Department of Pulmonary and Critical Care Medicine, The Second Xiangya Hospital, Central South University, Respiratory Disease Research Institute of Hunan Province, Changsha, China.
Respiratory Disease Diagnosis and Treatment Center of the Central South University, Changsha, China.
Respiration. 2019;98(4):347-356. doi: 10.1159/000502099. Epub 2019 Aug 15.
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal fibrotic lung disease with an unknown aetiology. Persistent myofibroblast differentiation is a prominent feature of IPF. Cigarette smoking is a risk factor for IPF and an indicator of poor prognosis. Cigarette smoking induces endoplasmic reticulum (ER) stress, and it has been shown that ER stress promotes fibroblast-to-myofibroblast differentiation in lung fibrosis. In this study, we investigated whether cigarette smoke extract (CSE) promotes lung myofibroblast differentiation via the induction of ER stress.
Our study concentrates on exploring the relationship between smoking and ER stress in the differentiation of lung fibroblasts to myofibroblasts.
Human embryonic lung fibroblasts (MRC-5 fibroblasts) were stimulated with various doses of CSE. Levels of α-smooth muscle actin (α-SMA) protein were evaluated by immunofluorescence and western blot analyses. ER stress was induced by thapsigargin (TG) and inhibited by 4-phenyl butyric acid (4-PBA). Protein levels of glucose-regulated protein-78 (GRP78), inositol-requiring enzyme 1 (IRE1), X box-binding protein-1 (XBP-1) and activating transcription factor 6 (ATF6) were determined by western blotting. GRP78 siRNA was transfected into MRC-5 cells using Lipofectamine RNAiMAX Reagent.
CSE at a concentration of 1.0% significantly increased α-SMA expression in MRC-5 cells. There was no significant cell apoptosis after cells were exposed to CSE. CSE treatment significantly increased the expression of GRP78, IRE1, XBP-1 and ATF6 at the protein level at 48 h. Pretreatment with TG enhanced, whereas pretreatment with 4-PBA inhibited, the CSE-induced expression of α-SMA, GRP78 and XBP-1. Furthermore, knockdown of GRP78 blocked α-SMA expression in MRC-5 cells exposed to CSE.
Our data suggested that CSE promotes lung fibroblast-to-myofibroblast differentiation by the induction of ER stress.
特发性肺纤维化（IPF）是一种病因不明的进行性致死性肺纤维化疾病。持续的成肌纤维细胞分化是 IPF 的一个显著特征。吸烟是 IPF 的一个危险因素，也是预后不良的一个指标。吸烟会引起内质网（ER）应激，已有研究表明 ER 应激会促进肺纤维化中纤维母细胞向肌成纤维细胞的分化。在本研究中，我们探讨了香烟烟雾提取物（CSE）是否通过诱导 ER 应激促进肺肌成纤维细胞分化。
本研究集中探讨吸烟与 ER 应激在肺成纤维细胞向肌成纤维细胞分化中的关系。
用不同剂量的 CSE 刺激人胚肺成纤维细胞（MRC-5 成纤维细胞）。通过免疫荧光和 Western blot 分析评估α-平滑肌肌动蛋白（α-SMA）蛋白水平。用他普西醇（TG）诱导 ER 应激，用 4-苯丁酸（4-PBA）抑制。Western blot 检测葡萄糖调节蛋白 78（GRP78）、肌醇需求酶 1（IRE1）、X 盒结合蛋白 1（XBP-1）和激活转录因子 6（ATF6）的蛋白水平。用 Lipofectamine RNAiMAX Reagent 将 GRP78 siRNA 转染入 MRC-5 细胞。
浓度为 1.0%的 CSE 显著增加了 MRC-5 细胞中α-SMA 的表达。细胞暴露于 CSE 后没有明显的细胞凋亡。CSE 处理显著增加了 48 小时时细胞内 GRP78、IRE1、XBP-1 和 ATF6 的蛋白表达。用 TG 预处理增强了 CSE 诱导的α-SMA、GRP78 和 XBP-1 的表达，而用 4-PBA 预处理则抑制了这种表达。此外，在暴露于 CSE 的 MRC-5 细胞中敲低 GRP78 会阻断α-SMA 的表达。
我们的数据表明，CSE 通过诱导 ER 应激促进肺成纤维细胞向肌成纤维细胞的分化。
