In vitro evaluation of ilexgenin A as a potential modulator of TGF-β/ALK5 signaling in idiopathic interstitial pneumonia-related fibrosis.

We aimed to evaluate the antifibrotic effects of ilexgenin A (ILA) in vitro and to determine its underlying mechanisms. Human pulmonary fibroblasts and alveolar epithelial cells were stimulated with transforming growth factor-β1 (TGF-β1) and treated with non-cytotoxic concentrations of ILA. Fibroblast activation, extracellular matrix (ECM) production, collagen contractility, epithelial barrier integrity, and epithelial-mesenchymal transition (EMT) were assessed. Smad signaling was analyzed by Western blot, and the site of action was investigated using molecular docking and a constitutively active ALK5 mutant (T204D). ILA dose-dependently suppressed α-SMA expression, collagen I/III accumulation, and matrix contractility in fibroblasts. In epithelial cells, barrier disruption and EMT induced by TGF-β1 were attenuated, as evidenced by restored TEER, reduced permeability, recovery of tight junction proteins, and modulation of E-cadherin and N-cadherin. Mechanistically, ILA reduced Smad2/3 phosphorylation without altering TGFBR1 abundance. Docking analysis suggested possible structural compatibility between ILA and ALK5. The loss of ILA's efficacy in ALK5 (T204D) models suggested that ILA may act at or upstream of the ligand-dependent receptor activation stage and thereby attenuate downstream Smad signaling. These findings support ILA as a potential natural modulator of TGF-β/ALK5/Smad signaling in idiopathic interstitial pneumonia for IIP-related fibrosis.