Alcohol-Induced Fatty Liver and Injury in Humanized CYP2E1 Knockin Mice.

描述（由申请人提供）：酒精引起细胞损伤的机制尚不清楚。脂质过氧化和氧化应激在酒精中毒中的作用是一个重要的机制，是相当多的研究的焦点。许多途径被认为在乙醇诱导“氧化应激”的过程中发挥了关键作用。我们假设体内CYP2E1诱导的氧化应激增加使肝细胞对慢性乙醇诱导的肝毒性和脂肪变性敏感。我们认为，氧化剂如过氧亚硝酸盐、MAP激酶如JNK和/或p38 MAPK的激活、NF-:B激活和生存因子合成的下降以及线粒体功能障碍是CYP2E1增强肝毒性的下游介质。我们认为，这些来自CYP2E1的下游介质与乙醇诱导的TNF1、同型半胱氨酸和内质网应激升高相关，激活了脂肪生成转录因子和酶，同时降低了脂肪分解转录因子和酶的激活，从而导致脂肪肝。支持这些假设的初步数据是发现乙醇诱导的脂肪肝在CYP2E1敲除小鼠中钝化，而在人源化的CYP2E1敲除小鼠中恢复。慢性乙醇喂养后，高水平人CYP2E1敲入的小鼠肝毒性和氧化/亚硝化应激升高。乙醇喂养的CYP2E1敲入小鼠似乎是酒精诱导肝毒性的有效口服模型。野生型、CYP2E1敲除型和CYP2E1敲除型小鼠分别饲喂不同时间的高脂Lieber-DeCarli饮食，例如1 - 6周。对照组用葡萄糖配对喂养。AIM1旨在评估CYP2E1表达升高导致乙醇喂养的CYP2E1敲入小鼠肝毒性的机制，包括氧化/亚硝化应激、线粒体功能障碍、CYP2E1、iNOS、TNF1、MAPK和NF:B的测定。AIM 2旨在评估CYP2E1促进酒精性脂肪肝的途径，包括转录因子水平的测定，如SREBP-1c和PPAR1，关键和限速的脂肪生成和脂肪分解酶的含量；将测定野生型、CYP2E1敲除小鼠和CYP2E1敲除小鼠饲喂乙醇或葡萄糖时的脂肪酸氧化率。下游CYP2E1效应物变化的时间过程将相对于乙醇诱导的脂肪变性和肝毒性而确定。CYP2E1、iNOS、氧化/亚硝化应激、MAPK、线粒体功能障碍、TNF1产生抑制剂对野生型和CYP2E1敲入小鼠乙醇诱导的脂肪变性和肝损伤的影响将被确定。我们相信这些实验将提供CYP2E1促进乙醇性肝损伤和脂肪肝的分子机制，并可能进一步开发新的治疗设计来治疗或最小化乙醇性肝损伤的进展，这是RFA-AA-006的既定目标。

DESCRIPTION (provided by applicant): The mechanism(s) by which alcohol causes cell injury are still not clear. A major mechanism that is a focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a key role on how ethanol induces "oxidative stress". We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to chronic ethanol induced hepatotoxicity and steatosis. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or p38 MAPK, a decline in activation of NF-:B and synthesis of survival factors, and mitochondrial dysfunction are downstream mediators of the CYP2E1 potentiated hepatotoxicity. We propose that such downstream mediators from CYP2E1 in association with ethanol-induced elevation of TNF1, homocysteine and ER stress activate lipogenic transcription factors and enzymes while decreasing activation of lipolytic transcription factors and enzymes, thereby resulting in fatty liver. Preliminary data in support of these hypotheses are the findings that ethanol-induced fatty liver is blunted in CYP2E1 knockout mice and restored in humanized CYP2E1 knockin mice. Hepatotoxicity and elevated oxidative/nitrosative stress in found in the CYP2E1 knockin mice with high levels of human CYP2E1 after chronic ethanol feeding. The ethanol-fed CYP2E1 knockin mice appear to be an effective ORAL model of alcohol-induced hepatotoxicity. Wild type, CYP2E1 knockout and CYP2E1 knockin mice will be fed the high fat Lieber-DeCarli diet for varying times, e.g. 1 to 6 weeks. Controls will be pair-fed with dextrose. AIM1 is designed to evaluate mechanisms by which elevated expression of CYP2E1 causes hepatotoxicity in the ethanol-fed CYP2E1 knockin mice and will include assays of oxidative/nitrosative stress, mitochondrial dysfunction, CYP2E1, iNOS, TNF1 , MAPK and NF:B. AIM 2 is designed to evaluate pathways by which CYP2E1 contributes to alcohol-induced fatty liver, including assays of levels of transcription factors such as SREBP-1c and PPAR1, content of key and rate- limiting lipogenic and lipolytic enzymes; rates of fatty acid oxidation will be determined in the wild type, CYP2E1 knockout and CYP2E1 knockin mice fed ethanol or dextrose. The time course of changes in the downstream CYP2E1 effectors will be determined relative to that for ethanol-induced steatosis and hepatotoxicity. The effect of inhibitors of CYP2E1, iNOS, oxidative/nitrosative stress, MAPK, mitochondrial dysfunction, TNF1 production on ethanol-induced steatosis and liver injury in wild type and CYP2E1 knockin mice will be determined. We believe these experiments will provide molecular mechanisms by which CYP2E1 promotes ethanol-induced liver injury and fatty liver and may further the development of new therapeutic designs to treat or minimize progression of ethanol-induced liver injury, a stated goal of RFA-AA-006. PUBLIC HEALTH RELEVANCE: Mechanisms responsible for alcohol-induced hepatotoxicity and fatty liver are not fully understood. With the novel use of CYP2E1 knockout mice and humanized CYP2E1 knockin mice, a role for CYP2E1 in these toxic actions of alcohol has been defined. Identification of downstream effectors of alcohol/CYP2E1 actions would be very informative in developing therapeutic interventions against alcohol-induced liver injury and fatty liver.