Lipid Peroxidation in Alcoholic Liver Disease

描述（由申请人提供）：在喂食乙醇的大鼠中，酒精性肝病的肝损伤机制多种多样。这些机制之一涉及当乙醇在肝脏中被CYP2E1代谢时产生自由基。产生的自由基导致脂质过氧化产物的形成增加，包括4羟基壬烯醛（4HNE）。4HNE与各种蛋白质形成加合物，包括26s蛋白酶体的一些亚基。慢性乙醇摄入还诱导蛋白酶体的翻译后修饰，如亚基的过度磷酸化。因此，我们假设，当乙醇持续消耗一段时间，CYP2E1被诱导，这产生了对脂质的自由基攻击。副产物改变了蛋白酶体的亚单位，导致催化功能的丧失.蛋白酶体蛋白水解的丧失导致改变的蛋白质在细胞中积累。具体目标1：测定灌胃乙醇1个月大鼠肝脏20 s和19 s蛋白酶体亚基的分子翻译后修饰（体内模型）。假设：4HNE加合物和蛋白酶体亚基上的特定位点上的磷酸化解释了慢性乙醇喂养大鼠中催化活性的丧失。具体目的2：确定[使用HepG2 CYP2E1转导细胞系体外乙醇抑制蛋白酶体的分子机制]。具体目标3：确定[通过微阵列分析，与蛋白酶体的乙醇抑制相关的基因表达变化，比较大鼠体内乙醇喂养与体外HepG2模型]。如果这种方法确定了乙醇诱导蛋白酶体抑制的[方式]，那么乙醇诱导的肝损伤的新机制将被建立，这可能为治疗干预提供靶点。

DESCRIPTION (provided by applicant): The mechanisms of liver injury in alcoholic liver disease in rats fed ethanol are numerous. One of these mechanisms involves free radical generation when ethanol is metabolized by CYP2E1 in the liver. The free radicals produced lead to the increase in formation of products of lipid peroxidation including 4 hydroxynonenal (4HNE). 4HNE forms adducts with various proteins including some of the subunits of the 26s proteasome. Chronic ethanol ingestion also induces post translational modification of the proteasomes such as hyperphosphorylation of the subunits. Therefore, we hypothesize that when ethanol is consumed continuously for a prolonged period, CYP2E1 is induced and this generates a free radical attack on lipids. The by- products alter the proteasomal subunits to cause loss of catalytic function. The loss of proteolysis by the proteasome leads to the accumulation of altered proteins in the cells. Specific Aim 1: To determine the molecular post translational modifications of the 20s and 19s proteasome subunits from the liver of rats-fed ethanol intragastrically for 1 month (in vivo model). Hypothesis: 4HNE adducts and phosphorylation on specific sites on subunits of the proteasome accounts for the loss of catalytic activity in chronic ethanol fed rats. Specific Aim 2: To determine [the molecular mechanisms of proteasome inhibition by alcohol in vitro using the HepG2 CYP2E1 transduced cell line]. Specific Aim 3: To determine [by microarray analysis, the changes in gene expression associated with ethanol inhibition of the proteasome, comparing the rat fed ethanol in vivo with the HepG2 model in vitro]. If this approach determines [how] ethanol-induces proteasomal inhibition, then a new mechanism of ethanol-induced liver injury will have been established which could provide a target for therapeutic intervention.