Role of Nrf2 signaling in modulating ethanol-induced teratogenesis

描述（由申请人提供）：母亲饮酒是智力迟钝的主要已知非遗传原因。越来越多的证据，包括提供保护，防止乙醇的致畸作用的外源性抗氧化剂，表明一个重要的贡献，活性氧（ROS）乙醇诱导的致畸作用。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作用的更有效策略的发展;基于乙醇暴露胚胎中内源性抗氧化剂上调的策略。作为实现这一目标的必要前提，在本申请中，我们建议研究Nrf 2信号转导在调节乙醇诱导的致畸作用。有待检验的假设是，化学诱导的Nrf 2转录激活和随后诱导的广谱解毒和抗氧化蛋白可以作为一种内源性保护系统，防止乙醇诱导的致畸作用。为此，将解决以下具体目标：目标1：阐明乙醇诱导的Nrf 2通路在早期小鼠胚胎中激活的分子机制。对于这项工作，我们将a）确定乙醇对Nrf 2 mRNA表达的影响，B）确定乙醇对Nrf 2蛋白稳定性的影响，c）研究乙醇促进Nrf 2核转位、Nrf 2-DNA结合和抗氧化反应元件（ARE）活化的潜力，和d）确定Nrf 2活化对其下游靶解毒和抗氧化基因的诱导的影响。目标二：使用Nrf 2基因敲除小鼠模型研究Nrf 2通路在乙醇诱导的氧化应激和致畸性中的保护作用。这将通过确定：a）Nrf 2信号传导的干扰是否导致乙醇对早期小鼠胚胎的放大效应，B）Nrf 2-/-小鼠中预期的畸形严重程度增加是否由抗氧化剂应答缺陷引起，以及c）抗氧化剂是否可以减弱Nrf 2-/-小鼠中乙醇诱导的致畸作用。目的3：确定母体膳食Nrf 2诱导剂在体内对乙醇诱导的致畸作用的保护作用。从这项研究中获得的见解将阐明Nrf 2途径在胚胎发育过程中乙醇损伤后氧化应激的调节作用。此外，这项研究的结果有望产生预防乙醇致畸作用的创新策略。本项目的主要目标是确定Nrf 2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.