Nrf2 as a Master Regulator in Liver Disease Prevention and Therapy

描述（由申请人提供）：目前治疗肝脏疾病的治疗干预措施非常有限；确定治疗靶点是开发治疗肝脏疾病新药的先决条件。氧化应激和炎症在肝脏疾病的发病机制中起关键作用。核因子2相关因子2 （Nrf2）是一种在肝细胞和免疫细胞中均表达的转录因子，是抗氧化和抗炎反应的关键因子。在静止的肝细胞中，Nrf2通过kelch样ech相关蛋白1 （Keap1）被隔离在细胞质中；在keap1敲低小鼠中，Nrf2核易位增加，导致Nrf2靶基因激活。Nrf2在细胞保护中的重要性已被认识。然而，Nrf2在肝脏病理生理中的作用最近才引起人们的关注。我们的长期目标是确定一种新的药物靶点，并开发一种治疗/预防肝脏疾病的策略。本R01基金提案的目的是深入研究Nrf2在肝脏病理生理保护中的作用，并了解其潜在的分子机制。基于我们强有力的初步数据，我们的中心假设是Nrf2是肝脏保护的主要调节因子，通过其在肝脏多种细胞类型的生物转化/处置、抗氧化、组织修复和抗炎症中的多方面作用。目的1将利用ChIP-Sequencing确定Nrf2调控的直接靶点，并在keap1敲低小鼠中确定Nrf2依赖的mRNA诱导。目的2将验证Nrf2激活对肝脏疾病的化学、饮食和遗传模型的保护。我们将使用3种具有不同Nrf2活性的小鼠遗传模型，即Nrf2-null、野生型和Nrf2-增强型小鼠，在13种不同的模型中测试肝脏疾病的易感性。Aim 3将与美国治疗学、发现和开发办公室合作，通过高通量筛选开发新型特异性Nrf2激活剂先导候选物。我们提出的研究是新颖的，因为它利用3个具有不同Nrf2活性的小鼠品系来研究Nrf2缺乏和Nrf2激活对通过不同毒性机制诱导的肝脏疾病的相反作用。本研究利用芯片测序、多重mRNA分析和核易位高通量筛选等创新技术来检测这三个目标。我们的研究具有重要意义，因为我们的实验结果将为Nrf2缺乏和Nrf2激活在肝脏病理生理中的重要性及其潜在的分子机制提供坚实的证据，为开发Nrf2激活剂作为治疗/预防各种肝脏疾病（如化学性肝损伤、炎症性肝病、脂肪性肝炎和肝纤维化/肝硬化）的新药奠定科学基础。公共卫生相关性：肝病是一种常见病，但与大多数其他疾病相比，治疗肝功能衰竭的药物很少。我们假设Nrf2，一个重要的转录因子，是一个药物靶点，可以用来预防和治疗广泛的肝脏疾病。这项研究将确定该药物靶点是否对广泛的肝脏疾病动物模型有效。

DESCRIPTION (provided by applicant): Current therapeutic interventions for treating liver diseases are very limited; identification of a therapeutic target is a prerequisite for developing new drugs to treat liver diseases. Oxidative stress and inflammation play key roles in the pathogenesis of liver diseases. Nuclear factor 2-related factor 2 (Nrf2), a transcription factor expressed in both hepatocytes and immune cells, is a key factor in anti-oxidative and anti-inflammatory responses. In quiescent hepatocytes, Nrf2 is sequestered in cytosol by kelch-like ECH-associated protein 1 (Keap1); in Keap1-knockdown mice, nuclear translocation of Nrf2 increases, resulting in activation of Nrf2 target genes. The importance of Nrf2 in cytoprotection is recognized. Nevertheless, the role of Nrf2 in liver pathophysiology has just recently gained attention. Our long-term goal is to identify a novel drug target and develop a therapeutic/preventive strategy for liver diseases. The objective of this R01 grant proposal is to investigate in-depth the role of Nrf2 in protecting against liver pathophysiology and understand its underlying molecular mechanisms. Based upon our strong preliminary data, our central hypothesis is that Nrf2 is a master regulator of liver protection through its multifaceted roles in biotransformation/disposition, antioxidation, tissue- repair, and anti-inflammation in multiple cell types in liver. Aim 1 will identify direct targets of Nrf2 regulation using ChIP-Sequencing and determine Nrf2-dependent mRNA induction in the Keap1-knockdown mouse. Aim 2 will test the hypothesis that Nrf2 activation protects against chemical, dietary, and genetic models of liver disease. We will use 3 genetic models of mice with differential Nrf2 activity, namely Nrf2-null, wild-type, and Nrf2-enhanced mice, to test liver disease susceptibility in 13 different models. Aim 3 will develop novel and specific Nrf2 activator lead candidates by high throughput screening in collaboration with the Office of Therapeutics, Discovery, and Development. Our proposed study is novel, because it utilizes 3 mouse lines with different Nrf2 activities to investigate the opposing effects of Nrf2 deficiency and Nrf2 activation on liver diseases induced via diverse mechanisms of toxicity. This proposal utilizes innovative techniques including ChIP-sequencing, multiplex mRNA analysis, and nuclear translocation high throughput screening in testing the 3 aims. Our proposed study is significant, because results from our experiments will provide solid evidence regarding the importance of Nrf2 deficiency and Nrf2 activation in liver pathophysiology and its underlying molecular mechanisms, setting the scientific foundation for developing Nrf2 activators as novel drugs to treat/prevent various liver diseases, such as chemical-induced liver injury, inflammatory liver diseases, steatohepatitis, and liver fibrosis/cirrhosis. PUBLIC HEALTH RELEVANCE: Liver disease is a common ailment, however in contrast to most other diseases, there are very few drugs for liver failure. We hypothesize that Nrf2, an essential transcription factor, is a drug target that can be used to prevent and treat a wide-range of liver ailments. This study will determine whether this drug target will be effective against a wide-spectrum of animal models of liver disease.