<p>RT-qPCR primer list.</p>

The involvement of Notch pathway-related genes (NPRGs) in idiopathic pulmonary fibrosis (IPF) remains inadequately understood. This study identified novel NPRG-associated biomarkers in IPF through integrated analysis of the GSE28042 dataset and NPRG gene sets, with the goal of uncovering potential therapeutic targets. Initially, 7 overlapping candidate genes were identified by intersecting 1,361 differentially expressed genes (DEGs) between IPF and control samples, 4,883 key module genes associated with IPF, and 428 known NPRGs. Four biomarkers—IL4, PLXND1, NBEA, and GATA2—were prioritized using machine learning methods. Immune infiltration analysis, conducted with the CIBERSORT algorithm (v2.0.4), revealed that IL4, NBEA, and GATA2 were significantly positively correlated with resting dendritic cells and negatively correlated with follicular helper T cells. Additionally, drug target prediction and pathway enrichment analyses suggested potential associations between these biomarkers and oxidative stress-related pathways. RT-qPCR validation using human blood samples confirmed significant down-regulation of IL4 and NBEA while PLXND1 was significantly up-regulated in patients with IPF compared to healthy controls. These biomarkers may contribute to the pro-fibrotic microenvironment, and their dysregulation is linked to the pathogenesis of pulmonary fibrosis. In summary, the identified NPRG-related biomarkers hold diagnostic potential for IPF. with further research needed to clarify their functional roles and assess their viability as therapeutic targets or as consequences of the fibrotic process.Notch通路相关基因（NPRGs）在特发性肺纤维化（IPF）中的作用仍未得到充分阐明。本研究通过对GSE28042数据集与NPRG基因集进行整合分析，筛选出IPF中新型的NPRG相关生物标志物，以期揭示潜在的治疗靶点。初始阶段，研究将IPF与对照样本间的1361个差异表达基因（DEGs）、4883个IPF相关关键模块基因以及428个已知NPRGs取交集，共获得7个重叠候选基因。随后通过机器学习方法筛选出4个核心生物标志物：IL4、PLXND1、NBEA及GATA2。采用CIBERSORT算法（v2.0.4）开展免疫浸润分析，结果显示IL4、NBEA与GATA2均与静息树突状细胞呈显著正相关，而与滤泡辅助性T细胞呈显著负相关。此外，药物靶点预测与通路富集分析结果表明，上述生物标志物与氧化应激相关通路存在潜在关联。通过人血液样本进行的实时定量聚合酶链反应（RT-qPCR）验证证实，与健康对照相比，IPF患者体内IL4与NBEA的表达显著下调，而PLXND1的表达显著上调。上述生物标志物可能参与促纤维化微环境的形成，其表达失调与肺纤维化的发病机制密切相关。综上，本研究鉴定的NPRG相关生物标志物具备IPF的诊断潜力，未来仍需进一步研究以明确其功能角色，并评估其作为治疗靶点或纤维化进程继发效应的可行性。