目的：通过体内外活性检测结合磷酸化蛋白质组学技术，探讨二陈汤治疗过敏性哮喘的活性及其作用机制。 方法：通过UPLC-MS/MS及HPLC对二陈汤中特征性成分进行定性定量检测；构建C48/80诱导的RBL-2H3肥大细胞脱颗粒模型及LPS诱导的RAW264.7巨噬细胞炎症模型，检测二陈汤的抗过敏及抗炎活性；构建OVA诱导的小鼠哮喘模型，观察二陈汤干预后小鼠行为学变化及炎症因子表达水平；采用Label-free磷酸化蛋白质组学技术筛选肺组织差异磷酸化修饰蛋白，通过GO、KEGG数据库进行生物信息学功能富集分析，解析二陈汤调控的核心信号通路，并通过Western blot对组学筛选的关键通路进行靶点验证。 结果：从二陈汤提取物中鉴定出13种特征性成分，并对其中五种进行了含量检测。二陈汤可剂量依赖性地抑制RBL-2H3细胞β-氨基己糖苷酶释放，降低RAW264.7细胞NO及炎症因子TNF-α、IL-1β的表达；并能显著缓解哮喘小鼠搔鼻打喷嚏等行为学改变，减轻气道炎性浸润，降低血清IgE及细胞炎症因子IL-4、IL-5及 IL-13水平。Label-free磷酸化蛋白质组学分析共鉴定出11870个差异磷酸化位点，KEGG富集分析显示差异蛋白显著富集于PI3K-Akt信号通路、AMPK信号通路及细胞骨架调节相关通路。WB结果表明二陈汤可显著下调RBL-2H3及RAW264.7细胞中p-Akt（Ser473）表达（p<0.01）。 结论：二陈汤具有显著的体内外抗哮喘活性，其作用机制可能与抑制PI3K-Akt信号通路激活，阻断肥大细胞脱颗粒及巨噬细胞活化有关。

Objective: To investigate the efficacy and mechanism of Erchen Decoction against allergic asthma, in vivo and in vitro experiments with phosphoproteomics were adopted. Methods: Qualitative and quantitative detection of characteristic components in Erchen Decoction was performed by UPLC-MS/MS and HPLC. In vitro, a C48/80-induced RBL-2H3 mast cell degranulation model and an LPS-induced RAW264.7 macrophage inflammation model were established to evaluate the anti-allergic and anti-inflammatory activities of Erchen Decoction. In vivo, an OVA-induced asthma mouse model was constructed to observe asthma-related behavioral changes and inflammatory cytokine expression following Erchen Decoction treatment. Label-free phosphoproteomics was employed to identify differentially phosphorylated proteins in lung tissues. Subsequently, bioinformatics analyses, including GO and KEGG pathway enrichment analyses, were performed to elucidate the core signaling pathways regulated by Erchen Decoction. Western blot validation was then conducted to confirm the key targets within the core signaling pathways identified by phosphoproteomic analysis. Results: Thirteen characteristic components were identified from the extract of Erchen Decoction, and five of them were quantitatively determined. Erchen Decoction dose-dependently inhibited β-hexosaminidase release from RBL-2H3 cells and reduced NO production , along with the mRNA expression of inflammatory cytokines TNF-α and IL-1β in RAW264.7 cells. In vivo, Erchen Decoction significantly attenuated airway inflammatory infiltration and decreased serum levels of IgE, IL-4, IL-5, and IL-13 in asthmatic mice. A total of 11,870 differentially phosphorylated sites were identified by Label-free phosphoproteomics. KEGG enrichment analysis revealed that the differentially phosphorylated proteins were significantly enriched in the PI3K-Akt signaling pathway, AMPK signaling pathway, and cytoskeleton regulation-related pathways. Western blot analysis demonstrated that Erchen Decoction significantly downregulated phosphorylated Akt at Ser473 (p-Akt Ser473) in both RBL-2H3 and RAW264.7 cells (p < 0.01). Conclusion: Erchen Decoction exhibits significant anti-asthmatic activity both in vivo and in vitro. Its underlying mechanism may involve the suppression of PI3K-Akt signaling pathway activation, thereby blocking mast cell degranulation and macrophage inflammatory responses.

（1）国家自然科学基金委员会青年科学基金项目（C类）（82505156）：二陈汤抑制IL-4/IL-13/STAT6环路调节肥大细胞-Th2细胞轴改善过敏性哮喘的作用机制研究；负责人：唐鼎；（2）湖北省自然科学基金青年项目（2024AFB377）：基于Syk靶点应用HEMT传感技术筛选二陈汤中抗过敏活性成分；负责人：唐鼎。