Euphol and Experimental Colitis Figure 6. Euphol prevents inflammatory/enterocyte cells proliferation and NF-kB activation after DSS-induced colitis. Expression of Ki67 and phosphorylation of NF-kB was performed 7 days after administration of DSS (3%) or with vehicle in colonic tissues. Pre-treatment with euphol (30 mg/kg, p.o.), significantly inhibited proliferation index (Ki67) (A) and phosphorylation of p65 NF-kB (B) in colon tissue after DSS-induced colitis in mice. (A–B) Representative images of Ki67 and phospho-p65 NF-kB immunoreactivity in colon tissue. Scale bar corresponds to 100 and 25 mm (black square) respectively, and applies throughout. Graphical representation of the immunostaining for Ki67 (C) and phospho-p65 NF-kB (D) expression evaluated in colon tissue. The mean intensity of Ki67 and p65 NF-kB staining were determined from image analysis and are represented as optical density. Each column represents the mean 6 S.E.M. of 8 to 10 mice per group and is representative of two independent experiments. #P,0.05 vs. control healthy group (non colitic); *P,0.05 vs. DSS-treated group. doi:10.1371/journal.pone.0027122.g006 and the migration of leucocytes at the site of the insult [21]. However, so far, there have been no reports about the effect of euphol on inflammatory bowel disease, such as UC and CD. Our results demonstrated the effectiveness of the isolated natural tetracyclic triterpene euphol in ameliorating two experimental model of colitis in mice. Euphol, given orally, consistently ameliorated the inflammatory symptoms associated with DSS- induced colitis, accompanied by a direct reduction in neutrophils migration, overproduction of pro-inflammatory mediators, mac- rophage infiltration and activation. Of interest, it was observed that euphol produced a marked decrease in adhesion molecule expression, such as integrins (ICAM-1, VCAM-1 and LFA-1) and selectins (P- and E-selectins), without compromising the integrity of the endothelial barrier, as seen by scanning electron microscopy. In addition, the present study clearly showed that euphol treatment consistently reduced activation of NOS-2, VEGF, Ki67 and p65 NF-kB. Furthermore, we also demonstrated that euphol treatment ameliorated TNBS-induced colitis, suggest- ing that the anti-inflammatory effect of euphol did not depend on the animal model of colitis used. Several studies support a crucial role for neutrophils in mediating tissue injury and clinical symptoms in colitis [7,8,43]. Here, DSS-mediated colitis induces MPO activity and relevantly treatment with euphol prevented the increase in MPO activity. The reduction in neutrophils influx after euphol treatment observed in this study was associated with a decrease in colon damage. Interestingly, euphol was able to modulate the release and/or expression of pro-inflammatory cytokines/chemokines in PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27122