2.3. Animals and grouping:
42 adult male Swiss albino mice, obtained from the Cairo university animal house, weighing between 35-40 g. Mice were housed and placed in a room; with controlled temperature of 22 ± 3°C and a 12 hour light/dark cycle. Animals had free access to food and water throughout the study. All experiments followed the guidelines for the Care and Use of Laboratory Animals 8th Edition (2011), that is adopted by the Institutional Animal Care and Use Committee (IACUC) of Cairo University, Approval number:(CU, III, F, 81, 18), and in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. Before conducting experiments, animals were acclimatized to laboratory conditions for 7 days and observed to exclude any mouse with prominent gastrointestinal dysfunction (i.e. Diarrhea, mucus secretion). Mice were randomly divided into 7 groups equally. Group 1 is control group. After fasting for 24 hours, and under isoflurane 2% anesthesia, mice in control group underwent colonic lavage with normal saline using a 4cm-polypropylene trocar cannula inserted through the rectum then left untreated all through the experiment. While groups 2-7 received 0.1 ml of acetic acid 4% intra-rectally that was left in contact with colonic tissue for 30 seconds to induce colitis, followed by colonic lavage with saline [12]. After 24 hours, all six groups continued treatment regimen for 10 days as follows: - Group 2: Acetic-acid/ A-A group: received saline by oral gavage. - Group 3: Liraglutide group: Mice in this group received liraglutide at a starting dose of 0.3 mg/kg/day S.C injection, increased to 0.6 mg/kg/day on day 2 then 1mg/kg/d from day 3 to day 10 [13]. - Group 4: Sitagliptin group: Mice in this group received sitagliptin 100 mg/kg/d by oral gavage once daily [14]. - Group 5: Sulfasalazine group: Mice in this group received sulfasalazine in a dose of 100 mg/kg/d by oral gavage once daily [15]. - Group 6: Combined Liraglutide-Sulfasalazine group/ CLS: Mice in this group received a combination of both drugs (liraglutide as mentioned in Liraglutide group) and (sulfasalazine 100 mg/kg/d) by oral gavage. - Group 7: Combined Sitagliptin-Sulfasalazine group/ CSS: Mice in this group received a combination of both drugs (sitagliptin 100 mg/kg/d) and (sulfasalazine 100 mg/kg/d) by oral gavage. Random blood glucose (RBG) was measured daily using a glucometer. Colitis activity was evaluated daily using a disease activity index (DAI) by recording weight loss, the incidence of diarrhea and rectal bleeding and scoring the findings in a range of 0-3, (0= unaffected, 1= mild, 2= moderate and 3= severe) to estimate the overall DAI [16]. After a 10 day course of treatment, mice were euthanized by cervical dislocation under 2% isoflurane inhalation. Length of colon segments were measured, dissected and cut into 2 parts each was about 2 cm in length. One part was immersed in 10% formalin for histopathological examination and the other part was frozen at -80°C, to be prepared as a tissue homogenate for testing the colonic tissue expression of NFκB, TNF-α, MDA and CRP.