Bromine Inhalation Induced Lung Injury: Novel Mechanisms and Treatment Strategies

 描述(申请人提供)：溴(BR2)是一种剧毒的暗红色液体，很容易蒸发成红色蒸汽，带有令人窒息的气味。世界溴2年产量超过30万吨。暴露于BR2会导致急性肺损伤、呼吸衰竭死亡和纤维化。由于工业和交通事故有可能在人口稠密的地区释放大量的溴2，溴2对公众健康构成明显和现实的威胁。很少有发表的研究评估吸入BR2的急性和慢性后遗症；治疗仍然是有症状的，没有有效的对策存在。与人类的病理相似，暴露在BR2下的小鼠会导致反应性呼吸道疾病综合征(RADS)，增加血气屏障对血浆蛋白的通透性，并导致炎症，继而导致上皮下呼吸道纤维化和显著死亡。这项应用的总体目的是确定导致这些事件的生化和分子机制，并制定适当的对策。我们认为Br2和次溴酸(HOBr2-)与高分子透明质酸(H-HA)相互作用并断裂，高分子透明质酸(H-HA)是一种普遍存在的基质糖胺聚糖，从而产生高度炎性的低分子透明质酸片段(L-HA)。L-HA与CD44和TLR4结合，增加细胞内钙离子浓度，激活肺上皮细胞和气道平滑肌细胞的转化生长因子--1和RhoA。这些事件导致RADS，上皮对血浆蛋白的通透性增加，呼吸道细胞上皮-间充质细胞转化(EMT)，上皮下纤维化，呼吸衰竭死亡。此外，我们首次展示了暴露于BR2的小鼠肺和血浆中溴化脂的形成。这些化合物是由BR2与肺血浆原相互作用形成的，它们介导和放大了BR2的肺损伤，并作为BR2暴露的生物标志物。基于可靠的数据，我们假设在Br2暴露后，雾化(H-HA的一种形式，目前正在欧洲进行哮喘临床试验)可以减轻肺损伤，促进修复并降低死亡率。第一个特定目标中提出的实验将评估暴露于br2并返回室内空气长达三周的小鼠的生理、生化和形态变化，并测试暴露后雾化亚布罗对减少肺损伤和死亡率的有效性。然后，我们将确定BR2损伤啮齿动物和人类呼吸道平滑肌(ASM)、支气管和肺泡II型(ATII)细胞的机制。我们推测Br2、溴化脂质和L-HA增加细胞内钙离子，激活RhoA，从而导致气道收缩和上皮通透性增加。实验将：(I)膜片钳测定膜电位；(Ii)Fura-2荧光测定细胞内钙离子；(Iii)RhoA和ROCK激活；(Iv)肌球蛋白轻链磷酸化；以及(V)(上皮细胞)对荧光葡聚糖的通透性。最后，在1、24和72小时分离小鼠气管环。暴露于BR2后，测量对乙酰甲胆碱反应的平滑肌收缩。

 DESCRIPTION (provided by applicant): Bromine (Br2) is a highly toxic dark-reddish liquid, which evaporates readily to a red vapor with a suffocating odor. World production of Br2 exceeds 300,000 tons per year. Exposure to Br2 causes acute lung injury, death from respiratory failure, and fibrosis. Because of the potential for industrial and transportation accidents to release of large amounts of Br2 in populated areas, Br2 presents a clear and present danger to public health. Few published studies have evaluated the acute and chronic sequelae of Br2 inhalation; treatment remains symptomatic and no effective countermeasures exist. Similar to human pathology, exposure of mice to Br2 causes reactive airway disease syndrome (RADS), increased permeability of the blood gas barrier to plasma proteins, and inflammation followed by sub-epithelial airway fibrosis and significant mortality. The overall purpose of this application is to identify the biochemical and molecular mechanisms responsible for these events and develop appropriate countermeasures. We propose that Br2 and hypobromous acid (HOBr-) interact with and fragment high molecular weight hyaluronan (H-HA), a ubiquitous matrix glycosaminoglycan, to generate highly inflammatory low molecular weight hyaluronan fragments (L-HA). L- HA binds to CD44 and Toll like receptor (TLR)-4, increases intracellular Ca+2 and activates TGF-1, and RhoA in lung epithelial and airway smooth muscle cells. These events lead to RADS, increased epithelial permeability to plasma proteins, epithelial-mesenchymal cell transition (EMT) of airway cells, sub-epithelial fibrosis, an death from respiratory failure. In addition, we demonstrate for the first time the formation of brominated lipids in the lungs and plasma of mice exposed to Br2. These compounds, formed by the interaction of Br2 with lung plasmalogens, mediate and amplify Br2 lung injury and act as biomarkers of Br2 exposure. Based on solid data we posit that post-Br2 exposure administration of aerosolized Yabro (a form of H-HA, currently in clinical trials in Europe for asthma), attenuates lung damage, enhances repair and decreases mortality. Experiments proposed in the first specific aim will assess physiological, biochemical, and morphological changes in mice exposed to Br2 and returned to room air for up to three weeks and test the effectiveness of aerosolized Yabro administered post exposure to decrease lung injury and mortality. We will then identify the mechanisms by which Br2 damages rodent and human airway smooth muscle (ASM), bronchial and alveolar type II (ATII) cells. We posit that Br2, brominated lipids, and L-HA increase intracellular Ca2+ and activate RhoA, which lead to increased airway contractility and epithelial permeability. Experiments will: (i) determine membrane potentials by patch clamp; (ii) intracellular Ca+2 by fura-2 fluorescence; (iii) RhoA and ROCK activation; (iv) myosin light chain phosphorylation; and (v) (for epithelial cells) permeability to fluorescent dextrans. Finally, we wll isolate mouse tracheal rings at 1, 24, and 72 hr. post Br2 exposure and measure smooth muscle contraction in response to methacholine.