新型手性氮氧自由基对Alzheimer病的保护作用研究

发布时间：2018-05-15 13:42

本文选题：活性氧自由基 + 阿尔兹海默病　；参考：《第四军医大学》2013年博士论文

【摘要】：目的: 阿尔兹海默病(Alzheimer's disease, AD)是老年期痴呆最常见的类型。认知能力的逐渐下降和淀粉样斑块及神经原纤维缠结的出现是其典型的临床病理特征。最终结果会导致脑功能的异常及神经元凋亡。随着我国人口老龄化的日益严重，AD越来越成为一个严重的社会问题。研究表明AD神经退行性病变过程中，氧化应激是除年龄外另一个重要因素。大量的研究表明DNA、RNA、脂质和蛋白质氧化程度在AD病变过程中不断升高，直至出现轻度认知损伤，这些都说明氧化应激出现在AD的早期阶段，即发生在淀粉样斑块和神经原纤维缠结之前出现。尽管在AD的治疗研究中有很多针对于对抗氧化应激介导损伤的神经保护策略，但是临床结果积极有效的只有很少一部分。氮氧自由基（Nitroxide radicals, NRs）作为一类含有自旋单电子的稳定的自由基化合物。早期被用作自旋示踪剂。近来研究表明NRs还具有其特殊的生物学活性，一些NRs具有抗肿瘤、辐射和缺血再灌注损伤等功能。它与有害自由基反应通过“催化剂量”方式进行，只需很少的剂量即可发挥高效、强效和长效的抗氧化活性。例如，NRs具有拟SOD的作用。它可以通过电子转移的方式参与到细胞内的线粒体的呼吸链式反应，非常快速的清除超氧阴离子自由基O2.-。正是由于NRs以催化方式分解不断产生的有害自由基，反应过程中本身不会被消耗，可以循环再生利用，这也是其它自由基清除剂所无法比拟的。同时因其特有的自旋示踪功能，可通过电子顺磁共振或核磁共振成像实时了解其在组织中的分布及变化反应。正是由于NRs这些独特的性质，我们推测其在AD的发病过程中会发挥很好的抗氧化的能力，起到预防、治疗甚至是诊断AD发生的作用。因此，有望将NRs制备成新型的应用于神经退行性疾病的抗氧化应激损伤的药物。方法：在本实验中，我们采用体外抗氧化模型，Aβ1-42诱导的细胞毒性损伤模型以及APP/PS1双转基因AD鼠模型，来评价NRs的抗氧化能力及对AD的保护作用。体外抗氧化实验中，我们分别采用光泽精诱导的发光体系、CHP诱导的脂质过氧化模型和F2-异前列素免疫试剂盒来评价NRs清除超氧阴离子和抑制脂质过氧化的能力；体外培养原代皮层神经元模型中，在培养到第10天的时候，分别给予10μM的姜黄素、Tempol和L-NNNBP，孵育24小时后，用25μM的寡聚态Aβ1-42处理12小时。换用原培养液继续培养24小时后收集细胞。分别用CCK-8试剂盒检测细胞的存活率；用Tunel试剂盒检测细胞的凋亡；用3-NT的ELISA试剂盒测定组织的硝化应激水平；用阳离子荧光染料-四甲基罗丹明乙酯检测线粒体的膜电位变化；用细胞的免疫荧光观察激活的Caspase-3的变化；最后采用APP/PS-1双转基因AD小鼠来评价NRs的自由基清除能力和对AD的保护作用。实验分为5组：姜黄素治疗组、Tempol治疗组、L-NNNBP治疗组、WT组和TG组。药物用饮用水溶解，终浓度均为1mM，WT组和TG组给予正常饮水。在小鼠6周时给予药物处理，连续给药1个月。用Morris水迷宫实验评价AD小鼠空间学习记忆能力；用刚果红染色方法观察Aβ斑块；用蛋白免疫印迹方法观察磷酸化Tau和GFAP蛋白含量变化；采用免疫组化方法观察星形胶质细胞的激活。结果：体外抗氧化实验中，我们发现L-NNNBP较姜黄素和Tempol能显著减少超氧阴离子和脂质过氧化水平(#P0.05，##P0.01)。培养皮层神经元模型中，不同剂量的L-NNNBP对Aβ1-42诱导的细胞毒性都有保护作用(*P0.05，**P0.01)，且是浓度依赖的关系。单独给予L-NNNBP，没有对细胞产生毒性作用。而Tempol的三种剂量都没有观察到神经元的保护作用；姜黄素只有在高剂量（10μM）时有细胞保护作用。Tunel法检测细胞凋亡的实验中，相对于姜黄素（42.9%±3.1%）和Tempol（51.1%±1.1%），L-NNNBP高剂量（10μM）处理组，神经元的凋亡显著降低（22.7%±2.6%，*P0.05,**P0.01,##P0.01）。细胞免疫荧光结果说明L-NNNBP的这种抗凋亡的作用是通过降低激活型Caspase-3的表达引起的。Aβ诱导的氧化应激损伤也会造成线粒体功能障碍，影响线粒体正常膜电位，使其去极化。L-NNNBP能显著降低TMRM+的密度，阻止线粒体膜的去极化，保护线粒体的正常功能，，且其作用强度显著高于姜黄素和Tempol（#P0.05）。除了氧化应激，硝化应激也是自由基产生的主要来源。L-NNNBP三个浓度都降低了3-NT的含量（**P0.01），且抑制率显著高于姜黄素和Tempol（#P0.05）。 Aβ斑块的沉积是AD的主要病理症状之一。在APP/PS1双转基因AD鼠模型中，6个月大的APP/PS-1小鼠给予L-NNNBP(1mM)1个月后，海马和皮层的Aβ斑块的沉积都显著减少，并且效果强于姜黄素和Tempol（##P0.05）。除了Aβ斑块沉积，AD的另一个病理特征就是Tau蛋白异常过度磷酸化后引起的神经原纤维缠结。APP/PS1小鼠Tau蛋白的Thr205和Ser235两个位点的磷酸化程度显著升高。相对于WT组和其它药物处理组，L-NNNBP显著的降低了Tau蛋白在两个位点的磷酸化程度（**P0.01，#P0.05，##P0.05）。星形胶质细胞的免疫组化和免疫蛋白印迹结果说明其在AD发病的早期就被激活，有可能进一步诱导β-淀粉样蛋白沉积加速和神经纤维缠结形成。而L-NNNBP在发挥抗氧化功效的同时抑制了星形胶质细胞的激活。Morris水迷宫的行为学结果也说明APP/PS1小鼠空间学习和记忆能力受到损伤，在给予L-NNNBP治疗后有显著改善，并且效果强于姜黄素和Tempol（**P0.01,##P0.01）。结论：本研究证明，无论是在氧化损伤的体外模型，还是Aβ1-42诱导的细胞毒性损伤模型，或是APP/PS1双转基因AD鼠模型中，新型的手性氮氧自由基L-NNNBP都表现出了令人兴奋的结果。它能清除超氧阴离子、抑制脂质过氧化、减轻Aβ1-42诱导的细胞毒性损伤、抗凋亡、降低氧化或硝化应激损伤、减少Aβ斑块沉积、降低Tau蛋白磷酸化水平、抑制星形胶质细胞激活和改善AD鼠的空间学习和记忆。总之，L-NNNBP很有可能开发成为临床AD治疗的候选药物。
[Abstract]:Purpose :

Alzheimer ' s disease ( AD ) is one of the most common types of Alzheimer ' s disease . The gradual decline of cognitive ability and the appearance of amyloid plaques and neurogenesis are typical clinical pathological characteristics . As the aging population of our country becomes more and more serious , AD has become a serious social problem .

NRs is a kind of stable free radical compound containing spin single electrons . It has been used as a spin tracer in the early stage . Recent studies have shown that NRs has its special biological activity , and some NRs have the functions of anti - tumor , radiation and ischemia - reperfusion injury .

Because of the unique nature of NRs , we have speculated that it plays a very good role in preventing , treating and even diagnosing AD during the onset of AD . Therefore , it is hopeful to prepare NRs into a new drug which can be applied to the anti - oxidative stress injury of neurodegenerative diseases .

Method :

In this experiment , we evaluated the anti - oxidation ability of NRs and the protective effect on AD by using in vitro antioxidant model , A尾1 - 42 induced cytotoxicity damage model and APP / PSl double transgenic AD rat model .

In vitro antioxidant experiments , we evaluated the ability of NRs to eliminate superoxide anion and inhibit lipid peroxidation by using the light - emitting system induced by glossiness , the lipid peroxidation model induced by CHP and the F2 - isoalprost immunokit .

In the model of primary cortical neurons cultured in vitro , 10.mu . M of curcumin , Tempol and L - NNNBP were administered to 10.mu . M of curcumin , Tempol and L - NNNBP , respectively , and incubated for 24 hours with 25 & mu ; M of the oligomerized A.beta . 1 - 42 for 12 hours . The cells were collected after continued culturing for 24 hours with the original culture broth . The survival rate of the cells was detected by CCK - 8 kit , respectively ;
The apoptosis of cells was detected by Tunel kit .
The level of nitrification stress was determined by ELISA kit of 3 - NT .
detecting mitochondrial membrane potential changes with cationic fluorescent dye - tetramethylrhodamine ethyl ester ;
The changes of Caspase - 3 were observed by immunofluorescence of cells .

The experiment was divided into five groups : curcumin treatment group , Tempol treatment group , L - NNNBP treatment group , WT group and TG group .
The A 尾 plaques were observed by Congo red staining .
The contents of phosphorylated Tau and GFAP were observed by Western blotting .
Immunohistochemical method was used to observe the activation of astrocytes .

Results :

In vitro antioxidant experiments , we found that L - NNNBP significantly reduced superoxide anion and lipid peroxidation ( P0.05 , P 0.01 ) .

In the cortical neuron model , the cytotoxicity of L - NNNBP induced by different doses of L - NNNBP on A尾1 - 42 was protective ( * P0.05 , ** P0.01 ) .
In the experiment of detecting apoptosis by Tunel method , curcumin ( 42.9 % 卤 3.1 % ) and Tempol ( 51.1 % 卤 1.1 % ) , L - NNNBP high dose ( 10 渭M ) treatment group , the apoptosis of neurons decreased significantly ( 22 . 7 % 卤 2.6 % , * P 0 . 05 , ** P0.01 , P P0.01 ) . L - NNNBP could significantly decrease the density of TMRM + , prevent depolarization of mitochondrial membrane , protect the normal function of mitochondria and protect the normal function of mitochondria , and the effect of L - NNNBP was significantly higher than that of curcumin and Tempol ( # P05 ) .

Compared with WT group and other drug treatment groups , L - NNNBP significantly decreased the phosphorylation of Tau protein in two sites ( ** P0.01 , # P05 , # P0 . 05 ) . Immunohistochemical and Western blot analysis of astrocytes showed that it was activated in the early stage of AD , and it was possible to further induce 尾 - amyloid deposition acceleration and neurofilament entanglement formation . L - NNNBP inhibited astrocytes from activation . The results of Morris water maze also showed that the spatial learning and memory abilities of APP / PSl mice were damaged , and the effect was stronger than curcumin and Tempol ( ** P0.01 , # P0.01 ) .

Conclusion :

In conclusion , the novel chiral nitrogen - oxygen free radical L - NNNBP showed an exciting result either in an in vitro model of oxidative damage , a cytotoxicity damage model induced by A.beta . 1 - 42 , or a novel chiral nitrogen - oxygen free radical L - NNNBP . It can scavenge superoxide anion , inhibit lipid peroxidation , reduce the damage of A尾1 - 42 induced cytotoxicity , reduce A尾 plaque deposition , decrease Tau protein phosphorylation level , inhibit astrocyte activation and improve the spatial learning and memory of AD rats .

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】