RT Journal Article SR Electronic T1 Molecular mechanisms of pirfenidone activity in human lung fibroblasts JF European Respiratory Journal JO Eur Respir J FD European Respiratory Society SP P825 VO 44 IS Suppl 58 A1 Enrico Conte A1 Mary Fruciano A1 Elisa Gili A1 Evelina Fagone A1 Maria Iemmolo A1 Carlo Vancheri YR 2014 UL http://erj.ersjournals.com/content/44/Suppl_58/P825.abstract AB Pirfenidone is a broad-spectrum anti-inflammatory and anti-fibrotic molecule that has been shown to inhibit the fibrosis progression in animal models and in patients with idiopathic pulmonary fibrosis. Even though the anti-fibrotic and anti-inflammatory properties of pirfenidone are well established both in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated.In this research, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs), and related molecular mechanisms.HLF cultures were established from surgical specimens and used up to passage eight. After 24 h serum starvation, cells were grown for 48h in 2% FCS medium resting or treated with pirfenidone (0.16-1.6 mM) in the absence or presence of TGF-β (5 ng/mL) added at the same time as pirfenidone treatment. Cell proliferation was evaluated by cell count as well as a BrdU incorporation assay. The expression levels of α-smooth muscle actin (SMA) and pro-collagen (Col)-I were evaluated by quantitative RT-PCR and western blot. Phosphorylation status of Smad3, p38, and Akt was assessed by specific immunoblot.Pirfenidone significantly inhibited fibroblast proliferation and attenuated TGF-β-induced α-SMA and Col-I expression at both mRNA and protein levels. Moreover, pirfenidone reduced TGF-β-induced phosphorylation of Smad3, p38, and Akt, key mediators of the TGF-β pathway.Considered together, these results demonstrate that pirfenidone regulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts as well as fibrogenic activity by inhibiting key TGF-β-induced signaling pathways' activation.