Animals

Adult male Sprague-Dawley rats weighing 140–150 g were from the National Research Centre (NRC) Animal House Colony, Egypt; they were placed under regular housing circumstances (60% humidity and 24-27 °C room temperature with alternating 12 hr cycles of light and dark). Water and standard food were provided ad libitum. All procedures were carried out in accordance with the protocol approved by the NRC (Giza, Egypt) which was obtained before starting the experiment (certificate no. 14145) and according to the Ethics Committee, Faculty of Pharmacy, Cairo University (no PT 1262).

Drugs and chemicals

CB1-receptor agonist NADA (N-arachidonoyl dopamine), CB1-receptor antagonist Rimonabant (SR141716A), CB2-receptor agonist GW 405833( 1,2,3-Dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-(4-morpholinyl)ethyl]-1H-indole), and CB2-receptor antagonist AM630 (6-Iodopravadoline) were all from Sigma-Aldrich Chemical, USA. Dimethyl sulfoxide (DMSO) was used to dissolve the agonists and antagonists.

Experimental design

Animals were located in six groups (6 rats each) as follows:

Group 1 :(vehicle): rats were given DMSO (0.1 ml, SC).

Group 2: (WIRS): rats were given DMSO (0.1 ml, SC) 30 min before the WIRS-induced ulcer.

Group 3-6: All agonists and antagonists of the cannabinoid receptors were given in the dose of (1 mg/kg SC in DMSO) 30 min before WIRS-induced ulcers.

Methods

Gastric mucosal injury studies

WIRS-induced gastric ulcer model

The water-immersion-restrain-stress-induced ulcer model requires animals to fast for 24–36 hr but have free admission to tap water before the beginning of the experiment. After that, ulcers are produced by confining the animals in a restricted cage (17 cm (H) ×4 cm (W)× 7 cm (L)), and immersing them vertically in a water tank (15–20 °C) for 4 hr to the level of xiphoid to prevent drowning, after completion of the stress, animals were then sacrificed and all stomachs were immediately removed. The stomachs were opened in the direction of the great curvature, cleaned, stretched out on a flat surface, and fixed with 10% formalin (19).

Assessment of gross mucosal damage

Determination of ulcer number and severity

The stomach was opened and fastened to a piece of plastic board along the greater curvature. Mucosal necrotic lesions and red erosions were examined on the mucosa. (20). The total number of ulcers was counted, and then the severity of the ulcers was assessed using the following scores:

0= no ulcer, 1 = lesion size ≤ than 1 mm, 2 = lesion of size 1-2 mm, 3 = lesion of size 2-3 mm, 4 = lesion of size 3-4 mm and 5= lesion of size > 4 mm.

Biochemical assessment

The stomach was immediately put over the ice-cold surface after gross lesion evaluation. The glandular mucosa was cut and washed by an ice-cold saline solution (0.9 % NaCl), weighed, and stored at (-80 °C). moreover, the tissues were homogenized using a Glas-Col homogenizer (Terre Haute, USA) with 0.1 M phosphate buffer saline (PBS) at pH 7.4 to obtain 20% w/v for the biochemical tests.

Determination of lipid peroxidation

The level of MDA was used to measure lipid peroxidation in gastric mucosal homogenates. Determination of MDA was by measuring thiobarbituric reactive species using the Uchiyama and Mihara method in which the thiobarbituric acid reactive substances react with thiobarbituric acid to give a red-colored complex having peak absorbance at 532 nm. The amount of lipid peroxidation was measured in nmol of MDA per gram tissue (21).

Determination of GSH

Ellman’s method was used to determine GSH. The procedure is based on Ellman ´s reagent reduction by–SH groups of GSH to form 2-nitro-s-mercaptobenzoic acid, the nitromercaptobenzoic acid anion which has a bright yellow color that can be detected spectrophotometrically at 412 nm. The amount of GSH in the stomach homogenate was measured in µmol per gram of tissue (22).

Determination of PON-1 activity

The arylesterase activity of PON-1 was measured spectrophotometrically by using phenylacetate. The cleavage of phenylacetate was catalyzed by PON-1 to produce phenol, which can be quantified using a Recording Spectrophotometer by measuring the increase in absorbance at 270 nm and 25 °C (Shimadzu Corporation). One unit of arylesterase activity is referred to as 1 μmol/l of phenol formed per minute. The activity of enzymes was estimated using the extinction coefficient of phenol of 1310 mol/cm at 270 nm, 25 °C, and pH 8.0 and then expressed as (kU/l) (23).

Determination of NO

Griess reagent was used to quantify NO as nitrite in tissue homogenates. Nitrate is converted to nitrite via nitrate reductase. The Griess reagent then converts nitrite to an azo compound that can be measured using a spectrophotometer (24). At 540 nm, the chromophoricazo derivative may be colorimetrically detected. The amount of total nitrite/nitrate (NOx) in each gram of tissue was measured in micromoles.

Pro-inflammatory cytokine assessment

Determination of tumor necrosis factor-alpha

TNF-α was measured in gastric homogenates using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (K0331196, Koma biotech, Gangseo-gu Seoul, Korea). All standards and samples were pipetted into wells containing immobilized antibodies specific for rat TNF-α and incubated at 37 °C for 30 min. The chromogens A & B were added to the wells and incubated at 37 °C for 15 min; the development of color is proportional to the TNF-α amount bounded. Color intensity was measured at 450 nm after 10 min.

Determination of Interleukin-1β

An enzyme-linked immunosorbent assay (ELISA) commercial kit (CEK1772, Cohesion Biosciences, London, UK) was used to measure IL-1 in stomach homogenate. 100 l of each standard and sample should be placed in the corresponding wells, covered tightly and incubated at room temperature for 90 min at 4 °C with moderate shaking. After removing the lid and discarding the solution, the plate was washed three times with the wash buffer working solution, allowing the solution to sit in the wells for 1 to 2 min each time. Using paper towels or another absorbent material blot the plate. At no point should the wells be entirely dry. In each well, pour 100 μl of biotin-labeled detection antibody working solution and incubate for 60 min at 37 °C. Wash the plate three times with a wash buffer working solution, allowing the solution to sit in the wells for one to two minutes each time. Pour off the wash buffer solution and wipe the plate with paper towels. In each well, pour 100 μl of Streptavidin-HRP working solution and incubate for 45 min at 37 °C. Wash the plate five times with the wash buffer working solution, allowing the wash solution to sit in the wells for 1 to 2 min each time. Remove the wash buffer from the plate and wipe it dry with paper towels or other absorbent material. In each well, pour 100 μl of TMB substrate solution and incubate it for 30 min at 37 °C in a dark place. Fill each well with 100 μl of stop solution. The color turns yellow almost instantly; the color intensity was measured at 450 nm after 10 min.

Neutrophil infiltration assessment

Determination of myeloperoxidase

Estimation of MPO in stomach homogenate was done by using enzyme-linked immunosorbent assay (ELISA) commercial kit (HK105-02, Hycult ®biotech, Uden, Netherlands). Before use, all reagents and samples were warmed to room temperature (20–25 °C). In the relevant wells, 100 μl of each standard and sample were introduced. The plate was firmly covered and incubated for 1 hr at room temperature. After discarding the solution, the wells were cleaned four times with a wash solution. Washing was accomplished by using a multi-channel pipette to fill each well with wash buffer (200 μl). Achieving good performance requires complete liquid elimination at each phase. Any leftover wash buffer was removed by flipping the plate against clean paper towels after the last wash. Each well received 100 l of diluted tracer. The plate was incubated for 1 hr with moderate shaking. The wash was repeated after the solution was dumped. Each well received 100 l of prepared Streptavidin solutions. At room temperature, the plate was incubated for 1 hr with moderate shaking. The solution was removed after the incubation period and the wash was repeated. After that, each well-received 100 l of TMB one-step substrate reagent. At room temperature, the plate was incubated also for 20–30 min. Each well received 100 l of stop solution. After adding a stop solution at 450 nm, the optical density was measured within 30 min.

Examination of histopathology

The stomachs of separate groups were dissected and preserved in (10%) formalin. Because formalin has a good hardening effect and produces less tissue shrinking, it was chosen. Dehydration in escalating degrees of alcohol (70 percent, 90 percent, and three changes in absolute alcohol) was followed by fixation for one or two days, clearing with xylene, and to create solid blocks containing the tissue, three consecutive changes in soft paraffin at 50 °C were used, followed by embedding in paraffin wax. Seven m thick serial transverse sections were cut. Paraffin slices were attached to albumin glycerin-coated slides and stained with hematoxylin and eosin. Light microscopy was used to assess the quality of hematoxylin and eosin sections (25).

Determination of inflammatory neutrophils count

A computer system using the Leica Qwin Plus software package version 3 (Switzerland) was used to count neutrophils in 5 fields in each group. Light microscopy at a magnification of 400 was used to transmit the image to the monitor screen.

Statistical analysis

The mean and standard error (SE) of the data were calculated. GraphPad Prism 5 Software version 5 (SanDiego, CA, USA) was used for statistical analysis. Using one-way analysis of variance test (ANOVA) followed by Dunnett’s multiple comparisons test. Ulcer scores were performed by Kruskal-Wallis non-parametric one-way analysis of variance (ANOVA) followed by Dunn’s multiple comparisons test. Data were expressed as median ± Interquartile Range (IQR). The significance criteria were set at 0.05 level of probability. Excel was used to create graphical representations and perform regression analysis.