Targeting Thioredoxin Reductase-1 to Prevent Bronchopulmonary Dysplasia

描述（由申请人提供）：在早产儿中，氧中毒和抗氧化剂缺乏有助于支气管肺发育不良（BPD）的发展。BPD每年影响多达10,000名婴儿，它代表了损伤的影响，包括O2毒性，对未成熟的肺发育导致肺发育停滞。虽然临床医生的氧气暴露有限，但BPD仍然是新生儿发病的重要原因。通过抗氧化治疗预防BPD的尝试也失败了。因此，有必要寻找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）激动剂可能增强内源性抗氧化表达，保持GSH水平，并预防o2介导的肺损伤。Nrf2显著影响新生小鼠肺泡化和高氧易感性。硫氧还蛋白还原酶-1 （TrxR1）最著名的功能是再生氧化硫氧还蛋白-1 （Trx1）的活性位点。越来越多的证据表明TrxR1抑制可能是Nrf2激动剂的共同特征。金硫葡萄糖（ATG）和金糠蛋白（AFN）能有效抑制TrxR1，临床上用于治疗类风湿性关节炎。我们之前的体内研究表明，ATG处理增加Nrf2激活，保持肺GSH水平，并防止成年小鼠的高氧性肺损伤。我们最近在体外证明，AFN处理增加nrf2介导的抗氧化反应和增加GSH水平。当谷胱甘肽系统被破坏时，ATG和AFN的保护作用就丧失了。我们新的初步数据表明，ATG减少了新生小鼠o2介导的肺发育缺陷。总的来说，我们的数据支持TrxR1抑制剂通过Nrf2和gsh依赖机制介导保护的工作模型。TrxR1抑制诱导Nrf2激活、提高GSH水平和预防o2介导的新生儿肺损伤的效用尚未得到验证。因此，这个应用程序的目标是

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.