Lipid Peroxidation in Alcoholic Liver Disease

描述（由申请人提供）：乙醇喂养的酒精性肝病大鼠肝损伤的机制是多方面的。其中一种机制涉及肝脏中CYP2E1代谢乙醇时自由基的产生。自由基的产生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。慢性乙醇摄入也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，当乙醇持续长时间消耗时，CYP2E1被诱导，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。蛋白酶体失去蛋白质水解作用导致细胞中改变蛋白的积累。目的1：观察大鼠灌胃乙醇1个月（体内模型）后肝脏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点的磷酸化是慢性乙醇喂养大鼠催化活性丧失的原因。特异性目的2：利用HepG2 CYP2E1转导细胞系确定乙醇体外抑制蛋白酶体的分子机制。特异性目的3：通过微阵列分析，比较体内喂乙醇的大鼠和体外喂乙醇的HepG2模型，确定与乙醇抑制蛋白酶体相关的基因表达变化。如果这种方法确定了乙醇诱导蛋白酶体抑制的机制，那么乙醇诱导肝损伤的新机制将被建立，这将为治疗干预提供一个靶点。

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.