Small extracellular vesicles from irradiated lung epithelial cells promote the activation of fibroblasts in pulmonary fibrosis

Alveolar epithelial injury and dysfunction are the risk factors for radiation-induced pulmonary fibrosis (RIPF). However, it is not clear about the relationship between RIPF and the small extracellular vesicles (sEV) secreted by irradiated alveolar epithelial cells. Based on the activation of fibroblasts, this study explored the role of sEV derived from alveolar epithelial cells in RIPF and the potential mechanisms. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were used to characterize sEV. Western blotting was used to detect fibrosis-associated proteins. Cell counts and transwell assays were used to evaluate the proliferation and migration ability of fibroblasts. RT-PCR was used to observe the extracellular matrix (ECM) synthesized by fibroblasts, miRNA changes in the sEV were determined by second-generation sequencing. TEM, NTA, and western blotting showed the extracellular vesicles with a double-layer membrane structure of approximately 100 nm in diameter. The sEV derived from irradiated A549, HBEC3-KT, and MLE12 cells upregulated FN1 and alpha-SMA proteins expression in fibroblasts and drove the fibroblast to myofibroblast transition, and the sEV from irradiated mouse bronchoalveolar lavage fluid (BALF) affirmed the same results. In addition, the sEV derived from irradiated alveolar epithelial cells significantly increased the migration ability of fibroblasts and the expression of extracellular matrix proteins such as FN1. The results of miRNA sequencing of sEV in BALF of rats with RIPF showed that the metabolic pathway may be important for miRNA to regulate the activation of fibroblasts. The sEV derived from radiated pulmonary epithelial cells promote the activation, migration and extracellular matrix proteins expression of lung fibroblasts; miRNA in sEV may be an important molecular that affects the activation of lung fibroblasts.

肺泡上皮损伤与功能异常是放射性肺纤维化（radiation-induced pulmonary fibrosis, RIPF）的危险因素。然而，该疾病与辐射暴露后肺泡上皮细胞分泌的细胞外小囊泡（small extracellular vesicles, sEV）之间的关联尚不明确。本研究以成纤维细胞激活为切入点，探讨了肺泡上皮细胞来源的细胞外小囊泡在放射性肺纤维化发生中的作用及其潜在机制。研究采用透射电子显微镜（transmission electron microscopy, TEM）、纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）以及蛋白质印迹法对细胞外小囊泡进行表型鉴定；通过蛋白质印迹法检测纤维化相关蛋白的表达水平；采用细胞计数法与Transwell实验评估成纤维细胞的增殖与迁移能力；采用实时荧光定量PCR（RT-PCR）观察成纤维细胞合成的细胞外基质（extracellular matrix, ECM），并通过二代测序检测细胞外小囊泡内的微小RNA（microRNA, miRNA）表达变化。透射电子显微镜、纳米颗粒追踪分析与蛋白质印迹法结果显示，所分离的细胞外小囊泡具有直径约100nm的双层膜结构。经辐射处理的A549、HBEC3-KT及MLE12细胞来源的细胞外小囊泡可上调成纤维细胞内纤连蛋白1（fibronectin 1, FN1）与α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）的表达水平，并诱导成纤维细胞向肌成纤维细胞转化；而来自辐射暴露小鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）的细胞外小囊泡也得到了一致的实验结果。此外，经辐射处理的肺泡上皮细胞来源的细胞外小囊泡可显著增强成纤维细胞的迁移能力，并上调纤连蛋白1等细胞外基质蛋白的表达水平。对放射性肺纤维化模型大鼠支气管肺泡灌洗液中细胞外小囊泡的微小RNA测序结果显示，代谢通路可能是微小RNA调控成纤维细胞激活的重要途径。经辐射的肺上皮细胞来源的细胞外小囊泡可促进肺成纤维细胞的激活、迁移以及细胞外基质蛋白的表达；细胞外小囊泡内的微小RNA可能是影响肺成纤维细胞激活的关键分子。