Amelioration of Mitochondrial Dysfunction by Thioredoxin in Hyperoxia

摘要 氧气治疗是一种常见的临床必需品，但它具有显着的负面影响。具体地说， 它产生的高氧条件产生了许多活性氧，包括超氧化物 导致线粒体功能障碍的阴离子。虽然这种毒性是氧气应用的关键因素 治疗，很少有人知道高氧如何影响线粒体能量产生，或蛋白质 再生机制，可以提供保护免受其影响。我们的研究计划侧重于 硫氧还蛋白（Trx），一种细胞质氧化还原蛋白，可以减少氧化应激和再生酶， 氧化作用已失活。最近，我们发表了硫氧还蛋白表达增加可保护肺 Trx基因表达降低的小鼠在高氧环境中的存活率明显高于Trx基因表达降低的小鼠， 对高氧敏感，死亡率很高。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， Trx 1易位线粒体在高氧，但这种运动不会发生在dnTrx-Tg小鼠。 这些发现促使我们假设Trx通过减少氧化应激来保护线粒体免受高氧损伤。 通过UCP-2依赖性解偶联应激，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关键机制 毒性因此，在目标1中，我们将确定易位的细胞质Trx 1是否以及如何保护细胞质中的Trx 1。 对抗高氧时的线粒体功能障碍在目标2中，我们将发现如果高水平的Trx阻止发动蛋白- 相关蛋白（Drp 1）激活，从而保护线粒体片段化和功能障碍。在 目的3：我们将确定Trx-Tg小鼠中PGC-1α向细胞核转位的增加是否能保护Trx-Tg小鼠免受 高氧引起的线粒体功能障碍。利用最先进的技术包括线粒体 通量分析，EPR光谱，生化酶测定和尖端分子方法，我们 将剖析Trx 1在高氧下保护功能失调的线粒体中的作用。我们还将创建一个 一种新的条件性Trx基因敲除小鼠，一种PGC 1a基因敲除小鼠， Trx以了解高水平Trx对高氧线粒体功能障碍的体内作用。该项目 预计将提供一个清晰的理解细胞溶质Trx 1影响线粒体功能的方式， 常氧和高氧。使用转基因小鼠（和来自它们的细胞）进行体内和体外研究， 机制的研究，我们希望揭示线粒体机制，是由Trx 1调制过程中， 高氧。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.