Waldow Thomas, Witt Wolfgang, Janke Andreas, Ulmer André, Buzin Anne, Matschke Klaus
Clinic for Cardiac Surgery, University Hospital Dresden, Germany.
J Surg Res. 2009 Nov;157(1):30-42. doi: 10.1016/j.jss.2008.07.042. Epub 2008 Sep 4.
Previous investigations have shown that short term inhalation of nitric oxide (NO) before ischemia and reperfusion (I/R) prevents I/R-related consequences on lung function. Here we correlate effects of NO-induced preconditioning, especially on the lung permeability barrier, with analysis of cell junction proteins and the level of vascular endothelial growth factor (VEGF).
A rat model of left lung in situ I/R was used. After left lateral thoracotomy, left lung ischemia was maintained for 60 min, followed by 30 min or 4 h (h) reperfusion (I/R groups). In the NO groups, inhalation of NO (10 min, 15 ppm) preceded I/R. Animals in control groups underwent sham surgery without NO inhalation and ischemia. The extent of I/R injury was assessed in terms of oxygenation (arterial PO(2)) and lung permeability (Evans blue extravasation). Expression of junctional proteins and phosphorylation was determined in complete protein extracts from lung tissue, whereas the adherens junction (AJ) core complex was analyzed in Triton extracts by co-immunoprecipitation using antibodies against E-cadherin and VE-cadherin.
The inhalation of NO prevented the I/R-induced increase of permeability at 30 min reperfusion, and the PO(2) increased from 27% of controls in the I/R group to 77% in the NO group. Left lung I/R correlated with a progressive loss of cadherins (VE-cadherin, E-cadherin, desmoglein 1) during reperfusion, whereas AJ catenins were largely preserved. Preconditioning with NO resulted in an increased ratio of catenins (alpha- and beta-catenin) to E-cadherin in immunoprecipitates and in reduced phosphorylation of beta-catenin. A reduction of VEGF in left lung lavage fluid was observed at 4 h but not at 30 min reperfusion.
The NO-induced changes of the AJ complex may have contributed to the stabilization of the lung permeability barrier during reperfusion.
先前的研究表明，在缺血再灌注（I/R）之前短期吸入一氧化氮（NO）可预防I/R对肺功能的相关影响。在此，我们将NO诱导的预处理效果，特别是对肺通透性屏障的影响，与细胞连接蛋白分析及血管内皮生长因子（VEGF）水平相关联。
采用大鼠左肺原位I/R模型。左胸外侧开胸后，左肺缺血维持60分钟，随后再灌注30分钟或4小时（I/R组）。在NO组中，I/R之前吸入NO（10分钟，15 ppm）。对照组动物接受假手术，不吸入NO且无缺血。根据氧合（动脉血氧分压）和肺通透性（伊文思蓝外渗）评估I/R损伤程度。在肺组织的完整蛋白提取物中测定连接蛋白的表达和磷酸化，而通过使用抗E-钙黏蛋白和VE-钙黏蛋白的抗体进行共免疫沉淀，在Triton提取物中分析黏着连接（AJ）核心复合物。
吸入NO可预防再灌注30分钟时I/R诱导的通透性增加，且氧分压从I/R组中对照组的27%升至NO组中的77%。左肺I/R与再灌注期间钙黏蛋白（VE-钙黏蛋白、E-钙黏蛋白、桥粒芯糖蛋白1）的逐渐丢失相关，而AJ连环蛋白基本保留。NO预处理导致免疫沉淀物中连环蛋白（α-连环蛋白和β-连环蛋白）与E-钙黏蛋白的比例增加，且β-连环蛋白的磷酸化减少。再灌注4小时时观察到左肺灌洗液中VEGF减少，但再灌注30分钟时未观察到。
NO诱导的AJ复合物变化可能有助于再灌注期间肺通透性屏障的稳定。
