ROLE OF NRF2 IN NITRERGIC MEDIAITED STOMACH MOTILITY

摘要 这项提议的长期目标是开发对糖尿病胃轻瘫的干预措施，这是一种 胃排空延迟主要影响女性。胃动力障碍是一种常见的 糖尿病的并发症，并可能涉及致病的氧化应激。在啮齿动物模型中，还原的一氧化氮 (NO)由于抑制神经元型一氧化氮合酶(NNOS)和增加反应性而获得 氧物种(ROS)已被证明在胃运动中起有害作用。转录因子 核因子(红系衍生的-2)样2(NRF2)调节第二相抗氧化剂和 解毒基因。NRF2蛋白的稳定性主要由两个E3泛素连接酶适配器调节： CUL3/Keap1和CUL1/SCF/TrCP。GSK3/TrCP轴独立于CUL3/Keap1影响核 排除和失活NRF2。初步研究表明，NRF2及其靶基因的表达， 在糖尿病的啮齿动物模型中，GCLC和GCLM受到抑制，而氧化应激则升高。我们有 证明了NRF2(NFe2/2-/-)的缺失导致了四氢生物蝶呤(BH4)水平的降低，BH4是一种关键的 NNOS二聚化和酶活性的辅助因素)，这导致氧化应激，抑制nNOS， NO水平降低与胃硝酸能神经元(神经元合成nNOS)运动和胃延迟 排空与年龄匹配的野生型小鼠相比。这些数据支持NRF2基因缺失的观点 糖尿病的表达损害了抗氧化基因的表达，从而解除了对NO合成的调控，从而 促进胃瘫的发展。我们提供的证据表明性激素雌二醇-17β(E2) 糖尿病发病过程中介导的NRF2/nNOS表达和胃动力受损。此外，我们还有 糖原合成酶-3β(GSK-3)β或E_2对女性胃NO合成的调节作用 组织。这一数据表明，高血糖时NRF2表达的丧失是一种激活的结果 GSK-3β/CUL1/SCF/TrCP/RBX1轴。我们的中心假设是氮能介导的胃动力 受NRF2调控。特定目标1将检验NRF2在高血糖期间表达缺失的假设 是GSK-3β/CUL1/SCF/TrCP/Rbx1轴激活的结果。《特定目标2》将测试 假设恢复NRF2和NRF2调节的抗氧化酶表达将导致正常 胃动力和胃排空；以及特定的目标3.将检验NRF2和nNOS的假设 女性性激素、17-雌二醇(E_2)、孕酮和/或其受体对介导的胃运动有调节作用 糖尿病动物的胃感受器。这些研究将有助于阐明NRF2介导的机制。 从而提高我们对胃轻瘫的病理生理学的认识。 概述的这项研究可能会确定糖尿病引起的胃动力障碍的新治疗方案， 尤其是在女性身上。

ABSTRACT The long term goal of this proposal is to develop interventions for diabetic gastroparesis, a syndrome of delayed gastric emptying predominantly affecting women. Gastric motility dysfunction is a common complication of diabetes and can involve pathogenic oxidative stress. In rodent models, reduced nitric oxide (NO) availability as a result of inhibition of neuronal nitric oxide synthase (nNOS) and increased reactive oxygen species (ROS) has been shown to play a detrimental role in gastric motility. The transcription factor nuclear factor (erythroid-derived-2)-like 2 (NRF2) regulates the expression of Phase II antioxidant and detoxification genes. NRF2 protein stability is mainly regulated by two E3 ubiquitin ligase adaptors: CUL3/Keap1 and CUL1/SCF/TrCP. Independent of CUL3/Keap1, the GSK3/TrCP axis influences the nuclear exclusion and inactivation of NRF2. Preliminary studies indicate that expression of NRF2 and its target genes, Gclc and Gclm, are suppressed while oxidative stress is elevated in a rodent model of diabetes. We have demonstrated that loss of NRF2 (Nfe2/2-/-) resulted in decreased levels of tetrahydrobiopterin (BH4, a critical cofactor for nNOS dimerization and enzyme activity) and that this led to oxidative stress, inhibition of nNOS, reduced NO levels & gastric nitrergic neuron (neurons synthesizes nNOS)motility, and delayed gastric emptying compared to age-matched wild-type mice. These data support the notion that loss of NRF2 expression in diabetes impairs antioxidant gene expression, which deregulates NO synthesis, thereby contributing to the development of gastroparesis. We provide evidence that sex hormone estradiol-17β (E2) mediated NRF2/nNOS expression and gastric motility is impaired in the onset of diabetes. In addition, we have shown that glycogen synthase kinase 3beta (GSK-3β) or E2 regulate the synthesis of NO in female gastric tissue. This data suggest that loss of NRF2 expression during hyperglycemia is a consequence of activation of a GSK-3β/CUL1/SCF/TrCP/RBX1 axis . Our central hypothesis is that nitrergic mediated gastric motility is regulated by NRF2. Specific Aim 1 will test the hypothesis that loss of NRF2 expression during hyperglycemia is a consequence of activation of a GSK-3β/CUL1/SCF/TrCP/RBX1 axis. Specific Aim 2 will test the hypothesis that restoration of NRF2 and NRF2-regulated antioxidant enzyme expression will result in normal gastric motility and gastric emptying; and Specific Aim 3. will test the hypothesis that NRF2 and nNOS mediated gastric motility are regulated by female sex hormones; 17-estradiol (E2), progesterone and/or their gastric receptors in diabetic animals. These studies will shed light on the mechanisms of NRF2-mediated gastric nNOS motility function and thereby enhance our understanding of the pathophysiology of gastroparesis. The research outlined will potentially identify novel treatment options for diabetes-induced gastric dysmotility, particularly in females.