目的 探讨小青龙汤通过抑制中性粒细胞胞外诱捕网（NETs）形成改善哮喘小鼠气道炎症的作用机制。方法 BALB/c小鼠随机分为对照组、模型组、小青龙汤（7.02 g·kg^-1）组，除对照组外，采用卵清蛋白（OVA）和完全弗氏佐剂（CFA）联合构建哮喘小鼠模型，造模14～20 d每天使用5% OVA进行雾化，每天进行40 min，小青龙汤组雾化前1 h ig小青龙汤7.02 g·kg^-1，对照组和模型组ig同等剂量的0.9%氯化钠溶液。通过苏木素-伊红（HE）、Masson染色观察小鼠肺组织气道炎症及胶原沉积； ELISA法检测血清免疫球蛋白E（IgE）、肿瘤坏死因子（TNF）-α、髓过氧化物酶（MPO）水平；转录组学测序结合K-means聚类筛选核心通路及差异基因；免疫荧光、免疫组化和Western blotting验证肺组织NETs形成。结果 与模型组比较，小鼠肺组织病理状态明显改善，肺泡扩张与结构破坏减轻，气道及肺间质炎症浸润减少，HE分数显著降低（P＜0.01）；气道纤维化病理改变有所缓解，表现为蓝染范围减小，胶原纤维沉积水平显著降低（P＜0.01）；血清中MPO、IgE和TNF-α含量明显降低（P＜0.001）； RNA-seq结合K-means聚类分析提示，小青龙汤干预效应与中性粒细胞活化及NETs相关通路密切相关，MPO、基质金属蛋白酶9（MMP9）、环磷酸腺苷（CAMP）、纤维蛋白原Α（FGA）等可能参与其调控过程；小青龙汤可显著降低肺组织气道周围MPO、中性粒细胞弹性蛋白酶（NE）荧光信号（P＜0.01、0.001）；显著降低肺组织气道周围MPO、肽基精氨酸脱亚氨酶4（PADI4）阳性面积（P＜0.01、0.001）；显著降低肺组织中NETs通路相关的NE、CAMP、MPO、PADI4、MMP9、FGA蛋白相对表达量（P＜0.05、0.01、0.001）。结论 小青龙汤可通过抑制NETs相关炎症反应改善哮喘小鼠的气道炎症。

Objective To investigate the effect and molecular mechanism of Xiaoqinglong Decoction in improving airway inflammation in asthmatic mice by regulating the neutrophil extracellular traps (NETs) pathway. Methods BALB/c mice were randomly divided into control, model, and Xiaoqinglong Decoction groups (7.02 g·kg^-1). Except for the control group, asthma models were induced using ovalbumin (OVA) and complete Freund’s adjuvant (CFA). From day 14 to 20 after modeling, mice received daily nebulization with 5% OVA for 40 min. The Xiaoqinglong Decoction group was administered Xiaoqinglong Decoction orally at 7.02 g·kg^-1 one hour before nebulization, while the control and model groups received an equivalent volume of 0.9% saline solution. Lung tissue was stained with hematoxylin-eosin (HE) and Masson trichrome to evaluate airway inflammation and collagen deposition. Serum levels of immunoglobulin E (IgE), tumor necrosis factor-alpha (TNF-α), and myeloperoxidase (MPO) were measured by ELISA. RNA sequencing combined with K-means clustering was used to identify key pathways and differentially expressed genes. Immunofluorescence, immunohistochemistry, and Western blotting were employed to validate NET formation in lung tissues. Results Compared with the model group, the pathological condition of lung tissue in the Xiaoqinglong Decoction group showed significant improvement, including reduced alveolar dilation and structural damage, decreased inflammatory infiltration in airways and interstitium, and significantly lower HE scores (P < 0.01). Pathological changes associated with airway fibrosis were alleviated, as indicated by reduced blue-stained areas and significantly decreased collagen fiber deposition (P < 0.01). Serum levels of MPO, IgE, and TNF-α were markedly reduced (P < 0.001). RNA-seq analysis combined with K-means clustering revealed that the therapeutic effect of Xiaoqinglong Decoction is closely related to pathways involving neutrophil activation and NET formation, with MPO, matrix metalloproteinase 9 (MMP9), cyclic AMP (CAMP), and fibrinogen alpha (FGA) potentially involved in its regulation. Xiaoqinglong Decoction significantly reduced fluorescent signals of MPO and neutrophil elastase (NE) around airways in lung tissue (P < 0.01, 0.001), significantly decreased positive staining areas of MPO and peptidylarginine deiminase 4 (PADI4) (P < 0.01, 0.001), and significantly downregulated the relative protein expression levels of NE, CAMP, MPO, PADI4, MMP9, and FGA in lung tissue (P < 0.05, 0.01, 0.001). Conclusion Xiaoqinglong Decoction can alleviate airway inflammation in asthmatic mice by inhibiting NETsrelated inflammatory responses.

R965

国家自然科学基金项目（82608616）；重庆市中医药创新团队项目（渝卫办白头〔2024〕570）；广东省基础与应用基础研究基金面上项目（批准号2026A1515011888）；广东省中医药局科研课题（20254036）；国家资助博士后研究人员计划（B档，GZB20250913）；中国博士后科学基金面上项目（2024M760670）；广州中医药大学2025年度校级创新创业训练项目（202510572334）