Roflumilast N-oxide, a PDE4 inhibitor, curbs bleomycin-induced lung fibroblast activation in vitro

Objective: Activated lung fibroblasts may foster small airway thickening in COPD. Roflumilast, an oral, selective PDE4 inhibitor approved in EU for severe COPD, mitigates bleomycin (BLM)-induced lung fibrosis in vivo . This study addressed whether roflumilast N-oxide (RNO), the active metabolite of roflumilast, modulates effects of BLM on human lung fibroblasts (HLF) in vitro . RNO was used at 2nM, corresponding to therapeutic plasma levels. Methods: HLF pre-incubated with RNO (2nM) were exposed to BLM for 24 or 48h. Reactive oxygen species (ROS) were quantified by 29,79-dichlorofluorescein (DCF) accumulation from dichlorodihydrofluorescein diacetate. Total glutathione (GSH) was measured with the GSH reductase DTNB protocol. Proliferation and collagen synthesis was assessed as [ 3 H] thymidine and [ 3 H] proline incorporation (*p # p Results: BLM (24h) at 50 and 100μg ml -1 enhanced DCF accumulation by 1.9* and 1.4*-fold of control. RNO (2nM) reduced this increment by 50% # and 85% # , respectively. In parallel, total GSH was reduced by BLM (100μg ml -1 ) yet rescued by RNO (2nM) (nmol mg protein -1 : Control 32.2±1.1, BLM 26.6±1.3*, BLM+2nM RNO 30±1.7 # ). BLM (50μg ml -1 ) increased [ 3 H] thymidine incorporation in HLF by 1.4*-fold that was abolished by RNO (2nM). Finally, RNO (2nM) reduced [ 3 H] proline incorporation that was increased to 179±35%* of control (100%) with BLM (50μg ml -1 ) to 83±13% # of control. RNO (2nM) did not affect basal ROS, total GSH, [ 3 H] thymidine or [ 3 H] proline incorporation. Conclusions: BLM augmented ROS formation, reduced total GSH and increased proliferation and collagen synthesis of HLF in vitro . RNO (2nM) prevented these effects.