After 1 week of adaptation, 6-week-old mice weighing 18–22 g were used for the experiments. Gastric ulcers were induced in female C57BL/6 mice (18–22 g) by intragastric administration of indomethacin. Mice had unlimited access to food and water. Randomized groups of mice (n = 10) were given either saline or indomethacin 20 mg/kg by gavage and sacrificed 24 h later. Experimental groups are shown in Table 1. SAC (3, 10, 30 mg/kg) and rebamipide (30 mg/kg) was administrated intragastrically 1 h before the indomethacin administration. After sacrifice, the isolated stomach was opened
gently and rinsed with ice-cold saline. Photographs were taken of specific areas of damage under a dissecting microscope with magnification. selleck kinase inhibitor selleck chemicals To investigate the degree of gross mucosal damage, the mucosal sides of the stomach were photographed using a digital camera. The area of damage was then fixed in 10% formalin for histological evaluation. For histopathological analysis, the stomach were fixed in 10% neutralized buffered formalin, processing using the standard method and embedded in paraffin. Sections of 4-μm thickness were then stained with HE. The glandular mucosae of corpus and antrum were examined histologically.
Pathologic index was graded according to criteria. Pathological data and slides were blindly reviewed by two independent GI specialists. The stomach mucosa was homogenized with ice-cold cell lysis buffer (Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich, St. Louis, MO, USA). After 20 min of incubation, samples were centrifuged at 10 000 × g for
10 min. Supernatants were then collected. Proteins in lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were incubated with primary antibodies, washed, incubated with peroxidase-conjugated secondary antibodies, rewashed, and then visualized using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The general procedure for Western blot analysis of cultured RGM-1 cells was similar to the procedures described above. Cultured cells were medchemexpress washed twice with cold phosphate-buffered saline (PBS) on ice and harvested by scraping with a rubber scraper. Cells were sedimented by centrifugation at 4°C and resuspended in cell lysis buffer (Cell Signaling Technology) containing 1 mM PMSF (Sigma Aldrich). After sacrifice of animals, blood was collected for ELISA assay. After centrifugation (9000 × g), the PGE2, IL-1β, TNF-α, and IL-6 levels in the supernatant was measured by ELISA, and the concentration is expressed as pg/mg protein. The processes were performed as prostaglandin E2 express EIA kit manuscript (Cayman, Ann Arbor, MI, USA), and IL-1β, TNF-α, and IL-6 kit manuscript (R&D SYSTEMS, Minneapolis, MN, USA).