Integrated UPLC-Q-Orbitrap-MS/MS, network pharmacology, and in vivo evaluation reveal bioactive constituents and mechanisms of Bushen Tongluo Decoction against idiopathic pulmonary fibrosis.

The active constituents and mechanisms of Bushen Tongluo Decoction (BTD) in idiopathic pulmonary fibrosis (IPF) remain unclear. We profiled BTD constituents and serum compounds by ultra‑high‑performance liquid chromatography‑quadrupole‑Orbitrap high‑resolution tandem mass spectrometry (UPLC-Q-Orbitrap-MS/MS) and combined network pharmacology, in vivo evaluation, molecular docking, molecular dynamics (MD) simulation, and surface plasmon resonance (SPR) analysis to identify candidate bioactive compounds and associated mechanisms.We identified 614 constituents in BTD and 100 serum compounds, including 50 prototype compounds and 50 metabolites; 32 met Level 1 identification criteria. Network pharmacology identified 171 shared targets and prioritized transforming growth factor‑beta (TGF-β)- and mitogen‑activated protein kinase (MAPK)-related pathways. BTD reduced histopathological injury, collagen accumulation, and α‑smooth muscle actin (α‑SMA) and fibronectin 1 (FN1) expression, with stronger effects at the higher dose. Treatment was associated with changes in TGF-β1, Smad2/3, extracellular signal‑regulated kinase (ERK), and their phosphorylated forms. Ellagic acid showed the most favorable predicted binding energy at the latency-associated peptide (LAP)- growth factor (GF) interface of TGF-β1. MD analyses supported relative complex stability and yielded an estimated binding free energy of -59.43 ± 4.79 kJ/mol. SPR analysis detected binding between ellagic acid and TGF-β1, yielding a KD of 3.63 × 10⁻⁶ M. These findings indicate that BTD attenuated experimental pulmonary fibrosis and nominate ellagic acid as a candidate active constituent, and support the involvement of TGF-β1/Smads/ERK-related signaling in its effects on experimental pulmonary fibrosis.