Lu Ling, Liu Ye-Dan, Liu Dong-Yun, Jin Rong, Qu Zheng-Hai
Department of Pediatrics, The Affiliated Hospital of Qingdao University, Qingdao, China.
Int Arch Allergy Immunol. 2025;186(8):769-783. doi: 10.1159/000543105. Epub 2024 Dec 18.
Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is an important mechanism for the onset and development of broncho-pulmonary dysplasia (BPD). Fibroblast growth factor 2 (FGF2) is involved in the development and repair of injury in many organs, particularly the lung. The role of FGF2 in BPD is currently unclear. The aim of our study was to investigate the expression of FGF2 in lung tissue of BPD mice, to further clarify the effect of FGF2 on EMT in alveolar epithelial cells and to actively search for possible signaling pathways.
The BPD model was induced by exposure to hyperoxia. Lung tissue samples were collected and hematoxylin and eosin staining was used to determine the modeling effect. Quantitative real-time polymerase chain reaction (QRT-PCR), immunohistochemistry was used to detect FGF2 expression in BPD mice. To further investigate the effect of FGF2 supplementation and deficiency on EMT in alveolar epithelial cells, A549 cells were cryopreserved, resuspended, cultured, and passaged. Transforming growth factor-β1 (TGF-β1) was used to induce EMT. FGF2 small interfering RNA fragments were synthesized and screened. Fibroblast growth factor receptor 1 (FGFR1) expression was inhibited by BGJ398. (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium) (MTS) assay was used to detect the effect of FGF2 and infigratinib (BGJ398) on cell proliferation. We used qRT-PCR and Western blot to detect the expression of epithelial cell markers, mesenchymal cell markers and EMT-related signaling pathway proteins.
Our results showed that the successful established hyperoxia mice model were characteristic by BPD. Hyperoxia decreased FGF2 on day 4, upregulated FGF2 on day 21, which resulted in EMT. In vitro, we found that FGF2 alone increased the expression of mesenchymal markers, decreased the expression of epithelial markers and activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), small mother against decapentaplegic (Smad), mitogen-activated protein kinase (P38), and extracellular signal-regulated kinase (ERK) signaling pathways. FGF2 could not reverse but synergistically promote Transforming growth factor-β1 (TGF-β1)-induced EMT of alveolar epithelial cells. Silencing FGF2 increased the expression of epithelial marker E-cadherin, inhibited the PI3K/AKT, Smad, and P38 signaling pathways activated by TGF-β1, but activated ERK signaling. FGF2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT, decreased the expression of FGFR1, and inhibited ERK signaling pathway activation.
FGF2 was closely associated with EMT in BPD mice. Both high and low levels of FGF2 promoted EMT in A549. The FGF2 receptor inhibitor BGJ398 reversed TGF-β1-induced EMT in A549 by inhibiting the FGFR1/P-ERK signaling pathway.
肺泡上皮细胞的上皮-间质转化（EMT）是支气管肺发育不良（BPD）发生发展的重要机制。成纤维细胞生长因子2（FGF2）参与许多器官尤其是肺的发育和损伤修复。FGF2在BPD中的作用目前尚不清楚。本研究旨在探讨FGF2在BPD小鼠肺组织中的表达，进一步阐明FGF2对肺泡上皮细胞EMT的影响，并积极寻找可能的信号通路。
通过暴露于高氧诱导建立BPD模型。收集肺组织样本，采用苏木精-伊红染色确定建模效果。运用定量实时聚合酶链反应（QRT-PCR）、免疫组织化学检测BPD小鼠中FGF2的表达。为进一步研究FGF2补充和缺乏对肺泡上皮细胞EMT的影响，将A549细胞冻存、重悬、培养及传代。用转化生长因子-β1（TGF-β1）诱导EMT。合成并筛选FGF2小干扰RNA片段。用BGJ398抑制成纤维细胞生长因子受体1（FGFR1）的表达。采用（3-(4, 5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H四唑）（MTS）法检测FGF2和英菲格拉替尼（BGJ398）对细胞增殖的影响。用qRT-PCR和蛋白质免疫印迹法检测上皮细胞标志物、间充质细胞标志物及EMT相关信号通路蛋白的表达。
我们的结果显示成功建立的高氧小鼠模型具有BPD特征。高氧在第4天降低FGF2表达，在第21天上调FGF2表达，从而导致EMT。在体外，我们发现单独的FGF2增加间充质标志物的表达，降低上皮标志物的表达，并激活磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）、小母亲抗五肢瘫（Smad）、丝裂原活化蛋白激酶（P38）和细胞外信号调节激酶（ERK）信号通路。FGF2不能逆转但协同促进转化生长因子-β1（TGF-β1）诱导的肺泡上皮细胞EMT。沉默FGF2增加上皮标志物E-钙黏蛋白的表达，抑制TGF-β1激活的PI3K/AKT、Smad和P38信号通路，但激活ERK信号通路。FGF2受体抑制剂BGJ398逆转TGF-β1诱导的EMT，降低FGFR1的表达，并抑制ERK信号通路激活。
FGF2与BPD小鼠的EMT密切相关。FGF2的高水平和低水平均促进A549细胞的EMT。FGF2受体抑制剂BGJ398通过抑制FGFR1/P-ERK信号通路逆转TGF-β1诱导的A549细胞EMT。
