Euphol and Experimental Colitis Figure 4. Euphol reduces pro-inflammatory cytokines and chemokines production in macrophages stimulates with lipopolysac- charide (LPS). Macrophage from bone marrow of na¨ıve mice were stimulated with LPS (1 mg/ml) in the presence or absence of euphol (1 and 10 mM) for 24 hours, and the culture supernatants were analyzed for cytokine levels using cytokine bead array kit (CBA). Euphol incubation in dose- related manner reduced production of MCP-1 (A), TNF-a (B), IL-6 (C), IFN-c (D), but increase the IL-10 levels (E). Data are reported as means 6 SEM (n = 4) and is representative of two independent experiments. #P,0.05 vs. control without LPS treatment (vehicle solution); *P,0.05 vs. LPS-treated group. Vehicle solution corresponds to 5% Tween 80 in medium. doi:10.1371/journal.pone.0027122.g004 through which this compound modulates intestinal inflammation (Fig. 6 B,D). Anti-inflammatory effect of euphol is mediated by inhibition of adhesion molecule expression in colonic tissue The process of leukocyte extravasations, a critical step in the inflammatory response, involves migration of leukocytes from the bloodstream toward colon tissue, which is orchestrated by the combined actions of cellular adhesion receptors (selectins and integrins) and chemotactic factors {Barreiro, #603}. In this set of experiments, we assessed whether oral treatment with euphol was able to decrease the expression of adhesion molecules in colonic tissue. In the first set of experiments, we assessed mRNA expression of adhesion molecules on day 7 after the induction of colitis. In the DSS group (untreated), a pronounced increase in colonic ICAM-1 (Fig. 7A), VCAM-1 (Fig. 7B) and LFA-1 (Fig. 7C) mRNA expression was observed. Interestingly, preventive treat- ment with euphol (30 mg/kg) markedly inhibited the up-regulated mRNA expression of ICAM-1, VCAM-1 and LFA-1 (Fig. 7) in colonic tissue. In addition, to determine the effect of euphol in regulating selectins, we performed immunofluorescence analysis of colon samples obtained from mice after DSS-induced colitis. Tissue sections from control healthy mice exhibited very low levels of specific staining for P-selectin (Fig. 7D) and E-selectin (Fig. 7E) in endothelial cells only. In contrast, 7 days after DSS administration, intense immunostaining for both P- and E-selectin expression in colon tissue was observed; however, preventive treatment with euphol (30 mg/kg, p.o.) during the 7 days after DSS administration resulted in a significant decrease in the positive immunostaining for P- and E-selectin (Fig. 7 D,E). Euphol treatment attenuates the severity of TNBS-induced colitis Recently, our group has shown that 72 h after TNBS administration, mice developed severe diarrhea, striking hyper- emia, necrosis and inflammation accompanied by an extensive wasting disease, rectal prolapse and sustained weight loss [6]. To assess whether or not euphol treatment would also prove to be beneficial in another chemical model of colitis, we tested its effects on some parameters of colitis induced by the hapten TNBS, which constitutes a Crohn’s disease model. Rectal administration of TNBS dissolved in ethanol induced severe colitis in CD1 mice that was characterized by weight loss and diarrhea. Treatment with euphol (30 mg/kg, p.o.), twice a day, starting 24 h after TNBS administration (i.e., at a time point when colitis was already established), significantly reduced body weight loss (Fig. 8A). At this same dose, euphol also reduced the macroscopic damage score (Fig. 8B) and the reduction in colon length (Fig. 8 C,D). Discussion Ulcerative colitis and Crohn’s disease are major forms of inflammatory bowel disease (IBD), a disease that affects millions of people worldwide and is characterized by chronic uncontrolled PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27122