Inhibitory effect of cannabichromene, a major non-psychotropic cannabinoid extracted from Cannabis sativa, on inflammation-induced hypermotility in mice

Abstract

Background and purpose: Cannabichromene (CBC) is a major non-psychotropic phytocannabinoid that inhibits endocannabinoid inactivation and activates the transient receptor potential ankyrin-1 (TRPA1). Both endocannabinoids and TRPA1 may modulate gastrointestinal motility. Here, we investigated the effect of CBC on mouse intestinal motility in physiological and pathological states.

Experimental approach: Inflammation was induced in the mouse small intestine by croton oil. Endocannabinoid (anandamide and 2-arachidonoyl glycerol), palmitoylethanolamide and oleoylethanolamide levels were measured by liquid chromatography-mass spectrometry; TRPA1 and cannabinoid receptors were analysed by quantitative RT-PCR; upper gastrointestinal transit, colonic propulsion and whole gut transit were evaluated in vivo; contractility was evaluated in vitro by stimulating the isolated ileum, in an organ bath, with ACh or electrical field stimulation (EFS).

Key results: Croton oil administration was associated with decreased levels of anandamide (but not 2-arachidonoyl glycerol) and palmitoylethanolamide, up-regulation of TRPA1 and CB₁ receptors and down-regulation of CB₂ receptors. Ex vivo CBC did not change endocannabinoid levels, but it altered the mRNA expression of TRPA1 and cannabinoid receptors. In vivo, CBC did not affect motility in control mice, but normalized croton oil-induced hypermotility. In vitro, CBC reduced preferentially EFS- versus ACh-induced contractions. Both in vitro and in vivo, the inhibitory effect of CBC was not modified by cannabinoid or TRPA1 receptor antagonists.

Conclusion and implications: CBC selectively reduces inflammation-induced hypermotility in vivo in a manner that is not dependent on cannabinoid receptors or TRPA1.

Figure 1

Anandamide (AEA) and 2-AG levels in the duodenum (A,D), jejunum (B,E) and ileum (C,F) of mice treated or not with croton oil. Some mice treated with croton oil were also treated with CBC (15 mg·kg⁻¹, i.p.). Data are mean ± SEM of four mice. *P < 0.05 versus control.

Figure 2

PEA and OEA levels in the duodenum (A,D), jejunum (B,E) and ileum (C,F) of mice treated or not with croton oil. Some mice treated with croton oil were also treated with CBC (15 mg·kg⁻¹, i.p.). Data are mean ± SEM of four mice.

Figure 3

Relative expression of NAPE PLD (A), GDE1 (B) and FAAH (C) mRNA in the jejunum of animals treated or not with croton oil. Some mice treated with croton oil were also treated with CBC (15 mg·kg⁻¹, i.p.). Total RNA extracted from the intestine of control and croton-oil-treated mice was subjected to quantitative (real-time) RT-PCR analysis as described in Methods. Data were analysed by GENEX software for groupwise comparisons and statistical analysis. The expression in control tissues for each target was considered as 1. Results are means ± SEM of four experiments. ***P < 0.001 versus control and ^##P < 0.01 versus croton oil.

Figure 4

Relative expression of cannabinoid (CB₁ and CB₂) receptors (A, B) and TRPA1 (C) mRNA in the duodenum, jejunum and ileum of animals treated or not with croton oil. Some mice treated with croton oil were also treated with CBC (15 mg·kg⁻¹, i.p.). Note that the effect of CBC was evaluated in the jejunum and ileum only (mRNA in the samples of the duodenum was degraded). Total RNA extracted from the intestine of control and croton-oil-treated mice was subjected to quantitative (real-time) RT-PCR analysis as described in Methods. Data were analysed by GENEX software for groupwise comparisons and statistical analysis. The expression in control tissues (duodenum, jejunum and ileum) for each target was considered as 1. Results are means ± SEM of four experiments. *P < 0.05 and **P < 0.01 versus control; ^#P < 0.05, ^#P < 0.01 and ^###P < 0.01 versus croton oil.

Figure 5

Effect of i.p. injected CBC (10 and 20 mg·kg⁻¹) and of the psychotropic cannabinoid receptor agonist WIN 55,212-2 (1 mg·kg⁻¹) on upper gastrointestinal transit (A), colonic propulsion (B) and whole gut transit in mice (C) (see Methods for details concerning the measurement of motility). Columns represent the mean ± SEM of 6–11 mice for each experimental group **P < 0.01 versus control. Note that a decreased transit is indicated by a decreased value of GC (A), by an increased value of ‘time of expulsion (min)’ (B) or by an increased time of ‘whole gut transit (min)’ (C).

Figure 6

Inhibitory effect of i.p.-injected CBC (1–20 mg·kg⁻¹) on intestinal transit in croton oil-treated mice in vivo. Transit was expressed as the GC of the distribution of a fluorescent marker along the small intestine. GC ranged from 1 (minimal motility) to 10 (maximal motility) (see Methods section). Columns represent the mean ± SEM of 10–12 mice for each experimental group. ^#P < 0.05 versus control and *P < 0.05 versus croton oil. Note that CBC (10 and 20 mg·kg⁻¹) did not affect transit in control mice (see Figure 5).

Figure 7

Croton oil-treated mice: effect of CBC (10 mg·kg⁻¹, i.p.) alone or in the presence of the cannabinoid CB₁ receptor antagonist rimonabant (0.1 mg·kg⁻¹, i.p.), the CB₂ receptor antagonist SR144528 (1 mg·kg⁻¹, i.p.) or the TRPA1 antagonists HC-030031 (30 mg·kg⁻¹, i.p.) and AP18 (100 mg kg⁻¹, i.p.) on upper gastrointestinal transit in vivo. Transit was expressed as the GC of the distribution of a fluorescent marker along the small intestine. GC ranged from 1 (minimal motility) to 10 (maximal motility) (see Methods section). Columns represent the mean ± SEM of 8–11 mice for each experimental group. ^#P < 0.05 versus control, *P < 0.05 versus croton oil.

Figure 8

Inhibitory effect of CBC (10⁻⁸–10⁻⁴ M) on the contractions induced by ACh (10⁻⁶ M) or EFS in the isolated mouse ileum of control and croton-oil-treated mice. Each point represents mean ± SEM of 7–8 experiments. Both in control mice and in croton oil-treated mice the curve representing the inhibitory effect of CBC on ACh-induced contractions was statistically different (P < 0.001) from the curve representing the inhibitory effect of CBC on EFS-induced contractions.

Figure 9

Electrically-induced contractions in the isolated mouse ileum of (A,C,E) control and (B,D,F) croton oil-treated mice: inhibitory effect of CBC (10⁻⁸–10⁻⁴ M) alone (vehicle) or in the presence of rimonabant (3 × 10⁻⁸ M) and SR144528 (10⁻⁷ M) (A,B), HC-030031 (10⁻⁵ M) and ruthenium red (3 × 10⁻⁶ M) (C,D) or L-NAME (3 × 10⁻⁴ M) and apamin (10⁻⁷ M) (alone or in combination (E,F). Each point represents mean ± SEM of 7–8 experiments. No significant differences among the curves were observed.

Figure 10

Electrically-induced contractions in the mouse isolated ileum of (A,C) control and (B,D) croton oil-treated mice: inhibitory effect of CBC (10⁻⁸–10⁻⁴ M) alone (vehicle) or in the presence of ω-conotoxin (10⁻⁸ M) (A,B), IBMX (10⁻⁷ M), rolipram (10⁻⁶ M) or forskolin (10⁻⁷ M) (C,D). Each point represents mean ± SEM of 7–8 experiments. Both in control mice (A) and in croton oil-treated mice (B), ω-conotoxin (but not IBMX, rolipram or forskolin) significantly (P < 0.01) reduced the inhibitory effect of CBC (significant difference between curves). The insert in (C) shows that IBMX (10⁻⁷ M), rolipram (10⁻⁶ M) and forskolin (10⁻⁷ M) significantly (P < 0.01) reduced the inhibitory effect of WIN55,212-2 (10⁻⁹–10⁻⁶ M) on electrically induced contractions in the ileum of control mice (n= 5).

Figure 11

ACh (10⁻⁶ M)-induced contractions in the isolated mouse ileum of (A) control and (B) croton oil-treated mice: inhibitory effect of CBC (10⁻⁸–10⁻⁴ M) alone (vehicle) or in the presence of ω-conotoxin (10⁻⁸ M), verapamil (10⁻⁶ M) or CPA (10⁻⁵ M). Each point represents mean ± SEM of 8–9 experiments. Both in control mice (A) and in croton oil-treated mice (B), verapamil (but not CPA or ω-conotoxin) significantly (P < 0.01) reduced the inhibitory effect of CBC (significant difference between curves).