Ophiopogon japonicus alleviates pulmonary fibrosis through modulation of HIF1A/TUBB3 axis-associated M2 macrophage polarization and arginine metabolic alterations.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogon japonicus (Thunb.) Ker Gawl. is a traditional Chinese medicinal herb commonly used to "nourish yin and moisten the lung", and has long been applied for the prevention and treatment of lung-related disorders in classical medical texts such as the Treatise on Typhoid Fever and Item Differentiation of Warm Febrile Diseases. Pulmonary fibrosis (PF) is a chronic progressive fibrotic lung disease with limited therapeutic options. Although Ophiopogon japonicus has shown anti-inflammatory and anti-fibrotic potential in respiratory diseases, its pharmacological mechanisms against PF remain unclear. AIM OF THE STUDY: This study aimed to evaluate the protective effects of Ophiopogon japonicus aqueous decoction against PF and to investigate whether its anti-fibrotic effects are associated with the modulation of hypoxia-inducible factor 1 subunit α (HIF1A)/tubulin beta 3 class III (TUBB3) axis-associated M2 macrophage polarization and arginine metabolic alterations. MATERIALS AND METHODS: Potential bioactive constituents of Ophiopogon japonicus aqueous decoction were first screened using UHPLC-Q Exactive Orbitrap-HRMS combined with Lipinski's rule of five. A bleomycin (BLM)-induced mouse model of PF was then established, and Ophiopogon japonicus aqueous decoction was administered as an intervention. Body weight, pulmonary function testing (PFT), micro-CT, histopathology, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) were used to evaluate its pharmacodynamic effects. Network pharmacology, Gene Expression Omnibus (GEO) mining, weighted gene co-expression network analysis (WGCNA), machine learning (ML), transcriptomics, metabolomics, and single-cell transcriptomics combined with virtual knockout analysis were further integrated to identify candidate targets, related pathways, and metabolic alterations. Finally, flow cytometry, western blotting, immunofluorescence (IF) co-staining, AlphaFold3, molecular docking, and molecular dynamics simulation were performed to further assess phenotypic changes, predicted molecular associations, and candidate active constituents. RESULTS: Ophiopogon japonicus aqueous decoction significantly alleviated BLM-induced PF, and 16 potential bioactive constituents were identified. In vivo experiments showed that Ophiopogon japonicus aqueous decoction attenuated body weight loss, pulmonary dysfunction, micro-CT abnormalities, histopathological injury, collagen deposition, inflammatory responses, and fibrosis scores in PF mice. Bioinformatic analysis identified HIF1A as a candidate hub target and TUBB3 as a key feature gene. Further multi-omics analysis showed that the HIF1A/TUBB3 axis-associated module was closely associated with M2 macrophage polarization and arginine metabolic dysregulation; combined with virtual knockout analysis, these results further suggested that HIF1A may be located upstream of TUBB3 at the predicted regulatory-network level. Experimental analyses showed that Ophiopogon japonicus aqueous decoction intervention was associated with decreased HIF-1α and TUBB3 protein expression, reduced M2 macrophage polarization, and partial correction of arginine metabolic dysregulation. Structural simulation suggested that Ophiopogonin D could stably bind to HIF-1α and TUBB3. CONCLUSION: This study demonstrated that Ophiopogon japonicus aqueous decoction alleviates BLM-induced PF. Integrated multi-omics analysis and experimental evidence suggested that its anti-fibrotic effects may be associated with modulation of HIF1A/TUBB3 axis-associated M2 macrophage polarization and arginine metabolic dysregulation. Ophiopogonin D may be one of the candidate active constituents associated with these protective effects.