Department of Physiology, Faculty of Medicine, University of Thessaly, BIOPOLIS, Larissa, Greece.
Department of Histopathology, Faculty of Medicine, University of Thessaly, BIOPOLIS, Larissa, Greece.
In Vivo. 2024 Sep-Oct;38(5):2294-2299. doi: 10.21873/invivo.13694.
BACKGROUND/AIM: Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle.
Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (I) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations.
The amiloride effect on normal epithelium led to a significant decrease in I [ΔI=33±5.92 μA/cm; p<0.001 versus control experiments (ΔI=1.44±0.71 μA/cm)]. After incubation with CSE, ENaC I was significantly reduced (ΔI=14.80±1.96 μA/cm; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells.
Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.
背景/目的：香烟烟雾已被证明会在人类中诱导出一种被称为“获得性囊性纤维化”的表型。这是因为烟雾成分的有害影响会损害囊性纤维化跨膜电导调节剂（CFTR）的全身功能。阐明香烟烟雾对气管上皮的影响很重要。本研究的目的是开发一种离体绵羊气管模型来研究气管离子功能。在该模型中，上皮钠通道（ENaC）在暴露于香烟烟雾提取物（CSE）后被抑制，作为原理的证明。
从健康绵羊中分离气管，并通过手术切除气管上皮。组织被安装在 Ussing 室中，并在 5% CSE 孵育 30 分钟后用 PBS 或 PBS 单独孵育后测量短电流（I）。通过在顶端添加阿米洛利（10M）来研究 ENaC 的功能。进行 Western blot 分析以评估 CSE 暴露后 ENaC 数量的差异。一些标本用 H&E 染色用于检测组织学改变。
正常上皮上阿米洛利的作用导致 I 显著降低[ΔI=33±5.92 μA/cm；p<0.001 与对照实验（ΔI=1.44±0.71 μA/cm）相比]。孵育 CSE 后，ENaC I 显著降低[ΔI=14.80±1.96 μA/cm；p<0.001]。在 CSE 暴露和正常气管上皮之间未观察到 αENaC 表达的差异。孵育 CSE 后组织学图像显示上皮高度降低，基底细胞增生和纤毛细胞丧失。
CSE 孵育后阿米洛利对 ENaC 的抑制作用减弱可能是由于气管上皮的改变。
