Callister M E, Pinhu L, Catley M C, Westwell A D, Newton R, Leaver S K, Quinlan G J, Evans T W, Griffiths M J, Burke-Gaffney A
Critical Care, Pulmonary Vascular and Sleep Science, Respiratory Science, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, UK.
Br J Pharmacol. 2008 Nov;155(5):661-72. doi: 10.1038/bjp.2008.258. Epub 2008 Jun 30.
Subtle changes in the intracellular reduction-oxidation (redox) state can modulate nuclear factor-kappaB (NF-kappaB) activity. Thioredoxin-1 (Trx) is a small, ubiquitous, redox-active thiol (-SH) protein that, with thioredoxin reductase-1 (TrxR), modifies the redox status of NF-kappaB pathway components. PMX464 is a novel thiol-reactive quinol thought to inhibit the Trx/TrxR system. The aim of this work was to investigate whether PMX464 inhibited NF-kappaB-mediated proinflammatory activation of human type II alveolar epithelial cells (A549).
Intercellular adhesion molecule-1 (ICAM-1), granulocyte-macrophage colony-stimulating factor (GM-CSF) and CXCL8, NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity were assessed in A549 cells stimulated with IL-1beta with or without PMX464 pretreatment. Effects of PMX464 on ICAM-1 expression in human lung microvascular endothelial cells (HLMVEC) were also investigated. For comparison, selected measurements (ICAM-1 and IkappaB-alpha phospho-IkappaB-alpha) were made on A549 cells after RNA interference-mediated silencing (siRNA) of Trx.
PMX464 reduced ICAM-1, GM-CSF and CXCL8 expression in IL-1beta-stimulated A549 cells and ICAM-1 in HLMVEC. PMX464 inhibited IL-1beta-induced NF-kappaB DNA binding, nuclear translocation of NF-kappaB p65 subunit and factors involved in NF-kappaB activation; specifically, IkappaBalpha degradation, IkappaB phosphorylation and IkappaB kinase (IKK) activity in A549. By contrast, Trx siRNA did not alter ICAM-1 expression or IkappaBalpha degradation/phosphorylation in IL-1beta-stimulated A549 cells.
PMX464 inhibits a proinflammatory response in A549 cells targeting the NFkappaB pathway above IKK. The lack of effect with Trx siRNA suggests that PMX464 acts on thiol proteins, in addition to Trx, to elicit anti-inflammatory responses in lung epithelial cells.
细胞内氧化还原状态的细微变化可调节核因子-κB（NF-κB）活性。硫氧还蛋白-1（Trx）是一种小的、普遍存在的、具有氧化还原活性的硫醇（-SH）蛋白，它与硫氧还蛋白还原酶-1（TrxR）一起改变NF-κB信号通路成分的氧化还原状态。PMX464是一种新型的硫醇反应性喹诺酮，被认为可抑制Trx/TrxR系统。本研究旨在探讨PMX464是否能抑制NF-κB介导的人II型肺泡上皮细胞（A549）的促炎激活。
在用或不用PMX464预处理的情况下，评估白细胞介素-1β刺激的A549细胞中细胞间黏附分子-1（ICAM-1）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和CXCL8、NF-κB DNA结合、NF-κB p65亚基的核转位、IκBα降解、IκB磷酸化和IκB激酶（IKK）活性。还研究了PMX464对人肺微血管内皮细胞（HLMVEC）中ICAM-1表达的影响。为作比较，在用RNA干扰介导的Trx沉默（siRNA）处理A549细胞后，对选定指标（ICAM-1和IκB-α磷酸化-IκB-α）进行检测。
PMX464可降低白细胞介素-1β刺激的A549细胞中ICAM-1、GM-CSF和CXCL8的表达以及HLMVEC中ICAM-1的表达。PMX464可抑制白细胞介素-1β诱导的NF-κB DNA结合、NF-κB p65亚基的核转位以及参与NF-κB激活的因子；具体而言，可抑制A549细胞中IκBα降解、IκB磷酸化和IκB激酶（IKK）活性。相比之下，Trx siRNA并未改变白细胞介素-1β刺激的A549细胞中ICAM-1的表达或IκBα降解/磷酸化。
PMX464通过靶向IKK以上的NF-κB信号通路抑制A549细胞中的促炎反应。Trx siRNA无效表明PMX464除作用于Trx外，还作用于硫醇蛋白，从而在肺上皮细胞中引发抗炎反应。
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