N-Acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis

Abstract

N-Palmitoylethanolamine or palmitoylethanolamide (PEA) is an anti-inflammatory compound that was recently shown to exert peroxisome proliferator-activated receptor-α-dependent beneficial effects on colon inflammation. The actions of PEA are terminated following hydrolysis by 2 enzymes: fatty acid amide hydrolase (FAAH), and the less-studied N-acylethanolamine-hydrolyzing acid amidase (NAAA). This study aims to investigate the effects of inhibiting the enzymes responsible for PEA hydrolysis in colon inflammation in order to propose a potential therapeutic target for inflammatory bowel diseases (IBDs). Two murine models of IBD were used to assess the effects of NAAA inhibition, FAAH inhibition, and PEA on macroscopic signs of colon inflammation, macrophage/neutrophil infiltration, and the expression of proinflammatory mediators in the colon, as well as on the colitis-related systemic inflammation. NAAA inhibition increases PEA levels in the colon and reduces colon inflammation and systemic inflammation, similarly to PEA. FAAH inhibition, however, does not increase PEA levels in the colon and does not affect the macroscopic signs of colon inflammation or immune cell infiltration. This is the first report of an anti-inflammatory effect of a systemically administered NAAA inhibitor. Because NAAA is the enzyme responsible for the control of PEA levels in the colon, we put forth this enzyme as a potential therapeutic target in chronic inflammation in general and IBD in particular.

Keywords: 2-AG; Crohn’s disease; N-acyltaurine; PF-3845; endocannabinoid.

Figure 1.

Impact of PEA administration and FAAH inhibition on colon inflammation in TNBS-induced colitis. Colitis is induced by intrarectal administration of TNBS to mice. PEA (10 mg/kg) and the FAAH inhibitor PF-3845 (PF; 10 mg/kg) were administered intraperitoneally. Mice were killed on d 3 after TNBS administration. A) Colon weight/length ratio, (B) feces weight in the cecum, (C) MPO activity, (D) IL-12 and F4-80 mRNA expression, and (E) proinflammatory cytokine production as measured by ELISA. Veh., vehicle. Results are expressed relative to the TNBS-untreated group set at 100%. Results are expressed as the mean ± SEM with n = 10 mice/group. *P < 0.05; **P < 0.005; ***P < 0.001 vs. the TNBS-untreated group; ^$P < 0.05 vs. the colitis group treated with PEA.

Figure 2.

Effect of colitis, FAAH inhibition, and NAAA inhibition on PEA and AEA levels measured by HPLC-MS. PEA and AEA levels from the colon and liver of the different colitis experiments are shown. A, B) PEA and AEA levels in (A) the colon and (B) the liver of mice with TNBS-induced colitis in the presence of the FAAH inhibitor PF-3845 (PF; 10 mg/kg). C, D) PEA and AEA levels in (C) the colon and (D) the liver of mice with TNBS-induced colitis in the presence of the NAAA inhibitor AM9053 [AM; 10 mg/kg twice a day (b.i.d.)]. E) PEA and AEA levels in the colon of mice with DSS-induced colitis. F) PEA and AEA levels in the colon of mice with TNBS-induced colitis in the presence of the FAAH inhibitor URB597 (5 mg/kg b.i.d.). CTL, control. Results are expressed relative to control mice except for (F), where results are expressed compared to the TNBS-untreated group set at 100%. Results are expressed as the mean ± SEM with n = 10 mice/group for TNBS-induced colitis and n = 8 mice/group for DSS-induced colitis. *P < 0.05; ***P < 0.001 vs. the TNBS-untreated group. ^##P < 0.005 for DSS-untreated mice vs. control mice.

Figure 3.

Beneficial effects of PEA administration and NAAA inhibition on colon inflammation in TNBS-induced colitis. Colitis is induced by intrarectal administration of TNBS to mice. PEA (10 mg/kg) and the NAAA inhibitor AM9053 (AM; 10 mg/kg b.i.d.) were administered intraperitoneally. Mice were killed on d 3 after TNBS administration. A) Colon weight/length ratio, (B) feces weight in the cecum, (C) MPO activity, (D) mRNA expression of F4-80 and CD11b, (E) IL-1β expression measured by ELISA, and (F) mRNA expression of IL-12, TNF-α, and MCP-1. prot., protein. Results are expressed as the mean ± SEM with n = 10 mice/group. *P < 0.05, **P < 0.005, ***P < 0.001 vs. the TNBS-untreated group.

Figure 4.

Effects of PEA administration and FAAH inhibition on systemic inflammation resulting from TNBS-induced colitis. TNBS-induced colitis is accompanied by increased proinflammatory cytokine expression in the liver and brain. Effects are shown for PEA (10 mg/kg) administration and FAAH inhibition with PF-3845 (PF; 10 mg/kg) on proinflammatory cytokine expression in (A) the liver and (B) brain. Results are expressed relative to the TNBS-untreated group set at 100%. Results are expressed as the mean ± SEM with n = 10 mice/group. *P < 0.05; **P < 0.005; ***P < 0.001 vs. the TNBS-untreated group.

Figure 5.

Effects of PEA administration and NAAA inhibition on systemic inflammation resulting from TNBS-induced colitis. TNBS-induced colitis is accompanied by increased proinflammatory cytokine expression in the liver and brain. Effects are shown for PEA administration and NAAA inhibition with AM9053 (AM; 10 mg/kg b.i.d.) on (A) proinflammatory cytokine mRNA expression and (B) IL-1β protein expression in the liver and (C) proinflammatory cytokine mRNA expression in the brain. Results are expressed as the mean ± SEM with n = 10 mice/group. *P < 0.05; **P < 0.005; ***P < 0.001 vs. the TNBS-untreated group.

Figure 6.

NATs in TNBS-induced colitis. A) NAT levels in the ileum of mice with TNBS-induced colitis in the presence of the FAAH inhibitor PF-3845 (PF; 10 mg/kg). Results are expressed relative to the control group set at 100% (dotted line). The length and degree of insaturation of the acyl chain are mentioned below their respective columns. B–D) Colitis is induced by intrarectal administration of TNBS to mice. N-Arachidonoyltaurine (NAT; 10 mg/kg) was administered intraperitoneally. Mice were killed on d 3 after TNBS administration. B) Colon weight/length ratio, (C) COX-2 mRNA expression, and (D) F4-80, MCP-1, IL-1β, and TNF-α mRNA expression in the colon. For (C) and (D), results are expressed relative to the control group set at 100%. Results are expressed as the mean ± SEM with n = 10 mice/group. *P < 0.05; **P < 0.005; ***P < 0.001 vs. the TNBS-untreated group.

Figure 7.

Effects of therapeutic administration of PEA and enzyme inhibitors in TNBS-induced colitis. Colitis is induced by intrarectal administration of TNBS to mice. PEA (10 mg/kg), the NAAA inhibitor AM9053 (AM; 10 mg/kg b.i.d.), and the FAAH inhibitor PF-3845 (PF; 10 mg/kg) were administered intraperitoneally, starting 24 h after colitis induction. Mice were killed on d 3 after TNBS administration. A) Colon weight/length ratio, (B) MPO activity in the colon, and (C) IL-1β and TNF-α protein expression measured by ELISA in the colon. Results are expressed as the mean ± SEM with n = 10 mice/group. *P < 0.05; ***P < 0.001 vs. the TNBS-untreated group.