Targeting Thioredoxin Reductase-1 to Prevent Bronchopulmonary Dysplasia

描述(申请人提供)：在早产儿中，氧气毒性和抗氧化剂缺乏导致支气管肺发育不良(BPD)的发展。BPD每年影响多达10,000名婴儿，代表损伤，包括氧气中毒，对发育中的未成熟肺造成的影响，导致肺发育停滞。尽管临床医生对氧气的暴露有限，但BPD仍然是新生儿发病率的重要原因。通过给予治疗性抗氧化剂来预防BPD的尝试也失败了。因此，需要新的方法来减轻氧气毒性的影响，促进早产儿的正常肺发育。最近的研究表明，核因子E2相关因子2(NRF2)激动剂有可能增强内源性抗氧化剂的表达，保持GSH水平，并防止O2介导的肺损伤。NRF2显著影响新生小鼠的肺泡化和高氧敏感性。硫氧还蛋白还原酶-1(TrxR1)最著名的功能是再生氧化的硫氧还蛋白-1(Trx1)的活性部位。越来越多的证据表明，TrxR1抑制可能是Nrf2激动剂的共同特征。硫代葡萄糖苷(ATG)和金诺芬(AFN)可有效抑制TrxR1，临床上用于治疗类风湿性关节炎。我们以前的体内研究表明，ATG治疗增加了Nrf2的激活，保持了肺GSH水平，并防止了成年小鼠的高氧性肺损伤。我们最近在体外证明，AFN治疗增加了Nrf2介导的抗氧化反应，并增加了GSH水平。ATG和AFN对GSH系统破坏的保护作用消失。我们新的初步数据表明，ATG可以减轻O2介导的新生小鼠肺发育缺陷。总体而言，我们的数据支持一个工作模型，在该模型中，TrxR1抑制剂的保护是通过Nrf2和GSH依赖的机制介导的。TrxR1抑制在诱导Nrf2激活、提高GSH水平和防止O2介导的新生儿肺损伤方面的效用尚未得到测试。因此，此应用程序的目标是 为了利用新生转基因动物、原代和永生化肺上皮细胞培养系统：1)确定肺TrxR1表达改变对Nrf2激活、GSH水平和O2介导的损伤的影响；2)评价TrxR1抑制对实验性O2介导的新生肺损伤的安全性和有效性；以及3)区分Nrf2和GSH对这些影响的贡献。我们的中心假设是，抑制TrxR1将通过Nrf2和GSH依赖的机制来减轻O2介导的新生儿肺损伤。为了验证这一假设，提出了以下特定目标：特定目标1将测试TrxR1基因剂量改变Nrf2激活、GSH水平和O2介导的新生儿肺损伤的假设。为了达到这个目的，TrxR1的表达将在体内和体外进行基因改变。我们将在BPD小鼠模型中确定TrxR1表达改变对Nrf2激活、GSH水平和O2介导的损伤的影响。利用杂合子和纯合子俱乐部(Clara)细胞特异性和肺泡2型(AT2)细胞特异性条件性TrxR1基因敲除小鼠，TrxR1的表达将在体内发生变化。使用杂合子和纯合子TrxR1基因敲除小鼠的原代培养的Club和AT2细胞，TrxR1的表达将在体外发生变化。在小鼠转化的俱乐部细胞(MtCC)和AT2细胞(MLE-12)中，将使用TrxR1特异的siRNA改变TrxR1。特定目标2将验证药物TrxR1抑制通过Nrf2和GSH依赖机制减轻O2介导的新生儿肺损伤的假设。这一目标将使用BPD小鼠模型来评估ATG预防O2介导的肺损伤的安全性和有效性。Nrf2和GSH的作用将通过遗传学和药理学方法确定。无论是1日龄新生仔鼠还是E19龄母鼠，ATG对新生Nrf2+/+和Nrf2-/-仔鼠的TrxR1均有抑制作用。在高氧暴露之前，将使用丁硫氨酸亚磺胺(BSO)来耗尽幼崽的GSH。在体外，AFN处理的Nrf2+/+和Nrf2-/-原代Club和AT2细胞以及AFN处理的Nrf2缺陷的mtCC和MLE-12细胞在有或没有BSO的情况下都会暴露在高氧中。该项目中概述的研究将是直截了当的，考虑到聚集的研究团队的专业知识，将确定TrxR1抑制作为一种减轻O2介导的新生儿肺损伤和肺发育受阻的新方法的安全性和有效性。我们的发现将为未来研究TrxR1抑制剂预防BPD奠定基础，BPD是早产儿发病率的重要和昂贵的原因。

DESCRIPTION (provided by applicant): In premature infants, O2 toxicity and antioxidant deficiencies contribute to the development of bronchopulmonary dysplasia (BPD). Affecting up to 10,000 infants annually, BPD represents the impact of injury, including O2 toxicity, to the immature developing lung resulting in arrested lung development. Though clinicians have limited O2 exposure, BPD remains a significant cause of neonatal morbidity. Attempts to prevent BPD by therapeutic antioxidant administration have also failed. Thus, there exists a need for novel approaches to lessen the impact of O2 toxicity and promote normal lung development in premature infants. Recent studies suggest a potential for nuclear factor E2-related factor 2 (Nrf2) agonists to enhance endogenous antioxidant expression, preserve GSH levels, and prevent O2-mediated lung injury. Nrf2 significantly influences alveolarization and hyperoxic susceptibility in newborn mice. Thioredoxin reductase-1 (TrxR1) is best known for regenerating the active site of oxidized thioredoxin-1 (Trx1). A growing body of evidence suggests that TrxR1 inhibition may be a common feature of Nrf2 agonists. Aurothioglucose (ATG) and auranofin (AFN) potently inhibit TrxR1 and are used clinically to treat rheumatoid arthritis. Our previous studies in vivo demonstrated that ATG treatment increases Nrf2 activation, preserves lung GSH levels, and prevents hyperoxic lung injury in adult mice. We recently demonstrated in vitro that AFN treatment increases Nrf2-mediated antioxidant responses and increases GSH levels. The protective effects of ATG and AFN are lost upon GSH system disruption. Our novel preliminary data indicate that ATG lessens O2-mediated lung developmental deficits in newborn mice. Collectively, our data support a working model in which protection by TrxR1 inhibitors are mediated via Nrf2 and GSH-dependent mechanisms. The utility of TrxR1 inhibition to induce Nrf2 activation, enhance GSH levels, and prevent O2-mediated neonatal lung injury has not been tested. The objective of this application, therefore, is to utilize newborn transgenic animal, primary and immortalized lung epithelial culture systems to: 1) determine the impact of altered lung TrxR1 expression on Nrf2 activation, GSH levels and O2-mediated injury; 2) evaluate the safety and efficacy of TrxR1 inhibition to attenuate experimental O2-mediated neonatal lung injury; and 3) distinguish the contributions of Nrf2 and GSH toward these effects. Our central hypothesis is that TrxR1 inhibition will attenuate O2-mediated neonatal lung injury via Nrf2 and GSH-dependent mechanisms. To test this hypothesis, the following specific aims are proposed: Specific Aim 1 will test the hypothesis that TrxR1 gene dosage alters Nrf2 activation, GSH levels, and O2-mediated neonatal lung injury. In this aim, TrxR1 expression will be genetically altered in vivo and in vitro. We will determine the effect of altered TrxR1 expression on Nrf2 activation, GSH levels, and O2-mediated injury in a BPD mouse model. TrxR1 expression will be altered in vivo using heterozygous and homozygous Club (Clara) cell-specific and alveolar type 2 (AT2) cell-specific conditional TrxR1 knockout mice. TrxR1 expression will be altered in vitro using primary cultured Club and AT2 cells from heterozygous and homozygous TrxR1 knockout mice. TrxR1 will be altered in murine transformed Club cells (mtCC) and AT2 cells (MLE-12) using TrxR1-specific siRNA. Specific Aim 2 will test the hypothesis that pharmacologic TrxR1 inhibition attenuates O2-mediated neonatal lung injury via Nrf2 and GSH-dependent mechanisms. This aim will use a BPD mouse model to evaluate the safety and efficacy of ATG to prevent O2-mediated lung injury. The contributions of Nrf2 and GSH will be determined using genetic and pharmacologic approaches. TrxR1 will be inhibited in newborn Nrf2+/+ and Nrf2-/- pups by ATG administration to either 1 d newborn pups or E19 dams. Buthionine sulfoximine (BSO) will be used to deplete GSH in the pups prior to hyperoxic exposure. In vitro, AFN-treated Nrf2+/+ and Nrf2-/- primary Club and AT2 cells and AFN-treated Nrf2-deficient mtCC and MLE-12 cells will be exposed to hyperoxia in the presence and absence of BSO. The studies outlined in this project, which will be straightforward given the expertise of the assembled research team, will determine the safety and efficacy of TrxR1 inhibition as a novel approach to attenuate O2-mediated neonatal lung injury and arrested lung development. Our findings will establish the rationale for future investigations of TrxR1 inhibitors to prevent BPD, a significant and costly cause of morbidity in preterm infants.