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

我的长期目标是成为一名独立的研究人员，研究包括酒精性肝病在内的肝脏疾病的发病机制。获得K01基金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R01的竞争对手。通过提供我的工资和研究资金，K01基金将把我从其他PI的项目中解放出来，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，我选择从事的科学领域是氧化还原生物学、信号转导和肝毒性的交界点，使用酒精作为肝脏损伤的模型。肿瘤坏死因子对肝细胞的损伤仍是酒精性肝损伤的中心环节。我们实验室的最新证据表明，通过GSH调节或氧化剂处理的氧化还原改变通过抑制核因子-β转录活性而使肝细胞对肿瘤坏死因子诱导的细胞凋亡敏感。这表明，在介导肝脏损伤的过程中，肿瘤坏死因子的分泌和氧化还原改变之间存在联系。需要检验的假设是酒精引起的氧化还原改变通过抑制依赖于核因子？B的信号通路而使肝细胞对肿瘤坏死因子诱导的细胞凋亡敏感。这一假说将以以下具体目标进行检验：1)确定酒精引起的细胞氧化还原状态的改变如何调节培养的原代肝细胞中的核因子-B信号和肿瘤坏死因子诱导的细胞凋亡。在原代培养的肝细胞中，在有肿瘤坏死因子存在的情况下，酒精引起的氧化还原改变是否抑制了核因子？B依赖基因的表达，这一问题将被探讨。2)研究慢性饮酒对大鼠肝脏氧化还原状态、核因子-β氧化还原状态、核因子-β信号通路、N与DNA结合及肝组织中核因子-β依赖基因表达的影响。在体内慢性酒精治疗后，将探讨核因子-B信号与氧化还原状态的关系。这一应用的一个重点是利用成像技术(例如，共聚焦显微镜、流式细胞术)来研究同一细胞中同时存在的氧化还原状态和核因子？B信号。这些方法将使我能够评估氧化还原状态和核因子-β信号可能的异质性，酒精可能会诱导肝细胞群。这些研究的结果将为酒精导致肝脏损伤的机制提供新的见解。 与公共健康相关：这些研究的结果将为酒精导致肝脏损害的机制提供新的见解。这一应用的发现可能会为使用硫醇抗氧化剂预防或治疗酒精相关疾病的新治疗策略提供帮助。

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.