Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Department of Radiation Oncology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Int Forum Allergy Rhinol. 2018 Jan;8(1):64-71. doi: 10.1002/alr.22035. Epub 2017 Oct 30.
Cholinergic stimulation plays a major role in inflammatory airway diseases. However, its role in airway surface liquid homeostasis and aquaporin 5 (AQP5) regulation remains unclear. In this study we sought to determine the effects of methacholine and dexamethasone on AQP5 expression in human nasal epithelial cells (HNEpC).
HNEpC were cultured with methacholine or dexamethasone at 4 concentrations in vitro. The subcellular distribution of AQP5 was explored using immunocytochemistry. The pharmacologic effects of methacholine and dexamethasone on the expression of the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein (p-CREB), AQP5, and nuclear factor-kappaB (NF-κB) were examined using Western blotting.
AQP5 was found to be located in cell membrane and cytoplasm and present in every group without a statistically significant difference. Methacholine inhibited expression of AQP5 and p-CREB in HNEpC, whereas dexamethasone increased these protein levels dose-dependently in a statistically significant manner. In turn, HNEpC treated with methacholine and dexamethasone showed the same trends as those intervened separately with these 2 drugs. Moreover, dexamethasone had the ability to reverse the inhibitory effect of methacholine. Western blotting revealed that, after incubation with 10 mol/L methacholine, NF-κB increased significantly, by 186.67%, compared with the untreated control group. Again, such an increase could be significantly reversed after dexamethasone treatment.
NF-κB activation is important for inhibition of p-CREB/AQP5 expression after methacholine intervention, and dexamethasone adjusts it to the opposite side. This observation could provide additional insight into the anti-inflammatory effects of glucocorticoids that contribute to maintaining airway surface liquid and mucosal defense.
胆碱能刺激在炎症性气道疾病中起着重要作用。然而，它在气道表面液体平衡和水通道蛋白 5（AQP5）调节中的作用尚不清楚。在这项研究中，我们试图确定乙酰甲胆碱和地塞米松对人鼻上皮细胞（HNEpC）中 AQP5 表达的影响。
体外将 HNEpC 分别用乙酰甲胆碱或地塞米松在 4 种浓度下培养。用免疫细胞化学法探索 AQP5 的亚细胞分布。用 Western 印迹法检测乙酰甲胆碱和地塞米松对环磷酸腺苷反应元件结合蛋白（p-CREB）、AQP5 和核因子-κB（NF-κB）磷酸化表达的药理作用。
AQP5 位于细胞膜和细胞质中，各组间无统计学差异。乙酰甲胆碱抑制 HNEpC 中 AQP5 和 p-CREB 的表达，而地塞米松则以剂量依赖性方式显著增加这些蛋白水平。相反，用乙酰甲胆碱和地塞米松处理的 HNEpC 表现出与这两种药物单独干预相同的趋势。此外，地塞米松具有逆转乙酰甲胆碱抑制作用的能力。Western 印迹显示，与未处理对照组相比，用 10 摩尔/升乙酰甲胆碱孵育后，NF-κB 显著增加了 186.67%。再次，地塞米松处理后，这种增加可以明显逆转。
NF-κB 激活对于乙酰甲胆碱干预后 p-CREB/AQP5 表达的抑制很重要，地塞米松将其调节到相反的方向。这一观察结果为糖皮质激素的抗炎作用提供了更多的见解，有助于维持气道表面液体和粘膜防御。
