目的 探讨失巢凋亡相关基因在重症哮喘嗜酸性粒细胞表型和中性粒细胞表型中气道重塑的潜在调控机制及生物标志物，并筛选靶向干预的中药化合物。方法 首先利用支气管活检样本的测序数据，通过加权基因共表达网络分析（weighted correlation network analysis， WGCNA）鉴定出重症哮喘嗜酸性粒细胞表型（eosinophilic asthma， EA） 和中性粒细胞表型（neutrophilic asthma， NA）高度相关的基因模块。进一步筛选出与失巢凋亡相关的基因，并与 WGCNA 结果映射以识别关键的调控基因。使用蛋白质分析通过进化关系（protein analysis through evolutionary relationships， PANTHER）进行通路富集，揭示了这些基因可能参与的信号通路及病理表型。再利用受试者工作特征（receiver operator characteristic， ROC）分析，鉴定出具有区分不同炎症表型和病理表型的潜在生物标志物。此外，利用人类蛋白质图谱数据库（human protein atlas,HPA） 数据库的单细胞测序数据，对标志物在组织和肺细胞中的表达模式进行注释。同时回归临床，借助 TcmBank 和 ETCM数据库，预测潜在调控这些标志物的中药化合物，评估其药动学和毒理学特性， 通过分子对接验证标志物与化合物的结合亲和力。最后构建预测模型探索年龄、性别、吸烟与否在重症哮喘患者不同炎症表型发病与否的价值。结果 WGCNA 提示包含 54 个基因的黑色模块与重症 EA 高度相关，包含 212 个基因的蓝色模块与重症 NA 高度相关。其中，黑色模块识别出 5个失巢凋亡基因，蓝色模块识别出 16 个失巢凋亡基因，这些基因均富集在与气道重塑显著相关的整合素信号通路。其中蛋白磷酸酶 2 调节亚基 B α 亚型（protein phosphatase 2 regulatory subunit balpha， PPP2R2A） 在重症 EA， 整合素亚基 β 5（integrinsubunit beta 5， ITGB5）、细胞周期蛋白 D1（cyclin D1， CCND1）、醛脱氢酶家族 1 成员 A1（aldehyde dehydrogenase 1 familymember A1， ALDH1A1）在重症 NA 的气道重塑中具有较高的 ROC 诊断价值，是潜在生物标志物。这些失巢凋亡基因在肺部高表达，且在肺部 1 型肺泡上皮细胞、 II 型肺泡上皮细胞、平滑肌细胞和成纤维细胞等在气道重塑中发挥重要作用的细胞中高表达。而水飞蓟素、花生四烯酸、熊果酸与这些促进重塑的失巢凋亡基因结合亲和力良好，具有潜在调控作用。此外，研究发现年龄、吸烟、性别均对重症 EA、重症 NA 发病有所影响，且年龄-吸烟-性别联合预测的影响大于任意单一因素的影响，是哮喘异质性的重要因素。结论 水飞蓟素、花生四烯酸、熊果酸可能通过靶向失巢凋亡基因 PPP2R2A、 ITGB5、CCND1 调控重症 EA、重症 NA 的气道重塑，为未来重症哮喘个体化治疗提供了新的策略。

Objective To explore the potential regulatory mechanisms and biomarkers of anoikis-related genes in airway remodeling in eosinophil and neutrophil phenotypes of severe asthma, and screen traditional Chinese medicine compounds for targeted intervention. Methods Sequencing data from bronchial biopsies were employed to discern gene modules correlated with eosinophilic asthma (EA) and neutrophilic asthma (EA) phenotypes of severe asthma via weighted gene co-expression network analysis (WGCNA). Anoikis-related genes were identified and integrated with WGCNA findings to delineate pivotal regulatory genes. The protein analysis through evolutionary relationships (PANTHER) pathway enrichment was used to elucidate the potential involvement of these genes in specific signaling pathways and pathological phenotypes. The receiver operator characteristic (ROC) facilitated the identification of biomarkers with the capacity to differentiate among various inflammatory and pathological profiles. Additionally, we annotated the expression patterns of these biomarkers in tissues and lung cells using single-cell sequencing data from the human protein atlas (HPA) database. Concurrently, we reverted to clinical relevance by predicting traditional Chinese medicine compounds that may regulate these biomarkers using the TcmBank and ETCM databases, assessing their pharmacokinetics and toxicological properties, and validating the binding affinity of biomarkers with compounds through molecular docking. Finally, a predictive model was constructed to explore the value of age, gender, and smoking status in the onset of different inflammatory phenotypes in patients with severe asthma. Results WGCNA analysis revealed a 54-gene black module strongly associated with EA and a 212-gene blue module associated with NA. Anoikis genes within these modules, five in the black and sixteen in the blue, were enriched in integrin pathways linked to airway remodeling. Protein phosphatase 2 regulatory subunit balpha (PPP2R2A) for EA and integrin subunit beta 5 (ITGB5), cyclin D1 (CCND1), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) for NA showed significant diagnostic potential in ROC analysis, indicating their potential as biomarkers. These genes were prominently expressed in lung tissue, particularly in cells pivotal to airway remodeling, such as type I and II alveolar epithelial cells, smooth muscle cells, and fibroblasts. Silymarin, arachidonic acid, and ursolic acid demonstrated strong binding affinity to these genes, suggesting regulatory potential. The study also identified age, smoking, and gender as influential factors in the pathogenesis of both EA and NA, with their combined effect being more substantial than any single factor, underscoring their role in asthma heterogeneity. Conclusion Silymarin, arachidonic acid, and ursolic acid may target the anoikis-related genes PPP2R2A, ITGB5, CCND1 to modulate airway remodeling in severe EA and NA, providing a novel strategy for personalized treatment of severe asthma in the future.

R285;Q811.4

国家自然科学基金面上项目(82204985);广东省教育厅项目青年创新人才项目(2022KQNCX013);广东省自然面上项目(2023A1515010807,2024A1515012183);广州市科技化项目(2023A04J1854);深圳市"医疗卫生三名工程"建设项目(SZZYSM202206013);国家中医优势专科建设项目(广州中医药大学第一附属医院肺病科);广东省重点科室(中西医协同科室)建设项目;深圳市中西医结合医院院内课题(YJ-2023-106);广州中医药大学青年拔尖人才(团队)培育"揭榜挂帅"项目;深圳市宝安区医疗卫生科研项目(BAYXH2024011);广州中医药大学第一附属医院青优人才项目;广东省中医药管理局项目(20251334);深圳市宝安区2024年度区属公立医院高质量发展研究项目(BAGZL2024053);深圳市科技创新局基础研究面上项目(JCYJ20240813114911016);深圳宝安区中医药临床研究专项(2023ZYYLCZX-9,2023ZYYLCZX-11);深圳市宝安区医疗卫生科研项目(2023JD124,2023JD107,2023JD105,2023JD110,2024JD289,2024JD293,2024JD316)