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

基本信息

批准号：
9322106
负责人：
Trent Tipple
金额：
$ 3.26万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-16 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9322106
关键词：
Active Sites Adult Affect Agonist Alveolar Antioxidants Antithymoglobulin Attenuated Auranofin Aurothioglucose Bronchopulmonary Dysplasia Buthionine Sulfoximine Cells Clara cell Clinical 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 Oxides Oxidoreductase Oxygen Pharmacology Predisposition Premature Infant Publishing Research Rheumatoid Arthritis Safety Small Interfering RNA System TXN gene Testing Therapeutic Toxic effect Transgenic Animals base cost 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 safety testing thioredoxin reductase 1

Active Sites Adult Affect Agonist Alveolar Antioxidants Antithymoglobulin Attenuated Auranofin Aurothioglucose Bronchopulmonary Dysplasia Buthionine Sulfoximine Cells Clara cell Clinical 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 Oxides Oxidoreductase Oxygen Pharmacology Predisposition Premature Infant Publishing Research Rheumatoid Arthritis Safety Small Interfering RNA System TXN gene Testing Therapeutic Toxic effect Transgenic Animals base cost 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 safety testing thioredoxin reductase 1

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项目摘要

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（由申请人提供）：在早产婴儿中，O2毒性和抗氧化剂缺乏症有助于开发支气管肺发育不良（BPD）。 BPD每年最多影响10,000名婴儿，代表了包括O2毒性在内的损伤影响，导致肺部发育不成熟，导致肺部发育。尽管临床医生的O2暴露有限，但BPD仍然是新生儿发病率的重要原因。通过治疗性抗氧化剂给药预防BPD的尝试也失败了。因此，存在一种新的方法来减轻O2毒性的影响并促进早产婴儿的正常肺发育。最近的研究表明，与核因子E2相关因子2（NRF2）激动剂的潜力可以增强内源性抗氧化剂表达，保持GSH水平并防止O2介导的肺损伤。 NRF2在新生小鼠中显着影响肺泡化和高氧化敏感性。硫氧还蛋白还原酶1（TRXR1）以再生氧化硫氧还蛋白-1（TRX1）的活性位点而闻名。越来越多的证据表明，TRXR1抑制可能是NRF2激动剂的共同特征。 Aurothioglucose（ATG）和Auranofin（AFN）有效抑制TRXR1，并在临床上用于治疗类风湿关节炎。我们先前在体内的研究表明，ATG治疗会增加NRF2激活，保留肺GSH水平并防止成年小鼠的高氧肺损伤。我们最近在体外证明了AFN治疗会增加NRF2介导的抗氧化剂反应并增加GSH水平。 ATG和AFN的保护作用在GSH系统中断时丢失了。我们的新型初步数据表明，ATG减少了新生小鼠中O2介导的肺发育缺陷。总体而言，我们的数据支持了一个工作模型，在该模型中，通过NRF2和GSH依赖性机制介导了TRXR1抑制剂的保护。 TRXR1抑制诱导NRF2激活，增强GSH水平并预防O2介导的新生儿肺损伤的实用性尚未进行测试。因此，此应用的目的是要利用新生儿转基因动物，原发性和永生的肺上皮培养系统：1）确定改变肺TRXR1表达对NRF2激活，GSH水平和O2介导的损伤的影响； 2）评估TRXR1抑制的安全性和功效，以减轻实验性O2介导的新生儿肺损伤； 3）区分NRF2和GSH对这些影响的贡献。我们的中心假设是TRXR1抑制作用将通过NRF2和GSH依赖性机制减轻O2介导的新生儿肺损伤。为了检验该假设，提出了以下特定目的：特定目标1将检验以下假设：TRXR1基因剂量会改变NRF2激活，GSH水平和O2介导的新生儿肺损伤。在此目标中，TRXR1表达将在体内和体外遗传改变。我们将确定在BPD小鼠模型中改变TRXR1表达对NRF2激活，GSH水平和O2介导的损伤的影响。使用杂合和纯合子俱乐部（CLARA）细胞特异性和肺泡2（AT2）细胞特异性条件TRXR1敲除小鼠，将在体内改变TRXR1表达。使用杂合和纯合TRXR1基因敲除小鼠的原代培养俱乐部和AT2细胞，将在体外改变TRXR1表达。使用TRXR1特异性siRNA，将在鼠转化的俱乐部细胞（MTCC）和AT2细胞（MLE-12）中改变TRXR1。具体目标2将检验以下假设：药理TRXR1抑制作用可通过NRF2和GSH依赖性机制减轻O2介导的新生儿肺损伤。该目标将使用BPD小鼠模型来评估ATG的安全性和功效，以防止O2介导的肺损伤。 NRF2和GSH的贡献将使用遗传和药理方法确定。 TRXR1将通过ATG给药抑制新生儿NRF2+/+和NRF2 - / - 幼崽在1 D新生幼犬或E19大坝中。丁香硫胺（BSO）将用于在暴露高氧之前耗尽幼崽的GSH。在体外，AFN处理的NRF2+/+和NRF2 - / - 原发俱乐部和AT2细胞以及AFN处理的NRF2缺乏的MTCC和MLE-12细胞将在存在和不存在BSO的情况下暴露于高氧。鉴于组装研究团队的专业知识，该项目中概述的研究将是直接的，它将确定TRXR1抑制作用的安全性和功效，是一种减弱O2介导的新生儿肺损伤并阻止肺发育的新方法。我们的发现将确定对TRXR1抑制剂的未来研究的基本原理，以防止BPD，这是早产儿的发病率的重大且昂贵的原因。