Calcaratarin D, a labdane diterpenoid, attenuates bleomycin-induced pulmonary fibrosis by blocking Wnt/β-catenin...

Idiopathic pulmonary fibrosis (IPF) is one of the most common interstitial lung diseases with a high mortality rate. Calcaratarin D (CalD), a labdane diterpenoid, has been shown to possess anti-inflammatory properties. The present study evaluated the therapeutic potential of CalD in pulmonary fibrosis. A single dose of bleomycin (BLM, 2.5 mg/kg) was instilled intratracheally in mice for up to 21 days to develop lung fibrosis. Oral CalD (50 mg/kg) reduced BLM-induced inflammatory cell infiltration, especially pro-fibrotic Arg1-expressing interstitial macrophages in the bronchoalveolar lavage fluid. During the late fibrotic phase, CalD decreased BLM-induced mortality and body weight loss. In addition, CalD ameliorated lung histopathology, reduced collagen deposition and mucus hypersecretion, and improved lung functions in BLM-exposed mice. Furthermore, CalD modulated the levels of pro-inflammatory cytokines, chemokines, and growth factors in BAL fluid and lung tissues. In mouse lungs, BLM selectively upregulated Wnt10A level and promoted β-catenin nuclear translocation. CalD not only blocked Wnt10A/β-catenin signaling pathway but also reduced pro-fibrotic markers such as collagens, α-SMA and FHL2. In normal human lung fibroblasts, CalD inhibited TGF-β1-stimulated pro-fibrotic markers and Wnt/β-catenin signaling pathway by reducing Wnt10A production, upregulating endogenous Wnt antagonist DKK1 level, dephosphorylating Wnt ligand co-receptor LRP6, and preventing β-catenin and YAP/TAZ nuclear translocation. The antifibrotic action of CalD was shown to be dependent on its α,β-unsaturated γ-butyrolactone structure that is essential for CalD to form covalent interaction with cellular protein targets. Our results imply that CalD could be a novel antifibrotic agent for IPF, acting through blockade of the Wnt/β-catenin signaling pathway.