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.