Roflumilast Ameliorates Isoflurane-Induced Inflammation in Astrocytes via the CREB/BDNF Signaling Pathway

Background and purpose: Astrocyte-mediated neuroinflammation plays an important role in anesthetic isoflurane-induced cognitive impairment. Roflumilast, a selective inhibitor of phosphodiesterase-4 (PDE-4) used for the treatment of chronic obstructive pulmonary disease (COPD), has displayed a wide range of anti-inflammatory capacity in different types of cells and tissues. In the current study, we aimed to investigate whether roflumilast possesses a protective effect against isoflurane-induced insults in mouse primary astrocytes. Methods: Primary astrocytes were isolated from the cerebral cortices of immature rats. The production of NO was determined using DAF-FM DA staining assay. QRT-PCR and western blot were used to evaluate the expression levels of iNOS, COX-2, and BDNF in the astrocytes treated with different therapies. The gene expressions and concentrations of IL-6 and MCP-1 released by the astrocytes were detected using qRT-PCR and ELISA, respectively. The expression levels of phosphorylated CREB and PGE₂ were determined using western blot and ELISA, respectively. H89 was introduced to evaluate the function of CREB. Recombinant human BDNF and ANA-12 were used to verify the role of BDNF. Results: The upregulated iNOS, excessive production of NO, IL-6, and MCP-1, and activated COX-2/PGE₂ signaling pathways in the astrocytes induced by isoflurane were significantly reversed by the introduction of roflumilast, in a dose-dependent manner. Subsequently, we found that BDNF could be upregulated by roflumilast, which was verified to be related to the activation of CREB and blocked by H89 (a CREB inhibitor). In addition, the COX-2/PGE₂ signaling pathway activated by isoflurane can be inactivated by recombinant human BDNF. Finally, the regulatory effect of roflumilast against the isoflurane-activated COX-2/PGE₂ signaling pathway was significantly blocked by ANA-12, which is a BDNF inhibitor. Conclusion: Roflumilast might ameliorate isoflurane-induced inflammation in astrocytes via the CREB/BDNF signaling pathway.