Modest supplemental oxygen worsens lung injury in a murine model of sepsis

描述(申请人提供)：急性肺损伤(ALI)和急性呼吸窘迫综合征(ARDS)与高水平的发病率和死亡率有关，给医疗保健系统带来了巨大的负担。脓毒症是ALI的常见病因，因此对脓毒症所致肺损伤的研究具有重要意义。应用全身性脂多糖(LPS)，我们观察到以间质性肺损伤为主，肺泡中性粒细胞稀少和损害，与直接肺损伤所见的明显的肺泡损伤和中性粒细胞内流明显不同。在了解小鼠直接损伤和间接损伤之间的差异的过程中，我们假设与脂多糖或单独使用50%氧气相比，50%氧气会增加全身创伤后小鼠的肺损伤。我们的损伤模型包括内毒素，12小时后暴露在50%或21%的氧气中60小时。在第3天，与其他组相比，暴露于脂多糖加氧气的小鼠表现出协同作用增加肺损伤，并伴有明显的肺泡成分。我们假设，在脂多糖中添加50%的氧气会增加氧化应激和炎性细胞因子的产生，从而加重肺损伤，而抗氧化治疗可以减轻损伤。我们计划通过以下两个目标来检验这一假说：1.检验通过联合暴露于内毒素和补充氧气而导致肺损伤加重的潜在机制。暴露于内毒素、50%氧气或内毒素加氧气的小鼠将被评估死亡率、体重减轻、BAL细胞计数、差异、总蛋白、肺湿重和组织学。此外，我们将测量：细胞内和细胞外ROS的产生；肺抗氧化酶水平；BAL/肺细胞因子；以及特定间隔的肺泡细胞凋亡。在体外，我们将幼稚的肺泡巨噬细胞(AM)和树突状细胞(DC)与脂多糖、50%氧气或其组合孵育，并测量ROS的产生和先前已确定的关键炎症介质。2.确定NAC联合补氧治疗是否能减轻肺损伤，并探讨其可能的作用机制。与目标1相同，将给予NAC或对照组，并将评估其对损伤模式的影响。此外，我们还将研究Aim 1发现的细胞因子和抗氧化潜在途径，以及特定时间和细胞的ROS和细胞凋亡活性。公共卫生相关性：系统性疾病可能导致肺部严重损伤，需要住院治疗和死亡。我们将在动物模型中研究中等氧气作为这种损伤的潜在放大因子的作用。这项工作有可能改善人类结局，减轻这种毁灭性疾病给卫生保健系统带来的负担。

DESCRIPTION (provided by applicant): Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high levels of morbidity and mortality, imposing a substantial burden on the health care system. Sepsis is a frequent cause of ALI making the study of sepsis-induced lung injury crucial. Using systemic lipopolysaccharide (LPS) we observed predominantly interstitial lung injury with a paucity of alveolar neutrophils and damage, dramatically different from the prominent alveolar injury and neutrophil influx seen with direct lung injury. Along the way to understanding the difference between direct and indirect injury in mice, we postulated that 50% oxygen would augment lung injury in mice after systemic insult compared to LPS or 50% oxygen alone. Our injury model consists of LPS followed 12 hrs later by exposure to 50% or 21% oxygen for 60 hrs. At day 3, mice exposed to LPS plus oxygen showed a synergistically increase lung injury, with a prominent alveolar component, compare to other groups. We hypothesize that the addition of 50% oxygen to LPS increases oxidative stress and inflammatory cytokine production, thereby augmenting lung injury, and that injury can be mitigated with antioxidant therapy. We plan to examine aspects of this hypothesis with following 2 aims: 1. To examine potential mechanisms driving the augmentation of lung injury by combined exposure to LPS and supplemental oxygen. Mice exposed to LPS, 50% oxygen, or LPS plus oxygen, will be assessed for mortality, weight loss; BAL cell counts, differentials, total protein, wet lung weight; and histology. In addition, we will measure: intracellular and extracellular ROS generation; lung anti-oxidant enzyme levels; BAL/Lung cytokines; and alveolar cell apoptosis at defined intervals. In vitro, we will incubate naive alveolar macrophages (AMs) and dendritic cells (DCs) with LPS, 50% oxygen, or in combination, and measure ROS production and key inflammatory mediators previously identified. 2. To determine if NAc therapy concurrent with supplemental oxygen abrogates lung injury, and examine potential mechanisms of that effect. NAc or control will be given to groups as in Aim 1, and the effect on injury patterns will be assessed. In addition, we will examine cytokine and anti-oxidant potential pathways indicated by Aim 1 findings, as well as time- and cell-specific ROS and apoptosis activity. Public Health Relevance: Significant injury to the lungs requiring hospitalization and death can occur from systemic illness. We will examine the role of moderate oxygen as a potential amplifier of that injury in an animal model. This work has the potential to improve human outcomes and decrease burden on the health care system from this devastating illness.