Lipid Peroxidation in Alcoholic Liver Disease

描述(由申请人提供)：在饮酒的大鼠酒精性肝病中，肝损伤的机制有多种。其中一个机制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生成增加，其中包括4-羟基壬烯醛(4HNE)。4HNE与多种蛋白质形成加合物，包括26S蛋白酶体的一些亚基。长期摄入乙醇也会导致蛋白酶体的翻译后修饰，例如亚基的过度磷酸化。因此，我们假设，当乙醇连续消耗较长时间时，会诱导细胞色素P450_2E_1，从而产生对脂质的自由基攻击。副产物改变蛋白酶体亚基，导致催化功能丧失。蛋白酶体失去蛋白分解作用会导致细胞内蛋白质的积累。具体目的1：测定酒精灌胃1个月(体内模型)大鼠肝脏20S和19S蛋白酶体亚基的分子翻译后修饰。假设：4HNE加合物和蛋白酶体亚基上特定位置的磷酸化是慢性酒精喂养的大鼠失去催化活性的原因。特定目的2：[利用转导人肝癌细胞系的细胞体外研究乙醇抑制蛋白酶体的分子机制]。具体目标3：[通过基因芯片分析，比较体内饲喂乙醇的大鼠和体外建立的HepG2模型，与乙醇抑制蛋白酶体相关的基因表达的变化]。如果这一方法确定了乙醇诱导蛋白酶体抑制的[如何]，那么酒精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.