Thiol Redox Modulation of NF-kB Pathway in Alcoholic Liver Injury.

描述（由申请人提供）：我的长期目标是成为一名独立的研究者，研究肝病（包括酒精性肝病）的发病机制。获得K 01资助将是我成为独立研究员的重要一步，因为它将为我提供机会和时间来克服许多弱点（缺乏酒精研究的初步数据和出版物，以前缺乏对临床重要模型系统的关注，在细胞信号传导，细胞成像方面缺乏经验，和分子生物学技术）在我的背景，成为竞争力的R 01在酒精研究在不久的将来。通过提供我的工资和研究资金，K 01赠款将使我从其他PI的项目中解脱出来，并使我能够真正专注于酒精，并在细胞信号传导，细胞成像和分子生物学方面变得更有经验。我选择在即将到来的时期追求的科学领域是氧化还原生物学，信号转导和肝毒性的界面，使用酒精作为肝损伤的模型。TNF诱导的肝细胞损伤仍然是酒精诱导的肝损伤的中心点。最近的证据表明，从我们的实验室通过谷胱甘肽调制或氧化剂处理氧化还原改变敏感的肝细胞肿瘤坏死因子诱导的细胞凋亡抑制NF-？B转录活性。这表明TNF分泌与介导肝脏损伤的氧化还原改变之间存在联系。要测试的假设是，氧化还原改变引起的酒精敏感肝细胞TNF诱导的细胞凋亡，通过抑制NF-？B依赖性信号通路。该假设将测试与以下具体目标：1）确定如何酒精诱导的细胞氧化还原状态的改变调节NF-？B信号和TNF诱导的原代肝细胞凋亡。酒精诱导的氧化还原变化是否抑制NF-？将探索在培养的原代肝细胞中存在TNF的情况下的B依赖性基因表达。2)确定慢性饮酒对肝脏氧化还原状态的影响，NF-？B氧化还原状态，NF-？B信号通路、N与DNA的结合以及NF-？肝脏中的B依赖性基因表达。NF-？B信号传导和氧化还原状态将在体内慢性酒精治疗后进行探索。本申请的焦点是利用成像技术（例如，共聚焦显微镜，流式细胞术），以研究氧化还原状态和NF-？B信号在同一细胞中同时发生。这些方法将使我能够评估可能的异质性氧化还原状态和NF-？B信号，酒精可能会诱导肝细胞群体。这些研究的结果将为酒精导致肝损伤的机制提供新的见解。 公共卫生相关性：这些研究的结果将为酒精导致肝损伤的机制提供新的见解。这项应用的发现可能揭示新的治疗策略，涉及硫醇抗氧化剂，用于预防或治疗酒精相关疾病。

DESCRIPTION (provided by applicant): It is my long term goal to become an independent investigator and study the pathogenesis of liver disease including alcoholic liver disease. Obtaining a K01 grant would be an important step for me to become an independent researcher since it will provide me an opportunity and time to overcome many weaknesses (lack of preliminary data and publications in alcohol research, previous lack of focus on a clinically important model system, inexperience in cell signaling, cell imaging, and molecular biology techniques) in my background to become competitive for an R01 in alcohol research in the near future. By providing my salary and research funding, the K01 grant will free me from working on other PIs' projects and allow me to really focus on alcohol, as well as become more experienced in cell signaling, cell imaging, and molecular biology. The area of science I have chosen to pursue in the upcoming period is at the interface of redox biology, signal transduction and hepatotoxicity using alcohol as a model of liver injury. TNF-induced damage to hepatocytes remains a central point in alcohol-induced liver damage. Recent evidence from our laboratory suggests that redox alterations through GSH modulation or oxidant treatment sensitize hepatocytes to TNF-induced apoptosis by inhibiting NF-?B transcriptional activity. This suggests a link between TNF secretion and redox alterations in mediating damage to liver. The hypothesis to be tested is that redox alterations caused by alcohol sensitize hepatocytes to TNF-induced apoptosis through inhibition of NF-?B dependent signaling pathways. The hypothesis will be tested with the following specific aims: 1) Determine how alcohol-induced alterations of cellular redox status modulates NF-?B signaling and TNF-induced apoptosis in cultured primary hepatocytes. The question of whether alcohol-induced redox changes inhibit NF-?B dependent gene expression in the presence of TNF in cultured primary hepatocytes will be explored. 2) Determine the effect of chronic alcohol consumption on hepatic redox status, NF-?B redox status, NF-?B signaling pathways, N binding to DNA, and NF-?B dependent gene expression in liver. The relationship between NF-?B signaling and redox status will be explored following chronic alcohol treatment in vivo. A focus of this application is to utilize imaging techniques (e.g., confocal microscopy, flow cytometry) to study redox status and NF-?B signaling simultaneous in the same cell. These methods will allow me to assess possible heterogeneity in redox status and NF-?B signaling that alcohol may induce to the hepatocyte population. The outcome of these studies will provide new insights into the mechanism by which alcohol causes liver damage. Public Health Relevance: The outcome of these studies will provide new insights into the mechanism by which alcohol causes liver damage. The findings of this application may shed light on new therapeutic strategies involving thiol antioxidants for prevention or treatment of alcohol related diseases.