【作者】 王彦芳；

【导师】 阚全程；

【作者基本信息】 郑州大学 ， 药理学， 2013， 硕士

【摘要】 研究背景肝功能衰竭(hepatic failure)是指肝细胞受到广泛、严重的损害,机体代谢功能发生严重紊乱而出现的临床综合症状。常伴有大量肝细胞坏死,血氨的恶性升高,严重的消化道症状,黄疸迅速加深,出血,脑水肿和肺水肿甚至肝性脑病(hepatic encephalopathy)。此综合征病情严重、临床症状复杂、病死率高,是严重的危害人们健康的肝脏疾病。其中肝性脑病是肝硬化失代偿期和肝功能衰竭的重要并发症之一,以代谢紊乱为基础的中枢神经系统功能失调的综合病征,其主要临床表现是意识障碍、行为失常和昏迷。而代谢紊乱引起的氨中毒学说一直在肝性脑病的发病机制中处于核心地位。同时,血氨在肝性脑病的诊断、治疗中也有十分重要的作用。胆红素作为一种生理性代谢产物并非绝对无用,许多临床及动物实验都证实胆红素可作为机体的抗氧化物质发挥极强的抗氧化作用。胆红素对脂类的抗氧化作用可以起到补充谷胱甘肽的作用。然而胆红素通过肝脏UGT1A1的分解代谢对胆红素的抗氧化特性起决定性作用。Notas等的研究结果显示,肝硬化早期肝代偿功能活跃,随着胆红素的增加,机体抗氧化功能呈增强的趋势,说明胆红素与机体抗氧化系统的潜在联系。另外,胆红素还具有抗脂质过氧化、抗炎、抑制细胞凋亡等作用。胆红素通过UGT酶催化的葡萄糖醛酸化作为肝脏代谢内源性物质最普遍的结合反应具有重要病理生理意义。目前证据提示氨可以作用于线粒体中三羧酸循环和苹果氨酸、天冬氨酸穿梭酶,导致ATP合成不足,发生氧化应激激。氧化应激主要通过启动膜脂质过氧化改变生物膜功能、与生物大分子共价结合及破坏酶的活性等。在细胞因子(如TNF-α, NF-κB)的共同作用下引起不同程度的肝损伤,所以抗氧化治疗对防治肝脏疾病起着重要的作用。活性氧类物质(Reactive oxygen species, ROS),通常是生物氧化过程中的副产物,由于分子中存在未配对的电子,所以十分活跃。过高的活性氧水平会对细胞和基因结构造成损坏,并且在细胞信号传导,和保持机体平衡中起很大作用。然而在外界环境影响下如：暴露于紫外线(UV exposure)或热源(heat exposure)下等,ROS的量会急剧增多,造成明显的细胞结构的损坏,这种现象被称为氧化应激。最近的研究表明氨可诱导星形胶质细胞中活性氧的产生。在急性氨中毒的动物模型中也显示出超氧化物产生的增加和各种抗氧化酶活性的下降。氧化应激的后果之一就是导致线粒体内膜上以钙离子依赖为特征的mPTP孔的开放。血氨作为一种生理性产物在病理条件下的恶性增高必然导致其在肝脏内的堆积,肝细胞内升高的氨浓度势必会作为一种病理性的毒物影响肝细胞的代谢。目前,国内外关于血氨升高如何引起星状胶质细胞形态改变机制的研究较多,包括有直接毒性作用,氧化应激作用,线粒体通透性转变和谷氨酰胺等学说。但是关于氨对肝细胞的毒性,尤其是肝脏中酶的活性的影响研究甚少；在我们之前的临床研究治疗中发现：肝衰竭过程中出现明显的血氨及胆红素升高,降氨后胆红素亦下降。在治疗急性肝衰竭和肝硬化基础上的慢加急性肝衰竭时,早期、全程联合应用降血氨药物能够显著缓解患者的肝脏损伤、降低患者死亡率、改善患者预后。胆红素的代谢过程中步步都涉及到能量的消耗,而在许多肝性脑病模型中都证实氨对线粒体氧化损伤及能量代谢的干扰,故推测氨可能通过氧化损伤影响胆红素代谢相关的酶或者是阻断肝细胞的能量供给来阻断胆红素的代谢,从而导致胆红素在体内的堆积。而高血氨状态下出现的胆红素水平的升高亦可能是组织处于氧化应激状态的标志。本实验通过高血氨动物模型和氨中毒细胞模型的建立,揭示氨对肝细胞氧化损伤及对胆红素代谢的阻碍作用,为临床治疗急慢性肝衰竭中早期、全程联合应用降氨药物提供理论依据。研究目的1.建立高血氨动物模型,检测胆红素代谢相关的酶HO-1和UGT1A1及NF-κB通路的激活情况；2.培养人正常的肝细胞chang liver,浓度梯度的氯化铵处理后检测ROS和ATP的变化,进一步阐明氨对肝脏的氧化损伤及能量代谢的干扰,证实氨与肝细胞氧化损伤的相关性；研究方法(1)高血氨动物模型：1. ELISA检测大鼠血清总胆红素(TBIL),直接胆红素(DBIL)水平；2. RT-PCR法检测UGTIA1的mRNA的变化；3.免疫组织化学染色检测HO-1和NF-κB的表达(2)氨中毒的细胞模型1.MTT法检测氨对肝细胞的毒性；2. TUNEL法测定NH4CL作用后肝细胞的凋亡3. ELISA法测细胞上清液中DBIL, GSSH, GSH水平的变化；4.钼磷酸比色法检测ATP的生成量；5.生化自动分析仪测定加入ATP后细胞内DBIL的水平；6. Western blot检测HO-1和UGT1A1的蛋白表达量的差异；7.荧光定量PCR检测HO-1和UGT1A1的mRNA的变化；8.电镜下观察线粒体形态的损伤；9.流式细胞技术观测ROS产生的多少。结果动物实验中,高血氨组大鼠血清TBIL、DBIL、ALT、AST较对照组明显升高,P<O.001,差异具有统计学意义；高血氨组的HO-1的组织蛋白表达升高,NF-κB的核转移增加；UGT1A1mRNA水平升高约为对照组的186%。细胞实验中,10mmol/L的氯化铵组细胞上清液中DBIL和GSH水平较对照组下降；胞内DBIL量较对照组升高；ATP的生成量下降；10mmol/L的氯化铵处理组在24小时ROS的产生显著增加并可见线粒体损伤；UGT1A1的mRNA和蛋白表达量均增加,但是HO-1的mRNA和蛋白表达量无差别。结论1.在氨升高的动物和细胞模型中均伴有胆红素和UGT1A1的升高；2.氨造成了肝细胞的氧化损伤和线粒体的形态改变；3.氨是通过线粒体的氧化损伤及能量供给障碍来影响胆红素的代谢。更多还原

【Abstract】 Research backgroundLiver failure (hepatic failure) is a clinical symptoms of serious metabolic dysfunction of disorder with liver cells Subjecting to extensive and serious damage. It Often accompanied by massive necrosis of liver cells, blood ammol/Lonia malignant rise, serious gastrointestinal symptoms, jaundice rapidly deepening, hemorrhage, cerebral edema and pμLmonary edema or hepatic encephalopathy (hepatic encephalopathy). This syndrome is a liver disease endangering peoples health seriously because of its serious condition, complex clinical symptom and high mortality. The hepatic encephalopathy is one of the most important complications of decompensated liver cirrhosis and liver failure, a syndrome based on central nervous system function in metabolic disorders. Its main clinical manifestation is consciousness disorder, conduct disorder and coma.The ammol/Lonia poisoning theory caused by metabolic disorder is at the core position in the pathogenesis of hepatic encephalopathy. At the same time, blood ammol/Lonia plays a very important role in the pathogenesis of hepatic encephalopathy.Bilirubin as a kind of physiological metabolism is not absolutely useless. Many clinical and animal experiments have confirmed that bilirubin, as antioxidant substances in the body, plays a strong role in the antioxidant [1]. Antioxidant effect of bilirubin on lipids can play a supplementary role to glutathione. However, the catabolism of bilirubin by the liver UGT1A1plays a decisive role for its antioxidant properties. The resμLts of Notas et al show that:the liver compensation function is active in the early cirrhosis and the antioxidant activities were enhanced, along with the increased bilirubin, Suggesting the potential connection between bilirubin and antioxidant [2]. In addition, bilirubin also carrys out the action of anti-lipid peroxidation, anti-inflammol/Latory, inhibiting cell apoptosis. The conjugation of bilirubin to glucuronic acid catalyzed by UGT as the most commol/Lon binding reaction of hepatic metabolism of endogenous substances has an important pathophysiological significance. The current evidence suggests that ammol/Lonia play a part in three carboxylic acid cycle and apple arginine, aspartate shuttle enzyme in the mitochondria, leading to the lack of ATP synthesis and the occurrence of oxidative stress. Oxidative stress changed the function of membrane, covalently combined with biological molecμLes and destroyed enzyme activity etc mainly through the start of membrane lipid peroxidation. Under the combined effect of cell factor (such as TNF-α, NF-κB), Oxidative stress caused different degree of liver injury. So the antioxidant treatment takes an important roles in the prevention and treatment of liver disease [3].Reactive oxygen species (Reactive oxygen, species, ROS), a byproduct of biological oxidation process is very active due to the presence of unpaired electrons in the molecμLe. The high level of active oxygen can cause damage to the cells and structure of gene, and lays a great role in the body balance and signal transduction in cells. However, the amount of ROS will increase rapidly when influenced by the outside environment such as:UV exposure or heat exposure and etc, causing obvious damage to cell structure. This phenomenon is known as oxidative stress. Recent studies indicate that ammol/Lonia can induce the generation of reactive oxygen species in astrocytes[4]. It also showed increased superoxide production and decrease of antioxidant enzyme activity in animal model of acute ammol/Lonia poisoning. One of the consequences of oxidative stress is the opening of Ca2+dependent mPTP pore in the mitochondrial. The liver is a major site for ammol/Lonia metabolism in vivo. Elevated blood ammol/Lonia and "liver cell anergy" phenomenon of chronic hepatitis or liver failure caused us great concern. Blood ammol/Lonia as a physiological product malignant increased in pathological conditions will inevitably lead to the accumμLation in liver. Elevated ammol/Lonia concentration in the liver will serve as a pathological poison and affects the metabolism of liver cells.At present, there are more research on the mechanisms increased blood ammol/Lonia cause astrocyte morphology at home and abroad, including direct toxicity, oxidative stress, mitochondrial permeability transition and glutamine theory. But little research is done on the toxicity of ammol/Lonia to liver cell, especially its effects to liver enzyme activity. in our previous clinical study, we found that:blood ammol/Lonia and bilirubin were significantly elevated in liver failure process and bilirubin decreased after reducing the ammol/Lonia level. On the basis of the treatment of acute liver failure and cirrhosis of the liver with acute-on-chronic liver failure, the combined application of lowering blood ammol/Lonia drugs can significantly relieve the liver injury in patients, reduce the mortality of patients and improve the prognosis of the patients. Metabolism of bilirubin is related to energy consumption in step by step. In many hepatic encephalopathy model, it is confirmed that:ammol/Lonia induced oxidative damage and interfered energy metabolism of mitochondria. ammol/Lonia may affects bilirubin metabolism related enzymes by oxidative damage or blocking the energy supply of liver cells to block the bilirubin metabolism, resμLting in the accumμLation of bilirubin in the body. Elevated levels of bilirubin in high blood ammol/Lonia case may also is the sign of oxidative stress. Through the establishment of high blood ammol/Lonia animal model and ammol/Lonia poisoning cell model this experiment reveals that:the ammol/Lonia caused oxidative damage to the liver and affected the metabolism of bilirubin, providing a theoretical basis for the combined and early use of reducing ammol/Lonia drugs in the clinical treatment of acute and chronic liver failure.Research purpose1. Establish animal model with high blood ammol/Lonia, detect the bilirubin metabolism related enzymes of HO-1and UGT1A1and the activation of NF-κB signal pathway; 2. CμLtured the normal hepatocytes Chang liver, detect the change of ROS and ATP after exposed to ammol/Lonium chloride of concentration gradient, further clarify the oxidative damage ammol/Lonia caused to the liver and interference to energy metabolism, confirmed the correlation between ammol/Lonia and oxidative damage in hepatocytes.Research methods(1) animal model with high blood ammol/Lonia:1. ELISA to detect the level of total bilirubin(TBIL), direct bilirubin(DBIL);2. Detect the Change of UGT1A1mRNAby RT-PCR;3. Expression of HO-1and NF-κB was detected by immol/Lunohistochemistry.(2) cell model of ammonia poisoning:1. Detect toxicity of ammonia to liver cells by method of MTT;2. Determinate the apoptosis of liver cells by TUNEL;3. ELISA to measure the change of DBIL, GSSH and GSH in the supernatant;4. Molybdophosphoric acid colorimetric method to detect the quantitative generation of ATP;5. Determination of intracellular DBIL levels by automatic biochemistry analyzer;6. Detect the changes of mRNA of HO-1and UGT1A1in quantitative PCR;7. Detect the expression difference of protein amount of HO-1and UGT1A1, using Western blot;8. observe the injury of mitochondrial by TEM;9. Flow cytometry to observe the amount of ROS.ResultSerum level of DBIL in high ammonia group rats was significantly higher than the control group in the animal experiment. P<0.001, the difference was statistically significant; The protein of HO-1in high blood ammol/Lonia group increased with NF-κB nuclear transfer increasing. The mRNA level of UGT1A1elevated about239%compared to the control group; in the Cells experiment:DBIL, GSH level in the control group is significantly lower than in the group treated with10mmol/L ammonium chloride; The intracellular DBIL is higher than that of control group; The production of ATP decreased in ammonium chloride group; The generation of ROS increased significantly when treated withlOmmol/L ammonium chloride for24hours and the damage of mitochondrial can be observed; The expression of mRNA and protein of UGT1A1were increased, but the expression of mRNA and protein of HO-1was no differenceConclusion1. There are increased level of bilirubin and UGT1A1in cell and animal models with Increased ammol/Lonia;2. The ammol/Lonia affected the metabolism of bilirubin by means of oxidative damage and obstacle to energy supply in liver cells;3. But the effect of ammol/Lonia on HO-1is a species difference.更多还原