本文選題：氧化應(yīng)激 + NO　； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：目的:阿爾茨海默病(Alzheimer’s disease,AD)是一種常見的慢性神經(jīng)退行性疾病,臨床往往表現(xiàn)為記憶力進行性下降,認(rèn)知功能出現(xiàn)障礙,同時伴有人格和行為異常等,對老年人身心健康造成嚴(yán)重威脅。神經(jīng)元凋亡是其最主要的病理特征之一。氧化應(yīng)激所產(chǎn)生的活性氧(reactive oxygen species,ROS)和一氧化氮(Nitric Oxide,NO)自由基在神經(jīng)退行性疾病、心腦血管疾病中發(fā)揮重要作用,過量的NO可以導(dǎo)致自由基損傷,并誘導(dǎo)神經(jīng)元的凋亡。一些天然抗氧化劑可通過拮抗氧化應(yīng)激,在預(yù)防和治療老年退行性疾病中發(fā)揮積極作用。大黃作為中醫(yī)傳統(tǒng)藥物具有重要的藥用價值,臨床應(yīng)用非常廣泛。近年研究表明大黃素具有抗氧化、免疫調(diào)節(jié)、抗菌、抗炎等功能,被廣泛應(yīng)用于腸道疾病、腎病、心血管疾病、胰腺炎等病癥的治療。叉頭轉(zhuǎn)錄因子O1(Forkhead box O1,FOXO1)是Forkhead家族的重要成員,在胰島素信號通路、氧化應(yīng)激拮抗、DNA損傷修復(fù)、細(xì)胞周期和凋亡調(diào)控等方面都有重要作用。目前,AD的發(fā)病機理和防治策略尚不明確,NO自由基對FOXO1及其誘導(dǎo)的細(xì)胞凋亡是否具有調(diào)控作用、哪些天然抗氧化劑可通過FOXO1發(fā)揮神經(jīng)細(xì)胞保護作用等還不十分清楚。本研究旨在以阿爾茨海默病為疾病模型,將與AD發(fā)病密切相關(guān)的NO、天然抗氧化劑等與FOXO1轉(zhuǎn)錄因子相聯(lián)系,研究三者可能的相互作用機制及對神經(jīng)細(xì)胞損傷和凋亡的影響,以期為AD等神經(jīng)退行性疾病的防治研究提供新的視角和線索。方法:本研究以C57BL/6小鼠原代神經(jīng)元和HT22細(xì)胞為細(xì)胞模型,用3×IRS-luciferase,Fas L-luciferase或Bim-luciferase轉(zhuǎn)染神經(jīng)元,在DIV7時分別加不同的試劑或藥物處理,包括特定濃度的NO供體GSNO(nitrosoglutathione,亞硝基谷胱甘肽)和L-精氨酸(L-arginine,L-Arg),58種天然中藥化合物(包括大黃素、鹽酸小檗堿、五味子酯甲等),以及L-Arg和大黃素的組合等。然后,通過熒光素雙報告基因檢測觀察FOXO1的轉(zhuǎn)錄活性及其下游促凋亡基因Fas L、Bim的表達變化,通過CCK-8細(xì)胞活性檢測觀察細(xì)胞活性變化,利用免疫熒光技術(shù)和細(xì)胞顯微圖像分析等觀察細(xì)胞形態(tài)變化,通過免疫印跡法測定FOXO1、AKT及相關(guān)蛋白等。通過一系列實驗,研究NO對FOXO1誘導(dǎo)細(xì)胞凋亡的影響及作用機制,天然抗氧化劑對FOXO1轉(zhuǎn)錄活性的影響,篩選所得大黃素對FOXO1誘導(dǎo)細(xì)胞凋亡的影響及作用機制,以及大黃素對NO和FOXO1作用效應(yīng)的影響。使用Graph Pad Prism 6和Adobe Illustrator CS4軟件進行數(shù)據(jù)分析和繪圖,采用了T檢驗和方差分析(α=0.05)。結(jié)果:實驗發(fā)現(xiàn),在小鼠原代神經(jīng)元和HT22細(xì)胞中,一定量的NO供體GSNO,L-Arg均可顯著激活FOXO1的轉(zhuǎn)錄活性,并可促進其下游促凋亡基因Fas L、Bim的表達,降低神經(jīng)細(xì)胞活力,誘導(dǎo)神經(jīng)細(xì)胞損傷。當(dāng)L-Arg濃度達到50mmol/L時,可誘導(dǎo)神經(jīng)元凋亡。在原代神經(jīng)元細(xì)胞,以FOXO1轉(zhuǎn)錄活性為靶向,對遴選的58種中藥化合物逐一實驗發(fā)現(xiàn),鹽酸嗎啉胍、五味子酯甲等能夠提高FOXO1轉(zhuǎn)錄活性,大黃素、鹽酸小檗堿和二苯乙烯苷等能夠降低其轉(zhuǎn)錄活性。其中,大黃素抑制作用明顯(P=0.0038),且臨床應(yīng)用廣泛、毒副作用小。對大黃素進行深入研究發(fā)現(xiàn),它在HT22細(xì)胞內(nèi)能夠明顯抑制FOXO1的轉(zhuǎn)錄活性,同時抑制FOXO1下游促凋亡基因Bim的表達,但沒有對神經(jīng)細(xì)胞形態(tài)產(chǎn)生影響。進一步實驗發(fā)現(xiàn),盡管大黃素能夠在一定程度上下調(diào)AKT的活性、進而調(diào)節(jié)FOXO1,但其最為主要的影響是顯著降低FOXO1總蛋白表達水平、抑制FOXO1的轉(zhuǎn)錄活性。在應(yīng)用L-Arg的同時使用大黃素,能夠明顯抑制NO過度激活導(dǎo)致的FOXO1核聚集和轉(zhuǎn)錄活性增強,并呈現(xiàn)數(shù)量相關(guān)關(guān)系。結(jié)論:我們的研究表明:1.在體外神經(jīng)細(xì)胞中,一定量的NO可導(dǎo)致FOXO1轉(zhuǎn)錄活性顯著增強,并能夠增加FOXO1下游促凋亡基因Fas L、Bim的表達。2.NO誘導(dǎo)的FOXO1調(diào)控的凋亡路徑的激活,可明顯降低神經(jīng)細(xì)胞活力,導(dǎo)致細(xì)胞損傷乃至凋亡。3.遴選的58種天然中藥化合物對FOXO1轉(zhuǎn)錄活性的影響不盡相同,鹽酸嗎啉胍、五味子酯甲等能夠提高FOXO1轉(zhuǎn)錄活性,大黃素、鹽酸小檗堿和二苯乙烯苷等能夠降低其轉(zhuǎn)錄活性,其中大黃素以更好的臨床應(yīng)用效應(yīng)更值得引起關(guān)注。4.在體外神經(jīng)細(xì)胞中,大黃素可顯著抑制FOXO1的轉(zhuǎn)錄活性,進一步下調(diào)其下游促凋亡基因Bim的表達,而且不會對神經(jīng)細(xì)胞產(chǎn)生損傷。5.大黃素能夠抑制FOXO1的轉(zhuǎn)錄活性,主要與顯著降低FOXO1總蛋白表達水平有關(guān)。6.大黃素能夠明顯緩解NO對FOXO1的過度激活,減輕其對神經(jīng)細(xì)胞的氧化損傷。我們的研究,首先揭示了NO自由基誘導(dǎo)神經(jīng)元損傷的新機制,有助于進一步深入了解NO自由基的神經(jīng)毒性。第二,該研究使得FOXO1可作為一個很好的藥物靶點,通過篩選抑制FOXO1活性的藥物來緩解氧化應(yīng)激導(dǎo)致的神經(jīng)元損傷。第三,我們從大量中藥化合物中篩選出可抑制FOXO1轉(zhuǎn)錄活性的大黃素,提供了天然抗氧化劑大黃素等抗氧化作用的新的實驗數(shù)據(jù),以及大黃素對神經(jīng)元凋亡的保護效應(yīng)和分子機制。整個研究將與AD發(fā)病與防治密切相關(guān)的天然抗氧化劑、NO和轉(zhuǎn)錄因子FOXO1相聯(lián)系,初步描繪了抗氧化劑-NO-FOXO1的信號轉(zhuǎn)導(dǎo)通路和聯(lián)合作用機制,為大黃素等中藥有效成分的臨床應(yīng)用提供了重要參考,也為阿爾茨海默病等神經(jīng)退行性疾病的發(fā)病機制及防治研究等提供了新的思路和依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R749.16

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