The Quassinoid Isobrucein B Reduces Inflammatory Hyperalgesia and Cytokine Production by Post-transcriptional Modulation Rangel L. Silva, † Alexandre H. Lopes, † Rafael O. Franc ̧ a, † Sílvio M. Vieira, †,‡ Ellen C. C. Silva, ‡ Rodrigo C. N. Amorim, ‡ Fernando Q. Cunha, † Adrian M. Pohlit, ‡ and Thiago M. Cunha* ,† † Department of Pharmacology, Ribeirã o Preto Medical School, University of Sã o Paulo, Sã o Paulo, Brazil ‡ National Institute for Amazon Research (INPA), Manaus, Amazonas, Brazil * S Supporting Information ABSTRACT: Isobrucein B (1) is a quassinoid isolated from the Amazonian medicinal plant Picrolemma sprucei. Herein we investigate the anti-inflammatory and antihyperalgesic effects of this quassinoid. Isobrucein B (1) (0.5-5 mg/kg) inhibited carrageenan-induced inflammatory hyperalgesia in mice in a dose-dependent manner. Reduced hyperalgesia was associated with reduction in both neutrophil migration and pronociceptive cytokine production. Pretreatment with 1 inhibited in vitro production/release of cytokines TNF, IL-1β, and KC/CXCL1 by lipopolysaccharide-stimulated macrophages. To investigate its molecular mechanism, RAW 264.7 macrophages with a luciferase reporter gene controlled by the NF-κB promoter were used (RAW 264.7-Luc). Quassinoid 1 reduced the lumines- cence emission by RAW 264.7-Luc stimulated by different compounds. Unexpectedly, NF-κB translocation to macrophage nuclei was not inhibited by 1 when evaluated by Western blotting and immunofluorescence. Furthermore, quassinoid 1 did not change the levels of TNF mRNA transcription in stimulated macrophages, suggesting post-transcriptional modulation. In addition, constitutive expression of luciferase in RAW 264.7 cells transiently transfected with a plasmid containing a universal promoter was inhibited by 1. Thus, isobrucein B (1) displays anti-inflammatory and antihyperalgesic activities by nonselective post- transcriptional modulation, resulting in decreased production/release of pro-inflammatory cytokines and neutrophil migration. I nflammation can be initiated by tissue injury or infection. Enhancement of pain sensitivity (hyperalgesia) is one of the most common symptoms of inflammatory processes. This symptom is mainly caused by the sensitization of primary nociceptive neurons, which is triggered by several mediators released during the inflammatory response. 1,2 Pro-inflammatory cytokines are some of the most important mediators involved in inflammatory hyperalgesia. For instance, a cascade of cytokines initiated by the release of tumor necrosis factor-α (TNF) and keratinocyte-derived chemokine (KC/CXCL1) plays a crucial role in the induction of inflammatory hyperalgesia. 3 In this cascade of pronociceptive mediators, TNF stimulates interleukin-1β (IL-1β) production, which in turn stimulates the production of prostaglandins. Besides triggering the IL-1β/prostanoid pathway, KC/CXCL1 is also able to stimulate the sympathomimetic component of inflammatory hyperalgesia. 3 Importantly, the peripheral prono- ciceptive action of these cytokines in acute inflammation depends on neutrophil recruitment. 4 The inhibition of pro- inflammatory cytokines/chemokine action/production or neu- trophil migration may reduce inflammatory hyperalgesia and is a potential target for the development of new antihyperalgesic drugs. 1-5 In fact, some cytokine-targeting immunobiologicals reduce pain sensitivity and are already used in the treatment of inflammatory diseases. 6-8 However, immunobiologicals have cost and pharmacokinetic parameters as limitations. There is still a need to develop drugs that reduce the release/action of these cytokines. Furthermore, natural products represent an important source of structurally diverse compounds for the development of anti-inflammatory, analgesic, and other classes of drugs. 9,10 Isobrucein B (1) is a quassinoid natural product that can be isolated from root, stem, and leaf extracts of Picrolemma sprucei Hook. f. (Simaroubaceae). 11,12 Quassinoids have a wide variety of biological activities in vitro and/or in vivo, including antitumor, antimalarial, antiviral, anti-inflammatory, insecticidal, amoebicidal, anthelmintic, and antiulcer activities. 13-17 In- fusions and other preparations of the roots, stems, and leaves of P. sprucei are used traditionally by the peoples of the Amazon region to treat gastropathies, malaria, and helminth infec- tions. 11,13,14 Recently, it was demonstrated that mice pretreated with 1 exhibited reduced gastric damage after acute administration of a nonsteroidal anti-inflammatory drug. 17 In addition, gastroprotective action of 1 was associated with a reduction in the production of pro-inflammatory cytokines, Received: October 17, 2014 Article pubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/np500796f J. Nat. Prod. XXXX, XXX, XXX-XXX