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

描述（由申请人提供）：酒精导致细胞损伤的机制仍不清楚。作为大量研究的焦点的一个主要机制是脂质过氧化和氧化应激在酒精毒性中的作用。许多途径已被认为在乙醇如何诱导“氧化应激”中起关键作用。我们假设体内CYP 2 E1诱导引起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的活化、NF-：B活化和存活因子合成的下降以及线粒体功能障碍是CYP 2 E1增强肝毒性的下游介质。我们认为，这种下游介质从CYP 2 E1与乙醇诱导的TNF-1，同型半胱氨酸和ER应激的升高激活脂肪生成转录因子和酶，同时减少脂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CYP 2 E1敲除小鼠中钝化，在人源化CYP 2 E1敲入小鼠中恢复。慢性乙醇喂养后，在CYP 2 E1基因敲入小鼠（人CYP 2 E1水平较高）中发现肝毒性和氧化/亚硝化应激升高。乙醇喂养的CYP 2 E1基因敲入小鼠似乎是酒精诱导的肝毒性的有效口服模型。野生型、CYP 2 E1敲除和CYP 2 E1敲入小鼠将喂食高脂肪Lieber-DeCarli饮食不同时间，例如1至6周。对照组将与葡萄糖成对饲喂。AIM 1旨在评价乙醇喂养的CYP 2 E1基因敲入小鼠中CYP 2 E1表达升高导致肝毒性的机制，并将包括氧化/亚硝化应激、线粒体功能障碍、CYP 2 E1、iNOS、TNF 1、MAPK和NF：B试验。AIM 2旨在评价CYP 2 E1导致酒精诱导的脂肪肝的途径，包括测定转录因子（如SREBP-1c和PPAR 1）的水平、关键和限速脂肪生成酶和脂解酶的含量;将在饲喂乙醇或葡萄糖的野生型、CYP 2 E1敲除和CYP 2 E1敲入小鼠中测定脂肪酸氧化速率。将相对于乙醇诱导的脂肪变性和肝毒性，确定下游CYP 2 E1效应物变化的时程。将确定CYP 2 E1、iNOS、氧化/亚硝化应激、MAPK、线粒体功能障碍、TNF 1产生的抑制剂对野生型和CYP 2 E1敲入小鼠中乙醇诱导的脂肪变性和肝损伤的影响。我们相信这些实验将提供CYP 2 E1促进乙醇诱导的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，以治疗或最大限度地减少乙醇诱导的肝损伤的进展，这是RFA-AA-006的既定目标。 公共卫生相关性：酒精诱导的肝毒性和脂肪肝的机制尚未完全了解。随着CYP 2 E1敲除小鼠和人源化CYP 2 E1敲入小鼠的新用途，CYP 2 E1在酒精的这些毒性作用中的作用已经被确定。识别酒精/CYP 2 E1作用的下游效应物将在开发针对酒精诱导的肝损伤和脂肪肝的治疗干预方面提供非常丰富的信息。

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.