Pirfenidone attenuates IL-1β-induced COX-2 and PGE2 production in orbital fibroblasts through suppression of NF-κB activity

The aim of this study was to determine the effect of pirfenidone on interleukin (IL)-1 beta-induced cyclooxygenase (COX)-2 and prostaglandin (PG)E-2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultures of orbital fibroblasts from patients with TAO (n = 4) and non-TAO subjects (n = 4) were prepared. The level of PGE(2) in orbital fibroblasts treated with IL-1 beta in the presence or absence of pirfenidone was measured using an enzyme-linked immunosorbent assay. The effect of pirfenidone on IL-1 beta-induced COX-2 expression in orbital fibroblasts from patients with TAO was evaluated by reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR analyses, and verified by Western blot. Activation of nuclear factor-kappa B (NF-kappa B) was evaluated by immunoblotting, for inhibitor of kappa B (I kappa B)alpha and phosphorylated I kappa B alpha, and DNA-binding activity of p50/p65 NF-kappa B was analyzed by electrophoretic mobility shift assay. In addition, IL-1 receptor type 1 (IL-1R1) expression was assessed by RT-PCR in IL-1 beta-treated cells with or without pirfenidone. Pirfenidone significantly attenuated IL-1 beta-induced PGE(2) release in both TAO and non-TAO cells. IL-1 beta-induced COX-2 mRNA and protein expression decreased significantly following co-treatment with pirfenidone. IL-1 beta-induced I kappa B alpha phosphorylation and degradation decreased in the presence of pirfenidone and led to decreased nuclear translocation and DNA binding of the active NF-kappa B complex. In our system, neither IL-1 beta nor pirfenidone co-treatment influenced IL-1R1 expression. Our results suggest that pirfenidone attenuates the IL-1 beta-induced PGE(2)/COX-2 production in TAO orbital fibroblasts, which is related with suppression of the NF-kappa B activation. (c) 2013 Elsevier Ltd. All rights reserved.