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，包括肝脂肪变性、脂肪性肝炎、纤维化和肝硬化。现在人们普遍认为，酒精性脂肪肝的分子机制是多因素的，包括抗氧化系统受损，导致反应性氧化剂物质过量产生，有可能损害肝细胞脂质、蛋白质和核酸。氧化剂介导的脂质修饰和由此产生的对肝细胞稳态至关重要的蛋白质修饰是促成ALD起始和/或进展的重要机制事件。所提出的实验的一般工作假设是，脂质过氧化反应导致的蛋白质修饰在ALD的发病机制中起着重要作用。在上一个资助期间，在开发最先进的蛋白质组学方法方面取得了重大进展，这些方法用于分离、鉴定和功能表征由脂质过氧化产物4-羟基壬烯醛（4-HNE）和4-氧代壬烯醛修饰的蛋白质。这些蛋白质组学方法将用于在以下三个具体目标中测试该假设：目标1中的实验将表征4-HNE修饰对先前鉴定的蛋白质靶标的功能后果，所述蛋白质靶标包括尿素循环酶、F0 F1 ATP酶和ER应激蛋白。目标2中提出的研究将鉴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-ONE修饰的蛋白质组，以鉴定在ALD的这些进展阶段中机械参与的蛋白质修饰。这些研究将采用保肝剂N-乙酰半胱氨酸的给药，以确定4-HNE或4-ONE修饰的蛋白质组对这种抗氧化剂的反应变化。目的3中提出的实验将采用该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-ONE修饰的宿主蛋白的自身抗体的产生和细胞毒性。结合这一目的提出的实验使用免疫分离技术和LC-MS/MS质谱法来鉴定起自身抗原作用的特异性宿主蛋白。总的来说，从这些实验中获得的机制信息将为减弱和/或逆转ALD的新治疗策略提供新的见解。7.项目叙述：慢性乙醇摄入损害肝脏的机制是多种多样的，相互作用的。我们对这种疾病的认识的进步将从系统研究导致这种复杂和进行性疾病的个体机制中发展出来。现在很清楚，氧化应激是ALD的重要组成部分。脂质过氧化是氧化应激的结果之一，导致产物脂质醛如4-HNE和4-ONE的产生，其是与ALD相关的肝损伤的重要因素。该更新申请的总体目标是扩展我们对这些内源性化合物参与早期（脂肪变性）炎症（脂肪性肝炎）以及ALD晚期（包括纤维化）病理生理学的机制的研究。 公共卫生相关性：细胞色素P450是一个大的蛋白质超家族，在人类中负责氧化大量的化合物，包括许多内源性化合物，如类固醇，胆汁酸，维生素和外源性物质，如药物，麻醉剂，溶剂和我们饮食中的化合物。这项研究将有助于更好地了解这些酶的功能，特别是它们的氧化还原伙伴细胞色素b5和细胞色素P450还原酶。

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.