Palmitoylethanolamide normalizes intestinal motility in a model of post-inflammatory accelerated transit: involvement of CB₁ receptors and TRPV1 channels

Abstract

Background and purpose: Palmitoylethanolamide (PEA), a naturally occurring acylethanolamide chemically related to the endocannabinoid anandamide, interacts with targets that have been identified in peripheral nerves controlling gastrointestinal motility, such as cannabinoid CB1 and CB2 receptors, TRPV1 channels and PPARα. Here, we investigated the effect of PEA in a mouse model of functional accelerated transit which persists after the resolution of colonic inflammation (post-inflammatory irritable bowel syndrome).

Experimental approach: Intestinal inflammation was induced by intracolonic administration of oil of mustard (OM). Mice were tested for motility and biochemical and molecular biology changes 4 weeks later. PEA, oleoylethanolamide and endocannabinoid levels were measured by liquid chromatography-mass spectrometry and receptor and enzyme mRNA expression by qRT-PCR.

Key results: OM induced transient colitis and a functional post-inflammatory increase in upper gastrointestinal transit, associated with increased intestinal anandamide (but not 2-arachidonoylglycerol, PEA or oleoylethanolamide) levels and down-regulation of mRNA for TRPV1 channels. Exogenous PEA inhibited the OM-induced increase in transit and tended to increase anandamide levels. Palmitic acid had a weaker effect on transit. Inhibition of transit by PEA was blocked by rimonabant (CB1 receptor antagonist), further increased by 5'-iodoresiniferatoxin (TRPV1 antagonist) and not significantly modified by the PPARα antagonist GW6471.

Conclusions and implications: Intestinal endocannabinoids and TRPV1 channel were dysregulated in a functional model of accelerated transit exhibiting aspects of post-inflammatory irritable bowel syndrome. PEA counteracted the accelerated transit, the effect being mediated by CB1 receptors (possibly via increased anandamide levels) and modulated by TRPV1 channels.

Figure 1

Effect of oil of mustard (OM) on upper gastrointestinal transit 28 days after its intracolonic administration (50 μL of a solution of 0.5% OM in 30% ethanol). Results are expressed as percentage of upper gastrointestinal transit (a low percentage indicates an anti-prokinetic effect). Bars represent the means ± SEM of 9–10 mice. *P < 0.05, significantly different from vehicle.

Figure 2

Inhibitory effect of PEA (1–10 mg·kg⁻¹, i.p.) on upper gastrointestinal transit in control mice (A) and (B) in mice treated with OM. Transit was measured 28 days after OM or vehicle (30% ethanol) administration. Results (the means ± SEM of 9–10 mice for each experimental group) are expressed as a percentage of upper gastrointestinal transit. *P < 0.05, **P < 0.01, significantly different from vehicle. In (C), the effect of PEA (1–10 mg·kg⁻¹, i.p.) on upper gastrointestinal transit is expressed as % of inhibition of corresponding control values. A statistically significant difference (P < 0.01) was observed between the two dose-response curves shown in (C). Note that in (A) the term ‘vehicle’ refers to the vehicle used to dissolve PEA, while in (B) the term ‘vehicle’ refers to the vehicle used to dissolve OM.

Figure 3

Effect of palmitic acid (1–10 mg·kg⁻¹, i.p.) on upper gastrointestinal transit in control mice (A) and in OM-treated mice (B). Transit was measured 28 days after OM or vehicle (30% ethanol) administration. Results (the means ± SEM of 9–10 mice for each experimental group) are expressed as a percentage of upper gastrointestinal transit. **P < 0.01, significantly different from vehicle. Note that in (A) the term ‘vehicle’ refers to the vehicle used to dissolve palmitic acid, while in (B) the term ‘vehicle’ refers to the vehicle used to dissolve OM.

Figure 4

Inhibitory effect of PEA (10 mg·kg⁻¹, i.p.) on upper gastrointestinal transit in control mice (A) and in OM-treated mice (B) in the presence of rimonabant (CB1 ant, 0.1 mg·kg⁻¹, i.p.) or SR144528 (CB2 ant, 1 mg·kg⁻¹, i.p.). Transit was measured 28 days after OM or vehicle (30% ethanol) administration. Results (the means ± SEM of six to eight mice for each experimental group) are expressed as a percentage of upper gastrointestinal transit. #P < 0.05, significantly different from vehicle; **P < 0.01, significantly different from OM; ††P < 0.01, significantly different from PEA. Note that in (A) the term ‘vehicle’ refers to the vehicle used to dissolve PEA, while in (B) the term ‘vehicle’ refers to the vehicle used to dissolve OM.

Figure 5

Inhibitory effect of PEA (10 mg·kg⁻¹, i.p.) on upper gastrointestinal transit in OM-treated mice or in the presence of the TRPV1 channel antagonist 5′-iodoresiniferatoxin (I-RTX, 0.17 mg·kg⁻¹, i.p.). Transit was measured 28 days after OM or vehicle (30% ethanol) administration. Results (the means ± SEM of six to eight mice for each experimental group) are expressed as a percentage of upper gastrointestinal transit. #P < 0.05, significantly different from vehicle, **P < 0.01, ***P < 0.001 significantly different from OM; ††P < 0.01, significantly different from PEA.

Figure 6

Inhibitory effect of PEA (10 mg·kg⁻¹, i.p.) on upper gastrointestinal transit in OM-treated mice alone or in the presence of PPARα antagonist GW6471 (1 mg·kg⁻¹, i.p.). Transit was measured 28 days after OM or vehicle (30% ethanol) administration. Results (the means ± SEM of six to eight mice for each experimental group) are expressed as a percentage of upper gastrointestinal transit. #P < 0.05, significantly different from vehicle; **P < 0.01, significantly different from OM.

Figure 7

Expression of mRNA for TRPV1 (A) and PPAR-α (B) in jejunoileal segments of in mice treated with OM or vehicle. Tissues were analysed 28 days after OM or vehicle administration. Results are the means ± SEM of three experiments. RT-PCR analysis was performed as described in methods, for statistical significance analysis Cq data were also analysed by the REST^R 2009 software(Pfaffl M.W., Nucleic Acid Research 2002, 30, E-36). *P < 0.05, significantly different from vehicle.

Figure 8

Levels of PEA in jejunoileal segments of mice treated with OM or vehicle. The insert shows the mRNA expression of N-acylethanolamine acid amidase (NAAA, a specific enzyme involved in palmitoylethanolamide degradation) in control and OM-treated mice. Tissues were analysed 28 days after OM or vehicle administration. Data are the means ± SEM of five mice (n = 3 for the data contained in the insert). No significant differences were found.

Figure 9

Anandamide (A) and 2-arachidonoylglycerol (B) levels in the small intestine of mice treated with OM or vehicle: effect of exogenous PEA. Tissues were analysed 28 days after OM or vehicle administration. PEA (10 mg·kg⁻¹, i.p.) was administered 30 min before the assay. Data are the means ± SEM of five mice. *P < 0.05, significantly different from vehicle (A: OM vs. OM plus PEA, P = 0.057; B: OM vs. vehicle, P = 0.245).

Inhibitory effect of PEA (1–10 mg·kg⁻¹, oral) on upper gastrointestinal transit in mice treated with OM. Transit was measured 28 days after OM administration. Results (the means ± SEM of 12–15 mice for each experimental group) are expressed as a percentage of upper gastrointestinal transit. #P < 0.05, significantly different from vehicle; *P < 0.05, significantly different from OM.