Palmitoylethanolamide, a naturally occurring lipid, is an orally effective intestinal anti-inflammatory agent

Abstract

Background and purpose: Palmitoylethanolamide (PEA) acts via several targets, including cannabinoid CB1 and CB2 receptors, transient receptor potential vanilloid type-1 (TRPV1) ion channels, peroxisome proliferator-activated receptor alpha (PPAR α) and orphan G protein-coupled receptor 55 (GRR55), all involved in the control of intestinal inflammation. Here, we investigated the effect of PEA in a murine model of colitis.

Experimental approach: Colitis was induced in mice by intracolonic administration of dinitrobenzenesulfonic acid (DNBS). Inflammation was assessed by evaluating inflammatory markers/parameters and by histology; intestinal permeability by a fluorescent method; colonic cell proliferation by immunohistochemistry; PEA and endocannabinoid levels by liquid chromatography mass spectrometry; receptor and enzyme mRNA expression by quantitative RT-PCR.

Key results: DNBS administration caused inflammatory damage, increased colonic levels of PEA and endocannabinoids, down-regulation of mRNA for TRPV1 and GPR55 but no changes in mRNA for CB1 , CB2 and PPARα. Exogenous PEA (i.p. and/or p.o., 1 mg·kg(-1) ) attenuated inflammation and intestinal permeability, stimulated colonic cell proliferation, and increased colonic TRPV1 and CB1 receptor expression. The anti-inflammatory effect of PEA was attenuated or abolished by CB2 receptor, GPR55 or PPARα antagonists and further increased by the TRPV1 antagonist capsazepine.

Conclusions and implications: PEA improves murine experimental colitis, the effect being mediated by CB2 receptors, GPR55 and PPARα, and modulated by TRPV1 channels.

Figure 1

DNBS-induced colitis in mice. Changes in body weight (A,B) and colon weight/colon length ratio (C,D) from control and DNBS-treated mice in the presence or absence of i.p. (A,C) or p.o. (B,D) PEA. Mice were weighed before DNBS (or vehicle) administration and immediately before killing. Tissues were analysed 3 days after vehicle or DNBS administration. PEA (0.1–10 mg·kg⁻¹) was administered once a day for three consecutive days starting 24 h after the inflammatory insult (therapeutic protocol). Bars are mean ± SEM of 12–15 mice for each experimental group. #P < 0.001 versus control (i.e. mice without intestinal inflammation). *P < 0.05, **P < 0.01 and ***P < 0.001 versus DNBS alone. The insert reports the percentage of inhibition of colon weight/colon length ratio after i.p. or p.o. PEA administration and demonstrates that i.p. PEA was significantly (P < 0.001) more active than p.o. PEA in the experimental model of colitis induced by DNBS.

Figure 2

Histological evaluations of inflamed (DNBS treated) and non-inflamed colons: effect of PEA. No histological modification was observed in the mucosa and submucosa of control mice receiving the vehicle used to dissolve PEA i.p. (A) or p.o. (B); mucosal injury induced by DNBS administration in mice receiving the vehicle used to dissolve PEA i.p. (C) or p.o. (D); i.p. (E) and p.o. (F) PEA reduced the erosion and the inflammation area in the mucosa and in the submucosa. Histological analysis was performed 3 days after DNBS administration. PEA (1 mg·kg⁻¹) was administered once a day for three consecutive days starting 24 h after the inflammatory insult (therapeutic protocol). Original magnification ×200. The Figure shows results representative of four experiments.

Figure 3

Inhibitory effect of PEA on MPO (a marker of intestinal inflammation) activity (A,B) and on serum FITC–dextran concentration (a measure of intestinal permeability; C,D) in DNBS-induced colitis in mice. Permeability and MPO activity were measured on colonic tissues 3 days after vehicle or DNBS administration. PEA [1 mg·kg⁻¹, i.p. (A,C) or p.o. (B,D)] was administered once a day for three consecutive days starting 24 h after the inflammatory insult (therapeutic protocol). Bars are mean ± SEM of four to five mice for each experimental group. #P < 0.001 versus control; **P < 0.01 and ***P < 0.001 versus DNBS alone.

Figure 4

Different patterns of Ki-67 immunoreactivity in the colonic mucosa of control mice [i.e. mice receiving the vehicle used to dissolve PEA i.p. (A) or p.o. (B)], DNBS-treated mice receiving the vehicle used to dissolve PEA [i.p. (C) or p.o. (D)] and mice treated with DNBS plus PEA [i.p. (E) or p.o. (F)]. (A,B) Ki-67-immunopositive cells were localized to the lower part of the crypts. (C,D) Ki-67-immunopositive cells were extended to the superficial part of the crypts. (E,F) Ki-67-immunopositive cells were observed in the two-thirds of the mucosa, only. PEA (1 mg·kg⁻¹) was administered for three consecutive days starting 24 h after the inflammatory insult (therapeutic protocol). The Figure shows results representative of four experiments.

Figure 5

Relative mRNA expression of GDE1, NAPE-PLD, FAAH and NAAA in the colon of control and DNBS-treated mice. Tissues were analysed 3 days after vehicle or DNBS administration. RT-PCR analysis was performed as described in Methods. Results (expressed as fold expression, compared with control, as unity) are mean ± SEM of six experiments. No significant effects of DNBS were found.

Figure 6

PEA (A), oleoylethanolamide (B), anandamide (C) and 2-arachydonoylglycerol (D) levels in colon of control mice and mice treated intracolonically with the pro-inflammatory agent DNBS: effect of PEA. Tissues were analysed 3 days after vehicle or DNBS administration. PEA (1 mg·kg⁻¹) was administered p.o. once a day for three consecutive days starting 24 h after the inflammatory insult. Data are mean ± SEM of four to five mice. *P < 0.05 versus control; #P < 0.05 versus DNBS alone.

Figure 7

Relative mRNA expression of CB₁ receptors, CB₂ receptors, GPR55, PPARα, and TRPV1 channels in the DNBS model of colitis: effect of PEA. (PEA, 1 mg·kg⁻¹) was administered p.o. once a day for three consecutive days starting 24 h after the inflammatory insult. Tissues were analysed 3 days after vehicle or DNBS administration. RT-PCR analysis was performed as described in Methods. Results (expressed as fold expression, compared with control equal to one) are mean ± SEM of six experiments. **P < 0.01 and ***P < 0.001 versus control; #P < 0.05 versus DNBS.

Figure 8

Effect of PEA alone or in the presence of the CB₁ receptor antagonist rimonabant (3 mg·kg⁻¹, i.p.) (A), the CB₂ receptor antagonist AM630 (10 mg·kg⁻¹, i.p.) (B), the GPR55 receptor antagonist ML-191 (0.5 mg·kg⁻¹, i.p.) (C), the PPARα receptor antagonist GW6471 (1 mg·kg⁻¹, i.p.) (D), or the TRPV1 antagonist capsazepine (10 mg·kg⁻¹, i.p.) (E) on colon weight/colon length ratio in mice with experimental colitis induced by DNBS. PEA (1 mg·kg⁻¹, p.o.) was administered once a day for three consecutive days starting 24 h after the inflammatory insult. The antagonists were given 30 min before PEA administration. Tissues were analysed 3 days after vehicle or DNBS administration. #P < 0.01–0.001 versus control, *P < 0.05 and **P < 0.01 versus DNBS, °P < 0.05 and °°P < 0.01 versus DNBS plus PEA (n = 6–8 mice for each experimental group).

Figure 9

Effect of PEA alone or in the presence of the CB₁ receptor antagonist rimonabant (3 mg·kg⁻¹, i.p.) (A), the CB₂ receptor antagonist AM630 (10 mg·kg⁻¹, i.p.) (B), the GPR55 receptor antagonist ML-191 (0.5 mg·kg⁻¹, i.p.) (C), the PPARα receptor antagonist GW6471 (1 mg·kg⁻¹, i.p.) (D) or the TRPV1 antagonist capsazepine (10 mg·kg⁻¹, i.p.) (E) on MPO activity in the murine model of colitis induced by DNBS. PEA (1 mg·kg⁻¹, p.o.) was administered once a day for three consecutive days starting 24 h after the inflammatory insult. The antagonists were given 30 min before PEA administration. MPO activity was measured on colonic tissues 3 days after vehicle or DNBS administration (therapeutic protocol). Bars are mean ± SEM of four to five mice for each experimental group. #P < 0.01–0.001 versus control, *P < 0.05 and **P < 0.01 versus DNBS, °P < 0.05 and °°P < 0.01 versus DNBS plus PEA.