OA对酒精性氧化损伤的保护作用及机制研究

发布时间：2018-05-12 09:19

本文选题：酒精 + 齐墩果酸　；参考：《第四军医大学》2012年硕士论文

【摘要】：【背景】酒精性相关疾病是一类由酒精代谢诱导机体发生代谢紊乱性疾病。酒精滥用日益成为现代社会严重的公共卫生问题。酒精对机体造成的损伤作用涉及多种病理机制。随着研究的日益深入，酒精滥用引起氧化损伤作用得到广泛认同和重视，认为ROS介导的氧化应激损伤是酒精性相关疾病的最主要致病因素。研究进展表明，酒精性相关疾病的预防和治疗还缺乏相应的药物和手段，抗氧化剂被认为是预防和治疗酒精性相关疾病具有良好应用前景的药物。齐墩果酸（Oleanolic acid，OA）是一种来源于植物的天然三萜系化合物，具有很多重要的生物药理活性。富含OA中药如龙胆科植物青叶胆、木樨科植物女贞子、五加科植物id木等在中医应用上历史悠久，现已经作为处方药主要用来肝炎类疾病的辅助治疗。研究发现OA具有一定程度的抗氧化功能，但是OA由于化学结构原因，其生物利用度低，限制了在临床上的应用。由于在酒精中溶解度较好，因此，将OA溶解于酒精后可能减轻酒精大量摄入导致的氧化应激损伤。阐明齐墩果酸的抗氧化保护作用相关机制，研发酒精类保健产品，对减轻酒精类相关疾病的发病过程以及减少酒精滥用导致的卫生经济负担等都具有重要意义。【目的】 (1)研究OA对酒精性氧化损伤的保护作用。 (2)探讨OA在酒精性氧化损伤中发挥保护作用的相关机制。【方法】 (1)细胞模型：通过建立酒精性肝细胞氧化损伤模型，评价OA在酒精性损伤模型中的抗氧化活性作用和保护效应。观察了不同浓度不同时间OA作用QZG细胞对Nrf-2等相关抗氧化酶蛋白表达的影响。还观察了5、10、15μM的OA干预对酒精诱导肝细胞增殖活性的影响，同时检测了细胞匀浆液内氧化应激相关指标，如SOD活性、MDA、GSH、GSSG和T-SH含量。测定了OA干预后对Nrf-2、SOD1、HO-1、Gpx的蛋白表达的改变。此外我们还观察了OA在体外DPPH、O2和OH三种自由基化学发光模型中的抑制作用。 (2)动物模型：通过4g/kg/day酒精灌胃大鼠30天诱导酒精性大鼠氧化损伤模型，同时给以10mg/kg/day和20mg/kg/day两种剂量的OA干预，取血清、肝、肌肉、脂肪匀浆，提取肝脏线粒体，测定实验动物体重变化和血糖改变，观察了肝体比的改变，转氨酶ALT和AST活性和乳酸脱氢酶学指标，血脂指标和线粒体活性和肿胀度改变，血清等组织器官内糖基化终末产物（AGEs）含量变化，同时还观察了血清、肝、脂肪和肌肉组织的氧化应激相关指标，如MDA、GSH、GSSG和T-SH含量，CAT、SOD、Gpx、T-AOC活性。提取肝脏总蛋白，测定了Nrf-2、SOD-1、GR、HO-1蛋白表达水平。提取肌肉总蛋白，测定了Nrf-2蛋白表达水平。同时提取肝脏组织的总RNA，测定了Nrf-2基因表达水平。做肝脏和胃脏病理切片，HE染色。系统观察了酒精性肝损伤模型中的氧化应激损伤反应性，以及抗氧化酶链的变化，重点研究了OA的抗氧化损伤的保护作用机制。【结果】成功构建了酒精干预QZG细胞的氧化损伤模型，同时结合文献报道，确定了200mM的EtOH作用6h作为创建QZG细胞酒精性氧化损伤模型的处理方法。同时验证了OA在体外DPPH、O2和OH三种化学发光模型中的抑制作用，结果表明OA直接清除自由基的能力不强。OA作用QZG细胞24h浓度在15.625μM以下时对细胞活力无显著影响，表明对细胞未产生明显毒作用。我们还发现5、10、15μM OA作用24h能显著提高Nrf-2蛋白表达，10μM OA作用不同时间能显著诱导Nrf-2、HO-1、CAT、PRX-1蛋白表达，在24h内表达量随时间延长而增加。15μMOA作用能显著抑制EtOH诱导的QZG细胞增殖活力损伤，统计学上有差异（P0.05）。OA对EtOH诱导的QZG细胞氧化损伤相关指标的影响结果表明结果表明200mMEtOH作用6h能诱发显著的活性氧介导的氧化应激损伤反应，表现为SOD活性明显降低、MDA含量升高，GSH含量降低、GSH/GSSH比值降低。给予不同剂量OA保护性干预后，细胞氧化应激水平明显降低，表现为MDA水平降低，抗氧化酶活性增强，GSH含量增高，同时GSH/GSSG比值也显著升高。蛋白表达水平实验结果表明，5、10、15μMOA干预后，能明显升高Nrf-2、SOD1、HO-1、Gpx的蛋白表达水平，与阳性对照组相比均有统计学意义。在酒精诱导氧化损伤的动物模型中，酒精灌胃后导致动物体重减轻明显，OA给药对酒精导致的大鼠体重降低无明显的改善作用，各组动物血糖无明显变化，但OA干预后显著改善了阳性对照组肝体比的增加，减轻了肝脏的脂肪变病理改变。给予10mg/kg/day和20mg/kg/day的OA后，血清ALT和AST活力值显著降低，表明OA给药后能显著保护酒精导致的肝功能受损。两种剂量的OA后明显地改善了总胆固醇和低密度脂蛋白水平的增高趋势，低剂量OA显著降低了甘油三酯的水平，， OA显著降低了高密度脂蛋白的水平，机制待阐明。OA给药后除对脂肪组织AGEs有一定程度的抑制作用外，其余如血清、肝脏和肌肉组织未发现明显地改变。OA有助于维持线粒体谷胱甘肽氧化还原平衡，明显改善了酒精诱导的线粒体的活力值减少，并减轻了线粒体的肿胀程度。在血清和肝脏匀浆测定氧化应激损伤指标结果表明，酒精作用可以导致血清和肝脏SOD和CAT活性下降，OA可提高以上两种抗氧化酶活性。阳性对照组的血清Gpx活性与阴性对照组相比无显著变化，OA后也未能改变其活性，阳性对照组肝匀浆Gpx活性无显著变化，但给予OA后，Gpx活性反而明显降低。阳性对照组血清T-AOC值上升，给药后该趋势减轻，肝脏内T-AOC的变化趋势同血清结果相反，OA干预后肝脏总抗氧化力水平明显增高。阳性对照组的血清和肝匀浆GSH含量降低、GSSG含量增加，在OA干预下，GSH显著增高，GSH/GSSG比值显著增高，这表明OA能通过恢复GSH系统发挥保护作用。T-SH结果表明酒OA后能显著保护酒精对巯基的耗竭作用。血清中MDA含量阳性对照组与阴性对照组相比无显著改变，OA干预后MDA含量降低。肝匀浆MDA含量阳性对照组明显增高，OA干预后明显降低。阳性对照组肝脏Nrf-2、HO-1、GR、SOD-1等抗氧化蛋白表达显著下调，肌肉Nrf-2也显著下调，OA干预后显著上调了上述蛋白的表达，同时还上调了肝脏Nrf-2的基因表达。组织病理学结果表明，OA给药都显著减轻了酒精诱导的胃脏和肝脏组织病理损伤。【结论】成功构建了酒精损伤性QZG细胞模型和动物模型，系统研究了酒精性氧化损伤机制，发现氧化应激在酒精性氧化损伤的细胞和动物模型中占有突出重要的地位，主要表现为自由基诱导的氧化损伤代谢产物增加，以Nrf2为核心的抗氧化酶链表达、活性异常，抗氧化防御系统功能降低；肝脏慢性氧化损伤与脂代谢异常具有密切相关性；利用体外抗氧化剂筛选技术模型，测定了具有保肝作用的单体化学药物OA的抗氧化反应能力，显示OA体外直接抗氧化作用相对较弱，可能与其明显的脂溶解性有关；但是，给酒精性肝损伤细胞和动物模型施加不同剂量的OA，则表现为明显的抗氧化作用；进一步研究表明，OA具有活化Nrf2为核心的抗氧化酶链作用，进而启动机体整体的抗氧化防御系统功能，有效抑制酒精引起的肝脏氧化应激损伤，可能是保护肝脏防止损伤的重要机制之一。本研究结果提示：OA直接清除自由基的作用较弱，但在酒精诱导的氧化损伤的体外细胞模型和体内动物模型中都发挥了良好的抗氧化保护作用。OA可能通过诱导Nrf-2相关抗氧化酶表达，保护谷胱甘肽氧化还原平衡，提高机体抗氧化酶活性，保护线粒体功能，减轻脂代谢紊乱发挥其抗氧化作用。本研究结果充分说明OA在酒精性肝损伤相关疾病中具有显著的研究、开发、应用前景。本研究为更深一步研究OA在酒精性相关疾病中的预防和治疗作用提供了一定的研究基础，为其进一步开发利用提供了有效的实验依据。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【参考文献】