2.9. DNBS-Induced Colitis in Mice

Colitis was induced by an intracolonic injection of DNBS, as described previously [55]. Briefly, mice were anaesthetized with inhaled 5% isoflurane (Centro Agrovete Campania, Scafati, Salerno, Italy) and successively, DNBS (150 mg kg⁻¹) was injected into the colon using a polyethylene catheter (1 mm in diameter) via the rectum (4.5 cm from the anus). Control mice received an intracolonic injection of water. Three days following DNBS or water injection, all animals were killed by asphyxiation with CO₂. Then, the abdomen of mice was opened by a midline incision and the colon was removed, isolated from surrounding tissues, opened along the anti-mesenteric border, rinsed, and weighed and the length was measured, in order to determine the colon weight/colon length ratio (mg cm⁻¹). The body weight of mice was measured every day throughout the treatment period. All measurements were performed by workers who were unaware of the particular treatment. The dose of DNBS was decided on the basis of preliminary experiments showing colonic damage associated with a high reproducibility and low mortality for a 150 mg kg⁻¹ dose. The time point of damage evaluation (i.e., 3 days after DNBS was injected) was chosen because the maximal DNBS-induced inflammation has been reported in mice after 3 days [56]. GLUPEA (1–10 mg kg⁻¹) or PEA-um (1–10 mg kg⁻¹) was administered by oral gavage three times (once a day) for three consecutive days starting 24 h after DNBS or vehicle injection. Animals were killed 2 h after the third administration of GLUPEA or PEA-um. DNBS was dissolved in 50% ethanol. GLUPEA or PEA-um was suspended in carboxymethyl cellulose (1.5%, 150 μL per mouse) for oral administration.