Role of CYP2E1 in TNFa-Ethanol -induced Liver Injury

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”中发挥关键作用，包括线粒体功能障碍、MAP激酶的激活、乙醇诱导的细胞因子形成的增加，特别是TNF1和CYP2E1的诱导。这些途径并不是相互排斥的，然而，它们之间的关联和相互作用，特别是在体内条件下，仍有待评估和澄清。我们假设体内CYP2E1诱导的氧化应激增加使肝细胞对tnf1诱导的肝毒性敏感。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-kB的失活和线粒体功能障碍是CYP2E1- TNF1增强肝毒性的下游介质。我们还假设CYP2E1的诱导在体内酒精治疗增强TNF1肝毒性的机制中起着核心作用。由于CYP2E1和TNF1被认为是酒精性肝损伤发展的关键危险因素，因此评估它们在体内可能的相互作用和增强作用非常重要。AIM 1将通过诱导CYP2E1来评估tnf1对肝损伤的增强作用。将TNF1或生理盐水给予具有基础水平CYP2E1的盐水处理小鼠，或具有高水平CYP2E1的吡唑处理小鼠，以及没有CYP2E1的CYP2E1敲除小鼠。给予不同浓度的tnf - 1，并在tnf - 1刺激后的不同时间杀死小鼠。我们将进行以下检测：肝毒性、氧化/亚硝化应激、NF-kB的激活、MAP激酶的激活、线粒体功能障碍。AIM 2将评估CYP2E1在乙醇诱导的TNF1肝损伤中的作用，并确定CYP2E1-乙醇- TNF1相互作用的下游靶点。急性乙醇模型评估的是酒精摄入后的TNF1或生理盐水刺激的暴饮模型。对于慢性乙醇喂养，小鼠将被喂食对照或乙醇Lieber-DeCarli饮食2、4或6周，然后用TNF1（或生理盐水）刺激。将进行类似上述的化验。为了证明CYP2E1在吡唑和乙醇模型中对损伤的增强起作用，我们将使用CYP2E1敲除小鼠和人源化CYP2E1敲除小鼠。公共卫生相关性：我们相信这些实验将有助于阐明两个被认为是酒精性肝损伤重要的独立危险因素，CYP2E1和TNF1相互作用如何促进这种肝损伤，将为CYP2E1在急性和慢性酒精/ TNF1增强肝损伤中的作用提供明确的证据，并有助于确定下游介质，信号通路和CYP2E1-乙醇- TNF1相互作用的靶点。后者可能具有潜在的治疗价值。小灵通398/2590 （Rev. 09/04，重新发布4/2006）页延续格式页

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", including mitochondrial dysfunction, activation of MAP kinases, ethanol-induced increases in cytokine formation, especially TNF1 and induction of CYP2E1. These pathways are not exclusive of each other, however, associations and interactions between them, especially under in vivo conditions, remain to be evaluated and clarified. We hypothesize that increased oxidative stress from CYP2E1 induction in vivo sensitizes hepatocytes to TNF1-induced hepatotoxicity. We propose that oxidants such as peroxynitrite, activation of MAP kinases such as JNK and/or P38 MAPK, inactivation of NF-kB and mitochondrial dysfunction are downstream mediators of the CYP2E1- TNF1 potentiated hepatotoxicity. We also hypothesize that induction of CYP2E1 plays a central role in mechanisms by which in vivo treatment with alcohol potentiates TNF1 hepatotoxicity. Since CYP2E1 and TNF1 are considered key risk factors in the development of alcoholic liver injury, possible interactions and potentiation of their actions in vivo is important to evaluate. AIM 1 will evaluate the potentiation of TNF1-induced liver injury by induction of CYP2E1. TNF1 or saline will be administrated to either saline-treated mice with basal levels of CYP2E1, or pyrazole-treated mice with high levels of CYP2E1, and to CYP2E1-knockout mice with no CYP2E1. Various concentrations of TNF1 will be given and mice killed at various times after TNF1 challenge. We will assay for the following: Hepatotoxicity, oxidative/nitrosative stress, activation of NF-kB, activation of MAP kinases, mitochondrial dysfunction. AIM 2 will evaluate the role of CYP2E1 in the potentiation of TNF1-induced liver injury by ethanol and to identify downstream targets for the CYP2E1-ethanol- TNF1 interactions. A binge model of alcohol intake followed by TNF1 or saline challenge will be the acute ethanol model evaluated. For chronic ethanol feeding, mice will be fed a control or an ethanol Lieber-DeCarli diet for 2, 4 or 6 weeks, followed by challenge with TNF1 (or saline). Assays similar to the above will be carried out. To prove that CYP2E1 is responsible for the potentiated injury in the pyrazole and ethanol models, CYP2E1 knockout mice and humanized CYP2E1 knockin mice will be used. PUBLIC HEALTH RELEVANCE: We believe that these experiments will help to clarify how two independent risk factors believed to be important for alcohol-induced liver injury, CYP2E1 and TNF1 interact to promote this liver injury, will provide clear evidence for a role for CYP2E1 in acute and chronic alcohol/ TNF1 potentiated liver injury, and help to identify downstream mediators, signaling pathways and targets of the CYP2E1-ethanol- TNF1 interactions. The latter may have potential therapeutic value. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page