higher piscicidal activity as compared with other synthetic pesticides, organophosphates and pyrethroids for the fish Hetero- pneustes fossilis [20]. Furthermore, a very recent study demonstrated that biopolymeric fraction (BET) from plant E. tirucalli showed dose dependent anti-arthritic activity and demonstrated in vivo suppression of CD4+ and CD8+ T cells associated with inhibition of intracellular IL-2 and IFN-c [21]. Our group has previously shown that euphol administered orally elicited pronounced and long-lasting analgesia when assessed in several rodent behavior models of inflammatory and neuropathic persistent pain and these actions were likely to be associated with the inhibition of TNF-a and IL-1b levels, as well as inhibition of transcription factors, such as nuclear factor-kB (NF-kB) and cyclic AMP response element- binding protein (CREB), both in the spinal cord and dorsal root ganglia (unpublished data). Despite great progress observed over the previous decades in understanding the cellular and molecular mechanisms involved in IBD, few effective and safe drugs have emerged to treat acute and chronic inflammatory bowel states. Therefore, new effective treatment for IBD is urgently needed. Hence, in the present study, we investigated the preventive and therapeutic potential effects of euphol in dextran sulfate sodium (DSS)- and 2,4,6- trinitrobenzene sulfonic acid (TNBS)-induced colonic inflamma- tion in mice. Herein, we report that the tetracyclic triterpene euphol can effectively ameliorate DSS- and TNBS-induced colitis by inhibiting pro-inflammatory mediators such as cytokines/ chemokines in the colonic tissue and in primary cultures of macrophages in vitro. Our data also indicated that the mechanisms underlying the anti-inflammatory activity of euphol was likely to be related with its ability to inhibit selectin and integrin expression in the endothelium, associated with the blocking of nitric oxide synthase-2 (NOS2), vascular endothelial growth factor (VEGF), and Ki67 expression in colonic tissue by modulating the nuclear transcription factor-kB (NF-kB). Results Euphol treatment attenuates the severity of DSS-induced acute colitis In mice with DSS-induced acute colitis, which resembles the acute phase of human ulcerative colitis, we observed hemorrhage in the colonic lumen, body weight loss and marked diarrhea with bloody stools, which ultimately resulted in a sharp increase of the disease activity index (DAI) from day 3 onwards, compared with control healthy (non-colitic) mice (Fig. 1). Euphol was adminis- tered orally at three different doses (3, 10 and 30 mg/kg) to detect potential dose-dependent effects. Preventive oral treatment with 30 mg/kg of euphol but not with 3 or 10 mg/kg significantly reduced the DAI from day 3 onwards (Fig. 1B). DSS administra- tion was associated with significant body weight loss on days 5, 6 and 7. Mice treated with euphol (30 mg/kg) were protected from marked body weight loss and recovered a healthy appearance that was similar to that of control healthy mice (non-colitic) (Fig. 1C). DSS administration resulted in colon inflammation associated with hyperemia, ulceration and bowel wall thickening, leading to an increase in macroscopic colon damage and decrease in colon length (Fig. 1 D,E). Oral euphol (30 mg/kg) treatment reduced macroscopic damage (Fig. 1D) and significantly prevented colon length reduction (Fig. 1E) on the 7th day after colitis induction. In contrast, treatment with euphol at doses of 3 or 10 mg/kg, p.o., was not able to reduce macroscopic damage, prevent colon length reduction and recover the loss in body weight. Based on these results, the dose of 30 mg/kg of euphol was used in subsequent Euphol and Experimental Colitis experiments to investigate some of the mechanisms underlying its anti-inflammatory effects. Next, we determined whether euphol also had a therapeutic effect on DSS-induced acute colitis by administering it after colitis symptoms were seen on day 3, since although preventive treatments are important, treatments that have efficacy after colitis symptoms have been established are more clinically relevant. The results for the disease activity index (Fig. 1B), body weight loss (Fig. 1C), macroscopic damage (Fig. 1D) and colon length (Fig. 1E) showed that the therapeutic effect of euphol (30 mg/kg, p.o.) was as effective as its preventive effect in alleviating the severity of DSS-induced acute colitis. Euphol treatment inhibited polymorphonuclear leukocyte influx and reduced colon damage Several studies have suggested that tissue damage and inflammatory signals in experimental colitis are mainly mediated by polymorphonuclear leukocytes (PMN), mainly neutrophils [6,8]. We therefore assessed whether the effect of euphol treatment in DSS-mediated colitis was associated with alterations in the composition of the neutrophil population in the intestinal mucosa. Seven days after the initiation of DSS treatment, mucosal neutrophils infiltration into the colon was indirectly assessed by measuring MPO activity. Colonic samples taken from untreated (DSS) mice displayed significantly increased MPO levels relative to control healthy mice (non-colitic) (Fig. 2A). Notably, preventive oral treatment with euphol at 10 and 30 mg/kg, but not 3 mg/kg, significantly reduced DSS-induced increase in colonic MPO levels (Fig. 2A). Of note, therapeutic treatment with euphol (30 mg/kg, p.o.) also inhibited MPO levels (Fig. 2A). To further investigate the effect of euphol treatment in the architecture and integrity of colonic structure and confirm results from MPO analysis, colons were processed for histological observation. Mice with colitis induced by DSS exhibited disruption of the epithelial barrier, a marked decrease in the number of crypts, and marked infiltration of inflammatory cells, predominantly neutrophils, into the mucosa and sub mucosa of the colon, corroborating the MPO assay (Fig. 2B). The histological evaluation of colons from euphol- treated (30 mg/kg, p.o.) mice revealed a pronounced reduction in the inflammatory response with moderate loss of epithelial cells and minimal inflammatory infiltration into the colonic tissue, resulting in a decreased microscopic damage score, compared with colons from DSS mice (untreated) (Fig. 2 B,C). Euphol prevents epithelial surface lesion in colonic tissue Next, we assessed whether euphol treatment could restore the morphological characteristics of inflamed colonic mucosa follow- ing DSS administration using scanning electron microscopy (SEM) [22]. SEM observations of the colonic mucosa in control healthy mice showed a normal epithelium with crypts and some granulated mast cells. The mucosal surface appeared to be subdivided by well-defined concave grooves and regular-shaped crypt openings containing mucin like material. A regular microvillus carpet makes the epithelial surface smooth and velvety (Fig. 2D). The DSS-group showed degenerated epithelium, severe inflammatory cell infiltration, widened grooves, dilatations of glandular crypts losing their regular shape by assuming fissure like aspects and depletion of goblet cells, leaving an irregular craterlike area (Fig. 2D). Relevantly, treatment with euphol (30 mg/kg, p.o.) following 7 days of DSS treatment significantly restored the architecture of the colon epithelium with a marked decrease in inflammatory cell infiltration compared with the DSS group (untreated mice). Similar to the control healthy group (non colitic), euphol treatment restored the microvillus carpet and made the epithelial surface smooth (Fig. 2D). PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27122