Preventive and Therapeutic Euphol Treatment Attenuates Experimental Colitis in Mice Rafael C. Dutra1., Rafaela F. Claudino1., Allisson F. Bento1, Rodrigo Marcon1, E´ der C. Schmidt2, Zenilda L. Bouzon2, Luiz F. Pianowski3, Joa˜ o B. Calixto1* 1 Department of Pharmacology, Centre of Biological Sciences, Universidade Federal de Santa Catarina, Floriano´ polis, Brazil, 2 Department of Cell Biology, Embryology and Genetics, Central Laboratory of Electron Microscopy, Universidade Federal de Santa Catarina, Floriano´ polis, Brazil, 3 Pianowski and Pianowski Ltda., Sa˜o Paulo, Brazil Abstract Background: The tetracyclic triterpene euphol is the main constituent found in the sap of Euphorbia tirucalli. This plant is widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer, including leukaemia, prostate and breast cancers. Here, we investigated the effect of euphol on experimental models of colitis and the underlying mechanisms involved in its action. Methodology/Principal Findings: Colitis was induced in mice either with dextran sulfate sodium (DSS) or with 2,4,6- trinitrobenzene sulfonic acid (TNBS), and the effect of euphol (3, 10 and 30 mg/kg) on colonic injury was assessed. Pro- inflammatory mediators and cytokines were measured by immunohistochemistry, enzyme-Linked immunoabsorbent assay (ELISA), real time-polymerase chain reaction (RT-PCR) and flow cytometry. Preventive and therapeutic oral administration of euphol attenuated both DSS- and TNBS-induced acute colitis as observed by a significant reduction of the disease activity index (DAI), histological/microscopic damage score and myeloperoxidase (MPO) activity in colonic tissue. Likewise, euphol treatment also inhibited colon tissue levels and expression of IL-1b, CXCL1/KC, MCP-1, MIP-2, TNF-a and IL-6, while reducing NOS2, VEGF and Ki67 expression in colonic tissue. This action seems to be likely associated with inhibition of activation of nuclear factor-kB (NF-kB). In addition, euphol decreased LPS-induced MCP-1, TNF-a, IL-6 and IFN-c, but increased IL-10 secretion from bone marrow-derived macrophages in vitro. Of note, euphol, at the same schedule of treatment, markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue. Conclusions/Significance: Together, these results clearly demonstrated that orally-administered euphol, both preventive or therapeutic treatment were effective in reducing the severity of colitis in two models of chemically-induced mouse colitis and suggest this plant-derived compound might be a potential molecule in the management of inflammatory bowel diseases. Citation: Dutra RC, Claudino RF, Bento AF, Marcon R, Schmidt E´C, et al. (2011) Preventive and Therapeutic Euphol Treatment Attenuates Experimental Colitis in Mice. PLoS ONE 6(11): e27122. doi:10.1371/journal.pone.0027122 Editor: Lucienne Chatenoud, Universite´ Paris Descartes, France Received May 12, 2011; Accepted October 11, 2011; Published November 2, 2011 Copyright: 2011 Dutra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Amazoˆ nia Fitomedicamentos Ltda., Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnolo´ gico (CNPq), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de N´ıvel Superior, Programa de Apoio aos Nu´ cleos de Exceleˆncia and the Fundac¸a˜o de Apoio a` Pesquisa Cient´ıfica Tecnol o´gica do Estado de Santa Catarina, all of Brazil. R.C.D., A.F.B., R.M. and E.C.S. are Ph.D. students and receiving grants from CNPq. R.F.C. holds a postdoctoral fellowship from CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Luiz Francisco Pianowski is proprietor of the contract research organization Pianowski & Pianowski Ltda. He has received consulting fees from Amazoˆ nia Fitomedicamentos. In addition, Luiz F. Pianowski performed previous chemical characterization of euphol and provided it for this study. Luiz Francisco Pianowski and Joa˜o Batista Calixto are assigned in the patent on the analgesic action of euphol as inventors. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: calixto@farmaco.ufsc.br . These authors contributed equally to this work. Introduction Inflammatory bowel diseases (IBD) are a group of chronic disease that affect the gastrointestinal tract and have been mainly subdivided as ulcerative colitis (UC) and Crohn’s disease (CD). Although its etiopathogenesis has not been definitively elucidated, it is currently considered an abnormal inflammatory response to intestinal microbial flora. However, there is still not consensus whether or not IBD should be considered as an autoimmunity, since the loss of tolerance is towards gut microbiota and the autoantigen in the human body is still missing [1]. IBD is now considered as a result of continuous microbial antigenic stimula- tion of pathogenic immune responses as a consequence of host genetic defects in mucosal barrier function, innate bacterial killing or immunoregulation [2]. CD is characterized by TH type-1 response, marked by up- regulation of interferon-c (IFN- c), a key cytokine, which mainly driven T cells. On the other hand, UC appears to exhibit an contribution of TH2 responses (characterized by secretion of interleukin (IL)-4 and IL-13), which driven NK-T cells [3], although this difference in immune response is not yet well established. Interestingly, it was observed that in both CD and UC some pro-inflammatory mediators such as tumour necrosis factor- a (TNF-a), IL-6 and IL-1b are released in inflamed mucosa. It is now well recognized that inflammation of the intestinal mucosa is characterized by chronic inflammatory cell infiltration PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27122