Role of Nrf2 signaling in modulating ethanol-induced teratogenesis

描述（由申请人提供）：母亲饮酒是已知的智力迟钝的主要非遗传原因。越来越多的证据，包括通过外源性抗氧化剂提供对乙醇致畸的保护，表明活性氧（ROS）在乙醇诱导的致畸中起主要作用。这些发现为实现我们的长期目标提供了基础，该目标是针对乙醇致畸制定更有效的策略；基于内源性抗氧化剂在乙醇暴露胚胎中的上调策略。作为实现这一目标的必要前提，在本应用中，我们建议研究Nrf2信号在调节乙醇诱导的畸胎性中的作用。需要验证的假设是，化学诱导的Nrf2转录激活和随后广泛的解毒和抗氧化蛋白的诱导可以作为内源性保护系统，防止乙醇诱导的畸胎症。为此，本研究的具体目的如下：目的1：阐明乙醇诱导小鼠早期胚胎中Nrf2通路激活的分子机制。在这项工作中，我们将a)确定乙醇对Nrf2 mRNA表达的影响，b)确定乙醇对Nrf2蛋白稳定的影响，c)研究乙醇促进Nrf2核易位、Nrf2- dna结合和抗氧化反应元件（ARE）激活的潜力，以及d)确定Nrf2激活对诱导其下游目标解毒和抗氧化基因的影响。目的2：通过Nrf2敲除小鼠模型，研究Nrf2通路在乙醇诱导的氧化应激和致畸中的保护作用。这将通过确定：a)对Nrf2信号的干扰是否会导致乙醇对早期小鼠胚胎的夸大作用，b) Nrf2-/-小鼠中预期的畸形严重程度的增强是否源于抗氧化反应的缺乏，以及c)抗氧化剂是否可以减轻乙醇诱导的Nrf2-/-小鼠的致畸。目的3：确定母体饮食中Nrf2诱导剂在体内对乙醇诱导的畸胎症的保护作用。从这项研究中获得的见解将阐明Nrf2途径在胚胎发生过程中乙醇损伤后氧化应激调节中的作用。此外，本研究的结果有望产生预防乙醇致畸的创新策略。该项目的主要目标是确定Nrf2信号在调节乙醇诱导的氧化损伤和出生缺陷中的作用。

DESCRIPTION (provided by applicant): Maternal alcohol consumption is the leading known non-genetic cause of mental retardation. Growing evidence, including provision of protection against ethanol's teratogenesis by exogenous antioxidants, suggests a major contribution of reactive oxygen species (ROS) to ethanol-induced teratogenesis. These findings provide a foundation for attaining our long-term goal which is directed toward the development of more effective strategies against ethanol's teratogenesis; strategies based on upregulation of endogenous antioxidants in ethanol-exposed embryos. As a necessary prerequisite to reaching this goal, in this application, we propose to investigate the role of Nrf2 signaling in modulating ethanol-induced teratogenesis. The HYPOTHESIS to be tested is that chemically-induced transcriptional activation of Nrf2 and subsequent induction of a broad spectrum of detoxifying and antioxidant proteins can act as an endogenous protective system against ethanol-induced teratogenesis. To this end, the following specific aims will be addressed: Aim1: To elucidate the molecular mechanisms underlying ethanol-induced activation of the Nrf2 pathway in early mouse embryos. For this work, we will a) determine the effects of ethanol on Nrf2 mRNA expression, b) determine the effects of ethanol on Nrf2 protein stabilization, c) investigate the potential of ethanol to promote Nrf2 nuclear translocation, Nrf2-DNA binding and activation of the antioxidant response element (ARE), and d) determine the effects of Nrf2 activation on the induction of its downstream target detoxifying and antioxidant genes. Aim 2: To investigate the protective role of the Nrf2 pathway in ethanol-induced oxidative stress and teratogenicity using a Nrf2 knockout mouse model. This will be accomplished by determining: a) whether interference with Nrf2 signaling leads to an exaggerated effect by ethanol on early mouse embryos, b) if the expected enhanced severity of dysmorphology in Nrf2-/- mice results from a deficiency in antioxidant response, and c) whether antioxidants can attenuate ethanol-induced teratogenesis in Nrf2-/- mice. Aim 3: To define the role of maternal dietary Nrf2 inducer in conferring in vivo protection against ethanol-induced teratogenesis. The insights gained from this study will elucidate the role of the Nrf2 pathway in modulation of oxidative stress following ethanol insult during embryogenesis. In addition, the results from this study are expected to yield innovative strategies for prevention of ethanol's teratogenesis. PUBLIC HEALTH RELEVANCE The primary goal of this project is to define the role of Nrf2 signaling in modulating ethanol-induced oxidative injury and birth defects.