Volume 85, Issue 9, 1 May 2013, Pages 1306–1316

Beneficial effect of the non-psychotropic plant cannabinoid cannabigerol on experimental inflammatory bowel disease

Francesca Borrelli ^a^,¹^,^,, Ines Fasolino ^a^,, Barbara Romano ^a^,, Raffaele Capasso ^a^,¹^,, Francesco Maiello ^b^,, Diana Coppola ^b^,, Pierangelo Orlando ^c^,¹^,, Giovanni Battista ^b^,, Ester Pagano ^a^,, Vincenzo Di Marzo ^d^,¹^,, Angelo A. Izzo ^a^,¹^,^,

^a Department of Pharmacy, University of Naples Federico II, via D Montesano 49, 80131 Naples, Italy
^b Ospedale dei Pellegrini, Department of Diagnostic Services (Anatomy and Pathologic Histology Service), ASL 1, Naples, Italy
^c Institute of Protein Biochemistry, National Research Council, Naples, Italy
^d Institute of Biomolecular Chemistry, National Research Council, Pozzuoli (NA), Italy

http://dx.doi.org/10.1016/j.bcp.2013.01.017, How to Cite or Link Using DOI

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Abstract

Inflammatory bowel disease (IBD) is an incurable disease which affects millions of people in industrialized countries. Anecdotal and scientific evidence suggests that Cannabis use may have a positive impact in IBD patients. Here, we investigated the effect of cannabigerol (CBG), a non-psychotropic Cannabis-derived cannabinoid, in a murine model of colitis. Colitis was induced in mice by intracolonic administration of dinitrobenzene sulphonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters (colon weight/colon length ratio and myeloperoxidase activity), by histological analysis and immunohistochemistry; interleukin-1β, interleukin-10 and interferon-γ levels by ELISA, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by western blot and RT-PCR; CuZn-superoxide dismutase (SOD) activity by a colorimetric assay. Murine macrophages and intestinal epithelial cells were used to evaluate the effect of CBG on nitric oxide production and oxidative stress, respectively. CBG reduced colon weight/colon length ratio, myeloperoxidase activity, and iNOS expression, increased SOD activity and normalized interleukin-1β, interleukin-10 and interferon-γ changes associated to DNBS administration. In macrophages, CBG reduced nitric oxide production and iNOS protein (but not mRNA) expression. Rimonabant (a CB₁ receptor antagonist) did not change the effect of CBG on nitric oxide production, while SR144528 (a CB₂ receptor antagonist) further increased the inhibitory effect of CBG on nitric oxide production. In conclusion, CBG attenuated murine colitis, reduced nitric oxide production in macrophages (effect being modulated by the CB₂ receptor) and reduced ROS formation in intestinal epithelial cells. CBG could be considered for clinical experimentation in IBD patients.

Graphical abstract

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Abbreviations

CB, cannabinoid;
CBD, cannabidiol;
CBG, cannabigerol;
CD, Crohn’s disease;
COX-2, cycloxygenase-2;
DNBS, 2,4,6-dinitrobenzene sulphonic acid;
H2DCF-DA, 2′,7′-dichlorfluorescein-diacetate;
IBD,Inflammatory bowel disease;
iNOS, inducible nitric oxide synthase;
MPO, myeloperoxidase;
ROS,reactive oxygen species;
SOD, superoxide dismutase;
UC, ulcerative colitis

Keywords

Cannabigerol;
Phytocannabinoids;
Inflammatory bowel disease;
Murine colitis;
Macrophages;
Dinitrobenzene sulphonic acid

Figures and tables from this article:

: Fig. 1. Dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Colon weight/length ratio (mg/cm) of colons from untreated and DNBS-treated mice in the presence or absence of cannabigerol (CBG). Tissues were analysed 3 days after vehicle or DNBS (150 mg/kg, intracolonically) administration. CBG (1–30 mg/kg) was administered (i.p.) once a day for six consecutive days starting 3 days before DNBS (preventive protocol, A) or for two consecutive days starting 24-h after the inflammatory insult (curative protocol, B). Bars are mean ± SEM of 12–15 mice for each experimental group. ^#p < 0.001 vs control, *p < 0.05 and **p < 0.01 vs DNBS alone.; Figure options

: Fig. 2. Histological evaluations of inflamed and non-inflamed colons: effect of cannabigerol (CBG). No histological modification was observed in the mucosa and submucosa of control mice (A); mucosal injury induced by dinitrobenzene sulfonic acid administration (B); treatment with CBG reduced colon injury by stimulating regeneration of the glands (C). Histological analysis was performed 3 days after DNBS administration. CBG (30 mg/kg) was administered (i.p.) for two consecutive days starting 24-h after the inflammatory insult (curative protocol). Original magnification x 200. The figure is representative of 4 experiments.; Figure options

: Fig. 3. Different patterns of Ki-67 immunoreactivity in the colonic mucosa of control mice (A), DNBS-treated mice (B) and mice treated with DNBS plus cannabigerol (C). (A) Ki-67 immunopositive cells were localized to the lower part of the crypts. (B) Ki-67 immunopositive cells were observed on inflammatory cells. (C) Ki-67 immunopositive cells were observed only in the expanded basal zone. CBG (30 mg/kg) was administered (i.p.) for two consecutive days starting 24-h after the inflammatory insult (curative protocol). The figure is representative of 4 experiments.; Figure options

: Fig. 4. Effect of cannabigerol (CBG) on intestinal permeability (evaluated as FITC-dextran permeability, see methods), myeloperoxidase (MPO, a marker of intestinal inflammation) activity (B) and superoxide dismutase (SOD) activity (C) in DNBS-induced colitis in mice. Colons (for MPO and SOD activities) and blood (for intestinal permeability) were analysed 3 days after vehicle or DNBS (150 mg/kg, intracolonically) administration. CBG (30 mg/kg) was administered (i.p.) for two consecutive days starting 24-h after the inflammatory insult (curative protocol). Bars are mean ± SEM of 5 mice for each experimental group. ^#p < 0.001 vs control and ***p < 0.001 vs DNBS alone.; Figure options

: Fig. 5. Inducible nitric oxide synthase (iNOS) (A) and cyclooxygenase-2 (COX-2) (B) expression in colonic tissues of animals treated or not with dinitrobenzene sulfonic acid (DNBS): effect of cannabigerol (CBG). Measurements were performed 3 days after DNBS (150 mg/kg, intracolonically) administration. CBG (30 mg/kg) was administered (i.p.) for two consecutive days starting 24-h after the inflammatory insult (curative protocol). Results are mean ± SEM of 3–4 experiments.*p < 0.05 and ^#p < 0.001 vs control; ***p < 0.001 vs DNBS alone.; Figure options

: Fig. 6. Effect of cannabigerol (CBG) on interleukin-1β (IL-1β) (A), interferon γ (IFN-γ) (B) and interleukin-10 (IL-10) (C) levels in mouse colons treated with dinitrobenzene sulfonic acid (DNBS). Measurements were performed 3 days after DNBS (150 mg/kg, intracolonically) administration. CBG (30 mg/kg) was administered (i.p.) for two consecutive days starting 24-h after the inflammatory insult (curative protocol). Results (expressed as picograms per ml of proteic extract) are mean ± SEM of 3–4 experiments. ^#p < 0.01–0.001 vs control, *p < 0.05 and **p < 0.01 vs DNBS alone.; Figure options

: Fig. 7. Effect of cannabigerol (CBG) on nitrite levels (A) in the cell medium of murine peritoneal macrophages incubated with lipopolysaccharide (LPS, 1 μg/ml) for 18 h. CBG (0.001–1 μM) was added to the cell media 30 min before LPS challenge. Results, expressed as nitrite concentration (nM), are mean ± SEM of four experiments (in triplicates). Panels B and C show the effect of CBG (1 μM) on inducible nitric oxide synthase (iNOS) expression in cell lysates, evaluated by western blot analysis (B, n = 5) or RT-PCR (C, n = 4), respectively. ^#p < 0.001 vs control; *p < 0.05, **p < 0.01 and ***p < 0.001 vs LPS alone.; Figure options

: Fig. 8. Effect of cannabigerol (CBG, 1 μM) alone or in presence of the cannabinoid CB₁ receptor antagonist rimonabant (Rim, 0.1 μM) (A) or the cannabinoid CB₂ receptor antagonist SR144528 (0.1 μM) (B) on nitrite levels in the cell medium of murine peritoneal macrophages incubated with lipopolysaccharide (LPS, 1 μg/ml) for 18 h. The antagonists were added to the cell media 30 min before CBG exposure. LPS was incubated 30 min after CBG. Results are means ± SEM of three experiments (in triplicates). ^#p < 0.001 vs control; *p < 0.05 and ***p < 0.001 vs LPS alone; °p < 0.001 vsLPS + CBG.; Figure options

: Fig. 9. Relative mRNA expression of cannabinoid CB₁ receptor (A), cannabinoid CB₂ receptor (B) in cell lysates from macrophages incubated or not with lipopolysaccharide (LPS, 1 μg/ml) for 18 h. Cannabigerol (CBG, 1 μM) was added alone to the cell media or 30 min before LPS challenge. Data were analysed by GENEX software for group wise comparisons and statistical analysis. Results are means ± SEM of four experiments. ^#p < 0.001 vs control.; Figure options

: Fig. 10. Reactive oxygen species (ROS) production produced by Fenton’s reagent (2 mM H₂O₂/Fe²⁺) in Ptk6 null colonic epithelial cells after 24-h exposure to cannabigerol (0.1–10 μM). Results are mean ± SEM of five experiments.^#p < 0.001 vs control, *p < 0.05 and **p < 0.01 vs H₂O₂/Fe²⁺alone.; Figure options

: Corresponding authors. Tel.: +39 081 678665; fax: +39 081 678403.

1: Endocannabinoid Research Group.

Beneficial effect of the non-psychotropic plant cannabinoid cannabigerol on experimental inflammatory bowel disease.

Beneficial effect of the non-psychotropic plant cannabinoid cannabigerol on experimental inflammatory bowel disease

Abstract

Graphical abstract

Abbreviations

Keywords

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