GADD45a in ventilator-induced lung injury: role of Akt signaling. Mus musculus

We explored the mechanistic involvement of the growth arrest and DNA damageinducible gene, GADD45a, in LPS- and ventilator-induced inflammatory lung injury (VILI). Multiple biochemical and genomic parameters of inflammatory lung injury indicated GADD45a-/- mice to be modestly susceptible to intratracheal LPS-induced lung injury and profoundly susceptible to high tidal volume ventilation-induced lung injury (VILI) with increases in microvascular permeability and levels of inflammatory cytokines in bronchoalveolar lavage. Expression profiling of lung tissues from GADD45a-/- mice revealed strong dysregulation in the B cell receptor signaling pathway suggesting involvement of PI3 kinase/Akt signaling components while the wild type controls depicted no observable changes. Western blot analyses of lung homogenates confirmed ~50% reduction in Akt protein levels in GADD45a-/- mice accompanied by marked increases in Akt ubiquitination. Electrical resistance measurements across human lung endothelial cell monolayers with either reduced GADD45a or Akt expression (siRNAs) revealed significant potentiation of LPS-induced human lung endothelial barrier dysfunction which was attenuated by overexpression of a constitutively active Akt1 transgene. These studies validate GADD45a as a novel candidate gene in inflammatory lung injury and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling Overall design: 4 experimental groups, each with 3 mouses. Wild type control group, VILI group, GADD-/- group, GADD-/- and VILI group.

本研究探讨了生长阻滞与DNA损伤诱导基因GADD45a（growth arrest and DNA damage-inducible gene GADD45a）在脂多糖（LPS）及呼吸机诱导的炎症性肺损伤（VILI）中的机制性作用。通过炎症性肺损伤的多项生化与基因组学参数分析可见，GADD45a基因敲除（GADD45a-/-）小鼠对气管内滴注LPS诱导的肺损伤仅表现为轻度易感性，但对高潮气量通气诱导的肺损伤（VILI）则呈现显著易感性，具体表现为支气管肺泡灌洗液中微血管通透性升高、炎症细胞因子水平上升。对GADD45a-/-小鼠肺组织的表达谱分析显示，B细胞受体信号通路存在显著失调，提示PI3激酶/Akt信号通路组分参与其中，而野生型对照组未观察到明显变化。肺组织匀浆的蛋白质印迹（Western blot）分析证实，GADD45a-/-小鼠的Akt蛋白水平下降约50%，同时伴随Akt泛素化水平显著升高。对采用小干扰RNA（siRNAs）敲低GADD45a或Akt表达的人肺内皮细胞单层进行跨膜电阻测量，结果显示LPS诱导的人肺内皮屏障功能障碍显著增强，而组成型激活的Akt1转基因过表达可削弱这一效应。本研究证实GADD45a是炎症性肺损伤的新型候选基因，并通过调控Akt介导的内皮信号通路，在血管屏障调节中发挥重要作用。实验设计：共设置4组实验组，每组各3只小鼠，分别为野生型对照组、VILI组、GADD45a基因敲除组、GADD45a基因敲除联合VILI组。