目的 探討失巢凋亡相關(guān)基因在重癥哮喘嗜酸性粒細(xì)胞表型和中性粒細(xì)胞表型中氣道重塑的潛在調(diào)控機制及生物標(biāo)志物，并篩選靶向干預(yù)的中藥化合物。方法 首先利用支氣管活檢樣本的測序數(shù)據(jù)，通過加權(quán)基因共表達網(wǎng)絡(luò)分析（weighted correlation network analysis， WGCNA）鑒定出重癥哮喘嗜酸性粒細(xì)胞表型（eosinophilic asthma， EA） 和中性粒細(xì)胞表型（neutrophilic asthma， NA）高度相關(guān)的基因模塊。進一步篩選出與失巢凋亡相關(guān)的基因，并與 WGCNA 結(jié)果映射以識別關(guān)鍵的調(diào)控基因。使用蛋白質(zhì)分析通過進化關(guān)系（protein analysis through evolutionary relationships， PANTHER）進行通路富集，揭示了這些基因可能參與的信號通路及病理表型。再利用受試者工作特征（receiver operator characteristic， ROC）分析，鑒定出具有區(qū)分不同炎癥表型和病理表型的潛在生物標(biāo)志物。此外，利用人類蛋白質(zhì)圖譜數(shù)據(jù)庫（human protein atlas,HPA） 數(shù)據(jù)庫的單細(xì)胞測序數(shù)據(jù)，對標(biāo)志物在組織和肺細(xì)胞中的表達模式進行注釋。同時回歸臨床，借助 TcmBank 和 ETCM數(shù)據(jù)庫，預(yù)測潛在調(diào)控這些標(biāo)志物的中藥化合物，評估其藥動學(xué)和毒理學(xué)特性， 通過分子對接驗證標(biāo)志物與化合物的結(jié)合親和力。最后構(gòu)建預(yù)測模型探索年齡、性別、吸煙與否在重癥哮喘患者不同炎癥表型發(fā)病與否的價值。結(jié)果 WGCNA 提示包含 54 個基因的黑色模塊與重癥 EA 高度相關(guān)，包含 212 個基因的藍(lán)色模塊與重癥 NA 高度相關(guān)。其中，黑色模塊識別出 5個失巢凋亡基因，藍(lán)色模塊識別出 16 個失巢凋亡基因，這些基因均富集在與氣道重塑顯著相關(guān)的整合素信號通路。其中蛋白磷酸酶 2 調(diào)節(jié)亞基 B α 亞型（protein phosphatase 2 regulatory subunit balpha， PPP2R2A） 在重癥 EA， 整合素亞基 β 5（integrinsubunit beta 5， ITGB5）、細(xì)胞周期蛋白 D1（cyclin D1， CCND1）、醛脫氫酶家族 1 成員 A1（aldehyde dehydrogenase 1 familymember A1， ALDH1A1）在重癥 NA 的氣道重塑中具有較高的 ROC 診斷價值，是潛在生物標(biāo)志物。這些失巢凋亡基因在肺部高表達，且在肺部 1 型肺泡上皮細(xì)胞、 II 型肺泡上皮細(xì)胞、平滑肌細(xì)胞和成纖維細(xì)胞等在氣道重塑中發(fā)揮重要作用的細(xì)胞中高表達。而水飛薊素、花生四烯酸、熊果酸與這些促進重塑的失巢凋亡基因結(jié)合親和力良好，具有潛在調(diào)控作用。此外，研究發(fā)現(xiàn)年齡、吸煙、性別均對重癥 EA、重癥 NA 發(fā)病有所影響，且年齡-吸煙-性別聯(lián)合預(yù)測的影響大于任意單一因素的影響，是哮喘異質(zhì)性的重要因素。結(jié)論 水飛薊素、花生四烯酸、熊果酸可能通過靶向失巢凋亡基因 PPP2R2A、 ITGB5、CCND1 調(diào)控重癥 EA、重癥 NA 的氣道重塑，為未來重癥哮喘個體化治療提供了新的策略。

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

國家自然科學(xué)基金面上項目(82204985);廣東省教育廳項目青年創(chuàng)新人才項目(2022KQNCX013);廣東省自然面上項目(2023A1515010807,2024A1515012183);廣州市科技化項目(2023A04J1854);深圳市"醫(yī)療衛(wèi)生三名工程"建設(shè)項目(SZZYSM202206013);國家中醫(yī)優(yōu)勢?？平ㄔO(shè)項目(廣州中醫(yī)藥大學(xué)第一附屬醫(yī)院肺病科);廣東省重點科室(中西醫(yī)協(xié)同科室)建設(shè)項目;深圳市中西醫(yī)結(jié)合醫(yī)院院內(nèi)課題(YJ-2023-106);廣州中醫(yī)藥大學(xué)青年拔尖人才(團隊)培育"揭榜掛帥"項目;深圳市寶安區(qū)醫(yī)療衛(wèi)生科研項目(BAYXH2024011);廣州中醫(yī)藥大學(xué)第一附屬醫(yī)院青優(yōu)人才項目;廣東省中醫(yī)藥管理局項目(20251334);深圳市寶安區(qū)2024年度區(qū)屬公立醫(yī)院高質(zhì)量發(fā)展研究項目(BAGZL2024053);深圳市科技創(chuàng)新局基礎(chǔ)研究面上項目(JCYJ20240813114911016);深圳寶安區(qū)中醫(yī)藥臨床研究專項(2023ZYYLCZX-9,2023ZYYLCZX-11);深圳市寶安區(qū)醫(yī)療衛(wèi)生科研項目(2023JD124,2023JD107,2023JD105,2023JD110,2024JD289,2024JD293,2024JD316)