Euphol and Experimental Colitis Figure 8. Therapeutic treatment with euphol protects mice against TNBS-induced acute colitis. Mice were given 100 mL of the TNBS (in 35% ethanol) and after 24 h, treated with euphol (30 mg/kg, p.o.). (A) The time-course of body weight changes on day 3 after TNBS-induced colitis. (B) Macroscopic score; (C) colon length after TNBS-induced colitis. (D) Representative photograph of colons from day 3 after the induction of TNBS-colitis. 1, Control healthy mice; 2, TNBS-treated (only vehicle administration); 3, TNBS plus euphol (30 mg/kg, p.o.). 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. TNBS-treated group. Vehicle corresponds to 5% Tween 80 in saline 0.9% NaCl. doi:10.1371/journal.pone.0027122.g008 expression and the euphol treatment consistently blocked their up- regulation after DSS-induced colitis, suggesting that blocking intestinal inflammation and patho-angiogenesis through inhibiting NOS2 and VEGF expression during IBD progression may be, at least in part, the explanation of how euphol attenuates experimental colonic inflammation. In addition, the pathway underlying the euphol protection of colon damage observed with preventive treatment could be associated with diminished of inflammatory and enterocyte cells proliferation (Ki67 immunostaining), which is a fundamental protein involved in cell proliferation [27]. This event seems to be associated with chronic inflammation and colon cancer and is increased under this condition [49]. In the present study, oral treatment with euphol notably reduced Ki67 protein activation in colonic tissue, suggesting that euphol ameliorates acute colitis, at least in parts, by reducing cell proliferation. However, these events might be an indirect consequence of the general improvement in inflammation mediated by euphol and need to be further clarified. Nuclear factor kB is a critical transcription factor involved in a broad range of biological processes, including the regulation of immune and inflammatory responses which has been associated to the pathogenesis of colitis and other IBDs [50]. Activation of the NF-kB transcription factor induces the expression of several pro- inflammatory mediators, such as cytokines, chemokines and adhesion molecules, which in turn mediate the recruitment and activation of immune cells [51]. Herein, our results using the phospho-p65 NF-kB antibody demonstrated that euphol is able to inhibit the translocation of p65 into the nucleus, thus strongly suggesting that inhibition of NF-kB activation should be a key mechanism through which this natural tetracyclic triterpene modulates intestinal inflammation. An increasing body of evidence has emerged indicating that cytokines and chemokines can up-regulate adhesion molecule PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27122