Da-yuan-yin decoction alleviates bleomycin-induced pulmonary injury by inhibiting epithelial-mesenchymal transition via E-cadherin/β-catenin complex restoration.

The Da-Yuan-Yin (DYY) decoction, a classical multi-herbal preparation documented in the Treatise on Pestilence (Wenyi Lun,⟪⟫), exhibits therapeutic potential in respiratory pathophysiology. Clinical studies during the COVID-19 pandemic have validated DYY's therapeutic efficacy in ameliorating respiratory symptoms in patients. However, its pharmacological mechanisms against lung injury remain unexplored. This investigation sought to delineate the medicinal efficacy and underlying pharmacological mechanisms of DYY through integrated experimental models encompassing animal studies and cellular assays. Pharmacological evaluation was conducted in a mouse model of pulmonary injury via intratracheal (i.t.) administration of bleomycin (BLM). Test cohorts received DYY extract at graded concentrations: low-dose (4.3 g/kg), medium-dose (8.6 g/kg), and high-dose (17.2 g/kg) via daily oral gavage. After 14 consecutive days of intervention, pulmonary specimens were harvested. Histomorphological alterations were quantified through Hematoxylin-eosin and Masson trichrome staining of pulmonary parenchyma. E-cadherin and α-SMA protein distribution in tissue samples was monitored by immunohistochemical or immunofluorescent staining techniques. Longitudinal fibrosis progression was assessed through HYP quantification on day 21 following BLM instillation. TGF-β1-elicited epithelial-mesenchymal transition (EMT) in A549 alveolar epithelial cells served as an in vitro paradigm. Proteomic characterization of EMT markers (E-cadherin, vimentin, N-cadherin, α-SMA) was conducted via immunoblotting. Migratory capacity was evaluated using standardized scratch assay protocols. Transcriptional dynamics of EMT-associated genes (CDH1, CDH2, VIM, ACTA2) were monitored by RT-qPCR. Subcellular β-catenin redistribution was visualized through confocal microscopy following fluorescence labeling. Histopathological analysis of day 14 specimens revealed that DYY intervention attenuated pulmonary histoarchitectural disruption, preserved epithelial integrity through E-cadherin maintenance, and suppressed α-SMA-mediated mesenchymal activation. Longitudinal evaluation at day 21 demonstrated DYY-mediated significant attenuation of fibrotic progression, evidenced by the reduction in HYP accumulation (p < 0.01) and collagen deposition regression. In cell-based models, DYY treatment upregulated CDH1 transcript/protein levels (p < 0.01) while downregulating mesenchymal markers (N-cadherin; vimentin; α-SMA; p < 0.05). Scratch-wound assays demonstrated that DYY treatment significantly suppressed TGF-β1-induced migration of A549 cells (p < 0.01). Mechanistically, DYY disrupted β-catenin nuclear localization, reducing transcriptionally active β-catenin in cytoplasmic fractions. Crucially, in vivo validation confirmed DYY's dose-dependent inhibition of EMT progression. This study delineates a molecular cascade through which DYY exerts its therapeutic effects: CDH1 transcriptional upregulation mechanistically mediates E-cadherin/β-catenin complex reconstitution at adherens junctions, achieving restoration of membrane-localized β-catenin. This stabilization effectively blocks β-catenin nuclear translocation, thereby suppressing α-SMA transcriptional activation. Crucially, early DYY intervention demonstrated alveolar epithelial preservation and significant attenuation of fibrotic progression, positioning it as a multi-target therapeutic strategy against EMT-driven pulmonary pathologies. This study demonstrates DYY's efficacy in mitigating bleomycin-induced alveolar injury within a single GMP-certified production batch; however, systematic validation of its long-term pharmacological stability under variable environmental conditions and multi-batch reproducibility remains necessary to confirm clinical translatability.