Transcription profiling of mouse lung after ventilator induced injury reveals genetic and pharmacologic evidence links oxidative stress

RATIONALE: Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear. OBJECTIVES: We hypothesized that disruption of the gene encoding Nrf2, a transcription factor which regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI), while antioxidant supplementation attenuates such effect. METHODS: To test our hypothesis and to examine the relevance of oxidative stress in VILI, here we have assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2(-/-)) mice and wildtype (Nrf2(+/+)) animals following acute (2 h) injurious model of MV with or without administration of antioxidant. MEASUREMENTS AND MAIN RESULTS: Nrf2(-/-) mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared to Nrf2(+/+) mice. Nrf2-deficieny enhances the levels of several pro-inflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2(-/-) mice; however antioxidant supplementation to Nrf2(-/-) mice remarkably attenuated VILI. When subjected to clinically relevant prolong period of MV, Nrf2(-/-) mice displayed greater levels of VILI than Nrf2(+/+) mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins and phosphatases in Nrf2(-/-) mice. CONCLUSIONS: Collectively, our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress. Experiment Overall Design: The Nrf2 wildtype (Nrf2+/+) and Nrf2-deficient (Nrf2-/-) CD-1/ICR strains of mice were subjected to mechanical ventilation with high (HVT) amounts of tidal volumes (VT) at 30 ml/kg for 2 hours. The animals subjected to spontaneous ventilation (SpV) for 2 hours were used as controls. Lungs were immediately removed and processed for total RNA isolation using TRIzol reagent (LifeTechnologies, Grand Island, NY). The isolated RNA was applied to Murine Genome 430A GeneChip arrays (Affymetrix, Santa Clara, CA), which contain probes for detecting ~14,500 well-characterized genes and 4371 expressed sequence tags according to standard microarray protocol. Scanned output files were analyzed by using Affymetrix GeneChip Operating Software and were independently normalized to an average intensity of 500.研究背景：机械通气（Mechanical ventilation, MV）是急性肺损伤与成人呼吸窘迫综合征重症患者不可或缺的治疗手段，但常规机械通气诱发肺损伤的具体机制尚未阐明。研究目的：本研究提出假说：编码核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）——一种可调控多种抗氧化酶表达的转录因子——的基因发生缺失，会加重呼吸机相关性肺损伤（ventilator-induced lung injury, VILI）的易感性，而抗氧化剂补充则可缓解这一效应。实验方法：为验证上述假说并探究氧化应激在VILI中的作用，本研究对Nrf2敲除（Nrf2(-/-)）小鼠与野生型（Nrf2(+/+)）小鼠实施2小时损伤性机械通气，并辅以或不辅以抗氧化剂干预，随后评估两组小鼠的肺损伤与炎症反应情况。检测指标与主要结果：与野生型小鼠相比，Nrf2(-/-)小鼠在机械通气后肺泡与血管通透性及炎症反应水平均显著升高。Nrf2缺失会升高多种参与VILI发病机制的促炎细胞因子水平。我们发现，在Nrf2(-/-)小鼠肺部，机械通气会导致关键抗氧化酶表达水平下调及氧化还原失衡；而向Nrf2(-/-)小鼠补充抗氧化剂可显著缓解VILI。当接受临床相关的长时间机械通气时，Nrf2(-/-)小鼠的VILI程度较野生型小鼠更为严重。转录组表达谱分析显示，Nrf2(-/-)小鼠中多种VILI相关基因、应激反应蛋白、溶质转运蛋白及磷酸酶均未被诱导表达。研究结论：综上，本研究首次证实Nrf2在VILI中发挥关键保护作用，其可通过调控氧化应激，对抗机械通气诱导的细胞反应。实验整体设计：将Nrf2野生型（Nrf2(+/+)）与Nrf2敲除（Nrf2(-/-)）的CD-1/ICR品系小鼠予以高潮气量通气（high tidal volume, HVT，潮气量VT为30 ml/kg），机械通气时长2小时；以自主通气（spontaneous ventilation, SpV）2小时的小鼠作为对照。即刻摘取肺组织，采用TRIzol试剂（LifeTechnologies，美国纽约州格兰德艾兰市）提取总RNA。将提取的RNA应用于小鼠基因组430A基因芯片（Affymetrix，美国加利福尼亚州圣克拉拉市），该芯片按照标准微阵列实验流程，搭载了可检测约14500个已充分注释的基因及4371个表达序列标签的探针。扫描得到的输出文件通过Affymetrix GeneChip Operating Software进行分析，并统一标准化至平均强度500。