柿叶提取物对阿尔茨海默病细胞模型的抗氧化作用及机制研究

发布时间：2018-05-29 13:20

本文选题：柿叶提取物 + 氧化应激　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景随着世界老龄化的不断加剧,衰老及相关退行性相关的疾病受到越来越多研究者的重视。神经系统退行性疾病在衰老疾病中占重要分支,如帕金森氏病,阿尔茨海默病(Alzheimer's Disease,AD)等。AD是一种以认知功能障碍、人格改变为主要临床症状的慢性进展性中枢神经系统退行性疾病,是痴呆的最常见原因。AD发病机制尚不明确,且无特效药。为了更好的防治衰老疾病、提高老年人生活质量、减轻社会负担,探讨退行性病变的病理机制、研发有效延缓退行进程的药物有重要意义。淀粉样蛋白级联反应是AD治病机制中的重要假说。不可溶的β淀粉样蛋白(β-amyloid peptide,Aβ)聚集而成的原纤维具有神经毒性,也是老年斑的组成成分。Aβ聚合会引发氧化应激反应,线粒体功能受损,使ROS增多。而氧化应激又会进一步促进Aβ聚集及微管相关蛋白tau的磷酸化,加重AD大脑中的氧化还原反应的失衡。氧化应激是将多种不同的致病机制联络在一起的共同关键点,因此寻找合适的抗氧化剂和多靶点的治疗方法,进行适当的临床干预,是防治AD的有效手段。Nrf2/HO-1在机体内源性抗氧化系统中有至关重要的地位,是最重要的抗氧化通路。Nrf2作为转录因子能启动机体的抗氧化通路。在受到氧化刺激时,Nrf2由胞质转位进入胞核,启动抗氧化酶相关基因的表达,通过调控下游氧化还原基因网,来维持细胞内氧化还原状态的平衡。HO-1是Nrf2下游的抗氧化酶之一,通过对线粒体功能的恢复起到保护神经元的作用。Nrf2/HO-1是机体维持氧化平衡的保护系统。近来发现,柿叶具有抗氧化,降血脂,降血糖,抗菌,止血等药效。临床病例分析报道,柿叶提取物在改善后循环缺血的疗效上优于银杏叶提取物。由柿叶提取物制成的脑心清片,可清除氧自由基,防止神经元兴奋性毒性作用,从而保护脑血管意外引起的脑损伤。在临床治疗中柿叶提取物已被广泛应用,尤其在脑血管病的相关领域中崭露头角。我们前期研究结果发现,柿叶提取物(Persimmon leaf extract,PLE)可以改善D-半乳糖致衰老小鼠的学习与记忆能力,提高抗氧化酶SOD,GSH-Px活性,那么在神经细胞中尤其是AD细胞模型中,是否也有相同的作用?是否可以通过减少Aβ的聚集、减轻ROS的活性,调节Nrf2/HO-1的表达来发挥神经保护作用?基于以上背景,本研究主要探讨柿叶提取物对AD细胞模型的是否有抗氧化作用,以及Nrf2和HO-1是否参与到抗氧化的调节中,为AD的药物研发提供依据。目的:采用携带瑞典突变APP基因的HEK293细胞(20E2)作为AD细胞模型,通过检测细胞上清Aβ1-42的水平,细胞内ROS的荧光强度,线粒体膜电位的变化等相关指标,探讨PLE对细胞抗氧化应激的作用,并从核Nrf2/HO-1信号通路研究其作用机制。方法:1.Western Blotting检测SH-SY5Y和20E2细胞中APP蛋白表达水平;ELISA检测两组细胞上清Aβ1-40,以确定该病理模型是否建立成功。2.CCK-8检测不同浓度的PLE对两组细胞活性的影响,选定干预最佳浓度。3.设分组:SH-SY5Y为空白对照组(NC组)20E2为模型组(20E2组),用柿叶提取物干预为加药组(20E2+PLE组)。4.DCFH-DA探针检测各组细胞内ROS的变化。5.JC-1检测线粒体膜电位的变化。6.ELISA检测各组细胞上清Aβ1-42的量。7.Western Blotting分析胞核Nrf2、胞浆Nrf2和全细胞HO-1蛋白的表达差异。结果:1.检测两种细胞中的APP、Aβ1-42蛋白表达:与SH-SY5Y细胞相比,20E2细胞APP表达量明显增加(P0.01),Aβ1-40的量明显增加(P0.01)。2.CCK-8检测不同浓度的PLE对两组细胞活性的影响:结果显示PLE在1.5-6μg/mL可增加SH-5Y5Y细胞活性(*P0.05);PLE在3-6μ g/mL可增加20E2细胞活性(#P0.05),超过6 μ g/mL后也有抑制细胞活性的趋势。3.DCFH-DA荧光探针检测各组细胞ROS的表达:与NC组相比,20E2组细胞内ROS荧光信号明显增强(P0.05);PLE干预后,20E2+PLE组ROS荧光信号减弱(P0.05)。4.JC-1检测线粒体膜电位结果:较NC组细胞,20E2组细胞,红色荧光较弱(P0.05),绿色荧光较强(P0.05),线粒体膜电位降低;用PLE干预后,20E2+PLE组细胞内的红色荧光强度增强(P0.05),绿色荧光较弱(P0.05),线粒体膜电位有上升。5.ELISA检测细胞外Aβ1-42浓度结果:与NC组细胞相比,20E2组细胞外Aβ1-42明显增多(P0.05);PLE干预后,20E2+PLE组细胞外Aβ1-42明显减少(P0.05)。6.分析胞核Nrf2、胞浆Nrf2和全细胞蛋白HO-1的表达差异:相比于NC组,20E2细胞中Nrf2蛋白和HO-1蛋白表达增多(P0.05);相比于模型组,加药组中胞核Nrf2表达增加,胞浆Nrf2表达减少,全细胞蛋白HO-1 增多(P0.05)结论PLE能明显降低细胞外Aβ的浓度,减少ROS的产生,恢复线粒体膜电位水平。可能是通过激活了 Nrf2/HO-1信号途径,促进Nrf2合成和核转位,从而促进下游抗氧化蛋白HO-1的表达来减弱氧化应激的损伤。因此,PLE能降低AD细胞模型的氧化应激的水平,对AD具有一定的治疗保护作用。
[Abstract]:Background with the growing aging of the world, more and more researchers have paid more attention to aging and related degenerative diseases. Neurodegenerative disease is an important branch of aging disease, such as Parkinson's disease, Alzheimer's Disease, AD, and so on, which is a kind of cognitive dysfunction and personality change. The main clinical symptoms of chronic progressive central nervous system degenerative disease, the most common cause of dementia, the pathogenesis of.AD is not clear, and there is no specific drug. In order to better prevent and cure aging disease, improve the quality of life of the elderly, reduce the social burden, explore the pathological mechanism of degenerative disease, research and develop drugs to effectively delay the process of degenerative process. It is of great significance. Amyloid cascade reaction is an important hypothesis in the mechanism of AD treatment. The fibrous fibrils of the insoluble beta amyloid (beta -amyloid peptide, A beta) aggregation are neurotoxic, and the components of the senile plaques,.A beta polymerization, can cause oxidative stress, mitochondrial function is damaged, and ROS increases. Oxidative stress will also occur. Further promoting the A beta aggregation and the phosphorylation of microtubule related protein tau, aggravating the imbalance of redox reaction in the AD brain. Oxidative stress is a common key point to connect various different pathogenic mechanisms. Therefore, it is effective to find appropriate antioxidants and multiple target treatments for the prevention and control of AD. .Nrf2/HO-1 plays an important role in the endogenous antioxidant system of the body. It is the most important antioxidant pathway,.Nrf2, as a transcription factor that can activate the body's antioxidant pathway. When stimulated by oxidation, Nrf2 is transferred from cytoplasm into the nucleus, initiates the expression of antioxidant enzyme related genes and regulates downstream redox genes. The balance.HO-1, to maintain the redox state of the cell, is one of the antioxidases in the downstream of Nrf2. By restoring the function of the mitochondria to protect the neurons,.Nrf2/HO-1 is the protective system for maintaining the body's oxidation balance. Recently, it has been found that persimmon leaves have antioxidation, blood fat, hypoglycemia, antiseptic, hemostasis and other efficacy. It is reported that the persimmon leaf extract is better than the extract of Ginkgo biloba in the treatment of posterior circulation ischemia. The brain Xin Qing tablet made from the extract of persimmon leaves can clear the oxygen free radical and prevent the excitotoxic effect of the neuron, thus protecting the brain injury caused by the cerebrovascular accident. In clinical treatment, the persimmon leaf extract has been widely used, especially in the brain blood. Our previous studies have shown that the persimmon leaf extract (Persimmon leaf extract, PLE) can improve the learning and memory ability of D- galactose induced aging mice and improve the antioxidant enzyme SOD, GSH-Px activity. Is there the same effect in the neural cells, especially in the AD cell model? In order to reduce the aggregation of A beta, reduce the activity of ROS and regulate the expression of Nrf2/HO-1 to play a neuroprotective role? Based on the above background, this study mainly discusses whether the persimmon leaf extract has antioxidant effect on the AD cell model, and whether Nrf2 and HO-1 are involved in the regulation of antioxidant activity, and provide the basis for the development of AD drugs. The HEK293 cell (20E2) with Swedish mutant APP gene (20E2) was used as a AD cell model. By detecting the level of A beta 1-42 in the cell supernatant, the fluorescence intensity of ROS in the cell, the changes of mitochondrial membrane potential and so on, the effect of PLE on the antioxidant stress of the cells was discussed and the mechanism of its action was studied from the HO-1 signal pathway of nuclear Nrf2/. Method: 1.Western Blotti Ng detected the expression level of APP protein in SH-SY5Y and 20E2 cells; ELISA detected A beta 1-40 in two groups of cell supernatants to determine whether the pathological model established a successful.2.CCK-8 detection of the effect of PLE on the activity of two groups, and selected the optimal concentration.3. set group: SH-SY5Y was the empty white control group (NC group) and the persimmon leaf The changes of ROS in each group were detected by.4.DCFH-DA probe in the treatment group (20E2+PLE group).5.JC-1 detection of mitochondrial membrane potential change.6.ELISA detection of A beta 1-42 in each group.7.Western Blotting analysis of Nrf2, cytoplasmic Nrf2 and whole cell HO-1 protein. Results: 1. detection of two kinds of cells of APP, beta 1 The expression of -42 protein: compared with SH-SY5Y cells, the expression of APP in 20E2 cells increased significantly (P0.01), and the amount of A beta 1-40 significantly increased (P0.01).2.CCK-8 to detect the effect of PLE on the activity of two groups of cells. The results showed that PLE in 1.5-6 micron could increase the activity of cells (*); at 3-6 micron, it could increase the activity of cells, more than 6 The expression of ROS in each group was detected by.3.DCFH-DA fluorescence probe after micron g/mL. Compared with group NC, the intracellular ROS fluorescence signal was obviously enhanced (P0.05); PLE dry prognosis, 20E2+PLE group ROS fluorescence signal weakened (P0.05).4.JC-1 detected mitochondrial membrane potential results: compared with group cells, cells, red fluorescence compared with 20E2 Weak (P0.05), strong green fluorescence (P0.05), mitochondrial membrane potential decreased; after PLE, the red fluorescence intensity in 20E2+PLE group increased (P0.05), green fluorescence was weaker (P0.05), mitochondrial membrane potential increased.5.ELISA detection of extracellular A beta 1-42 concentration fruit: compared with NC group cells, A beta 1-42 increased significantly in 20E2 group (P0.05); Prognosis, 20E2+PLE group A beta 1-42 significantly reduced (P0.05).6. analysis of nucleus Nrf2, cytoplasmic Nrf2 and whole cell protein HO-1 expression difference: compared with NC group, Nrf2 protein and HO-1 protein expression in 20E2 cells increased (P0.05). 05) conclusion PLE can obviously reduce the concentration of A beta, reduce the production of ROS, and restore the mitochondrial membrane potential level. It may be by activating the Nrf2/HO-1 signal pathway to promote Nrf2 synthesis and nuclear transposition, thus promoting the expression of the downstream antioxidant protein HO-1 to weaken the oxidative stress damage. Therefore, PLE can reduce the oxidative stress in the AD cell model. The level of stimulation has a certain protective effect on AD.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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