Tissue and immunostaining Seven days after DSS-induced experimental colitis, animals were sacrificed and each portion of the distal colon was fixed immediately in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Colons were removed and post-fixed 24 h in the same solution, following embedding in paraffin and sections (5 mm) mounted on glass slide and deparaffinized. Immunohistochemical analysis was performed on paraffin-embedded colon tissue (5 mm) using polyclonal rabbit anti-NOS2 (1:300), monoclonal mouse anti-VEGF (1:300), polyclonal rabbit anti-Ki-67 (1:300) and monoclonal mouse anti-phospho-p65 NFkB (1:50) according to the method described previously [74]. After quenching of endogenous peroxidase with 1.5% hydrogen peroxide in methanol (v/v) for 20 min, high-temperature antigen retrieval was per- formed by immersion of the slides in a water bath at 95 to 98uC in 10 mmol/L trisodium citrate buffer, pH 6.0, for 45 min. The slides were then processed using the VECTASTAINH Elite ABC reagent (Vector Laboratories, Burlingame, CA), according to the manufacturer’s instructions. After the appropriate biotinylated secondary antibody, sections were developed with 3,3’-diamino- benzidine (Dako Cytomation, Glostrup, Denmark) in chromogen solution for the exact same amount of time and counterstained with Harris’s hematoxylin. For immunofluorescence, sections 5 mm in thickness were deparaffinized and blocked with 2% BSA in 0.3% Triton X-100 for 1 h at room temperature and incubated overnight at 4uC with antibodies specific for P-selectin (polyclonal goat; 1:200 dilution) and E-selectin (polyclonal goat; 1:100 dilution), followed by fluorescein isothiocyanate-conjugated donkey goat-specific secondary antibody (1:50 dilution; Vector Laboratories, Burlingame, CA) for 2 h at room temperature. Nuclei were stained with Hoechst (0.5 ml/ml; Molecular Probes). Images were acquired using a Sight DS-5M-L1 digital camera connected to Eclipse 50i light microscope fluorescence (both from Nikon, Melville, NY, USA). Four ocular fields per section (5–10 mice per group) were captured and a threshold optical density that best discriminated staining from the background was obtained using the NIH ImageJ 1.36b imaging software (NIH, Bethesda, MD, USA). For immunohistochemistry analysis, total pixel intensity was determined and data are expressed as optical density (OD). For immunofluorescence, signal intensities were collected in triplicate and microscopy data were acquired by two investigators ‘blinded’ to the identity of the disease group. Murine bone marrow-derived macrophages CD1 mice were sacrificed by cervical dislocation. Total bone marrow was obtained from mice by flushing the femurs and tibiae with Dulbecco modified eagle medium (DMEM), as previously described [75]. Briefly, bone marrow mononuclear phagocyte precursor cells were propagated in suspension by culturing in macrophage medium (DMEM containing glucose, supplemented with 2 mM L-glutamine, 10% FCS, 10 mM Hepes, 100 mg/ml streptomycin, 100 U/ml penicillin (all from Sigma-Aldrich) sup- plemented with 20% L929 cell-conditioned medium (as a source of M-CSF). Cells were incubated at 37uC in 5% CO2 air and fed on day 5 by replacing the medium supplemented with 20% L929 cell- conditioned medium. Cells were harvested on day 7, and 16106 cells/ml were cultured in a 96-well cell culture plate for 24 h. Afterwards, adherent cells were stimulated for 24 h with LPS (1 mg/ml) in the presence or absence of euphol (1 and 10 mM) in a final volume of 250 ml/well. Control group corresponds to 5% Tween 80 in medium with/without LPS treatment. After stimulation, the plate was centrifuged (2006g/10 min) and the cell-free supernatant was collected and stored at 270uC for cytokine Euphol and Experimental Colitis determination. A cytokine bead array Mouse Kit Inflammation was used to measure MCP-1, TNF-a, IL-6, IFN-c and IL-10 secretion in the supernatant. The data were acquired using BD FACSCanto II (BD Biosciences, San Diego, CA, USA) and analyzed using FCAP Array software (BD Biosciences, San Diego, CA, USA). Drugs and reagents The sap from Euphorbia tirucalli was initially extracted with hexane and the resulting precipitate was extracted with n-butanol. The most lipophilic compounds present in the methanol fraction were purified by means of high performance liquid chromatog- raphy (HPLC) analysis. Further purification of the compounds was carried using Sephadex G-75 in a mixture of hexane-ethyl acetate. The euphol chemical characterization was conducted by means of nuclear magnetic resonance (NMR) and mass spectroscopic data. The tetracyclic triterpene euphol used in this study showed a purity .95%. Dexamethasone, H2O2, Tween 20, Tween 80, EDTA, aprotinin, phosphate buffered saline (PBS), H&E, 3,3,5,5- tetramethylbenzidine, H2O2 and TNBS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Formaldehyde was obtained from Merck (Frankfurt, Darmstadt, Germany). Anti mouse-KC and the DuoSet kits for IL-1b/IL-1F2, MCP-1 and MIP-2 were obtained from R&D Systems (Minneapolis, MN, USA). Bradford reagent was purchased from Bio-Rad Laborato- ries (Richmond, CA, USA). Polyclonal rabbit anti-NOS2 was purchased from Thermo Fisher Scientific Inc. (Fremont, CA, USA). Monoclonal mouse anti-VEGF was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal mouse anti-phospho-p65 NF-kB was purchased from Cell Signaling Technology (Danvers, MA, USA) and polyclonal rabbit anti-Ki-67 from purchased Abcam (Cambridge, MA, USA). Secondary antibody Envision Plus, streptavidin–HRP reagent, and 3,3- diaminobenzidine chromogen were purchased from DakoCyto- mation (Carpinteria, CA, USA). Trizol and M-MLV reverse transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). Primers and probes for mouse TNF-a (Mm00443258_m1), IL-1b (Mm01336189_m1), CXCL1/KC (Mm00433859_m1), IL- 6 (Mm99999064_m1), ICAM-1 (Mm005616024_g1), VCAM-1 (Mm01320970_m1), LFA-1 (Mm01278854_m1), GAPDH (NM_008084.2) and TaqManH Universal PCR Master Mix Kit were purchased from Applied Biosystems (Foster City, CA, USA). The CBA Mouse Inflammation Kit was purchased from BD Biosciences (San Diego, CA, USA). The compound euphol (3, 10 and 30 mg/kg) was diluted in a 5% Tween 80 solution made in saline (0.9% NaCl solution) and administered orally by gavage (p.o.). All other drugs were made in physiological saline (0.9% NaCl solution). Statistical analysis All data were expressed as means 6 S.E.M. (n = 8210 animals/ group). Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s test for non-parametric data, one-way ANOVA followed by Newman-Keuls test for parametric data. All analysis was conducted using GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA, USA). Differences with p#0.05 were considered to be statistically significant. Author Contributions Conceived and designed the experiments: RCD RFC AFB RM JBC. Performed the experiments: RCD RFC AFB RM ECS ZLB. Analyzed the data: RCD RFC AFB. Contributed reagents/materials/analysis tools: LFP JBC. Wrote the paper: RCD RFC JBC. PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e27122