Euphol and Experimental Colitis Figure 2. Treatment with euphol reduces cell influx and microscopic colon damage after DSS-induced acute colitis. At 7 days after euphol oral treatment, colon tissues were processed for histological evaluation, measurement of myeloperoxidase (MPO) activity and scanning electron microscopy. Preventive (3, 10, and 30 mg/kg, p.o.) or therapeutic (30 mg/kg, p.o.) treatment with euphol reduced MPO (A) activity. (B) Representative histological sections of colon from control healthy mice (non colitic), DSS-treated and euphol-treated mice (30 mg/kg, p.o.) were examined microscopically after H&E staining with original magnification x20. The images are representative of at least four mice per group. (C) Preventive treatment with euphol (30 mg/kg, p.o.) decreased the microscopic damage score in mouse colon. (D) Scanning electron microscopy photographs of the colon of the colon surfaces of control healthy mice, DSS-treated group, and DSS plus euphol (30 mg/kg, p.o.) treated mice after 7 days following DSS administration. Original magnification: x750 and x6,000, respectively. 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.g002 Euphol inhibits cytokine local release and expression during experimental ulcerative colitis It has been shown that selective blockade of IL-1R, CXCR2, CXCL1/KC, MCP-1, MIP-2, TNF-a and IL-6 significantly decreases severity of colitis and neutrophil/macrophage migration [4,6,23]. In this set of experiments, we investigated whether oral treatment with euphol could inhibit the levels and expression of pro-inflammatory cytokines/chemokine in the colonic tissues. Colonic levels of the pro-inflammatory cytokines IL-1b, CXCL1/ KC, MIP-2 and MCP-1 were markedly elevated 7 days after the initiation of DSS treatment (Fig. 3 A–D). Preventive treatment with euphol (30 mg/kg, p.o.) significantly inhibited the levels of IL-1b by 80% (Fig. 3A), KC by 48% (Fig. 3B), MIP-2 by 65% (Fig. 3C) and MCP-1 by 80% (Fig. 3D) in colon tissue (P,0.05). In addition, DSS administration resulted in a pronounced increase in colonic IL-1b, CXCL1/KC, TNF-a and IL-6 mRNA expression (Fig. 3 E–H). Interestingly, euphol treatment (30 mg/kg), given orally during 7 day following DSS administration, significantly inhibited the up-regulated mRNA expression of IL-1b by 95% (Fig. 3E), CXCL1/KC by 100% (Fig. 3F), TNF-a by 40% (Fig. 3G) and IL-6 by 75% (Fig. 3H) (P,0.05). Such data suggest that the anti-inflammatory action of euphol is likely to be associated with its abilities to inhibit the release and expression of cytokines/chemokines as well as inhibit neutrophils influx into colonic tissue after DSS administration. Euphol reduces cytokine release from LPS-stimulated macrophages The reduction in IL-1b, CXCL1/KC, MCP-1 and MIP-2 levels induced by euphol in DSS-treated colon could be just a consequence of decreased cellular adhesion or migration; inflam- matory cells produce cytokines and chemokines, thus a decrease in the presence of these cells could also correspond to a decrease in their secreted cytokines/chemokines. To clarify whether euphol could also diminish the production of inflammatory mediators or modify the profile of cytokines produced by the inflammatory cells present in colonic tissue after inflammatory stimulus, we cultured primary bone marrow-derived macrophages and evaluated the influence of euphol on MCP-1, TNF-a, IL-6, IFN-c and IL-10 production after in vitro stimulation with LPS (1 mg/ml, for 24 h), an PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27122