大黄素缓解一氧化氮对神经细胞损伤作用机制的研究

发布时间：2018-06-06 06:21

本文选题：氧化应激 + NO　；参考：《吉林大学》2017年博士论文

【摘要】：目的:阿尔茨海默病(Alzheimer’s disease,AD)是一种常见的慢性神经退行性疾病,临床往往表现为记忆力进行性下降,认知功能出现障碍,同时伴有人格和行为异常等,对老年人身心健康造成严重威胁。神经元凋亡是其最主要的病理特征之一。氧化应激所产生的活性氧(reactive oxygen species,ROS)和一氧化氮(Nitric Oxide,NO)自由基在神经退行性疾病、心脑血管疾病中发挥重要作用,过量的NO可以导致自由基损伤,并诱导神经元的凋亡。一些天然抗氧化剂可通过拮抗氧化应激,在预防和治疗老年退行性疾病中发挥积极作用。大黄作为中医传统药物具有重要的药用价值,临床应用非常广泛。近年研究表明大黄素具有抗氧化、免疫调节、抗菌、抗炎等功能,被广泛应用于肠道疾病、肾病、心血管疾病、胰腺炎等病症的治疗。叉头转录因子O1(Forkhead box O1,FOXO1)是Forkhead家族的重要成员,在胰岛素信号通路、氧化应激拮抗、DNA损伤修复、细胞周期和凋亡调控等方面都有重要作用。目前,AD的发病机理和防治策略尚不明确,NO自由基对FOXO1及其诱导的细胞凋亡是否具有调控作用、哪些天然抗氧化剂可通过FOXO1发挥神经细胞保护作用等还不十分清楚。本研究旨在以阿尔茨海默病为疾病模型,将与AD发病密切相关的NO、天然抗氧化剂等与FOXO1转录因子相联系,研究三者可能的相互作用机制及对神经细胞损伤和凋亡的影响,以期为AD等神经退行性疾病的防治研究提供新的视角和线索。方法:本研究以C57BL/6小鼠原代神经元和HT22细胞为细胞模型,用3×IRS-luciferase,Fas L-luciferase或Bim-luciferase转染神经元,在DIV7时分别加不同的试剂或药物处理,包括特定浓度的NO供体GSNO(nitrosoglutathione,亚硝基谷胱甘肽)和L-精氨酸(L-arginine,L-Arg),58种天然中药化合物(包括大黄素、盐酸小檗碱、五味子酯甲等),以及L-Arg和大黄素的组合等。然后,通过荧光素双报告基因检测观察FOXO1的转录活性及其下游促凋亡基因Fas L、Bim的表达变化,通过CCK-8细胞活性检测观察细胞活性变化,利用免疫荧光技术和细胞显微图像分析等观察细胞形态变化,通过免疫印迹法测定FOXO1、AKT及相关蛋白等。通过一系列实验,研究NO对FOXO1诱导细胞凋亡的影响及作用机制,天然抗氧化剂对FOXO1转录活性的影响,筛选所得大黄素对FOXO1诱导细胞凋亡的影响及作用机制,以及大黄素对NO和FOXO1作用效应的影响。使用Graph Pad Prism 6和Adobe Illustrator CS4软件进行数据分析和绘图,采用了T检验和方差分析(α=0.05)。结果:实验发现,在小鼠原代神经元和HT22细胞中,一定量的NO供体GSNO,L-Arg均可显著激活FOXO1的转录活性,并可促进其下游促凋亡基因Fas L、Bim的表达,降低神经细胞活力,诱导神经细胞损伤。当L-Arg浓度达到50mmol/L时,可诱导神经元凋亡。在原代神经元细胞,以FOXO1转录活性为靶向,对遴选的58种中药化合物逐一实验发现,盐酸吗啉胍、五味子酯甲等能够提高FOXO1转录活性,大黄素、盐酸小檗碱和二苯乙烯苷等能够降低其转录活性。其中,大黄素抑制作用明显(P=0.0038),且临床应用广泛、毒副作用小。对大黄素进行深入研究发现,它在HT22细胞内能够明显抑制FOXO1的转录活性,同时抑制FOXO1下游促凋亡基因Bim的表达,但没有对神经细胞形态产生影响。进一步实验发现,尽管大黄素能够在一定程度上下调AKT的活性、进而调节FOXO1,但其最为主要的影响是显著降低FOXO1总蛋白表达水平、抑制FOXO1的转录活性。在应用L-Arg的同时使用大黄素,能够明显抑制NO过度激活导致的FOXO1核聚集和转录活性增强,并呈现数量相关关系。结论:我们的研究表明:1.在体外神经细胞中,一定量的NO可导致FOXO1转录活性显著增强,并能够增加FOXO1下游促凋亡基因Fas L、Bim的表达。2.NO诱导的FOXO1调控的凋亡路径的激活,可明显降低神经细胞活力,导致细胞损伤乃至凋亡。3.遴选的58种天然中药化合物对FOXO1转录活性的影响不尽相同,盐酸吗啉胍、五味子酯甲等能够提高FOXO1转录活性,大黄素、盐酸小檗碱和二苯乙烯苷等能够降低其转录活性,其中大黄素以更好的临床应用效应更值得引起关注。4.在体外神经细胞中,大黄素可显著抑制FOXO1的转录活性,进一步下调其下游促凋亡基因Bim的表达,而且不会对神经细胞产生损伤。5.大黄素能够抑制FOXO1的转录活性,主要与显著降低FOXO1总蛋白表达水平有关。6.大黄素能够明显缓解NO对FOXO1的过度激活,减轻其对神经细胞的氧化损伤。我们的研究,首先揭示了NO自由基诱导神经元损伤的新机制,有助于进一步深入了解NO自由基的神经毒性。第二,该研究使得FOXO1可作为一个很好的药物靶点,通过筛选抑制FOXO1活性的药物来缓解氧化应激导致的神经元损伤。第三,我们从大量中药化合物中筛选出可抑制FOXO1转录活性的大黄素,提供了天然抗氧化剂大黄素等抗氧化作用的新的实验数据,以及大黄素对神经元凋亡的保护效应和分子机制。整个研究将与AD发病与防治密切相关的天然抗氧化剂、NO和转录因子FOXO1相联系,初步描绘了抗氧化剂-NO-FOXO1的信号转导通路和联合作用机制,为大黄素等中药有效成分的临床应用提供了重要参考,也为阿尔茨海默病等神经退行性疾病的发病机制及防治研究等提供了新的思路和依据。
[Abstract]:Objective: Alzheimer 's disease (AD) is a common chronic neurodegenerative disease. Clinical manifestations are often manifested by progressive memory decline, cognitive impairment and abnormal personality and behavior, which pose a severe threat to the physical and mental health of the elderly. Neuron apoptosis is one of the most important pathological features of the disease. The reactive oxygen species (reactive oxygen species, ROS) and nitric oxide (Nitric Oxide, NO) free radicals produced by oxidative stress play an important role in neurodegenerative diseases, cardiovascular and cerebrovascular diseases. Excessive NO can lead to free radical damage and induce neuronal apoptosis. Some natural antioxidants can be prevented by antagonizing oxidative stress. And it plays an active role in the treatment of senile degenerative diseases. Rhubarb, as traditional Chinese medicine, has important medicinal value and is widely used in clinical practice. In recent years, emodin has the functions of antioxidation, immunomodulation, antibacterial and anti-inflammatory. It is widely used in the treatment of intestinal diseases, kidney disease, cardiovascular disease, pancreatitis and other diseases. The head transcription factor O1 (Forkhead box O1, FOXO1) is an important member of the Forkhead family. It plays an important role in the insulin signaling pathway, oxidative stress antagonism, DNA damage repair, cell cycle and apoptosis regulation. At present, the pathogenesis and prevention strategy of AD are not clear. NO free radical has a good effect on FOXO1 and its induced apoptosis. The purpose of this study is to study the possible mechanisms of interaction between the three and the three possible mechanisms of interaction and damage to the nerve cells. The aim of this study is to connect the NO, natural antioxidants, and other FOXO1 transcription factors, which are closely related to the pathogenesis of AD. The effects of injury and apoptosis are expected to provide new perspectives and clues for the prevention and control of AD and other neurodegenerative diseases. Methods: the primary neurons and HT22 cells of C57BL/6 mice were used as cell models, neurons were transfected with 3 x IRS-luciferase, Fas L-luciferase or Bim-luciferase, and different reagents or drugs were added to DIV7, respectively. Including the specific concentration of NO donor GSNO (nitrosoglutathione, nitroso glutathione) and L- arginine (L-arginine, L-Arg), 58 natural Chinese herbal compounds (including emodin, berberine hydrochloride, schisandrin), and the combination of L-Arg and emodin. Then, the transcriptional activity of FOXO1 and the activity of FOXO1 are observed and observed by the fluorescein double reporter gene. The changes in the expression of apoptotic gene Fas L, Bim, the activity of cell activity were observed by the activity of CCK-8 cells, and the changes of cell morphology were observed by immunofluorescence and cell microscopic image analysis, and FOXO1, AKT and related proteins were measured by immunoblotting. The effects of NO on apoptosis induced by FOXO1 were studied by a series of experiments. The effect of action mechanism, the effect of natural antioxidants on the transcriptional activity of FOXO1, the effect of emodin on FOXO1 induced apoptosis and the effect of emodin on the effect of NO and FOXO1. Using Graph Pad Prism 6 and Adobe Illustrator CS4 software for data analysis and mapping, T test and variance analysis (alpha = = =) 0.05). Results: the results showed that in the primary and HT22 cells of the mice, a certain amount of NO donor GSNO, L-Arg could significantly activate the transcriptional activity of FOXO1, and could promote the expression of Fas L, Bim, reduce the vitality of the nerve cells and induce the injury of nerve cells in the downstream of the apoptosis gene. When the concentration of L-Arg reached 50mmol/L, it could induce neuronal apoptosis. In the primary neuronal cells, the FOXO1 transcriptional activity was targeted. One by one selection of the selected 58 kinds of Chinese medicine compounds showed that morphine guanidine hydrochloride and schisandrin could improve the transcriptional activity of FOXO1. Emodin, berberine hydrochloride and two styrene glycosides could reduce their transcriptional activity. The inhibitory effect of emodin was obvious (P=0.0038) and clinical It is widely used and has small toxic and side effects. In deep study of emodin, it has been found that it can inhibit the transcription activity of FOXO1 in HT22 cells and inhibit the expression of Bim in the downstream of FOXO1, but it does not affect the morphology of nerve cells. Further experiments have found that emodin can down regulate the activity of AKT to a certain extent. And then regulate FOXO1, but its most important effect is to significantly reduce the total protein expression level of FOXO1 and inhibit the transcriptional activity of FOXO1. The use of emodin at the same time with L-Arg can obviously inhibit the FOXO1 nuclear aggregation and transcriptional activity enhancement caused by NO overactivation, and there is a quantitative correlation. Conclusion: our study showed that 1. in vitro In nerve cells, a certain amount of NO can lead to a significant increase in the transcriptional activity of FOXO1, and can increase the activation of the apoptotic pathway of FOXO1 regulated by.2.NO induced by the expression of Fas L in the downstream FOXO1, which can obviously reduce the vitality of the nerve cells and lead to the cell damage and the apoptosis.3. selection of 58 natural Chinese medicine compounds for FOXO1 transcriptional activity. The effects of morphine guanidine hydrochloride and schisandral ester on FOXO1 transcriptional activity, emodin, berberine hydrochloride and two styrene glycosides can reduce their transcriptional activity. The emodin is more worthy of attention to.4. in the in vitro deity cells, and rhubarb can significantly inhibit the transcription of FOXO1. Sex, further down-regulation of the expression of the downstream apoptotic gene Bim, and no damage to the nerve cells,.5. emodin can inhibit the transcriptional activity of FOXO1. It is mainly related to the significant reduction of the expression level of FOXO1 total protein by.6. emodin, which can significantly alleviate the excessive activation of NO to FOXO1 and reduce its oxidative damage to the nerve cells. Our The study, first of all, reveals a new mechanism of NO free radical induced neuronal damage, which helps to further understand the neurotoxicity of NO free radicals. Second, this study makes FOXO1 a good drug target, by screening drugs that inhibit the activity of FOXO1 to alleviate neuronal damage caused by oxidative stress. Third, we are from a large number of The drug compounds screening emodin that inhibits FOXO1 transcriptional activity, provides new experimental data on antioxidant effects of natural antioxidants and emodin, as well as the protective effects and molecular mechanisms of emodin to neuronal apoptosis. The whole study will be associated with natural antioxidants, NO and transcription factor FOXO1, which are closely related to the pathogenesis and Prevention of AD It provides an important reference for the clinical application of the effective components of emodin and other Chinese medicine, and provides a new idea and basis for the pathogenesis and prevention and treatment of Alzheimer's disease, such as Alzheimer's disease, as well as a preliminary description of the signal transduction pathway and mechanism of the antioxidant -NO-FOXO1.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R749.16

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