Department of Physiology, School of Basic Medical Science, Central South University, Changsha 410013.
Department of Nephrology Medicine, Third Xiangya Hospital, Central South University, Changsha 410013.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2021;46(9):949-957. doi: 10.11817/j.issn.1672-7347.2021.210210.
Many studies have shown that respiratory syncytial virus persistent infection may be the main cause of chronic respiratory pathology.However, the mechanism is unclear. Cystic fibrosis transmembrane conduction regulator (CFTR) is an apical membrane chloride channel, which is very important for the regulation of epithelial fluid, chloride ion, and bicarbonate transport. CFTR dysfunction will lead to changes in bronchial secretions and impair mucus clearance, which is related to airway inflammation. In our previous study, we observed the down-regulation of CFTR in airway epithelial cells in respiratory syncytial virus (RSV) infected mouse model. In this study, we further investigated the expression and function of CFTR by constructing an airway epithelial cell model of RSV persistent infection.
16HBE14o- cells were infected with RSV at 0.01 multiplicity of infection (MOI). The expression of CFTR was detected by real-time RT-PCR, immunofluorescence, and Western blotting. The intracellular chloride concentration was measured by N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE) and the chloride current was measured by whole-cell patch clamp recording.
16HBE14o- cells infected with RSV were survived to successive passages of the third generation (G3), while the expression and function of CFTR was progressively decreased upon RSV infection from the first generation (G1) to G3. Exposure of 16HBE14o- cells to RSV led to the gradual increase of TGF-β1 as well as phosphorylation of Smad2 following progressive RSV infection. Disruption of TGF-β1 signaling by SB431542 prevented Smad2 phosphorylation and rescued the expression of CFTR.
RSV infection can lead to defective CFTR function in airway epithelial cells, which may be mediated via activation of TGF-β1 signaling pathway.
许多研究表明，呼吸道合胞病毒（RSV）持续感染可能是慢性呼吸道病理的主要原因。然而，其机制尚不清楚。囊性纤维化跨膜电导调节因子（CFTR）是一种顶端膜氯离子通道，对调节上皮液、氯离子和碳酸氢盐转运非常重要。CFTR 功能障碍会导致支气管分泌物改变，并损害黏液清除，这与气道炎症有关。在我们之前的研究中，我们观察到 RSV 感染小鼠模型中气道上皮细胞 CFTR 的下调。在这项研究中，我们通过构建 RSV 持续感染的气道上皮细胞模型，进一步研究 CFTR 的表达和功能。
以 0.01 感染复数（MOI）感染 16HBE14o-细胞 RSV。通过实时 RT-PCR、免疫荧光和 Western blot 检测 CFTR 的表达。通过 N-（乙氧羰基甲基）-6-甲氧基喹啉溴化物（MQAE）测量细胞内氯离子浓度，通过全细胞膜片钳记录测量氯离子电流。
RSV 感染的 16HBE14o-细胞可连续传代至第三代（G3），而 CFTR 的表达和功能在从第一代（G1）到 G3 的 RSV 感染过程中逐渐降低。16HBE14o-细胞暴露于 RSV 导致 TGF-β1 的逐渐增加以及随着 RSV 感染的进行 Smad2 的磷酸化。SB431542 阻断 TGF-β1 信号通路可防止 Smad2 磷酸化并恢复 CFTR 的表达。
RSV 感染可导致气道上皮细胞 CFTR 功能缺陷，这可能是通过激活 TGF-β1 信号通路介导的。
