Fan Yunxin, Feng Xiumin, Zhu Guanglin, Zhang Jingxi, Dong Yuchao, Bai Chong
Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital, Naval Medical University, Shanghai 200433, China.
Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital, Naval Medical University, Shanghai 200433; Department of Respiratory and Critical Care Medicine, Changji Branch of the First Affiliated Hospital of Xinjiang Medical University, Changji 831100, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Jan;39(1):1-8.
Objective To explore how alveolar macrophages from chronic obstructive pulmonary disease (COPD)-model rats affect proliferation and secretion of 16HBE human bronchial epithelial cells and investigate the associated mechanism. Methods Alveolar macrophages were extracted from COPD rats induced by cigarette smoke exposure and LPS instillation through bronchoalveolar lavage, then co-cultured with 16HBE cells in vitro. Exosomes were extracted from alveolar macrophages of rats with exosome isolation kit. The differentially expressed miRNA in exosomes derived from macrophages of rats in COPD group and control group was detected by PCR. miR-380 was overexpressed with miR-380 mimic while the expression of cystic fibrosis transmembrane transduction regulator (CFTR) was knocked down with siRNA in 16HBE cells. The proliferation of 16HBE cells was detected with CCK-8 assay. The migration ability of 16HBE cells was evaluated with Transwell migration assay. The levels of mucins (MUC5AC, MUC5B, MUC2) and CFTR expressed by 16HBE cells were detected with Western blot analysis. The expression of TNF-α and IL-6 in the supernatant of 16HBE cells was detected with ELISA. Results The alveolar macrophages from COPD rats enhanced the proliferation and migration of 16HBE cells. The production of mucins and TNF-α as well as IL-6 in 16HBE cells were increased by COPD macrophages. The expression of miR-380 was significantly elevated in exosomes derived from COPD alveolar macrophages. Both overexpression of miR-380 and inhibition of CFTR decreased the expression of CFTR, resulting in the significantly enhanced proliferation and migration of 16HBE cells as well as increased expression of MUC5AC, MUC5B, MUC2 and TNF-α, IL-6. Conclusion The alveolar macrophages from COPD rats can enhance the proliferation and mucin expression as well as inflammatory cytokine secretion of 16HBE cells. This process may be involved with abnormal expression of miR-380 in exosomes of COPD alveolar macrophages and down-regulation of CFTR in bronchial epithelial cells.
目的 探讨慢性阻塞性肺疾病（COPD）模型大鼠的肺泡巨噬细胞如何影响人支气管上皮细胞16HBE的增殖和分泌，并研究其相关机制。方法 通过支气管肺泡灌洗从香烟烟雾暴露联合脂多糖（LPS）诱导的COPD大鼠中提取肺泡巨噬细胞，然后与16HBE细胞进行体外共培养。用外泌体分离试剂盒从大鼠肺泡巨噬细胞中提取外泌体。采用PCR检测COPD组和对照组大鼠巨噬细胞来源外泌体中差异表达的微小RNA（miRNA）。在16HBE细胞中用miR-380模拟物过表达miR-380，并用小干扰RNA（siRNA）敲低囊性纤维化跨膜转导调节因子（CFTR）的表达。用CCK-8法检测16HBE细胞的增殖情况。用Transwell迁移实验评估16HBE细胞的迁移能力。用蛋白质免疫印迹法检测16HBE细胞表达的黏蛋白（MUC5AC、MUC5B、MUC2）和CFTR的水平。用酶联免疫吸附测定（ELISA）法检测16HBE细胞培养上清中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的表达。结果 COPD大鼠的肺泡巨噬细胞增强了16HBE细胞的增殖和迁移能力。COPD巨噬细胞使16HBE细胞中黏蛋白、TNF-α以及IL-6的产生增加。COPD肺泡巨噬细胞来源的外泌体中miR-380的表达显著升高。miR-380过表达和CFTR抑制均降低了CFTR的表达，导致16HBE细胞的增殖和迁移显著增强，同时MUC5AC、MUC5B、MUC2以及TNF-α、IL-6的表达增加。结论 COPD大鼠的肺泡巨噬细胞可增强16HBE细胞的增殖、黏蛋白表达以及炎性细胞因子分泌。这一过程可能与COPD肺泡巨噬细胞外泌体中miR-380的异常表达及支气管上皮细胞中CFTR的下调有关。
