Roflumilast N-oxide, a PDE4 inhibitor, inhibits fibrotic changes induced by isoprostanes in human lung fibroblasts

Oxidative stress plays an critical role in the pathogenesis of pulmonary fibrosis. Isoprostanes, the most proximal products of lipid peroxidation, are known mediators of important biological effects and have been found to be increased in fibrosis. Roflumilast, a PDE4 inhibitor, has been demonstrated to mitigate the oxidative stress-induced lung fibrotic response in vivo. In this study we evaluated in vitro the effect of roflumilast N-oxide (RNO), the active metabolite of roflumilast, on oxidative stress and some markers of fibrotic response induced by isoprostanes. Human lung fibroblasts (HLF) were incubated in the absence or presence of RNO (2nM-1M) for 30 min and then treated with 8-epi-PGE2the most represented isomer of the species). 8-epi-PGE2 (10nM) induced a 1.3-fold increase of radical oxygen species (ROS) generation that was abolished by RNO both at the low (2nM) and at the high (1M) concentration (p<0.05). RNO did not change baseline ROS levels. The time course for total glutathione (GSH) levels showed that 8-epi-PGE2 (10nM) induced an increase (by 19% vs respective control, p<0.05) in total GSH levels after 6 h of incubation. RNO did not add to this effect. On the other hand, RNO (1µM) alone increased total GSH levels by 16% (p<0.05). Based on the latter findings the notion was raised that PDE4 inhibition may enhance total glutathione production possibly by inducing an increased expression of glutamate cysteine ligase (GCLC). In fact, RNO 1µM but also 8-epi-PGE2 (10nM) increased GCLC mRNA expression by 1.3 (p<0.05) and 1.2 fold (p<0.05), respectively. Furthermore, 8-epi-PGE2alpha (10nM) stimulated cell proliferation (+17%; p<0.05) and collagen synthesis (+27%; p<0.05) and RNO (2nM) completely abolished such an increase. Taken together, inhibition of PDE4 (by roflumilast N-oxide) curbs ROS formation, collagen synthesis and proliferation of human lung fibroblasts triggered by the isoprostane 8-epi-PGE2alpha. In addition, the PDE4 inhibitor enhanced total GSH.