Melatonin, but not melatonin receptor agonists Neu-P11 and Neu-P67, attenuates TNBS-induced colitis in mice

Abstract

Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory bowel diseases. The aim of the present study was to characterize the role of melatonin receptors (MT) in the anti-inflammatory effect of melatonin and to assess the anti-inflammatory potential of two novel MT receptor agonists, Neu-P11 and Neu-P67, in the mouse model of trinitrobenzenesulfonic acid (TNBS)-induced colitis. Colitis was induced on day 1 by intracolonic (i.c.) administration of TNBS in 30 % ethanol in saline. Melatonin (4 mg/kg, per os (p.o.)), Neu-P11 (20 mg/kg, p.o.; 50 mg/kg, intraperitoneally (i.p.), 50 mg/kg, i.c.), and Neu-P67 (20 mg/kg, p.o.) were given twice daily for 3 days. Luzindole (5 mg/kg, i.p.) was injected 15 min prior to melatonin administration. On day 4, macroscopic and microscopic damage scores were assessed and myeloperoxidase (MPO) activity quantified using O-dianisidine-based assay. Melatonin significantly attenuated colitis in mice, as indicated by the macroscopic score (1.90 ± 0.34 vs. 3.82 ± 0.62 for melatonin- and TNBS-treated mice, respectively), ulcer score (0.87 ± 0.18 vs. 1.31 ± 0.19, respectively), and MPO activity (4.68 ± 0.70 vs.6.26 ± 0.94, respectively). Luzindole, a MT receptor antagonist, did not inhibit the anti-inflammatory effect of melatonin (macroscopic score 1.12 ± 0.22, ulcer score 0.50 ± 0.16); however, luzindole increased MPO activity (7.57 ± 1.05). MT receptor agonists Neu-P11 and Neu-P67 did not improve inflammation induced by TNBS. Melatonin, but not MT receptor agonists, exerts potent anti-inflammatory action in acute TNBS-induced colitis. Our data suggests that melatonin attenuates colitis by additional, MT receptor-independent pathways.

Keywords: Colitis; Inflammatory bowel diseases; Melatonin; Melatonin receptor agonists.

Fig. 1

Oral administration of melatonin at the dose of 4 mg/kg twice daily significantly attenuated colitis in mice but blockage of MT receptors did not cause worsening of the intestinal inflammation. Figure shows data for macroscopic score (a), MPO activity (b), ulcer score (c), and colon length (d). ***p < 0.001 as compared with control, ##p < 0.01, ###p < 0.001 as compared with TNBS-treated animals, @@@ as compared with melatonin-treated mice. Data represent mean ± SEM, n = 6–8 mice per group

Fig. 2

Oral administration of Neu-P11 at the dose of 20 mg/kg twice daily did not improve colitis in mice. Figure shows data for macroscopic score (a), MPO activity (b), ulcer score (c), and colon length (d). *p < 0.05, ***p < 0.001 as compared with control. Data represent mean ± SEM, n = 6–8 mice per group

Fig. 3

Intraperitoneal injection of Neu-P11 at the dose of 50 mg/kg twice daily did not improve colitis in mice. Figure shows data for macroscopic score (a), MPO activity (b), ulcer score (c), and colon length (d). **p < 0.01, ***p < 0.001 as compared with control. Data represent mean ± SEM, n = 6–8 mice per group

Fig. 4

Intracolonic injection of Neu-P11 at the dose of 50 mg/kg twice daily did not improve colitis in mice. Figure shows data for macroscopic score (a), MPO activity (b), ulcer score (c), and colon length (d). **p < 0.01, ***p < 0.001 as compared with control. Data represent mean ± SEM, n = 6–8 mice per group

Fig. 5

Oral administration of Neu-P67 at the dose of 20 mg/kg twice daily did not improve macroscopic score, but decreased MPO activity in TNBS-treated mice. Figure shows data for macroscopic score (a), MPO activity (b), ulcer score (c), and colon length (d). **p < 0.01, ***p < 0.001 as compared with control, ##p < 0.01, as compared with TNBS-treated animals. Data represent mean ± SEM, n = 6–8 mice per group

Fig. 6

Melatonin attenuated microscopic damage score. Microscopic total damage score and representative micrographs of hematoxylin and eosin-stained sections of distal colon from a control, b TNBS, c TNBS and melatonin (4 mg/kg, p.o., twice daily), d TNBS and melatonin (4 mg/kg, p.o., twice daily) co-administered with luzindole (5 mg/kg, i.p.), e TNBS and Neu-P11 (20 mg/kg, p.o., twice daily), f TNBS and Neu-P11 (50 mg/kg, i.p., twice daily), g TNBS and Neu-P11 (50 mg/kg, i.c., twice daily) treated mice, (H) TNBS and Neu-P67 (20 mg/kg, p.o., twice daily), and i total microscopic damage score. Scale bar = 100 μm. ***p < 0.001, as compared with control. #p < 0.05, ###p < 0.001, as compared with TNBS-treated mice. @@p < 0.01, as compared with melatonin-treated mice. Data represent mean ± SEM, n = 6–8 mice per group