Figure 1.
Euphol ameliorates DSS-induced acute colitis.
(A), Chemical structure of euphol. Mice received DSS for 5 days and drinking water for the next 2 days. Animals were orally treated by gavage with 3, 10, or 30 mg/kg of euphol twice a day from day 0 to day 7 (preventive treatment) or with 30 mg/kg from day 3 to day 7 (therapeutic treatment). Preventive or therapeutic oral treatment with euphol improved the disease activity index (DAI) score (B), reduced body weight loss (C) and colon macroscopic damage (D), and enhanced colon length (E) when compared with mice from the DSS group. Data are reported as means ± S.E.M. of 8 to 10 mice per group and is representative of three independent experiments. #P<0.05 vs. control healthy group; *P<0.05 vs. DSS-treated group.
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 ± 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.
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-1β (IL-1β) (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-1β (E), CXCL1/KC (F), tumor necrosis factor-α (TNF-α) (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 ± 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.
Figure 4.
Euphol reduces pro-inflammatory cytokines and chemokines production in macrophages stimulates with lipopolysaccharide (LPS).
Macrophage from bone marrow of naïve mice were stimulated with LPS (1 µg/ml) in the presence or absence of euphol (1 and 10 µM) for 24 hours, and the culture supernatants were analyzed for cytokine levels using cytokine bead array kit (CBA). Euphol incubation in dose-related manner reduced production of MCP-1 (A), TNF-α (B), IL-6 (C), IFN-γ (D), but increase the IL-10 levels (E). Data are reported as means ± SEM (n = 4) and is representative of two independent experiments. #P<0.05 vs. control without LPS treatment (vehicle solution); *P<0.05 vs. LPS-treated group. Vehicle solution corresponds to 5% Tween 80 in medium.
Figure 5.
Euphol treatment inhibits NOS2 and VEGF expression in colonic tissue.
After a 7-day euphol treatment, colon samples were processed for immunohistochemistry analysis. Preventive treatment with euphol (30 mg/kg, p.o.) significantly reduced NOS2 (A) and VEGF (B) immunostaining in the colon tissue after DSS-induced colitis in mice. Graphical representation of the immunostaining for NOS2 (C) and VEGF (D) expression evaluated in colon tissue. The mean intensity of NOS2 and VEGF staining were determined from image analysis and are represented as optical density. Scale bar corresponds to 100 µm and applies throughout. Each column represents the mean ± 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.
Figure 6.
Euphol prevents inflammatory/enterocyte cells proliferation and NF-κB activation after DSS-induced colitis.
Expression of Ki67 and phosphorylation of NF-κB was performed 7 days after administration of DSS (3%) or with vehicle in colonic tissues. Pre-treatment with euphol (30 mg/kg, p.o.), significantly inhibited proliferation index (Ki67) (A) and phosphorylation of p65 NF-κB (B) in colon tissue after DSS-induced colitis in mice. (A–B) Representative images of Ki67 and phospho-p65 NF-κB immunoreactivity in colon tissue. Scale bar corresponds to 100 and 25 mm (black square) respectively, and applies throughout. Graphical representation of the immunostaining for Ki67 (C) and phospho-p65 NF-κB (D) expression evaluated in colon tissue. The mean intensity of Ki67 and p65 NF-κB staining were determined from image analysis and are represented as optical density. Each column represents the mean ± 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.
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 µl/ml). Scale bar corresponds to 50 µm and applies throughout. Data are reported as means ± 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.
Figure 8.
Therapeutic treatment with euphol protects mice against TNBS-induced acute colitis.
Mice were given 100 µL of the TNBS (in 35% ethanol) and after 24 h, treated with euphol (30 mg/kg, p.o.). (A) The time-course of body weight changes on day 3 after TNBS-induced colitis. (B) Macroscopic score; (C) colon length after TNBS-induced colitis. (D) Representative photograph of colons from day 3 after the induction of TNBS-colitis. 1, Control healthy mice; 2, TNBS-treated (only vehicle administration); 3, TNBS plus euphol (30 mg/kg, p.o.). Each column represents the mean ± 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. TNBS-treated group. Vehicle corresponds to 5% Tween 80 in saline 0.9% NaCl.