The cannabinoid TRPA1 agonist cannabichromene inhibits nitric oxide production in macrophages and ameliorates murine colitis

Abstract

Background and purpose: The non-psychotropic cannabinoid cannabichromene is known to activate the transient receptor potential ankyrin-type1 (TRPA1) and to inhibit endocannabinoid inactivation, both of which are involved in inflammatory processes. We examined here the effects of this phytocannabinoid on peritoneal macrophages and its efficacy in an experimental model of colitis.

Experimental approach: Murine peritoneal macrophages were activated in vitro by LPS. Nitrite levels were measured using a fluorescent assay; inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2) and cannabinoid (CB1 and CB2 ) receptors were analysed by RT-PCR (and/or Western blot analysis); colitis was induced by dinitrobenzene sulphonic acid (DNBS). Endocannabinoid (anandamide and 2-arachidonoylglycerol), palmitoylethanolamide and oleoylethanolamide levels were measured by liquid chromatography-mass spectrometry. Colonic inflammation was assessed by evaluating the myeloperoxidase activity as well as by histology and immunohistochemistry.

Key results: LPS caused a significant production of nitrites, associated to up-regulation of anandamide, iNOS, COX-2, CB1 receptors and down-regulation of CB2 receptors mRNA expression. Cannabichromene significantly reduced LPS-stimulated nitrite levels, and its effect was mimicked by cannabinoid receptor and TRPA1 agonists (carvacrol and cinnamaldehyde) and enhanced by CB1 receptor antagonists. LPS-induced anandamide, iNOS, COX-2 and cannabinoid receptor changes were not significantly modified by cannabichromene, which, however, increased oleoylethanolamide levels. In vivo, cannabichromene ameliorated DNBS-induced colonic inflammation, as revealed by histology, immunohistochemistry and myeloperoxidase activity.

Conclusion and implications: Cannabichromene exerts anti-inflammatory actions in activated macrophages - with tonic CB1 cannabinoid signalling being negatively coupled to this effect - and ameliorates experimental murine colitis.

Figure 1

Inhibitory effect of cannabichromene on nitrite levels in the cell medium of murine peritoneal macrophages incubated with lipopolysaccharide (LPS, 1 μg·mL⁻¹) for 18 h. Cannabichromene (CBC, 0.001–1 μM) was added to the cell media 30 min before LPS challenge (i.e. 18.5 h before nitrites assay). Results are mean ± SEM of six experiments (in triplicates). ^#P < 0.001 versus control; *P < 0.05 and ***P < 0.001 versus LPS alone. The insert (on top of the figure) shows the effect of CBC (expressed as percentage of inhibition of the corresponding control values, with the difference between LPS and control considered as 100%) when given 30 min before LPS (CBC before LPS) or 15 h after LPS (CBC after LPS). No statistically significant difference was observed between the two concentration–response curves reported in the insert.

Figure 2

Inducible nitric oxide synthase (iNOS) (A, B) and cyclooxygenase-2 (COX-2) (C, D) mRNA and protein levels in cell lysates from macrophages incubated or not with lipopolysaccharide (LPS, 1 μg·mL⁻¹) for 18 h. mRNA expression was evaluated by RT-PCR. The expression levels, normalized with respect to the reference genes, were scaled to the expression value of the control, considered as 1. The means of the quantitative-cycles (Cq) for the control were: 26.00 and 25.58 for iNOS and COX-2 respectively. The reaction background was N/A (see text) at 40 reaction cycles. Protein expression was evaluated by Western blot analysis. Cannabichromene (CBC, 1 μM) was added to the cell media 30 min before LPS challenge. ^#P < 0.001 versus control (n = 4–5 experiments).

Figure 3

Effect of cannabichromene (CBC) on interleukin-1β (IL-1β) (A), interleukin-10 (IL-10) (B) and interferon-γ (C) levels detected in the cell media of macrophages incubated with lipopolysaccharide (LPS, 1 μg·mL⁻¹) for 18 h. CBC (1 μM) was added to the media 30 min before LPS challenge. Results are means ± SEM of four experiments (in quadruplicates). ^#P < 0.001 versus control, *P < 0.05 and **P < 0.01 versus LPS.

Figure 4

Effect of cannabichromene (CBC, 1 μM) alone and in presence of the cannabinoid CB₁ receptor antagonists rimonabant (0.1 μM) (A) and AM251 (1 μM) (B) as well as in the presence of the cannabinoid CB₂ receptor antagonist SR144528 (0.1 μM) (C) on nitrite levels in the cell medium of murine peritoneal macrophages incubated with lipopolysaccharide (LPS, 1 μg·mL⁻¹) for 18 h. The antagonists were added to the cell media 30 min before CBC exposure. LPS (1 μg·mL⁻¹for 18 h) was incubated 30 min after CBC. Results are means ± SEM of three experiments (in triplicates). ^#P < 0.001 versus control; *P < 0.05, **P < 0.01 and ***P < 0.001 versus LPS; ^°P < 0.05 versus LPS + CBC.

Figure 5

Effect of cannabichromene (CBC, 1 μM) alone and in combination with AM251 (1 μM, CB₁ receptor antagonist, A) or rimonabant (SR1, 0.1 μM, CB₁ receptor antagonist, B) on [³⁵S]GTPγS binding to hCB₁- CHO cell membranes (n = 12–16). CBC was added 30 min after the CB₁ antagonists. Symbols represent mean values ± SEM.

Figure 6

Inhibitory effect of the selective transient receptor potential ankyrin type 1 (TRPA1) agonists carvacrol (0.001–0.1 μM) and cinnamaldehyde (0.001–0.1 μM) (A, B) and of the selective TRPA1 antagonists AP18 (10–20 μM) and HC-030031 (HC, 10–20 μM) (C) on nitrite levels detected in the cell media of macrophages incubated with lipopolysaccharide (LPS, 1 μg·mL⁻¹) for 18 h. Both TRPA1 agonists and antagonists were added to the medium 30 min before LPS challenge. Results are mean ± SEM of two experiments (in triplicates). ^#P < 0.001 versus control; *P < 0.05, and ***P < 0.001 versus LPS.

Figure 7

Relative mRNA expression of cannabinoid CB₁ receptor (A) and cannabinoid CB₂ receptor (B) in cell lysates from macrophages incubated or not with lipopolysaccharide (LPS, 1 μg·mL⁻¹) for 18 h: effect of cannabichromene (CBC, 1 μM, added to the cell media or 30 min before LPS challenge). The expression levels of mRNA, evaluated by qRT-PCR and normalized with respect to the reference genes, was scaled for all conditions to the expression value of the control, considered as 1. The means of the quantitative-cycles (Cq) for the control values were: 31.2 (CB₁ receptor) and 24.48 (CB₂ receptor). The reaction background was 37.30 Cq and 36.60 Cq for CB₁ receptor and CB₂ receptor, respectively, at 40 reaction cycles. ^#P < 0.001 versus control (n = 4).

Figure 8

Dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Colon weight/colon length ratio of colons from control and DNBS-treated mice in the presence or absence of cannabichromene (CBC). Tissues were analysed 3 days after vehicle or DNBS (150 mg·kg⁻¹, intracolonically) administration. CBC (0.1 and 1 mg·kg⁻¹) was administered (i.p.) once a day for 2 consecutive days starting 24-h after the inflammatory insult. Bars are mean ± SEM of 12–15 mice for each experimental group. ^#P < 0.001 versus control; **P < 0.01 versus DNBS alone.

Figure 9

Inhibitory effect cannabichromene (CBC) on serum FICT-dextran concentration (a measure of intestinal barrier function) (A) and myeloperoxidase (MPO, a marker of intestinal inflammation) activity (B) in dinitrobenzene (DNBS)-induced colitis in mice. Permeability and MPO activity were measured on colonic tissues 3 days after vehicle or DNBS (150 mg·kg⁻¹, intracolonically). CBC (1 mg·kg⁻¹) was administered (i.p.) for 2 consecutive days starting 24 h after the inflammatory insult. Bars are mean ± SEM of five mice for each experimental group. ^#P < 0.001 versus control; *P < 0.05 and **P < 0.01 versus DNBS alone.

Figure 10

Histological evaluations of inflamed and non-inflamed colons: effect of cannabichromene (CBC). No histological modification was observed in the mucosa and submucosa of control mice (A); mucosal injury induced by dinitrobenzene sulfonic acid (DNBS) administration (B); treatment with CBC reduced colon injury stimulating a regeneration of the glands (C). CBC (1 mg·kg⁻¹) was administered (i.p.) for 2 consecutive days starting 24 h after the inflammatory insult. Histological analysis was performed 3 days after DNBS (150 mg·kg⁻¹, intracolonically). Original magnification ×200. The figure is representative of three experiments.

Figure 11

Different patterns of Ki-67 immunoreactivity in the colonic mucosa of control mice (A), dinitrobenzene sulfonic acid (DNBS)-treated mice (B) and mice treated with DNBS plus cannabichromene (C). (A) Ki-67 immunopositive cells localized to the lower of the crypts. (B) Ki-67 immunoreactivity was observed on inflammatory cells. (C) Ki-67 immunopositive cells observed only in the expanded basal zone. CBC (1 mg·kg⁻¹) was administered (i.p.) for 2 consecutive days starting 24 h after the inflammatory insult. The figure is representative of three experiments.