Preventive and Therapeutic Euphol Treatment Attenuates Experimental Colitis in Mice

BACKGROUND: The tetracyclic triterpene euphol is the main constituent found in the sap of Euphorbia tirucalli. This plant is widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer, including leukaemia, prostate and breast cancers. Here, we investigated the effect of euphol on experimental models of colitis and the underlying mechanisms involved in its action. METHODOLOGY/PRINCIPAL FINDINGS: Colitis was induced in mice either with dextran sulfate sodium (DSS) or with 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the effect of euphol (3, 10 and 30 mg/kg) on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, enzyme-Linked immunoabsorbent assay (ELISA), real time-polymerase chain reaction (RT-PCR) and flow cytometry. Preventive and therapeutic oral administration of euphol attenuated both DSS- and TNBS-induced acute colitis as observed by a significant reduction of the disease activity index (DAI), histological/microscopic damage score and myeloperoxidase (MPO) activity in colonic tissue. Likewise, euphol treatment also inhibited colon tissue levels and expression of IL-1β, CXCL1/KC, MCP-1, MIP-2, TNF-α and IL-6, while reducing NOS2, VEGF and Ki67 expression in colonic tissue. This action seems to be likely associated with inhibition of activation of nuclear factor-κB (NF-κB). In addition, euphol decreased LPS-induced MCP-1, TNF-α, IL-6 and IFN-γ, but increased IL-10 secretion from bone marrow-derived macrophages in vitro. Of note, euphol, at the same schedule of treatment, markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue. CONCLUSIONS/SIGNIFICANCE: Together, these results clearly demonstrated that orally-administered euphol, both preventive or therapeutic treatment were effective in reducing the severity of colitis in two models of chemically-induced mouse colitis and suggest this plant-derived compound might be a potential molecule in the management of inflammatory bowel diseases

Treatment with euphol reduces cell influx and microscopic colon damage after DSS-induced acute colitis.

<p>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.</p

Euphol reduces pro-inflammatory cytokines and chemokines production in macrophages stimulates with lipopolysaccharide (LPS).

<p>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.</p

Preventive treatment with euphol blocks integrins and selectins expression in the colonic tissue after DSS-induced colitis.

<p>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.</p

Euphol ameliorates DSS-induced acute colitis.

<p>(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.</p

Therapeutic treatment with euphol protects mice against TNBS-induced acute colitis.

<p>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.</p

Preventive treatment with euphol changes colonic protein levels and mRNA expression of inflammatory mediators.

<p>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.</p

Euphol treatment inhibits NOS2 and VEGF expression in colonic tissue.

<p>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.</p