2.9. Assessment of Antiulcer Activity of AFE

The test was performed using earlier described protocols [23–25] with slight modifications. Animals fasted for 24 hrs before the administration of test agents. The fasted animals were pretreated with AFE (30, 100, and 300 mg/kg, p.o.), omeprazole (10 mg/kg, p.o.), or normal saline (10 mL/kg, p.o.). Sixty (60) min after the treatment, the ulcer was induced via oral administration of 5 mL/kg of 98% ethanol. The animals were humanely sacrificed under diethyl ether anesthesia 60 min after ulcer induction.

The content of the stomach was washed with 10 mL of normal saline into plain test tubes and centrifuged at 4000 rpm for 10 min. The pH of the supernatant was measured using a Digital pH Meter (MODEL # 152–R, Reena Instruments Company, New Delhi, India).

The stomach was dissected along the greater curvature, washed, and gently stretched on a white sheet of paper. Gross examination of the stomach was carried out to assess the degree of ulceration by looking out for lesions, hemorrhages, erosions, and thickening of the gastric epithelia. The ulcer index was determined using the earlier described criteria [26] as follows: 0 = no lesion, 0.5 = hemorrhage, 1 = 1–3 small lesions < 10 mm length, 2 = 1–3 large lesions > 10 mm length, 3 = 1–3 thickened lesions, 4 = more than 3 small lesions, 5 = more than 3 large lesions, and 6 = more than 3 thickened lesions. The ulcer index (UI) was then used to calculate the percentage protection of the treatments by the following formula:

The stomach was fixed in 10% phosphate-buffered formalin for 24 hrs. Portions of the fixed tissues were processed for routine histopathology, embedded in paraffin, sectioned at 5 μm, and stained in hematoxylin and eosin [27]. Sections were examined using an Olympus microscope mounted with a live view digital SLR camera (E-330), and photomicrographs were taken.

Formalin-fixed paraffin-embedded tissue (3-4 micron) was cut and mounted on positively charged slides and air-dried for 20 min at a temperature of 80°C. Afterward, the tissue was deparaffinized (using 3 changes of xylene), dehydrated (using 5 changes of alcohol of increasing concentration), and rehydrated (using distilled water). Heat-induced antigen retrieval was performed using citrate buffer solution at 98°C to reveal a masked epitope. Subsequently, nonspecific protein reaction blocking was performed in 10% normal goat serum in 10 mM PBS+0.03% Triton X-100 and 1% bovine serum albumin (BSA) for 2 hrs at room temperature. The endogenous peroxidase block was done using 0.3% hydrogen peroxide in TBS (15 min). Sections were then incubated in 500 μL primary monoclonal anti-mouse antibody for Ki67 protein (code: sc-23900, dilution 1 : 200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted in the blocking buffer overnight at 4°C. After adequate washes, appropriate HRP-conjugated secondary antibodies were diluted in TBS+1% BSA and applied to slides for an incubation period of 1 hr at room temperature. The immunogenic reaction was developed using 3′3′ diaminobenzidine tetrachloride (DAB). Sections were then counterstained in hematoxylin, washed, dehydrated in absolute alcohol, cleared in xylene, and mounted in DPX. Cells immunoreactive for Ki67 were detected by the presence of a dark reddish-brown chromogen in the nucleus and quantified using the public domain software ImageJ.

About 3 mL of blood was collected by venipuncture of the heart into Vacutainer Hemogard serum separator tubes (SST) (Becton, Dickinson and Company, Temse, Belgium). Separation of serum in SST was accomplished by centrifugation at 4000 rpm for 5 min. Serum was collected and stored at -80°C until they were used for the cytokine assays. Serum levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (INF-γ) were determined using the DuoSet ELISA reagents (R&D Systems Inc., Minneapolis, USA) following the manufacturer's instructions.