Down-Regulation of NOX4 Expression by Roflumilast N-oxide Reduces Markers of Fibrosis in Lung Fibroblasts

The phosphodiesterase 4 inhibitor rolumilast prevents bleomycin- (BLM-) induced lung ibrosis in animal models. However, its mechanism of action remains unknown.We investigated whether rolumilast N-oxide (RNO), the active metabolite of rolumilast, can modulate in vitro the oxidative efects of BLMon human lung ibroblasts (HLF). In addition, since BLMincreases the production of F2-isoprostanes that have per se ibrogenic activity, the effect of RNO on oxidative stress and fibrogenesis induced by the F2-isoprostane 8-epi-PGF2alpha was investigated. HLF were preincubated either with the vehicle or with RNO and exposed to either BLM or 8-epi-PGF2alpha. Proliferation and collagen synthesis were assessed as [3H]-thymidine and [3H]-proline incorporation. Reactive oxygen species (ROS) and F2-isoprostanes were measured. NADPH oxidase 4 (NOX4) protein and mRNA were also evaluated. BLMincreased both cell proliferation and collagen synthesis and enhanced ROS and F2-isoprostane production. These effects were significantly prevented by RNO. Also, RNO significantly reduced the increase in both NOX4 mRNA and protein, induced by BLM. Finally, 8-epi-PGF2alpha per se stimulated HLF proliferation, collagen synthesis, and NOX4 expression and ROS generation, and RNO prevented these effects. Thus, the antiibrotic effect of RNO observed in vivo may be related to its ability to mitigate ROS generation via downregulation of NOX4.