Pharmacological effects and mechanisms of danlong oral liquid in asthma airway remodeling: Insights from serum medicinal chemistry, network pharmacology, and experimental validation

Ethnopharmacological relevance

Danlong oral liquid (DLOL) is a traditional Chinese proprietary medicine commonly used to treat chronic respiratory diseases, including bronchial asthma and chronic obstructive pulmonary disease. However, the therapeutic effects and pharmacological mechanisms of DLOL in improving airway remodeling remain unclear.

Aims of the study

This study utilizes in vivo and in vitro experiments, serum pharmacological analysis, and network-based pharmacology approaches to investigate the effects and mechanisms of DLOL on airway remodeling and epithelial-mesenchymal transition (EMT) in asthma.

Methods

An asthma model was established through ovalbumins (OVA) sensitization and challenge in BALB/c mice to observe the effects of DLOL on airway hyperresponsiveness (AHR), inflammation, remodeling, and molecular markers of EMT. The absorbed chemical prototype constituents of DLOL were analyzed using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS), and targets for asthma and airway remodeling were predicted using a network pharmacology approach. Key biological processes and signaling pathways were analyzed. Additionally, TGF-β1 was used to induce EMT in BEAS-2B cells. TGF-β1 and DLOL-containing serum were screened to determine the optimal time and concentration in BEAS-2B cells using CCK8 assays. The cell scratch assay was used to assess cell migration, while immunofluorescence and immunohistochemistry were employed to evaluate protein expression levels.

Results

DLOL improved AHR in asthmatic mice, reduced inflammatory cell infiltration in lung tissue, decreased airway wall and smooth muscle thickness, and reduced collagen deposition. It also down-regulated mesenchymal markers (N-cadherin, vimentin, α-SMA) and key remodeling factors (TGF-β1, MMP9), while up-regulating the epithelial marker E-cadherin. A total of 17 absorbed chemical prototype constituents were identified, predicting 54 core targets involved in airway remodeling. Following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the key targets were found to be associated with the regulation of cell migration, cell-cell adhesion, and cell adhesion molecular processes, with the PI3K-Akt signaling pathway likely playing a critical role. Cellular experiments confirmed that DLOL-containing serum inhibited TGF-β1-induced EMT in BEAS-2B cells and suppressed the phosphorylation of Akt and GSK-3β.

Conclusion

This study identifies, for the first time, the serum medicinal chemistry of DLOL using UPLC-MS. Combining network pharmacology, in vivo and in vitro experiments, it elucidates the effects and potential mechanisms of the drug on airway remodeling and EMT. DLOL may offer a novel therapeutic approach for asthma-related airway remodeling.