Amelioration of Mitochondrial Dysfunction by Thioredoxin in Hyperoxia

摘要 氧疗是临床上常见的一种治疗方法，但它也伴随着显著的副作用。具体来说， 它产生的高氧状态会产生许多活性氧物种，包括超氧化物 导致线粒体功能障碍的阴离子。尽管这种毒性是氧气应用中的一个关键因素 治疗方面，人们对高氧血症如何影响线粒体能量生产或蛋白质知之甚少。 再生机制，可以提供保护免受其影响。我们的研究项目侧重于 硫氧还蛋白(TRX)，一种细胞质氧化还原蛋白，可以减少氧化应激并再生酶 氧化作用已经失活。最近我们发表了硫氧还蛋白表达增加对肺的保护作用 TRX-TG小鼠在高氧环境下的损伤和存活增加，但TRX表达较低的小鼠 对高氧敏感，并遭受重大死亡。然而，高水平的 硫氧还蛋白对肺损伤的保护作用尚不清楚。我们的初步数据表明，细胞质 Trx1在高氧期间移位到线粒体，但这种移动在dnTrx-TG小鼠中不发生。 这些发现促使我们假设，Trx通过减少氧化来保护线粒体免受高氧的影响 应激通过UCP-2依赖的解偶联，并进一步通过保护线粒体功能障碍 线粒体中产生超氧阴离子的高氧是肺氧的一个关键机制 毒性。因此，在目标1中，我们将确定移位的细胞质Trx1是否以及如何保护 抗高氧性线粒体功能障碍。在目标2中，我们将发现如果高水平的Trx阻止动力- 相关蛋白(Drp1)被激活，从而防止线粒体碎裂和功能障碍。在……里面 目的3我们将确定在trx-tg小鼠中增加pgc-1α移位到细胞核是否可以保护小鼠免受 高氧引起的线粒体功能障碍。使用最先进的技术，包括线粒体 通量分析、EPR光谱分析、生化酶分析和前沿分子方法，我们 将剖析Trx1在高氧中保护功能障碍的线粒体中所起的作用。我们还将创建一个 一种新的条件性Trx基因敲除小鼠--PGC1a基因敲除小鼠 以了解体内高水平的Trx在高氧下线粒体功能障碍中的作用。该项目是 希望提供一个清楚的了解胞质Trx1影响线粒体功能的方式 常氧和高氧。利用转基因小鼠(及其衍生的细胞)进行体内和体外实验 机制研究，我们希望揭示线粒体机制是由Trx1在 高氧症。我们相信，该项目产生的结果将激发新的干预策略来保护患者 对抗氧疗引起的肺部毒性。

Abstract Oxygen therapy is a common clinical necessity, but it comes with significant negative side effects. Specifically, the hyperoxic condition it produces generates a number of reactive oxygen species, including superoxide anions that cause mitochondrial dysfunction. Although this toxicity is a key factor in the application of oxygen therapy, little is known about how hyperoxia impacts mitochondrial energy production, or the protein regeneration mechanisms that can offer protection from its effects. Our research program focuses on thioredoxin (Trx), a cytoplasmic redox protein that can reduce oxidative stress and regenerate enzymes that oxidation has inactivated. Recently we published that increased expression of Thioredoxin protects the lung injury and increased survival of Trx-Tg mice in hyperoxia, but mice with lower expression of Trx were more sensitive to hyperoxia and suffered significant mortality. However, the mechanism by which high levels of Thioredoxin protects against lung injury remains unknown. Our preliminary data establish that cytoplasmic Trx1 translocates to mitochondria during hyperoxia, but this movement does not occur in dnTrx-Tg mice. These findings propel us to hypothesize that Trx protects mitochondria from hyperoxia by reducing oxidative stress through UCP-2-dependent uncoupling, and furthermore by protecting mitochondrial dysfunction in hyperoxia as superoxide anion generation in the mitochondria is a key mechanism of pulmonary oxygen toxicity. Accordingly, in Aim 1 we will determine whether and how translocated cytoplasmic Trx1 protects against mitochondrial dysfunction in hyperoxia. In Aim 2 we will find if high levels of Trx prevents dynamin- related protein (Drp1) activation and thereby protects against mitochondrial fragmentation and dysfunction. In Aim 3 we will determine if increased translocation of PGC-1α to the nucleus in Trx-Tg mice can protect against the mitochondrial dysfunction caused by hyperoxia. Using state-of-the-art techniques that include mitochondrial flux analysis, EPR spectroscopy, biochemical enzymatic assay, and cutting-edge molecular approaches, we will dissect the role Trx1 plays in protecting the dysfunctional mitochondria in hyperoxia. We will also create a novel conditional Trx knockout mouse, a PGC1a-knockout mouse with increased or decreased expression of Trx to understand in vivo role of high levels of Trx on mitochondrial dysfunction in hyperoxia. The project is expected to provide a clear understanding of the way cytosolic Trx1 affects mitochondrial function during normoxia and hyperoxia. Using the transgenic mice (and cells derived from them) for in vivo and in vitro mechanistic studies, we expect to uncover mitochondrial mechanisms that are modulated by Trx1 during hyperoxia. We believe results produced by the project will incite novel intervention strategies to protect patients against pulmonary toxicity resulting from oxygen therapy.