Roflumilast prevents ischemic stroke-induced neuronal damage by restricting GSK3β-mediated oxidative stress and IRE1α/TRAF2/JNK pathway

Inhibition of phosphodiesterase 4 (PDE4) protects against neuronal apoptosis induced by cerebral ischemia. However, the exact mechanisms responsible for the protection of PDE4 inhibition have not been completely clarified. Roflumilast (Roflu) is an FDA-approved PDE4 inhibitor for the treatment of chronic obstructive pulmonary disease. The potential protective role of Roflu against ischemic stroke-associated neuronal injury remains unexplored. In this study, we investigated the effect and mechanism of Roflu against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) and in vivo rat middle cerebral artery occlusion (MCAO) models. We demonstrated that Roflu significantly reduced the apoptosis of HT-22 cells exposed to OGD/R, enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2), and reduced oxidative stress. Treatment with Roflu increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3 beta) but decreased the level of phosphorylated inositol requiring enzyme 1 alpha (IRE1 alpha). Interestingly, constitutively active GSK3 beta (S9A) mutation abolished the effects of Roflu on oxidative stress and IRE1 alpha phosphorylation. Moreover, Roflu decreased the binding of IRE1 alpha to tumor necrosis factor receptor-associated factor 2 (TRAF2) and attenuated the phosphorylation of c-Jun N-terminal kinase (JNK). We also found that PDE4B knockdown reduced the phosphorylation of both IRE1 alpha and JNK, while overexpression of PDE4B antagonized the role of PDE4B knockdown on the activation of IRE1 alpha and JNK. Besides, the inhibition of PDE4 by Roflu produced similar effects in primary cultured neurons. Finally, Roflu ameliorated MCAO-induced cerebral injury by decreasing infarct volume, restoring neurological score, and reducing the phosphorylation of IRE1 alpha and JNK. Collectively, these data suggest that Roflu protects neurons from cerebral ischemia reperfusion-mediated injury via the activation of GSK3 beta/Nrf-2 signaling and suppression of the IRE1 alpha/TRAF2/JNK pathway. Roflu has the potential as a protective drug for the treatment of cerebral ischemia.