Targeting Thioredoxin Reductase-1 to Prevent Bronchopulmonary Dysplasia

靶向硫氧还蛋白还原酶 1 预防支气管肺发育不良

• 批准号: 8695635
• 负责人: Trent Tipple
• 金额: $ 36.63万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695635
• 关键词: Active Sites; Adult; Affect; Agonist; Alveolar; Alveolar Cell; Antioxidants; Attenuated; Auranofin; Aurothioglucose; Bronchopulmonary Dysplasia; Buthionine Sulfoximine; Cells; Clara cell; Data; Development; Epithelial; Exposure to; Funding; Future; Gene Dosage; Genetic; Glutathione; Goals; Health; Human; Hyperoxia; In Vitro; Infant; Injury; Innovative Therapy; Investigation; Knockout Mice; Lung; Lung diseases; Mediating; Modeling; Morbidity - disease rate; Mus; Natural regeneration; Newborn Infant; Nuclear; Oxygen; Predisposition; Premature Infant; Publishing; Research; Rheumatoid Arthritis; Safety; Small Interfering RNA; System; Testing; Therapeutic; Thioredoxin; Toxic effect; Transgenic Animals; Work; base; efficacy testing; in vivo; inhibitor/antagonist; lung development; lung injury; lung maturation; mouse model; neonatal lung injury; neonatal morbidity; novel; novel strategies; prevent; protective effect; public health relevance; pup; response; thioredoxin reductase 1

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.

描述（由申请方提供）：在早产儿中，O2毒性和抗氧化剂缺乏会导致支气管肺发育不良（BPD）的发生。BPD每年影响多达10，000名婴儿，代表损伤（包括O2毒性）对未成熟发育的肺的影响，导致肺发育停滞。虽然临床医生有有限的氧气暴露，BPD仍然是新生儿发病的一个重要原因。通过抗氧化剂治疗来预防BPD的尝试也失败了。因此，需要新的方法来减轻O2毒性的影响并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（Nrf 2）激动剂的潜力，以提高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。nrf 2显著影响新生小鼠肺泡形成和高氧易感性。硫氧还蛋白还原酶-1（TrxR 1）最为人所知的是再生氧化硫氧还蛋白-1（Trx 1）的活性位点。越来越多的证据表明，TrxR 1抑制可能是Nrf 2激动剂的共同特征。金硫葡萄糖（ATG）和金诺芬（AFN）有效抑制TrxR 1，并在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Nrf 2激活，保持肺GSH水平，并防止成年小鼠高氧肺损伤。我们最近在体外证明，AFN治疗增加Nrf 2介导的抗氧化反应，并增加GSH水平。当GSH系统被破坏时，ATG和AFN的保护作用丧失。我们的新的初步数据表明，ATG减轻新生小鼠O2介导的肺发育缺陷。总的来说，我们的数据支持一个工作模型，其中TrxR 1抑制剂的保护作用是通过Nrf 2和GSH依赖性机制介导的。尚未测试TrxR 1抑制诱导Nrf 2活化、提高GSH水平和预防O2介导的新生儿肺损伤的效用。因此，本申请的目的是 利用新生转基因动物、原代和永生化肺上皮培养系统：1）确定改变的肺TrxR 1表达对Nrf 2活化、GSH水平和O2介导的损伤的影响; 2）评价TrxR 1抑制减轻实验性O2介导的新生儿肺损伤的安全性和功效; 3）区分Nrf 2和GSH对这些效应的贡献。我们的中心假设是TrxR 1抑制将通过Nrf 2和GSH依赖性机制减弱O2介导的新生儿肺损伤。为了检验这一假设，提出了以下具体目标：具体目标1将检验TrxR 1基因剂量改变Nrf 2激活、GSH水平和O2介导的新生儿肺损伤的假设。在这个目标中，TrxR 1的表达将在体内和体外进行遗传改变。我们将在BPD小鼠模型中确定TrxR 1表达改变对Nrf 2激活、GSH水平和O2介导的损伤的影响。使用杂合和纯合Club（Clara）细胞特异性和肺泡2型（AT 2）细胞特异性条件性TrxR 1敲除小鼠，将在体内改变TrxR 1表达。使用来自杂合和纯合TrxR 1敲除小鼠的原代培养的Club和AT 2细胞，在体外改变TrxR 1表达。使用TrxR 1特异性siRNA，将在鼠转化的Club细胞（mtCC）和AT 2细胞（MLE-12）中改变TrxR 1。具体目标2将检验以下假设：药理学TrxR 1抑制通过Nrf 2和GSH依赖性机制减弱O2介导的新生儿肺损伤。该目的将使用BPD小鼠模型来评价ATG预防O2介导的肺损伤的安全性和有效性。将使用遗传学和药理学方法确定Nrf 2和GSH的贡献。通过对1 d新生幼崽或E19母鼠给予ATG，新生Nrf 2 +/+和Nrf 2-/-幼崽中的TrxR 1将受到抑制。在高氧暴露前，将使用丁硫酰亚胺亚砜（BSO）消耗幼仔中的GSH。在体外，在存在和不存在BSO的情况下，将AFN处理的Nrf 2 +/+和Nrf 2-/-原代Club和AT 2细胞以及AFN处理的Nrf 2缺陷型mtCC和MLE-12细胞暴露于高氧。该项目中概述的研究将直接考虑到研究团队的专业知识，将确定TrxR 1抑制作为减轻O2介导的新生儿肺损伤和肺发育停滞的新方法的安全性和有效性。我们的研究结果将为未来研究TrxR 1抑制剂预防BPD奠定基础，BPD是早产儿发病的重要且昂贵的原因。