Euphol and Experimental Colitis Figure 3. Preventive treatment with euphol changes colonic protein levels and mRNA expression of inflammatory mediators. At the end of 7 days, colon tissue was collected and processed for cytokine levels and mRNA expression. (A–D) Enzyme-linked immunosorbent assay. Preventive treatment with euphol (30 mg/kg, p.o.) reduced colonic levels of interleukin-1b (IL-1b) (A), keratinocyte-derived chemokine (CXCL1/KC) (B), macrophage inflammatory protein-2 (MIP-2) (C) and monocyte chemoattractant protein-1 (MCP-1) (D). (E–H) Real-time PCR. The same scheme of treatment with euphol also impaired the increase colonic mRNA expression of IL-1b (E), CXCL1/KC (F), tumor necrosis factor-a (TNF-a) (G) and interleukin-6 (IL-6) (H). The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. 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.g003 important component of colitis-induced damage. Primary macro- phages stimulated with LPS for 24 h increased MCP-1, TNF-a, IL- 6 and IFN-c levels (Fig. 4 A–E). In vitro pre-treatment (30 min) with euphol (1 and 10 mM) markedly blocked MCP-1, TNF-a, IL-6 and IFN-c levels after LPS administration (Fig. 4). In addition, LPS administration decrease IL-10 levels and, interestingly, euphol (1 and 10 mM) increased IL-10 production in the macrophage culture after LPS administration (Fig. 4E). Euphol inhibits NOS2 and VEGF expression induced by DSS Ulcerative colitis appears to be caused by a disruption of intestinal homeostasis and integrity, while up-regulated NOS2 expression in gut mucosa has been shown to cause apoptosis of epithelial cells [24]. Furthermore, it has been suggested that NOS2 is also involved in angiogenesis [25] a relevant phenomenon that has recently been demonstrated to be one of the major contributors to the pathogenesis of IBD [26]. Our present data corroborated with this observation by demonstrating that DSS- induced colitis increased NOS2 (Fig. 5 A,C) and VEGF expression (Fig. 5 B,D). Interestingly, preventive treatment with euphol (30 mg/kg) significantly blocked the increase in NOS2 and VEGF expression in colonic tissue (Fig. 5). Euphol inhibits inflammatory and enterocyte cells proliferation during inflammation bowel induced by DSS Ki-67 is a nuclear protein necessary for cell proliferation and is expected to play a central role in the inflammatory process [27]. To explore whether or not euphol could interfere with inflammatory and enterocyte cells proliferation we evaluated Ki- 67 staining in colon tissue after DSS administration. Tissue sections from control mice exhibited very low levels of specific staining for Ki-67 in epithelial cells (Fig. 6). In contrast, at 7 days after DSS administration, the untreated (DSS) group showed intense immunostaining for Ki67 expression in colon tissue, mainly in the inflammatory cells, but also in enterocyte cells (Fig. 6 A,C). On the other hand, the preventive oral treatment with euphol (30 mg/kg, twice a day) during 7 days following DSS administration, significantly inhibited proliferation index in colon tissue, using monoclonal antibodies against ki67 (Fig. 6 A,C). Euphol prevents colonic NF-kB activation in the colon tissue after DSS administration The transcriptional factor NF-kB is among the major pro- inflammatory signaling pathways involved in colitis [28,29]. Herein, in order to further evaluate the possible mechanisms involved in the anti-inflammatory action of euphol, we next assessed whether the oral pre-treatment with euphol was able to decrease the activation of transcriptional factors NF-kB, in the colonic tissue after DSS administration. As shown in Fig. 6, low levels of NF-kB p65 was detected in the naive mouse colon; however, DSS-induced inflammation bowel, as expected, lead to a pronounced phosphorylation of p65 NF-kB in the colonic tissue after 7 days (Fig. 6 B,D). Interestingly, the pre-treatment with euphol (30 mg/kg, p.o.), significantly reduced the p65 NF-kB activation in the mouse colon tissue, thus strongly suggesting that inhibition of NF-kB activation seems to be the key mechanism PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27122