Ding Ting, Zhao Shunshun, Gu Yanhui, He Guiqiang, Lang Yanzhu, Rao Ximin, Chen Jie, Ou-Yang Yao
Department of Respiratory and Critical Care Medicine, Affiliated Hospital of Zunyi Medical University, Guizhou, 563003, China.
Sci Rep. 2025 May 13;15(1):16546. doi: 10.1038/s41598-025-00458-9.
The airway and lung tissue inflammation and structural changes caused by COPD lead to persistent and irreversible airflow limitation in patients. Several studies have associated IL-17A with COPD airway inflammation and collagen deposition, while autophagy is essential for maintaining normal cell function. Based on these findings, we propose a hypothesis that IL-17 affecting the autophagy of macrophages through the PI3K/AKT/mTOR pathway may contribute to the regulation of the airway remodeling process in COPD. The COPD airway remodelling model was confirmed by pulmonary function tests and HE and Masson staining. IL-17A, IL-6 and CCL20 were detected by ELISA, autophagosomes (APs) were observed by transmission electron microscopy, and western blotting was used to detect PI3K/AKT/mTOR-related proteins, autophagy-related proteins and collagen. Immunofluorescence revealed colocalization of LC3 in mouse bronchial fibroblasts (MBFs). MBFs were isolated and cultured via lentiviral transfection, IL-17RA overexpression or silencing, and quantitative analysis of PI3K/AKT/mTOR pathway-related proteins and autophagy-related proteins via western blotting. The results validated the establishment of the COPD model. Increased IL-17A in the COPD airway and increased IL-6 and CCL20 expression were observed in the COPD mice supplemented with the autophagy inhibitor 3MA. Using TEM, we observed a significant reduction in the number of APs in the airways in both the COPD and 3MA groups. Moreover, the PI3K/AKT/mTOR pathway phospho-p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR ratios were also increased in the COPD group. Moreover, as P62 increased, Beclin-1 and LC3II/I expression decreased correspondingly, while Collagen I and Collagen III also increased. In our study of cultured mouse MBFs, the results showed that overexpression (OE) virus-mediated transfection of MBFs overexpressing IL-17RA increased the p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR ratios, increased the P62 content and reduced the expression of Beclin-1 and LC3II/I. However, compared with the OE group, the silencing (sh)-mediated transfection of MBFs with IL-17RA had almost opposite effects. Increased collagen production and the expression of IL-17A, IL-6 and CCL20 in the airways of COPD mice. Autophagy decreased, and the PI3K/AKT/mTOR pathway was activated in COPD airway tissue. In primary cultured MBFs from COPD mice, IL-17A combined with IL-17RA activates the PI3K/AKT/mTOR pathway, thereby inhibiting autophagy.
慢性阻塞性肺疾病（COPD）引起的气道和肺组织炎症及结构改变导致患者出现持续性、不可逆的气流受限。多项研究已将白细胞介素 - 17A（IL - 17A）与COPD气道炎症和胶原沉积联系起来，而自噬对于维持正常细胞功能至关重要。基于这些发现，我们提出一个假设，即IL - 17通过PI3K/AKT/mTOR途径影响巨噬细胞的自噬，这可能有助于调控COPD中的气道重塑过程。通过肺功能测试以及苏木精 - 伊红（HE）和马松（Masson）染色确认了COPD气道重塑模型。采用酶联免疫吸附测定（ELISA）检测IL - 17A、IL - 6和CCL20，通过透射电子显微镜观察自噬体（APs），并使用蛋白质免疫印迹法检测PI3K/AKT/mTOR相关蛋白、自噬相关蛋白和胶原蛋白。免疫荧光显示小鼠支气管成纤维细胞（MBFs）中微管相关蛋白1轻链3（LC3）的共定位。通过慢病毒转染、IL - 17RA过表达或沉默分离并培养MBFs，并通过蛋白质免疫印迹法对PI3K/AKT/mTOR途径相关蛋白和自噬相关蛋白进行定量分析。结果验证了COPD模型的建立。在补充自噬抑制剂3 - 甲基腺嘌呤（3MA）的COPD小鼠中，观察到COPD气道中IL - 17A增加以及IL - 6和CCL20表达增加。使用透射电子显微镜，我们观察到COPD组和3MA组气道中APs数量均显著减少。此外，COPD组中PI3K/AKT/mTOR途径的磷酸化p - PI3K/PI3K、p - AKT/AKT和p - mTOR/mTOR比值也增加。此外，随着P62增加，Beclin - 1和LC3II/I表达相应降低，而I型胶原蛋白和III型胶原蛋白也增加。在我们对培养的小鼠MBFs的研究中，结果表明过表达（OE）病毒介导的MBFs转染过表达IL - 17RA会增加p - PI3K/PI3K、p - AKT/AKT和p - mTOR/mTOR比值，增加P62含量并降低Beclin - 1和LC3II/I的表达。然而，与OE组相比，IL - 17RA的沉默（sh）介导的MBFs转染具有几乎相反的效果。COPD小鼠气道中胶原蛋白生成增加以及IL - 17A、IL - 6和CCL20表达增加。自噬减少，且COPD气道组织中PI3K/AKT/mTOR途径被激活。在来自COPD小鼠的原代培养MBFs中，IL - 17A与IL - 17RA结合激活PI3K/AKT/mTOR途径，从而抑制自噬。
