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）：酒精会导致细胞损伤的机制尚不清楚。大量研究重点的主要机制是脂质过氧化和氧化应激在酒精毒性中的作用。已经建议许多途径在乙醇如何诱导“氧化应激”方面发挥关键作用。我们假设体内CYP2E1诱导中增加的氧化应激增加了肝细胞对慢性乙醇诱导的肝毒性和脂肪变性的敏感性。我们提出，氧化剂，例如过氧亚硝酸盐，诸如JNK和/或p38 MAPK等MAP激酶的激活，NF-：B的激活下降和生存因子的合成以及线粒体功能障碍是CYP2E1的下游介体的CYP2E1促进了HEPATOTOTOXICITIC。我们提出，来自CYP2E1的下游介质与乙醇诱导的TNF1升高，同型半胱氨酸和ER应力相关，同时激活脂肪性转录因子和酶，同时降低脂肪溶解转录因子和酶的激活，从而导致脂肪脂裂。支持这些假设的初步数据是乙醇诱导的脂肪肝在CYP2E1基因敲除小鼠中钝化并在人源化CYP2E1敲除小鼠中恢复的发现。在慢性乙醇喂养后，肝毒性和氧化/亚硝化应激升高在具有高水平的人CYP2E1的CYP2E1敲击蛋白中发现。乙醇喂养的CYP2E1敲击蛋白小鼠似乎是酒精诱导的肝毒性的有效口服模型。野生型，CYP2E1敲除和CYP2E1敲除小鼠将在不同的时间喂养高脂lieber-decarli饮食，例如1至6周。控件将与葡萄糖配对。 AIM1旨在评估机制，通过这种机制，CYP2E1的表达升高会在乙醇喂养的CYP2E1敲蛋白小鼠中引起肝毒性，并将包括氧化/硝化应力的测定，线粒体功能障碍，CYP2E1，CYP2E1，CYP2E1，INOS，INOS，TNF1，MAPK，MAPK和NF：b：b：b：b：b。 AIM 2旨在评估CYP2E1对酒精诱导的脂肪肝的贡献的途径，包括转录因子水平（例如SREBP-1C和PPAR1）的测定，关键和限制脂肪生成酶和脂肪性酶的含量；脂肪酸氧化的速率将在野生型，CYP2E1基因敲除和喂食乙醇或葡萄糖的CYP2E1敲除小鼠中确定。下游CYP2E1效应子的变化时间过程将相对于乙醇诱导的脂肪变性和肝毒性的时间。 CYP2E1，iNOS，氧化/亚硝化应激的抑制剂，MAPK，线粒体功能障碍，TNF1产生对乙醇诱导的脂肪变性和野生型和CYP2E1敲击蛋白小鼠中乙醇诱导的脂肪性损伤的影响。我们认为，这些实验将提供分子机制，通过这些机制CYP2E1促进乙醇诱导的肝损伤和脂肪肝，并可能进一步发展新的治疗设计，以治疗或最小化乙醇诱导的肝损伤的进展，这是RFA-AA-006的指定目标。公共卫生相关性：尚未完全了解饮酒引起的肝毒性和脂肪肝的机制。随着CYP2E1基因敲除小鼠和人源化CYP2E1敲除小鼠的新颖使用，CYP2E1在这些酒精的这些有毒作用中的作用已被定义。鉴定酒精/CYP2E1作用的下游效应因子在开发针对酒精引起的肝损伤和脂肪肝的治疗干预方面非常有用。