Hydronidone Mitigates Pulmonary Fibrosis by Regulating the TGF-β/Smad2/3 Axis in in Vitro and in Vivo Models

Pulmonary fibrosis (PF) is a progressive, life-threatening lung disease marked by excessive accumulation of extracellular matrix (ECM), especially collagen, which leads to lung stiffening and impaired respiratory function. Although antifibrotic drugs like nintedanib and pirfenidone can slow disease progression, their clinical benefits remain modest. Hydronidone, a structural analogue of pirfenidone, has shown reduced/no hepatotoxicity, improved tolerability (in humans), and proven antifibrotic effects in liver fibrosis. This study investigated hydronidone's therapeutic potential in pulmonary fibrosis through both in vitro and in vivo models. In vitro studies utilizing transforming growth factor-β (TGF-β)-induced fibrotic differentiation in LL29 and DHLF cells revealed that hydronidone (62.5, 125, and 250 μM) significantly attenuated the expression of fibrotic markers at both the gene and protein levels, demonstrating greater efficacy compared with pirfenidone (500 μM). In an in vivo manner, a bleomycin (BLM)-induced PF mouse model was employed. BLM administration led to significant physiological and histological alterations, including body weight loss, elevated lung index, alveolar wall thickening, and excessive collagen deposition. Hydronidone treatment significantly attenuated these pathological changes in a dose-dependent manner. Moreover, it improved the lung functional capacity and suppressed BLM-induced upregulation of fibrotic gene and protein expression in lung tissues. Mechanistic studies revealed that hydronidone binds to TGF-β receptor-1 kinase with a better docking score of -7.245 kcal/mol compared to the ligand binding score and inhibits the TGF-β/SMAD (Suppressor of mothers against Decapentaplegic) signaling pathway, similar to pirfenidone. Hydronidone effectively reduces pulmonary fibrotic marker expression and improves lung function at lower doses (25 and 50 mg/kg) than pirfenidone (100 mg/kg) without compromising the safety profile. These findings support its potential as a promising antifibrotic agent and warrant further clinical investigation.