本文選題：酒精 + 齊墩果酸��； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：【背景】 酒精性相關(guān)疾病是一類(lèi)由酒精代謝誘導(dǎo)機(jī)體發(fā)生代謝紊亂性疾病。酒精濫用日益成為現(xiàn)代社會(huì)嚴(yán)重的公共衛(wèi)生問(wèn)題。酒精對(duì)機(jī)體造成的損傷作用涉及多種病理機(jī)制。隨著研究的日益深入，酒精濫用引起氧化損傷作用得到廣泛認(rèn)同和重視，認(rèn)為ROS介導(dǎo)的氧化應(yīng)激損傷是酒精性相關(guān)疾病的最主要致病因素。研究進(jìn)展表明，酒精性相關(guān)疾病的預(yù)防和治療還缺乏相應(yīng)的藥物和手段，抗氧化劑被認(rèn)為是預(yù)防和治療酒精性相關(guān)疾病具有良好應(yīng)用前景的藥物。 齊墩果酸（Oleanolic acid，OA）是一種來(lái)源于植物的天然三萜系化合物，具有很多重要的生物藥理活性。富含OA中藥如龍膽科植物青葉膽、木樨科植物女貞子、五加科植物id木等在中醫(yī)應(yīng)用上歷史悠久，現(xiàn)已經(jīng)作為處方藥主要用來(lái)肝炎類(lèi)疾病的輔助治療。研究發(fā)現(xiàn)OA具有一定程度的抗氧化功能，但是OA由于化學(xué)結(jié)構(gòu)原因，其生物利用度低，限制了在臨床上的應(yīng)用。由于在酒精中溶解度較好，因此，將OA溶解于酒精后可能減輕酒精大量攝入導(dǎo)致的氧化應(yīng)激損傷。闡明齊墩果酸的抗氧化保護(hù)作用相關(guān)機(jī)制，研發(fā)酒精類(lèi)保健產(chǎn)品，對(duì)減輕酒精類(lèi)相關(guān)疾病的發(fā)病過(guò)程以及減少酒精濫用導(dǎo)致的衛(wèi)生經(jīng)濟(jì)負(fù)擔(dān)等都具有重要意義。 【目的】 (1)研究OA對(duì)酒精性氧化損傷的保護(hù)作用。 (2)探討OA在酒精性氧化損傷中發(fā)揮保護(hù)作用的相關(guān)機(jī)制。 【方法】 (1)細(xì)胞模型：通過(guò)建立酒精性肝細(xì)胞氧化損傷模型，評(píng)價(jià)OA在酒精性損傷模型中的抗氧化活性作用和保護(hù)效應(yīng)。觀察了不同濃度不同時(shí)間OA作用QZG細(xì)胞對(duì)Nrf-2等相關(guān)抗氧化酶蛋白表達(dá)的影響。還觀察了5、10、15μM的OA干預(yù)對(duì)酒精誘導(dǎo)肝細(xì)胞增殖活性的影響，同時(shí)檢測(cè)了細(xì)胞勻漿液內(nèi)氧化應(yīng)激相關(guān)指標(biāo)，如SOD活性、MDA、GSH、GSSG和T-SH含量。測(cè)定了OA干預(yù)后對(duì)Nrf-2、SOD1、HO-1、Gpx的蛋白表達(dá)的改變。此外我們還觀察了OA在體外DPPH、O2和OH三種自由基化學(xué)發(fā)光模型中的抑制作用。 (2)動(dòng)物模型：通過(guò)4g/kg/day酒精灌胃大鼠30天誘導(dǎo)酒精性大鼠氧化損傷模型，同時(shí)給以10mg/kg/day和20mg/kg/day兩種劑量的OA干預(yù)，取血清、肝、肌肉、脂肪勻漿，提取肝臟線(xiàn)粒體，測(cè)定實(shí)驗(yàn)動(dòng)物體重變化和血糖改變，觀察了肝體比的改變，轉(zhuǎn)氨酶ALT和AST活性和乳酸脫氫酶學(xué)指標(biāo)，血脂指標(biāo)和線(xiàn)粒體活性和腫脹度改變，血清等組織器官內(nèi)糖基化終末產(chǎn)物（AGEs）含量變化，同時(shí)還觀察了血清、肝、脂肪和肌肉組織的氧化應(yīng)激相關(guān)指標(biāo)，如MDA、GSH、GSSG和T-SH含量，CAT、SOD、Gpx、T-AOC活性。提取肝臟總蛋白，測(cè)定了Nrf-2、SOD-1、GR、HO-1蛋白表達(dá)水平。提取肌肉總蛋白，測(cè)定了Nrf-2蛋白表達(dá)水平。同時(shí)提取肝臟組織的總RNA，測(cè)定了Nrf-2基因表達(dá)水平。做肝臟和胃臟病理切片，HE染色。系統(tǒng)觀察了酒精性肝損傷模型中的氧化應(yīng)激損傷反應(yīng)性，以及抗氧化酶鏈的變化，重點(diǎn)研究了OA的抗氧化損傷的保護(hù)作用機(jī)制。 【結(jié)果】 成功構(gòu)建了酒精干預(yù)QZG細(xì)胞的氧化損傷模型，同時(shí)結(jié)合文獻(xiàn)報(bào)道，確定了200mM的EtOH作用6h作為創(chuàng)建QZG細(xì)胞酒精性氧化損傷模型的處理方法。同時(shí)驗(yàn)證了OA在體外DPPH、O2和OH三種化學(xué)發(fā)光模型中的抑制作用，結(jié)果表明OA直接清除自由基的能力不強(qiáng)。OA作用QZG細(xì)胞24h濃度在15.625μM以下時(shí)對(duì)細(xì)胞活力無(wú)顯著影響，表明對(duì)細(xì)胞未產(chǎn)生明顯毒作用。我們還發(fā)現(xiàn)5、10、15μM OA作用24h能顯著提高Nrf-2蛋白表達(dá)，10μM OA作用不同時(shí)間能顯著誘導(dǎo)Nrf-2、HO-1、CAT、PRX-1蛋白表達(dá)，在24h內(nèi)表達(dá)量隨時(shí)間延長(zhǎng)而增加。15μMOA作用能顯著抑制EtOH誘導(dǎo)的QZG細(xì)胞增殖活力損傷，統(tǒng)計(jì)學(xué)上有差異（P0.05）。OA對(duì)EtOH誘導(dǎo)的QZG細(xì)胞氧化損傷相關(guān)指標(biāo)的影響結(jié)果表明結(jié)果表明200mMEtOH作用6h能誘發(fā)顯著的活性氧介導(dǎo)的氧化應(yīng)激損傷反應(yīng)，表現(xiàn)為SOD活性明顯降低、MDA含量升高，GSH含量降低、GSH/GSSH比值降低。給予不同劑量OA保護(hù)性干預(yù)后，細(xì)胞氧化應(yīng)激水平明顯降低，表現(xiàn)為MDA水平降低，抗氧化酶活性增強(qiáng)，GSH含量增高，同時(shí)GSH/GSSG比值也顯著升高。蛋白表達(dá)水平實(shí)驗(yàn)結(jié)果表明，5、10、15μMOA干預(yù)后，能明顯升高Nrf-2、SOD1、HO-1、Gpx的蛋白表達(dá)水平，與陽(yáng)性對(duì)照組相比均有統(tǒng)計(jì)學(xué)意義。 在酒精誘導(dǎo)氧化損傷的動(dòng)物模型中，酒精灌胃后導(dǎo)致動(dòng)物體重減輕明顯，OA給藥對(duì)酒精導(dǎo)致的大鼠體重降低無(wú)明顯的改善作用，各組動(dòng)物血糖無(wú)明顯變化，但OA干預(yù)后顯著改善了陽(yáng)性對(duì)照組肝體比的增加，減輕了肝臟的脂肪變病理改變。給予10mg/kg/day和20mg/kg/day的OA后，血清ALT和AST活力值顯著降低，表明OA給藥后能顯著保護(hù)酒精導(dǎo)致的肝功能受損。兩種劑量的OA后明顯地改善了總膽固醇和低密度脂蛋白水平的增高趨勢(shì)，低劑量OA顯著降低了甘油三酯的水平，， OA顯著降低了高密度脂蛋白的水平，機(jī)制待闡明。OA給藥后除對(duì)脂肪組織AGEs有一定程度的抑制作用外，其余如血清、肝臟和肌肉組織未發(fā)現(xiàn)明顯地改變。OA有助于維持線(xiàn)粒體谷胱甘肽氧化還原平衡，明顯改善了酒精誘導(dǎo)的線(xiàn)粒體的活力值減少，并減輕了線(xiàn)粒體的腫脹程度。在血清和肝臟勻漿測(cè)定氧化應(yīng)激損傷指標(biāo)結(jié)果表明，酒精作用可以導(dǎo)致血清和肝臟SOD和CAT活性下降，OA可提高以上兩種抗氧化酶活性。陽(yáng)性對(duì)照組的血清Gpx活性與陰性對(duì)照組相比無(wú)顯著變化，OA后也未能改變其活性，陽(yáng)性對(duì)照組肝勻漿Gpx活性無(wú)顯著變化，但給予OA后，Gpx活性反而明顯降低。陽(yáng)性對(duì)照組血清T-AOC值上升，給藥后該趨勢(shì)減輕，肝臟內(nèi)T-AOC的變化趨勢(shì)同血清結(jié)果相反，OA干預(yù)后肝臟總抗氧化力水平明顯增高。陽(yáng)性對(duì)照組的血清和肝勻漿GSH含量降低、GSSG含量增加，在OA干預(yù)下，GSH顯著增高，GSH/GSSG比值顯著增高，這表明OA能通過(guò)恢復(fù)GSH系統(tǒng)發(fā)揮保護(hù)作用。T-SH結(jié)果表明酒OA后能顯著保護(hù)酒精對(duì)巰基的耗竭作用。血清中MDA含量陽(yáng)性對(duì)照組與陰性對(duì)照組相比無(wú)顯著改變，OA干預(yù)后MDA含量降低。肝勻漿MDA含量陽(yáng)性對(duì)照組明顯增高，OA干預(yù)后明顯降低。陽(yáng)性對(duì)照組肝臟Nrf-2、HO-1、GR、SOD-1等抗氧化蛋白表達(dá)顯著下調(diào)，肌肉Nrf-2也顯著下調(diào)，OA干預(yù)后顯著上調(diào)了上述蛋白的表達(dá)，同時(shí)還上調(diào)了肝臟Nrf-2的基因表達(dá)。組織病理學(xué)結(jié)果表明，OA給藥都顯著減輕了酒精誘導(dǎo)的胃臟和肝臟組織病理?yè)p傷。 【結(jié)論】 成功構(gòu)建了酒精損傷性QZG細(xì)胞模型和動(dòng)物模型，系統(tǒng)研究了酒精性氧化損傷機(jī)制，發(fā)現(xiàn)氧化應(yīng)激在酒精性氧化損傷的細(xì)胞和動(dòng)物模型中占有突出重要的地位，主要表現(xiàn)為自由基誘導(dǎo)的氧化損傷代謝產(chǎn)物增加，以Nrf2為核心的抗氧化酶鏈表達(dá)、活性異常，抗氧化防御系統(tǒng)功能降低；肝臟慢性氧化損傷與脂代謝異常具有密切相關(guān)性；利用體外抗氧化劑篩選技術(shù)模型，測(cè)定了具有保肝作用的單體化學(xué)藥物OA的抗氧化反應(yīng)能力，顯示OA體外直接抗氧化作用相對(duì)較弱，可能與其明顯的脂溶解性有關(guān)；但是，給酒精性肝損傷細(xì)胞和動(dòng)物模型施加不同劑量的OA，則表現(xiàn)為明顯的抗氧化作用；進(jìn)一步研究表明，OA具有活化Nrf2為核心的抗氧化酶鏈作用，進(jìn)而啟動(dòng)機(jī)體整體的抗氧化防御系統(tǒng)功能，有效抑制酒精引起的肝臟氧化應(yīng)激損傷，可能是保護(hù)肝臟防止損傷的重要機(jī)制之一。 本研究結(jié)果提示：OA直接清除自由基的作用較弱，但在酒精誘導(dǎo)的氧化損傷的體外細(xì)胞模型和體內(nèi)動(dòng)物模型中都發(fā)揮了良好的抗氧化保護(hù)作用。OA可能通過(guò)誘導(dǎo)Nrf-2相關(guān)抗氧化酶表達(dá)，保護(hù)谷胱甘肽氧化還原平衡，提高機(jī)體抗氧化酶活性，保護(hù)線(xiàn)粒體功能，減輕脂代謝紊亂發(fā)揮其抗氧化作用。本研究結(jié)果充分說(shuō)明OA在酒精性肝損傷相關(guān)疾病中具有顯著的研究、開(kāi)發(fā)、應(yīng)用前景。本研究為更深一步研究OA在酒精性相關(guān)疾病中的預(yù)防和治療作用提供了一定的研究基礎(chǔ)，為其進(jìn)一步開(kāi)發(fā)利用提供了有效的實(shí)驗(yàn)依據(jù)。
[Abstract]:[background]
Alcohol related diseases are a class of metabolic disorders induced by alcohol metabolism. Alcohol abuse is becoming a serious public health problem in modern society. Alcohol damage to the body involves a variety of pathological mechanisms. With the deepening of the study, alcohol abuse leads to extensive recognition and emphasis on the effect of oxidative damage. It is considered that the oxidative stress induced by ROS is the most important pathogenic factor of alcohol related diseases. Research progress shows that the prevention and treatment of alcohol related diseases are still lack of corresponding drugs and means. Antioxidants are considered to be a promising drug for the prevention and treatment of alcohol related diseases.
Oleanolic acid (OA) is a natural three terpenoid compound derived from plants and has many important biological pharmacological activities. It is rich in OA, such as Gentiana, Ligustrum lucidum, and ID wood of the family of acanthopanacoidaca. It has been used as a prescription drug for hepatitis diseases. Adjuvant therapy. The study found that OA has a certain degree of antioxidant function, but the bioavailability of OA is low because of chemical structure, which restricts its clinical application. Because of the good solubility in alcohol, the dissolving of OA to alcohol may reduce oxidative stress damage caused by heavy alcohol intake. The antioxidant activity of oleanolic acid is clarified. It is of great significance to reduce the process of alcohol related diseases and reduce the burden of alcohol abuse on the health and economic burden.
[Objective]
(1) to study the protective effect of OA on alcoholic oxidative injury.
(2) to explore the protective mechanism of OA in alcoholic oxidative injury.
[method]
(1) cell model: To evaluate the antioxidant activity and protective effect of OA in alcoholic injury models by establishing an alcoholic liver cell oxidative damage model, the effects of QZG cells on the expression of Nrf-2 and other related antioxidant enzymes were observed at different concentrations and different concentrations of OA at different concentrations at different concentrations. The OA intervention of 5,10,15 micron M was also observed for alcohol induced liver refinement. The effects of cell proliferation activity, and the oxidative stress related indexes in cell homogenate, such as SOD activity, MDA, GSH, GSSG and T-SH, were detected. The changes in the protein expression of Nrf-2, SOD1, HO-1 and Gpx after OA intervention were measured. Furthermore, we also observed the inhibitory effects of OA on the three kinds of free radical chemiluminescence models.
(2) animal model: the rats were induced by 4g/kg/day alcohol for 30 days to induce the oxidative damage model of alcoholic rats. At the same time, two doses of 10mg/kg/day and 20mg/kg/day were given to intervention. The serum, liver, muscle, fat homogenate, liver mitochondria were extracted, the body weight change and blood glucose change were measured, the changes of the liver body ratio and the transaminase ALT were observed. The changes of AST activity and lactate dehydrogenase, blood lipid index and mitochondrial activity and swelling degree, changes in the content of glycosylated end products (AGEs) in the tissues and organs of the serum, and the oxidative stress related indexes of serum, liver, fat and muscle tissue, such as MDA, GSH, GSSG and T-SH, CAT, SOD, Gpx, T-AOC activity. Protein, the expression level of Nrf-2, SOD-1, GR, HO-1 protein was measured. The total protein of muscle was extracted and the expression level of Nrf-2 protein was measured. The total RNA of liver tissue was extracted and the expression level of Nrf-2 gene was measured. The pathological section of liver and stomach and HE staining. The oxidative stress reactivity in the alcoholic liver injury model was systematically observed and the resistance to oxidative stress was observed and the resistance to oxidative stress in alcoholic liver injury model was systematically observed. The change of oxidase chain has focused on the protective mechanism of OA against oxidative damage.
[results]
The oxidative damage model of alcohol interfered with QZG cells was successfully constructed. At the same time, combined with the literature, the EtOH action of 200mM was determined as a method to create an alcoholic oxidative damage model of QZG cells. The inhibition effect of OA in three chemiluminescence models of DPPH, O2 and OH in vitro was verified. The results showed that OA directly scavenged free radicals. There was no significant effect on cell viability when the concentration of QZG cell 24h was below 15.625 M, indicating that there was no significant toxic effect on cells. We also found that 24h can significantly increase the expression of Nrf-2 protein by the action of 5,10,15 micron M OA, and that the expression of the protein expression at different time could be significantly induced by the action of 24h in the action of 24h, and the expression of the protein was expressed with time. The effect of prolonging and increasing.15 mu MOA can significantly inhibit the proliferation damage of QZG cells induced by EtOH, and the effect of P0.05.OA on the oxidative damage related indexes of QZG cells induced by EtOH shows that 200mMEtOH action 6h can induce significant reactive oxygen mediated oxidative stress damage reaction, showing SOD activity. The content of MDA increased, the content of GSH decreased, and the ratio of GSH/GSSH decreased. The level of oxidative stress decreased significantly after OA protective drying at different doses. The level of MDA decreased, the activity of antioxidant enzymes increased, the content of GSH increased, and the ratio of GSH/GSSG increased significantly. The results of protein expression showed that the prognosis of 5,10,15 u MOA was the prognosis. The protein expression level of Nrf-2, SOD1, HO-1 and Gpx was significantly increased compared with the positive control group.
In the animal model of alcohol induced oxidative damage, the weight loss of the animals was obviously reduced after alcohol irrigate, and the OA administration had no obvious improvement on the weight loss of rats induced by alcohol. There was no significant change in blood glucose in each group, but the OA intervention significantly improved the increase of the liver body ratio in the positive control group and alleviated the pathological changes of the liver. After OA of 10mg/kg/day and 20mg/kg/day, serum ALT and AST activity decreased significantly, indicating that OA could significantly protect the liver function caused by alcohol. Two doses of OA significantly improved the increase trend of total cholesterol and low density lipoprotein levels, low dose OA significantly reduced triglyceride levels, OA significantly decreased The level of high density lipoprotein (HDL), the mechanism needs to be elucidated that after.OA is given to a certain degree of inhibition of the adipose tissue AGEs, the rest, such as serum, liver and muscle tissue, did not significantly change.OA to maintain the redox balance of mitochondrial glutathione, significantly improved the decrease of alcohol induced mitochondrial activity and reduced the activity of mitochondria. The results of oxidative stress damage in serum and liver homogenate showed that alcohol could lead to the decrease of SOD and CAT activity in serum and liver, and OA could increase the activity of the above two antioxidases. The activity of Gpx in the positive control group was not significantly different from that of the negative control group, and the activity of OA could not change its activity after OA. In the positive control group, there was no significant change in the activity of Gpx in the liver homogenate, but the activity of Gpx decreased obviously after the treatment of OA. The serum T-AOC value of the positive control group increased and the trend was reduced after the administration. The change trend of T-AOC in the liver was opposite to the serum results. After OA intervention, the total antioxidant capacity of the liver increased significantly. The serum and liver homogenate GSH of the positive control group were GSH. The content of GSSG increased and the content of GSH increased significantly with the intervention of OA, and the ratio of GSH/GSSG increased significantly. This indicates that OA can protect the exhaustion effect of alcohol on the sulfhydryl group by restoring the GSH system with the protective effect of.T-SH. The serum MDA content positive control group has no significant changes compared with the negative control group, and the OA dry prognosis is MDA. The content of MDA content in the liver homogenate was significantly higher than that in the positive control group. The expression of Nrf-2, HO-1, GR, SOD-1 and other antioxidative proteins in the liver of the positive control group decreased significantly and the muscle Nrf-2 was down significantly. The expression of the above protein was significantly up-regulated after the intervention of OA, and the gene expression of the liver Nrf-2 was up regulated. Histopathological junctions of the liver were also up. The results showed that OA could significantly reduce the pathological damage of alcohol induced gastric and liver tissues.
[Conclusion]
The alcohol damaged QZG cell model and animal model were successfully constructed, and the mechanism of alcohol induced oxidative damage was systematically studied. It was found that oxidative stress plays an important role in the cell and animal models of alcoholic oxidative damage, which is mainly manifested by the increase of oxidative damage metabolites induced by free radical, and the antioxidant enzyme chain at the core of Nrf2. Expression, activity abnormality, function of antioxidant defense system decreased, liver chronic oxidative damage was closely related to abnormal lipid metabolism; the antioxidant activity of OA, a single chemical drug with protective effect, was determined by using antioxidant screening model in vitro. It showed that the direct antioxidant activity of OA in vitro was relatively weak and possible. However, the effect of different doses of OA on alcoholic liver damage cells and animal models was shown to be an obvious antioxidant activity. Further studies showed that OA has the antioxidant enzyme chain that activates Nrf2 as the core, and then activates the whole body's antioxidant defense system function and effectively inhibits alcohol. Oxidative stress injury in the liver may be one of the important mechanisms to protect the liver from injury.
The results of this study suggest that the effect of OA directly on scavenging of free radicals is weak, but in vitro and in vivo animal models of alcohol induced oxidative damage, a good antioxidant protective effect of.OA may be induced by inducing the expression of Nrf-2 related antioxidant enzymes, protecting the redox balance of Gu Guanggan peptide and improving the activity of antioxidant enzymes in the body. This study provides a further research basis for the study of the role of OA in the prevention and treatment of alcohol related diseases. This study provides a further basis for the study of the role of OA in the prevention and treatment of alcohol related diseases. It provides an effective experimental basis for further development and utilization.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R114

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