Euphol and Experimental Colitis Figure 7. Preventive treatment with euphol blocks integrins and selectins expression in the colonic tissue after DSS-induced colitis. At the end of 7 days, colon tissue was collected and processed for mRNA expression and immunofluorescence. Preventive treatment with euphol (30 mg/kg, p.o.) reduced colonic mRNA expression of inter-cellular adhesion molecule 1 (ICAM-1) (A), vascular cell adhesion molecule-1 (VCAM-1) (B) and lymphocyte function-associated antigen 1 (LFA-1) (C). The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. The same scheme of treatment with euphol also impaired the increase of P-selectin (D) and E-selectin (E). Representative images of P-selectin and E-selectin immunofluorescent stains were obtained on day 7 from control healthy mice, DSS-treated group and euphol (30 mg/kg, p.o.) treated group. Nuclei were stained with Hoechst (0.5 ml/ml). Scale bar corresponds to 50 mm and applies throughout. Data are reported as means 6 S.E.M. of 8 to 10 mice per group and is representative of three independent experiments. #P,0.05 vs. control healthy group (non colitic); *P,0.05 vs. DSS-treated group. doi:10.1371/journal.pone.0027122.g007 colonic tissue. In this context, there is now a considerable amount of experimental evidence indicating that cytokines, such as MIP-2 and CXCL1/KC, play a pivotal role in the regulation of cell migration in colon tissue [6]. Taken together, these results suggest that euphol treatment contributes to a decrease in cell influx by diminishing the production of chemotactic factors, thus, in turn, ameliorating colon inflammation. As discussed earlier, given that inflammatory cells up-regulated the production of cytokines/chemokines in pathological condi- tions, we next investigated whether the decreased levels and/or expression of colonic cytokines demonstrated by euphol treat- ment could be just a consequence of decreased cell migration. To further strengthen this view, we cultured primary bone marrow- derived macrophages and showed that euphol significantly reduced MCP-1, TNF-a, IL-6, and IFN-c production in macrophages. Interestingly, in vitro pre-treatment (30 min) with euphol (1 and 10 mM) markedly increased IL-10 production, a relevant anti-inflammatory cytokine, in the macrophage culture after LPS administration. Considering these data, it is possible to suggest that euphol could modulate macrophage activation and in consequence decrease cytokine production, an effect which might contribute to the reduction of adhesion molecule expression. Nitrosative stress caused by nitric oxide synthase 2 (NOS2 or iNOS)-derived nitric oxide (NO) is strongly associated with IBD progression and contributes to the pathogenesis of human IBD and experimental colitis [44]. Blocking NOS2 expression using gene knockout or specific inhibitors ameliorates the severity of experimental colitis [45,46]. In addition, another relevant study has shown that NOS2 is implicated in the induction of VEGF [25], a cytokine that has a relevant role in angiogenesis and is increased in patients with IBD [26]. Additional evidence has indicated that local microvasculature and inflammation-dependent angiogenesis exert a relevant role in both human and murine IBD [26,47,48]. Here, we demonstrated that induction of colitis was associated with a significant increase in NOS2 and VEGF PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27122