All animal care and experimental procedures complied with the guidelines of Animal Care and were approved by Use Committee of the University of Perugia and by the Italian Minister of Health and Istituto Superiore di Sanità (Italy) and was in agreement with the European guidelines for use of experimental animals (permissions n. 583/2017-PR and 1126/2016-PR). The general health conditions of the animals were monitored daily by the veterinarian in the animal facility. The study protocol caused minor suffering. However, mice with an hight grade of pain were eutanized. AhR knock out mice (Ahr−/−) on C57BL/6 background and their C57BL/6 congenic littermates wild type (Ahr+/+) were originally supplied by Charles River (Wilmington, MA, USA). Colonies were housed under controlled temperature (22 °C) and photoperiods of 12:12-h light/dark cycle in restricted access area and abled to acclimate to these conditions for at least 7 days before inclusion in an experiment.

To reproduce a mouse model of Acute Colitis a 2,4,6-trinitrobenzenesulfonic acid (Sigma Chemical Co, St Louis, MO) (TNBS)-colitis model is used as previously described [27]. Briefly, according to this 8–10 weeks old male mice C57BL/6J wild-type and Ahr+/+ and Ahr−/− on C57BL/6J genetic background were administrated. Briefly, mice were fasted for 12 h overnight (Day −1). The next day (Day 0), mice were sedated by administration of Zoletil at a dose of 50 mg/Kg of body weight. Then a 3.5F = 11.55 mm catheter was inserted into the colon such that it was up to 4 cm from the anus and 1 mg of TNBS in 50% ethanol administered via the catheter into the colon lumen using a 1 mL syringe (injection volume of 100 μL). Control mice received 50% ethanol alone. Pelargonidins were administered by oral gavage (1, 5 or 10 mg/kg) daily from day 0 to the 4 day, that is the day of sacrifice. The severity of colitis was scored daily for each mouse by assessing body weight, the fecal occult blood, and stool consistency. Each parameter was scored from 0 to 4 and the sum represents the Colitis Disease Activity Index (CDAI). The scoring system was as follows: Percent of body weight loss: None = 0; 1–5% = 1; 5–10% = 2; 10–20% = 3; >20% = 4. Stool consistency: Normal = 0; soft but still formed = 1; very soft = 2; diarrhea = 3; liquid stools that stick to the anus or anal occlusion = 4. Fecal blood: None = 0; visible in the stool = 2; severe bleeding with fresh blood around the anus and very present in the stool = 4. On day 4, surviving mice were sacrificed, blood samples collected by facial vein, and the colon excised, measured length and weight, and evaluated for macroscopic damage.

To investigate the effects of Pelargonidins in a mouse model of NASH, 10–12 weeks old C57BL/6 male mice, Ahr wild type (Ahr+/+) and their congenic littermates AhR null mice (Ahr−/−) were fed a high fat diet containing 59 KJ% fat plus 1% cholesterol, w/o sugar (ssniff, Soest, GE) and fructose in drinking water (42 g/L) for 8 weeks as previously described [28]. Food intake was valued as the difference of weight between the provided and the remnant amount of food at 2-day intervals. The food was provided as pressed pellets and the residual spillage was not considered. After 8 days, Ahr+/+ and Ahr−/− HFD-F mice were randomized to receive HFD-F alone or plus Pelargonidin (5 mg/Kg/die) by oral gavage for remaining 7 weeks. On the sacrifice day, fed mice were deeply anesthetized with Zoletil at a dose of 50 mg/Kg and sacrificed before 12 AM. Thus, blood, liver, small intestine, colon, spleen, epididymal adipose tissue (eWAT), brown adipose tissue (BAT) and gastrocnemius were collected. The abdominal circumference (AC) (immediately anterior to the forefoot), body weight, and body length (nose-to-anus or nose–anus length), were measured in anaesthetized mice at the day of sacrifice. The body weight and body length were used to calculate the Body mass index (BMI) (=body weight (g)/length2 (cm2)).

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