expression [52,53,54,55,56]. The characteristic steps taken by leukocytes to extravasate from blood to the site of inflammation caused by either exogenous or endogenous stimuli have been recognized as the ‘three-step’ paradigm of inflammatory cell recruitment that involve rolling, activation and adhesion [16]. The interaction between leukocytes and the endothelium comprises a variety of adhesive and migratory molecular events, including low affinity transient and reversible rolling adhesion, integrin-depen- dent firm adhesive interaction and migratory events of leukocytes through the endothelium and beyond that, such as the penetration of the membrane and migration into the interstitial space [16]. The initial process of active leukocyte recruitment is the tethering or rolling of leukocytes, described as the initial selectin-mediated interaction between leukocytes and endothelial cells [57]. Multiple studies have indicated that antibody blockade of selectins inhibit leukocyte rolling in vivo [16], P-selectin-/- mice show no leukocyte rolling in vivo [58] and double knockout mice for P- and E-selectin show a decrease in neutrophils mobilization [59]. The most important ligand for selectins is the P-selectin glycoprotein ligand-1 (PSGL-1), which is present on leukocytes and can bind to both P- and E-selectin [60]. Interestingly, PSGL-1 ligation in neutrophils by both P-selectin and E-selectin can result in activation of integrins, thus providing a link between rolling and the subsequent integrin-mediated firm adhesion [60]. Neutrophils and macrophages both use lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18; aLb2) and Mac-l (CD11b/ CD18; aMb2) for adhesion [61] in endothelial ICAMs, such as ICAM-1 and ICAM-2. Moreover, ICAM-1 expression is further increased after endothelial activation [62], and neutrophils- mediated adhesion to endothelial cells. In contrast, endothelial VCAM-1 is recognized by b1-integrin receptors predominantly found on lymphocytes and monocytes [63]. Another study demonstrated that induction of colitis in rats by TNBS is followed by up-regulation of endothelial VCAM-1, and suggests that VCAM-1 and constitutive ICAM-1 are major determinants of leukocyte recruitment to the inflamed intestine [64]. On the other hand, a relevant report established that VCAM-1 plays a central role in leukocyte recruitment in colitis and blockade of this adhesion molecule has higher therapeutic effect than immune neutralization of ICAM-1 or MAdCAM-1 in colitis experimental model. In the same study, chronic administration of anti-VCAM-1 antibody, but not anti-ICAM-1 or anti-MAdCAM-1, resulted in significant attenuation of colitis in terms of disease activity index, colon length, ratio of colon weight to length, and myeloperoxidase activity [65]. In this context, to gain further insight into the mechanisms through which euphol modulates cell migration and improves DSS-induced colitis in mice, we assessed the content of chemotactic factors and the expression of adhesion molecules after induction of colitis. Our results demonstrated that euphol almost totally suppressed the increase in mRNA expression of all integrins, accompanied by a significant decrease in the positive immunostaining of both selectin in colon tissue. Our present data suggest that euphol affects recruitment of leucocytes, especially neutrophils and macrophages, by regulating adhesion molecule expression in leucocytes and colonic endothelial cell. However, as described above the anti-inflammatory effect of euphol in the experimental colitis, could be justified mainly by the blockade of VCAM expression in the colonic tissue, however further studies are necessary to clarify this hypothesis. In conclusion, our results demonstrate that orally-administered euphol has both preventive and therapeutic anti-inflammatory properties when assessed in two models of colitis in mice. The beneficial action of euphol in ameliorating colitis seems likely associated with its ability to prevent the expression of pro- Euphol and Experimental Colitis inflammatory mediators and/or release in colonic tissue through inhibiting leukocyte influx (mainly neutrophils and macrophages) into inflammatory foci by blocking adhesion molecule expression, such as selectins (P- and E-selectins) and integrins (ICAM-1, VCAM-1 and LFA-1), without compromising the integrity of the endothelial barrier. Moreover, euphol treatment markedly inhib- ited the activation of NF-kB in the mouse colon tissue. These results suggest that euphol might constitute a potential candidate for the treatment of IBD. Materials and Methods Experimental animals Male CD1 mice (8–10 weeks of age) were obtained from the Laborato´ rio de Farmacologia Experimental (LAFEX), Universi- dade Federal de Santa Catarina (UFSC, Floriano´polis, SC, Brazil) and housed in collective cages at 22 6 1uC under a 12-h light/ dark cycle (lights on at 07:00 h), with free access to food and tap water. All experiments were performed during the light phase of the cycle. The experimental procedures were previously approved by UFSC’s Committee on the Ethical Use of Animals and were carried out in accordance with Brazilian regulations on animal welfare (CEUA/UFSC protocol number 23080.030926/2010-62). DSS-induced colitis This model of colitis was employed as previously described [66] and consisted of adding 3% w/v dextran sodium sulfate (30– 50 kD, MP Biomedicals, Cleveland, OH, USA) to the animals’ drinking water for 5 days, followed by another 2 days during which they were offered DSS-free (i.e., plain) drinking water alone. Control healthy mice (non colitic) received plain drinking water at all times. TNBS-induced colitis This colitis model employed here was originally described [67] and slightly modified [6]. After being deprived of food for 18–24 h with free access to a 5% glucose solution, mice were randomly divided into control healthy and colitis groups. Briefly, mice deprived of food for 1 day were lightly anaesthetized by administration of xylazine (10 mg/kg, i.p.) and ketamine (80 mg/kg, i.p.), and then a catheter (polyethylene PE-50) was carefully inserted into the colon (4 cm proximal to the anus). To induce colitis, TNBS (1.5 mg in 100 mL of 35% ethanol solution) was slowly administered (day 0). To assure the distribution of TNBS within the entire colon, mice were carefully maintained at a 45u angle (head down position) for 2 min and then returned to their cages. Four hours later, the animals were given free access to food and water. Throughout the experiments, mice were monitored for body weight loss and overall mortality. At 72 h following TNBS administration (day 3), i.e., 4 h after receiving the last injection, the animals were killed, and the colon was removed, dissected and opened lengthwise. Treatment protocols The compound euphol was diluted in 5% Tween 80 solution made in saline (0.9% NaCl solution) and administered orally by gavage (p.o.). In the experiments involving DSS-induced colitis, mice received euphol (3, 10 and 30 mg/kg, p.o.) or vehicle (5% Tween 80 in saline 0.9% NaCl) twice a day from day 0 to day 7 (preventive treatment) or with 30 mg/kg from day 3 to day 7 (therapeutic treatment), and were sacrificed 4 h after receiving the last administration. After that the dose of 30 mg/kg (p.o.) euphol was used in subsequent experiments. To evaluate the potential therapeutic effect of euphol in TNBS-induced colitis, animals were PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27122