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

人源化 CYP2E1 敲入小鼠中酒精诱导的脂肪肝和损伤。

基本信息

批准号：
8127679
负责人：
ARTHUR I CEDERBAUM
金额：
$ 34.27万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-15 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8127679
关键词：
3-nitrotyrosine 4 hydroxynonenal ADD-1 protein Acetylcysteine Alcoholic Fatty Liver Alcoholic Liver Diseases Alcohols Antibodies Biological Assay CYP2E1 gene Chlormethiazole Chronic Cyclosporine Data Development Diet Enzymes Ethanol Ethanol toxicity Event Family member Fatty Liver Fatty acid glycerol esters Glucose Goals Hepatocyte Hepatotoxicity Histopathology Homocysteine Homocystine Human Hydrogen Peroxide In Situ Nick-End Labeling Injury Knock-out Knockout Mice Lipid Peroxidation Liver MAP Kinase Gene MAPK14 gene MAPK8 gene Malondialdehyde Mediator of activation protein Membrane Potentials Mitochondria Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinases Modeling Molecular Mus Oral Oxidants Oxidative Stress Oxygen Consumption Pathway interactions Pentoxifylline Permeability Peroxisome Proliferator-Activated Receptors Peroxonitrite Play Principal Investigator Production Proteins Relative (related person)Research Role S-Adenosylmethionine SB 203580 SP600125 Serum Stress Superoxides TNF gene Therapeutic Intervention Time Toxic Actions Toxic effect Transaminases adduct alcohol effect caspase-3 cell injury cytochrome c cytokine design endoplasmic reticulum stress fatty acid oxidation feeding gadolinium chloride in vivo inhibitor/antagonist insight mitochondrial dysfunction mitochondrial membrane nitrosative stress novel novel therapeutics prevent programs public health relevance research study transcription factor

项目摘要

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.

描述（由申请人提供）：酒精引起细胞损伤的机制仍不清楚。大量研究关注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。许多途径被认为在乙醇如何诱导“氧化应激”方面发挥着关键作用。我们假设体内 CYP2E1 诱导导致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。我们认为过氧亚硝酸盐等氧化剂、JNK 和/或 p38 MAPK 等 MAP 激酶的激活、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，CYP2E1 的此类下游介质与乙醇诱导的 TNF1、同型半胱氨酸和 ER 应激升高相关，可激活脂肪生成转录因子和酶，同时减少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，乙醇诱导的脂肪肝在 CYP2E1 敲除小鼠中减弱，而在人源化 CYP2E1 敲除小鼠中恢复。在长期摄入乙醇后，人类 CYP2E1 水平较高的 CYP2E1 敲入小鼠中发现肝毒性和氧化/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 敲除和 CYP2E1 敲除小鼠将被喂食高脂肪 Lieber-DeCarli 饮食不同的时间，例如1至6周。对照将与葡萄糖配对喂养。 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，并将包括氧化/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导致酒精性脂肪肝的途径，包括检测SREBP-1c和PPAR1等转录因子的水平、关键和限速脂肪生成酶和脂肪分解酶的含量；将在喂食乙醇或葡萄糖的野生型、CYP2E1 敲除和 CYP2E1 敲除小鼠中测定脂肪酸氧化速率。下游 CYP2E1 效应器变化的时间过程将根据乙醇诱导的脂肪变性和肝毒性的时间过程来确定。将确定 CYP2E1、iNOS、氧化/亚硝化应激、MAPK、线粒体功能障碍、TNF1 产生等抑制剂对野生型和 CYP2E1 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 CYP2E1 促进乙醇引起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来治疗或最大程度地减少乙醇引起的肝损伤的进展，这是 RFA-AA-006 的既定目标。公共卫生相关性：酒精引起的肝毒性和脂肪肝的机制尚不完全清楚。随着 CYP2E1 敲除小鼠和人源化 CYP2E1 敲除小鼠的新用途，CYP2E1 在酒精毒性作用中的作用已被明确。识别酒精/CYP2E1 作用的下游效应器对于制定针对酒精性肝损伤和脂肪肝的治疗干预措施非常有用。