起的作用与中等浓度水平苦参碱(1.0-50μM)与Aβ42寡聚体相互作用的结果相当,更重要的是,当Mat-NPs累积释放出的苦参碱的量高于游离苦参碱中等水平、达到相应的较高水平时,它们不但能够抑制Aβ42寡聚体的细胞毒性,而且仍能够增强Aβ42对细胞的营养作用。这表明,两种苦参碱纳米粒子阻抑Aβ42寡聚体细胞毒性的作用均效果更好,持续的时间也更长,充分体现了纳米粒子缓释意义和应用优势。四、苦参碱及其纳米粒子能有效降低AD模型鼠脑内Aβ42的沉积和淀粉样斑块的负荷,减轻海马区组织病理学的改变。1.建立了体外血脑屏障(BBB)模型,利用此模型检测证明,Mat-PLGA-NPs和Mat-PEG-PLGA-NPs均能够有效实现脑转运。与游离苦参碱相比,两类苦参碱纳米粒子均更加持久地维持过血脑屏障的苦参碱水平,其24小时内的苦参碱的累积释放率分别为15.0±0.53%与14.3±0.78%。2.使用AD模型鼠开展了苦参碱及其纳米粒子Mat-PLGA-NPs和Mat-PEG-PLGA-NPs的动物试验:(1)HE染色证明:苦参碱及其纳米粒子Mat-PLGA-NPs和Mat-PEG-PLGA-NPs均能够减轻AD模型鼠海马区组织病理学的改变,而苦参碱纳米粒子较游离苦参碱更有效。Mat-PLGA-NPs和Mat-PEG-PLGA-NPs给药的AD模型鼠的海马区,细胞排列整齐,大部分细胞核仁明显,核膜清晰,染色质丰富,只有少部分细胞胞浆和细胞核固缩,染色变深,体积变小,整体状况更接近阴性对照鼠海马区的状况;(2)免疫组化结果表明:苦参碱及其纳米粒子Mat-PLGA-NPs和Mat-PEG-PLGA-NPs均能够使AD模型小鼠海马区中淀粉样斑块数量减少以及斑块体积变小,可有效降低了AD模型鼠脑组织内淀粉样斑块的负荷,而后者的效果更明显。这说明,Mat-PLGA-NPs和Mat-PEG-PLGA-NPs能够通过持续而有效地抑制AD小鼠脑海马中Aβ42的沉积,进而减轻了大脑海马区的病理学改变。(3)斑点印迹结果显示:苦参碱及其纳米粒子Mat-PLGA-NPs和Mat-PEG-PLGA-NPs均能够使AD模型小鼠海马区中淀粉样蛋白的水平降低,而后者的效果更显著,这表明Mat-PLGA-NPs和Mat-PEG-PLGA-NPs能够通过缓释苦参碱,更能长时间有效地降低海马区淀粉样蛋白的水平。本论文体外、体内研究结果证明,苦参碱及其纳米粒子形式能够正向增强Aβ42单体的有益生理作用、负向阻抑Aβ42的聚集及其聚集体的神经细胞毒性和老年斑的形成。在本论文确定的最适条件下制备的苦参碱纳米粒子形式,要较游离苦参碱更能够持久地、有效地发挥这些正向和负向的功效。这些研究结果为苦参碱的临床用药奠定了一定的基础,提供了有力的依据。更多还原

【Abstract】 The formation of β-amyloid protein 1-42(Aβ42)oligomers and the cytotoxicity of Aβ42 oligomers are two important pathological characteristics of Alzheimer’s disease(AD),which play a key role in the pathogenesis of AD.Selective inhibition of the formation of Aβ42 aggregates and the cytotoxicity of Aβ42 oligomers provide the best target for AD therapy.Some natural compounds from Chinese herbal medicine(CHM)have the effects of anti-Aβ42 aggregation and inhibiting the neurotoxicity of Aβ42,which can protect nerve cells from the damage of the cytotoxicity of Aβ42 aggregates.In our early studies and literatures by other scholars,we have found that matrine(Mat)can inhibit the toxicity of Aβ42 oligomer to human neuroblastoma cells(SH-SY5Y).In our previous study,we further found that the inhibitory effect of Mat on the cytotoxicity of Aβ42 oligomer is not completely proportional to the concentration of Mat,which suggests that the inhibitory effect of Mat on Aβ42 oligomer may not be single,but may have diversity,or may not be unidirectional.However,the content of these aspects has not been further revealed and reported before.In this study,we found that the inhibitory effect of Mat on the toxicity of Aβ42oligomer was not parallel in concentration,in order to further reveal the(multiple)mechanism of Mat on the target of Aβ42 oligomer,and to reveal its dose-response relationship,and we further studied the low-dose and high-efficiency inhibition of Matrine on the cytotoxicity of Aβ42 oligomer.We used nanotechnology to study and determine the optimal preparation system of Mat nanoparticles by using safe and biodegradable polymer compounds.With the help of the sustained-release characteristics of Mat nanoparticles,we used App/PS1 transgenic mice(AD model mice)to determine the sustained efficacy of Mat nanoparticles in vivo.The results are as follows:Ⅰ.For the first time,it was found that Mat could maintain or even enhance the cellular nutritional efficacy of Aβ42 monomer by inhibiting the aggregation of Aβ42 monomer and cooperating with Aβ42 monomer.1.Mat can selectively bind to Aβ42 oligomer and Aβ42 monomer at a concentration far below its IC50 value(such as 10 μM),rather than Aβ42 fiber.2.When Mat is directly combined with Aβ42 monomer or the new Aβ42 oligomer formed by Aβ42 monomer,Mat can not only reduce or even eliminate the cytotoxicity of Aβ42 oligomer,but also promote the proliferation or increase the survival rate of SH-SY5 Y cells in vitro.At the same time,it was found for the first time that there was no absolute linear correlation between the protective effects of Mat on SH-SY5 Y cells and the concentration of Mat used.Only at a certain concentration(1.0-50μM)can it play a better role.Based on the results of molecular docking of Mat with Aβ42 monomer or newly formed Aβ42 oligomer,it was revealed for the first time that Mat can enhance the cellular nutritional function of Aβ42 monomer,which may be realized by the positive synergistic effect of Mat with Aβ42 monomer.It is speculated that there may be Mat-like metabolites in the human brain,which can act as a molecular chaperone of Aβ42 monomer to stabilize Aβ42 monomer.The functional properties of Mat to Aβ42 or the dose-response relationship of Mat to Aβ42 have not been reported before,which should be related to the amphotericity and integrated conformation of Aβ42.Ⅱ.It was found for the first time that the cytotoxicity of Mat to Aβ42 oligomer also showed the characteristics of regional concentration.The interaction between Mat(1.0-50 μM)and Aβ42 oligomer not only inhibited the cytotoxicity of Aβ42 oligomer,but also promoted the depolymerization of Aβ42 oligomer into Aβ42 monomer,which played the role of cell nutrition and cell protection on SH-SY5 Y cells.However,higher concentrations of Mat(100 μM)only inhibited the cytotoxicity of Aβ42 oligomer.According to the docking results of Mat and Aβ42 oligomer,it is proposed for the first time by conformational analysis that these differences in the efficacy of Mat are due to the interaction of Mat and Aβ42 oligomer at different molecular ratios,and also point to the same speculation that there may be Mat-like metabolites in human brain,which can be used as molecular chaperones of Aβ42 monomer.However,in the absence of this chaperone,Aβ42 monomer will gradually aggregate to form Aβ42 oligomer with cytotoxicity.Ⅲ.Mat can effectively achieve brain transport,and its nanoparticle forms are conducive to maintaining its effective concentration in the brain.The results of animal experiments showed that although Mat could be rapidly absorbed into the brain after oral administration,the level of Mat in the blood and brain tissue continued to decline with the extension of time.In order to make Mat continuously inhibit the cytotoxicity of Aβ42 oligomer,and cooperate with Aβ42 monomer to exert the effect of cell nutrition,this paper adopts nanotechnology,using the poly(lactic-co-glycolic acid)(PLGA)and Polyethylene glycol-poly(lactic-co-glycolic acid)(PEG-PLGA),which have biological safety and degradability to be prepared Mat loaded nanoparticles(Mat-PLGA-NPs and Mat-PEG-PLGA-NPs)respectively,At the same time,the effective brain transport and sustained release in brain of Mat were realized.1.Use emulsion solvent evaporation method,and base on single factor analysis and orthogonal test analysis,and be detected by high performance liquid chromatography(HPLC),dynamic light scattering(DLS)and scanning electron microscope(SEM)to determine the optimum conditions for the preparation of two kind of Mat nanoparticles.The optimum conditions for the preparation of Mat-PLGA-NPs were as follows: the dosage of PLGA was 50 mg,the concentration of Polyvinylalcohol(PVA)was 2%,the stirring time was 3 h,the ultrasonic time was 8min,and the volume of Mat solution was 200 μL;The optimum conditions for the preparation of Mat-PEG-PLGA-NPs were as follows: the dosage of PEG-PLGA was 50 mg,the concentration of Polyvinylalcohol(PVA)was 1%,the stirring time was 3 h,the ultrasonic time was 8 min,and the volume of Mat solution was200 μL.The preparation conditions of the two kinds of Mat-NPs are very similar,but the weight of each factor is different.The optimized conditions are the most suitable for the corresponding Mat-PLGA-NPs or Mat-PEG-PLGA-NPs,and the conditions and processes are simple,easy to operate and feasible.2.Ten batches of Mat-PLGA-NPs or Mat-PEG-PLGA-NPs were prepared under their respective optimum conditions,and their important performance indexes were analyzed: the encapsulation efficiency of Mat-PLGA-NPs was 80.67±2.5%,drug loading was 3.24±0.06%,and the average particle size was 190.5±2.43 nm;the encapsulation efficiency of Mat-PEG-PLGA-NPs was 81.75±3.96%,drug loading was 3.27±0.16%,and the average particle size was 190.5±2.43 nm The diameter is 121.2±3.15 nm.Mat-PLGA-NPs and Mat-PEG-PLGA-NPs were observed by SEM(50.00 KX).The particles are well dispersed and their average particle size was controlled within 200 nm(the average particle size of Mat-PEG-PLGA-NPs was 121.2 ± 3.15 nm,while the average particle size of Mat-PLGA-NPs was 190.5 ± 2.43 nm).The appearance is spherical,the surface is smooth and the size is uniform.The above results show that PEG not only introduces its efficacy,but also reduces the size of nanoparticles,which is not only conducive to the absorption and transportation of nanoparticles in vivo,but also helps to improve the brain transport efficiency of nanoparticles and improves the drug loading of nanoparticles.3.Sustained release is one of the most important purposes of nanoparticles as drug delivery system.Drug release performance of drug loaded nanoparticles in vitro is one of the important indicators to evaluate the quality of the prepared nanoparticles.In vitro release test showed that the effective release of Mat-PLGA-NPs lasted for 9 d,and the cumulative release rate was 93.2±0.26%;the effective release of Mat-PEG-PLGA-NPs lasted for 10 d,and the cumulative release rate was 92.4±0.69%.According to the release results,the release equation fitted by Mat-PLGA-NPs was Q=33.956t（？）-4.849(R2 = 0.9958);the release equation fitted by Mat-PEG-PLGA-NPs was Q=31.795t（？）-5.166(R2 = 0.9978).Therefore,we prepared two kinds of Mat nanoparticles are in line with the Higuchi equation,which belongs to the sustained-release drug delivery system,in line with the expected preparation of sustained-release Mat nanoparticles.The in vitro release process of Mat-PLGA-NPs and Mat-PEG-PLGA-NPs can be roughly divided into the first two days of sudden release process and 3-9/10 d of sustained release process.It is speculated that the sudden release in the first two days is mainly due to the direct release of Mat carried on the surface of the prepared nanoparticles into the medium.The slow release in the next 3-9/10 d was due to the gradual degradation of the polymer(PLGA or PEG-PLGA)skeleton and the release of Mat into the medium.This is in line with the basic requirements of drug release mode of drug loaded nanoparticles.In addition,compared with Mat-PLGA-NPs,Mat-PEG-PLGA-NPs has better sustained-release effect,longer release duration and better bioavailability of Mat.4.Two Mat nanoparticles(Mat-PLGA-NPs and Mat-PEG-PLGA-NPs)were similar to free Mat in general,and could inhibit the cytotoxicity of Aβ42 in a certain level range.However,compared with free Mat,the effect of Mat released from the two kinds of Mat nanoparticles is similar to that of the interaction of medium concentration level Mat(1.0-50 μM)with Aβ42 oligomer.More importantly,when the cumulative release of Mat from Mat-NPs was higher than that of free Mat,they could not only inhibit the cytotoxicity of Aβ42 oligomer,but also enhance the nutritional effect of Aβ42 on cells.These results indicate that the inhibition effect of two Mat nanoparticles on cytotoxicity of Aβ42 oligomer is better and lasts longer,which fully reflects the significance and application advantages of sustained-release nanoparticles.Ⅳ.Mat and its nanoparticles can effectively reduce the load of Aβ42 deposition in the brain of AD model rats and alleviate the histopathological changes in the hippocampus.1.The blood-brain barrier(BBB)model in vitro was established,and it was proved that Mat-PLGA-NPs and Mat-PEG-PLGA-NPs could effectively transport in the brain.Compared with free Mat,the two kinds of Mat nanoparticles maintained the level of Mat across the BBB more persistently.The cumulative release rates of Mat in 24 h were15.0±0.53% and 14.3±0.78%,respectively.2.Animal experiments of Mat and its nanoparticles Mat-PLGA-NPs and Mat-PEG-PLGA-NPs were carried out in AD model rats.(1).HE staining showed that Mat and its nanoparticles Mat-PLGA-NPs and Mat-PEG-PLGA-NPs could alleviate the histopathological changes in hippocampus of AD model rats,and Mat nanoparticles were more effective than free Mat.In the hippocampus of AD model rats treated with Mat-PLGA-NPs and Mat-PEG-PLGA-NPs,the cells were arranged in order,most of them had obvious nucleolus,clear nuclear membrane and abundant chromatin,only a few cells had pyknosis in cytoplasm and nucleus,and the staining became deeper and smaller,and the overall situation was closer to that of negative control rats.(2).The results of immunohistochemistry showed that Mat and its nanoparticles Mat-PLGANPs and Mat-PEG-PLGA-NPs could reduce the number and volume of amyloid plaques in the hippocampus of AD model rats,and could effectively reduce the burden of Aβ42 plaques in the brain of AD model rats,and the effect of the latter was more obvious.These results suggest that Mat-PLGA-NPs and Mat-PEG-PLGA-NPs can inhibit the deposition of Aβ42in hippocampus of AD rats continuously and effectively,thereby alleviating the pathological changes in hippocampus.(3).Dot blotting results showed that Mat and its nanoparticles(Mat-PLGA-NPs and Mat-PEG-PLGA-NPs)could reduce the level of amyloid protein in the hippocampus of AD model rats,and the effect of the latter was more significant.These results show that Mat-PLGA-NPs and Mat-PEG-PLGA-NPs can reduce the total level of amyloid protein in hippocampus for a long time by releasing Mat from its nanoparticles.The results of in vitro and in vivo studies show that Mat and its nanoparticles can positively enhance the beneficial physiological effects of Aβ42 monomer,negatively inhibit the aggregation of Aβ42 and the neurotoxicity of Aβ42 aggregates and the formation of senile plaques.Under the optimal conditions,the prepared Mat nanoparticles can exert these positive and negative effects more persistently and effectively than free Mat.These results lay a foundation for the clinical use of Mat and provide a strong basis.更多还原