發(fā)布時(shí)間：2018-05-29 13:20

  本文選題：柿葉提取物 + 氧化應(yīng)激��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：研究背景隨著世界老齡化的不斷加劇,衰老及相關(guān)退行性相關(guān)的疾病受到越來越多研究者的重視。神經(jīng)系統(tǒng)退行性疾病在衰老疾病中占重要分支,如帕金森氏病,阿爾茨海默病(Alzheimer's Disease,AD)等。AD是一種以認(rèn)知功能障礙、人格改變?yōu)橹饕R床癥狀的慢性進(jìn)展性中樞神經(jīng)系統(tǒng)退行性疾病,是癡呆的最常見原因。AD發(fā)病機(jī)制尚不明確,且無特效藥。為了更好的防治衰老疾病、提高老年人生活質(zhì)量、減輕社會(huì)負(fù)擔(dān),探討退行性病變的病理機(jī)制、研發(fā)有效延緩?fù)诵羞M(jìn)程的藥物有重要意義。淀粉樣蛋白級(jí)聯(lián)反應(yīng)是AD治病機(jī)制中的重要假說。不可溶的β淀粉樣蛋白(β-amyloid peptide,Aβ)聚集而成的原纖維具有神經(jīng)毒性,也是老年斑的組成成分。Aβ聚合會(huì)引發(fā)氧化應(yīng)激反應(yīng),線粒體功能受損,使ROS增多。而氧化應(yīng)激又會(huì)進(jìn)一步促進(jìn)Aβ聚集及微管相關(guān)蛋白tau的磷酸化,加重AD大腦中的氧化還原反應(yīng)的失衡。氧化應(yīng)激是將多種不同的致病機(jī)制聯(lián)絡(luò)在一起的共同關(guān)鍵點(diǎn),因此尋找合適的抗氧化劑和多靶點(diǎn)的治療方法,進(jìn)行適當(dāng)?shù)呐R床干預(yù),是防治AD的有效手段。Nrf2/HO-1在機(jī)體內(nèi)源性抗氧化系統(tǒng)中有至關(guān)重要的地位,是最重要的抗氧化通路。Nrf2作為轉(zhuǎn)錄因子能啟動(dòng)機(jī)體的抗氧化通路。在受到氧化刺激時(shí),Nrf2由胞質(zhì)轉(zhuǎn)位進(jìn)入胞核,啟動(dòng)抗氧化酶相關(guān)基因的表達(dá),通過調(diào)控下游氧化還原基因網(wǎng),來維持細(xì)胞內(nèi)氧化還原狀態(tài)的平衡。HO-1是Nrf2下游的抗氧化酶之一,通過對(duì)線粒體功能的恢復(fù)起到保護(hù)神經(jīng)元的作用。Nrf2/HO-1是機(jī)體維持氧化平衡的保護(hù)系統(tǒng)。近來發(fā)現(xiàn),柿葉具有抗氧化,降血脂,降血糖,抗菌,止血等藥效。臨床病例分析報(bào)道,柿葉提取物在改善后循環(huán)缺血的療效上優(yōu)于銀杏葉提取物。由柿葉提取物制成的腦心清片,可清除氧自由基,防止神經(jīng)元興奮性毒性作用,從而保護(hù)腦血管意外引起的腦損傷。在臨床治療中柿葉提取物已被廣泛應(yīng)用,尤其在腦血管病的相關(guān)領(lǐng)域中嶄露頭角。我們前期研究結(jié)果發(fā)現(xiàn),柿葉提取物(Persimmon leaf extract,PLE)可以改善D-半乳糖致衰老小鼠的學(xué)習(xí)與記憶能力,提高抗氧化酶SOD,GSH-Px活性,那么在神經(jīng)細(xì)胞中尤其是AD細(xì)胞模型中,是否也有相同的作用?是否可以通過減少Aβ的聚集、減輕ROS的活性,調(diào)節(jié)Nrf2/HO-1的表達(dá)來發(fā)揮神經(jīng)保護(hù)作用?基于以上背景,本研究主要探討柿葉提取物對(duì)AD細(xì)胞模型的是否有抗氧化作用,以及Nrf2和HO-1是否參與到抗氧化的調(diào)節(jié)中,為AD的藥物研發(fā)提供依據(jù)。目的:采用攜帶瑞典突變APP基因的HEK293細(xì)胞(20E2)作為AD細(xì)胞模型,通過檢測(cè)細(xì)胞上清Aβ1-42的水平,細(xì)胞內(nèi)ROS的熒光強(qiáng)度,線粒體膜電位的變化等相關(guān)指標(biāo),探討PLE對(duì)細(xì)胞抗氧化應(yīng)激的作用,并從核Nrf2/HO-1信號(hào)通路研究其作用機(jī)制。方法:1.Western Blotting檢測(cè)SH-SY5Y和20E2細(xì)胞中APP蛋白表達(dá)水平;ELISA檢測(cè)兩組細(xì)胞上清Aβ1-40,以確定該病理模型是否建立成功。2.CCK-8檢測(cè)不同濃度的PLE對(duì)兩組細(xì)胞活性的影響,選定干預(yù)最佳濃度。3.設(shè)分組:SH-SY5Y為空白對(duì)照組(NC組)20E2為模型組(20E2組),用柿葉提取物干預(yù)為加藥組(20E2+PLE組)。4.DCFH-DA探針檢測(cè)各組細(xì)胞內(nèi)ROS的變化。5.JC-1檢測(cè)線粒體膜電位的變化。6.ELISA檢測(cè)各組細(xì)胞上清Aβ1-42的量。7.Western Blotting分析胞核Nrf2、胞漿Nrf2和全細(xì)胞HO-1蛋白的表達(dá)差異。結(jié)果:1.檢測(cè)兩種細(xì)胞中的APP、Aβ1-42蛋白表達(dá):與SH-SY5Y細(xì)胞相比,20E2細(xì)胞APP表達(dá)量明顯增加(P0.01),Aβ1-40的量明顯增加(P0.01)。2.CCK-8檢測(cè)不同濃度的PLE對(duì)兩組細(xì)胞活性的影響:結(jié)果顯示PLE在1.5-6μg/mL可增加SH-5Y5Y細(xì)胞活性(*P0.05);PLE在3-6μ g/mL可增加20E2細(xì)胞活性(#P0.05),超過6 μ g/mL后也有抑制細(xì)胞活性的趨勢(shì)。3.DCFH-DA熒光探針檢測(cè)各組細(xì)胞ROS的表達(dá):與NC組相比,20E2組細(xì)胞內(nèi)ROS熒光信號(hào)明顯增強(qiáng)(P0.05);PLE干預(yù)后,20E2+PLE組ROS熒光信號(hào)減弱(P0.05)。4.JC-1檢測(cè)線粒體膜電位結(jié)果:較NC組細(xì)胞,20E2組細(xì)胞,紅色熒光較弱(P0.05),綠色熒光較強(qiáng)(P0.05),線粒體膜電位降低;用PLE干預(yù)后,20E2+PLE組細(xì)胞內(nèi)的紅色熒光強(qiáng)度增強(qiáng)(P0.05),綠色熒光較弱(P0.05),線粒體膜電位有上升。5.ELISA檢測(cè)細(xì)胞外Aβ1-42濃度結(jié)果:與NC組細(xì)胞相比,20E2組細(xì)胞外Aβ1-42明顯增多(P0.05);PLE干預(yù)后,20E2+PLE組細(xì)胞外Aβ1-42明顯減少(P0.05)。6.分析胞核Nrf2、胞漿Nrf2和全細(xì)胞蛋白HO-1的表達(dá)差異:相比于NC組,20E2細(xì)胞中Nrf2蛋白和HO-1蛋白表達(dá)增多(P0.05);相比于模型組,加藥組中胞核Nrf2表達(dá)增加,胞漿Nrf2表達(dá)減少,全細(xì)胞蛋白HO-1 增多(P0.05)結(jié)論P(yáng)LE能明顯降低細(xì)胞外Aβ的濃度,減少ROS的產(chǎn)生,恢復(fù)線粒體膜電位水平。可能是通過激活了 Nrf2/HO-1信號(hào)途徑,促進(jìn)Nrf2合成和核轉(zhuǎn)位,從而促進(jìn)下游抗氧化蛋白HO-1的表達(dá)來減弱氧化應(yīng)激的損傷。因此,PLE能降低AD細(xì)胞模型的氧化應(yīng)激的水平,對(duì)AD具有一定的治療保護(hù)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749.16

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