Lipid Aldehydes and Ethanol Induced Liver Damage

描述（摘自申请人的摘要）： 本项目旨在确定以下产品的机械作用： 脂质过氧化（4-羟基壬烯醛[4-HNE]和丙二醛[MDA]， 酒精性肝病（ALD）越来越明显的是， 化学反应性醛具有干扰光谱的潜力 从酶抑制到基因启动 表情我们的假设是，当生产过剩时， 酒精诱导的氧化应激、4-HNE和MDA与肝细胞相互作用 导致蛋白质功能改变、蛋白质降解受损的蛋白质 以及参与各种纤维化的基因的功能失调表达 流程.这一假设将评估它的三个具体目标使用肝脏 组织和细胞从给予酒精的大鼠和小鼠获得， 胃内输注。在具体目标1中，我们将继续描述 从长期治疗的大鼠中分离的肝磷脂加合蛋白 用酒精对于这些研究，将对蛋白质进行免疫分离， 进行基质辅助激光解吸电离-飞行时间 （MALDI-TOF）分析用于氨基酸序列分析和鉴定 特定的加合氨基酸残基。具体目标2中提出的实验 将研究4-HNE和MDA减少 肝星状细胞中NF κ B复合物的反式激活，导致 下调IL-6表达。NF-κ B中的特异性机制事件 将在分离的肝细胞和星状细胞中评价信号传导途径， 包括1 KB蛋白的磷酸化和蛋白水解， p65和PKAc。我们还将使用转基因技术检测IL-6在ALD中的作用。 IL-6敲除小鼠。在具体目标3中，我们将评估 4-HNE和MDA与蛋白质和多催化蛋白酶/蛋白体（MCP） 改变蛋白质降解。这些实验还将使用 免疫沉淀/免疫印迹程序以及MALDI-TOF分析， 使用体外系统研究加合模型蛋白的降解， 确定参与肝损害的内收的分子靶点， MCP降解蛋白质。这些研究将大大提高我们的 了解4-HNE和MDA在酒精诱导的 肝损伤识别敏感细胞靶点和特异性 机制可以提供信息，导致制定有效的 用于改变ALD中涉及的特定事件的治疗干预。

DESCRIPTION (Adopted from the Applicant's Abstract): The long-range goal of this project is to establish the mechanistic role(s) of aldehydic products of lipid peroxidation (4-hydroxynonenal [4-HNE] and malondialdehyde [MDA] in alcoholic liver disease (ALD). It is becoming more apparent that these chemically reactive aldehydes have the potential to interfere with a spectrum of cellular functions ranging from enzyme inhibition to initiation of gene expression. It is our hypothesis that when produced in excess as a result of alcohol-induced oxidative stress, 4-HNE and MDA interact with hepatocellular proteins resulting in altered protein function, impaired protein degradation and the dysfunctional expression of genes involved in various fibrotic processes. This hypothesis will be evaluated it three specific aims using liver tissue and cells obtained from rats and mice administered alcohol by intragastric infusion. In Specific Aim 1, we will continue our characterization of hepatic aldehyde-adducted proteins isolated from rats treated chronically with alcohol. For these studies, the proteins will be immunoisolated, and subjected to matrix assisted laser desorption ionization-time of flight (MALDI-TOF) analysis for amino acid sequence analysis and identification of specific adducted amino acid residues. Experiments proposed in Specific Aim 2 will investigate the potential of 4-HNE and MDA to diminish the trans-activation of NFKB complex in hepatic stellate cells, resulting in down-regulation of IL-6 expression. The specific mechanistic events in NF-KB signaling pathway will be evaluated in isolated hepatocytes and stellate cells, including phosphorylation and proteolysis of 1KB proteins, phosphorylation of p65 and PKAc. We will also examine the role of IL-6 in ALD using transgenic IL-6 knockout mice. In Specific Aim 3 we will evaluate how the interactions of 4-HNE and MDA with proteins and the multicatalytic proteinase/proteosome (MCP) alters protein degradation. These experiments will also employ immunoprecipitation/immunoblotting procedures as well as MALDI-TOF analysis to investigate degradation of adducted model proteins using in vitro systems and to identify the molecular targets of adduction involved in impaired hepatic protein degradation by the MCP. These studies will greatly enhance our understanding of the mechanistic role(s) of 4-HNE and MDA in alcohol-induced liver damage. Identification of susceptible cellular targets and specific mechanisms could provide information leading to the development of effective therapeutic interventions for altering specific events involved in ALD.