title: Pirfenidone attenuates IL-1β-induced COX-2 and PGE2 production in orbital fibroblasts through suppression of NF-κB activity.
abstract: The aim of this study was to determine the effect of pirfenidone on interleukin (IL)-1β-induced cyclooxygenase (COX)-2 and prostaglandin (PG)E2 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 PGE2 in orbital fibroblasts treated with IL-1β in the presence or absence of pirfenidone was measured using an enzyme-linked immunosorbent assay. The effect of pirfenidone on IL-1β-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-κB (NF-κB) was evaluated by immunoblotting for inhibitor of κB (IκB)α and phosphorylated IκBα, and DNA-binding activity of p50/p65 NF-κB was analyzed by electrophoretic mobility shift assay. In addition, IL-1 receptor type 1 (IL-1R1) expression was assessed by RT-PCR in IL-1β-treated cells with or without pirfenidone. Pirfenidone significantly attenuated IL-1β-induced PGE2 release in both TAO and non-TAO cells. IL-1β-induced COX-2 mRNA and protein expression decreased significantly following co-treatment with pirfenidone. IL-1β-induced IκBα phosphorylation and degradation decreased in the presence of pirfenidone and led to decreased nuclear translocation and DNA binding of the active NF-κB complex. In our system, neither IL-1β nor pirfenidone co-treatment influenced IL-1R1 expression. Our results suggest that pirfenidone attenuates the IL-1β-induced PGE2/COX-2 production in TAO orbital fibroblasts, which is related with suppression of the NF-κB activation.
authors.affiliation: Department of Physiology, Tissue Injury Defense Research Center, Ewha Womans University School of Medicine, Seoul, Republic of Korea.
authors.forename: Youn-Hee
authors.initials: YH
authors.lastname: Choi
authors.forename: Keum Ok
authors.initials: KO
authors.lastname: Back
authors.forename: Hee Ja
authors.initials: HJ
authors.lastname: Kim
authors.forename: Sang Yeul
authors.initials: SY
authors.lastname: Lee
authors.forename: Koung Hoon
authors.initials: KH
authors.lastname: Kook
mesh_terms: D000328:Adult; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000235:genetics / Q000378:metabolism*; D004357:Drug Synergism; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D049970:Graves Ophthalmopathy / Q000473:pathology; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009915:Orbit / Q000473:pathology*; D011728:Pyridones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D053573:Receptors, Interleukin-1 Type I / Q000235:genetics
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D053583:Interleukin-1beta; D016328:NF-kappa B; D011728:Pyridones; D012333:RNA, Messenger; D053573:Receptors, Interleukin-1 Type I; C093844:pirfenidone; D051546:Cyclooxygenase 2; D015232:Dinoprostone
keywords: COX; COX-2; IL; IL-1β; NF; NF-κB; PG; PGE(2); TAO; cyclooxygenase; interleukin; nuclear factor; orbital fibroblast; pirfenidone; prostaglandin; thyroid-associated ophthalmopathy