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，研究包括酒精性肝病在内的肝脏疾病的发病机制。获得K01资助将是我成为一名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我背景中的许多弱点（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，细胞成像和分子生物学技术方面缺乏经验），以便在不久的将来成为酒精研究中的R01竞争对手。通过提供我的薪水和研究经费，K01的资助将使我从其他pi的项目中解脱出来，让我真正专注于酒精，并在细胞信号、细胞成像和分子生物学方面变得更有经验。在接下来的一段时间里，我选择的科学领域是在氧化还原生物学、信号转导和肝毒性的界面，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。我们实验室最近的证据表明，通过GSH调节或氧化处理的氧化还原改变通过抑制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。需要验证的假设是，酒精引起的氧化还原改变通过抑制NF-？B依赖的信号通路。该假设将以以下具体目标进行验证：1)确定酒精诱导的细胞氧化还原状态的改变如何调节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-？在培养的原代肝细胞中，TNF存在时B依赖性基因表达将被探索。2)确定慢性饮酒对肝氧化还原状态NF-？B氧化还原态，NF-？B信号通路，N与DNA结合，NF-？B依赖性基因在肝脏中的表达。NF-？B信号和氧化还原状态将探讨慢性酒精治疗后的体内。该应用的一个重点是利用成像技术（如共聚焦显微镜、流式细胞术）来研究氧化还原状态和NF-？B信号同时在同一个细胞中传递。这些方法将允许我评估氧化还原状态和NF-？B信号，酒精可诱导肝细胞群。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。

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.