Lipid Aldehydes and Ethanol Induced Liver Damage

描述(摘自申请者摘要)：远景目标 本项目旨在确立乙醛产品的机械作用(S) 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，这些 化学反应的醛有可能干扰光谱 从酶抑制到基因启动的细胞功能 表情。我们的假设是，当过量生产时， 酒精氧化应激、4-HNE和丙二醛与肝细胞的相互作用 蛋白质导致蛋白质功能改变，蛋白质降解受损 以及参与各种纤维化的基因的功能异常表达 流程。这一假设将被评估它的三个具体目标使用肝脏 酒精染毒大鼠和小鼠的组织和细胞 胃内输液。在具体目标1中，我们将继续我们的角色 慢性给药大鼠肝脏乙醛加合物蛋白的分离 带着酒精。在这些研究中，这些蛋白质将被免疫分离，并且 受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。在具体目标2中提出的实验 将研究4-HNE和丙二醛是否有可能降低 肝星状细胞中NFKB复合体的反式激活，导致 下调IL-6的表达。核因子-知识库中的特定机制事件 信号通路将在分离的肝细胞和星状细胞中进行评估， 包括1KB蛋白的磷酸化和蛋白水解性，磷酸化 P65和PKAc。我们还将利用转基因技术研究IL-6在ALD中的作用 IL-6基因敲除小鼠。在具体目标3中，我们将评估 4-HNE、丙二醛与蛋白质和多催化蛋白酶/蛋白体(MCP) 改变蛋白质的降解。这些实验还将使用 免疫沉淀/免疫印迹程序以及MALDI-TOF分析 利用体外实验系统研究加合物模型蛋白的降解 肝损伤时内收相关分子靶点的确定 MCP对蛋白质的降解。这些研究将大大提高我们的 对4-HNE和丙二醛在酒精诱导中的作用的认识(S) 肝脏受损。识别易感细胞靶点和特异性 机制可以提供信息，导致制定有效的 改变ALD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.