Lipid Aldehyde and Ethanol Induced Liver Damage

描述(由申请人提供)：酒精性肝病(ALD)是美国发病率和死亡率的主要来源。ALD是一种进行性疾病，包括肝脏脂肪变性、脂肪性肝炎、纤维化和肝硬变。目前已被广泛接受的ALD的分子机制是多因素的，包括抗氧化系统受损导致活性氧化剂的过量产生，从而有可能损害肝细胞的脂质、蛋白质和核酸。氧化剂介导的脂类修饰和由此导致的对肝细胞稳态至关重要的蛋白质修饰是导致ALD发生和/或进展的重要机制事件。提出的实验的一般工作假设是，作为脂质过氧化的结果而发生的蛋白质修饰在ALD的发病机制中发挥着重要作用。在前一次筹资期间，在分离、鉴定和鉴定被脂质过氧化产物4-羟基壬烯醛(4-HNE)和4-羟基酮烯醛修饰的蛋白质的最先进的蛋白质组学方法方面取得了重大进展。这些蛋白质组学方法将在以下三个特定目标中用于验证这一假说：目标1中的实验将表征4-HNE修饰对先前识别的蛋白质靶标的功能影响，包括尿素循环酶、F0F1 ATPase和内质网应激蛋白质。AIM 2中提出的研究将确定酒精诱导的脂肪变性、脂肪性肝炎或纤维化的全肠内营养(TEN)模型中的4-HNE和4-酮修饰蛋白质组，以确定与ALD这些进展阶段机械相关的蛋白质修饰。这些研究将使用肝脏保护剂N-乙酰半胱氨酸来确定4-HNE或4-酮修饰蛋白质组对这种抗氧化剂的反应变化。目标3中提出的实验将使用相同的动物模型来表征在以脂肪变性、脂肪性肝炎和纤维化为特征的ALD期间，针对4-HNE和4-酮修饰宿主蛋白的自身抗体的产生和细胞毒性。结合这一目标提出的实验使用免疫分离技术和LC-MS/MS质谱仪来鉴定作为自身抗原的特定宿主蛋白。总的来说，从这些实验中获得的机制信息将为减轻和/或逆转ALD的新治疗策略提供新的见解。7.项目叙述：长期摄入酒精损害肝脏的机制是多方面的和相互作用的。我们对这种疾病的认识的进步将从对导致这种复杂和进行性疾病的个体机制的系统研究中演变出来。现在很清楚，氧化应激是ALD的一个重要组成部分。脂质过氧化是氧化应激的结果之一，导致产物脂醛的产生，如4-HNE和4-酮，它们是与ALD相关的肝损伤的重要因素。这一更新应用的总体目标是扩大我们对这些内源性化合物参与ALD早期(脂肪变性)、炎症性(脂肪性肝炎)以及晚期ALD(包括纤维化)的病理生理机制的研究。 与公共卫生相关：细胞色素P450是一个蛋白质超家族，在人类中负责氧化大量化合物，包括许多内源性化合物，如类固醇、胆汁酸、维生素，以及外源化合物，如药物、麻醉剂、溶剂和我们饮食中的化合物。这项拟议的研究将有助于更好地了解这些酶的功能，特别是与它们的氧化还原伙伴细胞色素b5和细胞色素P450还原酶的作用。

DESCRIPTION (provided by applicant): Alcoholic liver disease (ALD) is a prominent source of morbidity and mortality in the United States. ALD is a progressive disease encompassing hepatic steatosis, steatohepatitis, fibrosis and cirrhosis. It is now widely accepted the molecular mechanisms underlying ALD are multifactorial including compromised antioxidant systems resulting in overproduction of reactive oxidant species having the potential to damage hepatocellular lipids, proteins and nucleic acids. The oxidant-mediated modification of lipids and the resulting modification of proteins critical for hepatocellular homeostasis are important mechanistic events contributing to initiation and/or progression of ALD. The general working hypothesis underlying the proposed experiments is that protein modification occurring as a consequence of lipid peroxidation plays a significant role in the pathogenesis of ALD. During the previous funding period, significant progress has been made in the development of state-of-the-art proteomic approaches for isolating, identifying and functionally characterizing proteins modified by the lipid peroxidative products 4-hydroxynonenal (4-HNE) and 4-oxononenal. These proteomic approaches will be used to test this hypothesis in the following three specific aims: Experiments in Aim 1 will characterize the functional consequences of 4-HNE modification on proteins targets previously identified including urea cycle enzymes, F0F1 ATPase and ER stress proteins. Studies proposed in Aim 2 will identify the 4-HNE- and 4-ONE-modified proteome in the Total Enteral Nutrition (TEN) model of alcohol-induced steatosis, steatohepatitis or fibrosis to identify protein modifications that are mechanistically involved in these progressive stages of ALD. These studies will employ administration of the hepatoprotectant N-acetylcysteine to identify changes in the 4-HNE or 4-ONE-modified proteomes responsive to this antioxidant. Experiments proposed in Aim 3 will employ this same animal model to characterize the production and cytotoxicity of autoantibodies against 4-HNE and 4-ONE-modified host proteins during ALD characterized by steatosis, steatohepatitis and fibrosis. Experiments proposed in conjunction with this aim use immunoisolation techniques and LC-MS/MS mass spectrometry to identify the specific host proteins which function as autoantigens. Collectively, the mechanistic information derived from these experiments will provide new insight into novel therapeutic strategies to attenuate and or reverse ALD. 7. Project Narrative: The mechanisms by which chronic ethanol ingestion damages the liver are multiple and interactive. The advancement in our knowledge of this disease will evolve from systematic investigations of individual mechanisms which contribute to this complex and progressive disease. It is now clear that oxidative stress is an important component of ALD. Lipid peroxidation is one consequence of oxidative stress resulting in the production of products lipid aldehydes such as 4-HNE and 4-ONE that are important factors in liver injury associated with ALD. The overall goal of this renewal application is to extend our investigation of the mechanisms by which these endogenous compounds are involved in the pathophysiology of early (steatosis) inflammatory (steatohepatitis) as well as late stages of ALD, including fibrosis. Public Health Relevance: The cytochromes P450 are a large superfamily of proteins that in humans are responsible for oxidizing a vast array of compounds, including many endogenous compounds such as steroids, bile acids, vitamins, and xenobiotics such as drugs, anesthetics, solvents, and compounds in our diet. The proposed research will lead to a better understanding of how these enzymes function, especially with their redox partners, cytochrome b5 and cytochrome P450 reductase.