Induction of airway smooth muscle proliferation and airway hyperreactivity after exposure to airborne particles

在体外和体内实验中，暴露于环境超细颗粒诱导气道炎症反应和组织重塑，以及气道高反应性。为了确定超细炭黑（ufCB）是否影响气道平滑肌细胞的增殖，如果有，作用机制是什么，我们研究了人原代支气管平滑肌细胞培养。在含ufCB的无血清培养基中培养细胞，[3H]-胸苷和[3H]-亮氨酸的掺入量呈时间和剂量依赖性增加。自由基清除剂和NADH/NADPH氧化酶抑制剂可减弱这种作用，表皮生长因子受体（EGF-R）酪氨酸激酶抑制剂AG1478和BIBX1382以及丝裂原活化蛋白激酶（MEK）抑制剂PD98059预处理可完全抑制这种作用。转染H-Ras显性阴性突变体同样消除了ufCB的作用。ufCB刺激还诱导膜锚定的前肝素结合(HB) -EGF的加工，可溶性HB-EGF释放到培养基中，磷酸化的EGF-R和Shc与谷胱甘肽- s -转移酶- grb2融合蛋白的关联，以及细胞外信号调节激酶（ERK）的磷酸化。预处理AG1478、[Glu^52]白喉毒素（HB-EGF特异性抑制剂）和中和HB-EGF抗体均可抑制ufcb诱导的ERK活化。此外，暴露于ufCB的小鼠对吸入甲基胆碱的反应性增加，这种效应同样被EGFR抑制剂抑制。因此，ufCB引起氧化应激介导的气道平滑肌增殖和由此产生的气道高反应性，可能是通过处理HB-EGF和EGF-R以及ERK级联激活。

In this in vitro and in vivo experiments, exposure to ambient ultrafine particles induced airway inflammatory reactions and tissue remodelling, as well as airway hyperresponsiveness. To determine whether ultrafine carbon black (ufCB) affects proliferation of airway smooth muscle cells and, if so, what the mechanism of action is, we studied human primary bronchial smooth muscle cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]-thymidine and [3H]-leucine into cells in a time- and dose dependent manner. This effect was attenuated by a free radical scavenger and a NADH/NADPH oxidase inhibitor, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG1478 and BIBX1382, and the mitogen-activated protein kinase (MEK) inhibitor PD98059. Transfection of a dominant negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane anchored pro-heparin-binding (HB) -EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione-S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK) . Pretreatment with each AG1478, [Glu^52]Diphtheria toxin, a specific inhibitor of HB-EGF, and neutralizing HB-EGF antibody inhibited ufCB-induced ERK activation. Furthermore, exposure of mice to ufCB induced an increased responsiveness to inhaled methacholine, and effect that was likewise inhibited by EGFR inhibitors. Thus, ufCB causes oxidative stress-mediated proliferation of airway smooth muscle and a resultant airway hyperreactivity, presumably through the processing of HB-EGF and EGF-R and ERK cascade activation.