Animal preparations
Male Wistar rats weighing 250–300 g were used in this study. They were maintained under normal conditions such as free access to food and water, 23 ± 2º C room temperature, 12 h light and 12 h dark cycles and standard humidity. One day before experiment, all rats were deprived of food but had free access to water. All experiments were carried out according to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The Ethical Committee of Tehran University of Medical Sciences, approved all experiment details and animal study. (IR.TUMS.VCR.REC.1398.225).
Drugs
Dapsone powder was a gift from Gilaranco Co, Tehran, Iran and dissolved in normal saline. Absolute ethanol (Merck, Germany) was diluted 1:1 (vol/ vol) in water. Indomethacin (Sigma-Aldrich, United States) was suspended in 1% carboxymethyl cellulose solution.
Experimental procedures
120 male Wistar rats were used in this study and were randomly divided into 3 experimental models:
Model 1: Ethanol-induced gastric damage: Ethanol-induced erosion was produced by administration of 5 ml/kg of ethanol (1:1 v/v) via oral gavage [26].
Model 2: Water immersion stress model: Stress erosion were induced by immersing restrained rats in cold water (23 º C) up to the level of animal necks for 3 and a half an hours as described [27].
Model 3: Indomethacin-induced gastric damage; Indomethacin-induced erosion was produced by oral administration of indomethacin at a single dose of 30 mg/kg by gavage [28].
Each of three experimental models were divided into 5 groups of eight rats, which included 1. Normal group (rats without any intervention), 2. Control (peptic erosion-induced group + vehicle), 3. Gastric erosion + pretreated dapsone 1 mg/kg, 4. Gastric erosion + pretreated dapsone 3 mg/kg, 5. Gastric erosion+ pretreated dapsone 10 mg/kg.
In group 1 animals received only intraperitoneal injection of normal saline without gastric damage. Treated groups in all 3 models received either 1, 3, or 10 mg/kg intraperitoneal injection of dapsone 30 minutes before gastric damage induction.
After 4 hours of indomethacin and 1 hour of ethanol erosion administration, rats were anesthetized with ketamine (80 mg/kg; i.p.) and xylaxine (8 mg/kg; i.p.), then the stomach was separated and observed for macroscopic evaluation. After that, their blood was collected for measurement of inflammatory factors such as TNF-α and IL1-betausingenzyme-linked immunosorbent assay (ELISA) method.
Finally, the gastric tissues were fixed in buffered formaldehyde for histopathological analysis or restored in -80◦C for measurement of myeloperoxidase (MPO) activity.
Assessment of gastric erosions
Macroscopic assessment: The stomachs were washed with 1 ml of phosphate-buffered. The severity of erosion was examined according to J-Scoring method and the score of each group was determined as the ulcer index. Macroscopic measurements were performed blindly. J- Score for each group was calculated by evaluating the erosions in size order: (0–1mm) in diameter = 1; (1–2 mm) = 2; greater than 2 mm in diameter = 3. The sum of these measured area in each animal was described as the ulcer index [29].
Microscopic assessment: Histological studies were performed on stomach by hematoxylin and eosin (H&E) staining method. In brief, tissues were fixed in 10% of buffered formalin solution for 24 hours, embedded in paraffin wax to form blocks and produced sections into 4 μm thicknesses. These sections were hydrated and stained for evaluating morphological damages by a blinded histopathologist. The observation was performed under a light microscope.
Inflammatory Cytokines measurement
Serum levels of TNF-α and IL-1β were measured by ELISA method using rat tumor necrosis factor α ELISA Kit (RAB0479, Sigma Aldrich, United States) and rat IL-1β ELISA Kit (RAB0277, Sigma Aldrich, United States). Serums were separated by centrifugation at 2500 (xg) for 10 min and then samples were kept in aliquots at -80◦C until the time of assay. Serum levels were assessed for TNF-α and IL-1β using (Bio-Tek Synergy HT, US) ELISA plate reader. The levels of TNF-α and IL-1β are presented in pg/ml.
Measurement of gastric Myeloperoxidase (MPO) activity
Gastric tissues were used for measurement of MPO activity. Tissues were homogenized by a mechanical homogenizer and centrifuged at 15,000 x g for 20 min. The supernatantswere used for measurement of MPO activity analysis using a plate reader as described by the manufacturer's instruction (Sigma Aldrich, United States). Tissue MPO activities are presented as U/g tissue.
Immunohistochemical assessment of P-NF-κB
The primary and secondary antibodies used were rabbit polyclonal NF-κB p65 (phospho S536) antibody (at 1:100 dilution, Ab 86299, Cambridge, USA) and FITC Goat anti-Rabbit IgG (at 1:200 dilution, Ab 6717, Cambridge, USA), respectively. To evaluation of p-NF-κB protein localization in stomach tissue, 4-µm thick paraffin-embedded tissue pieces were deparafinized in xylene. Slides were incubated sequentially overnight at 4◦ C with primary antibodies and in secondary antibody for 30 min at room temperature and then washed in PBS. The slides were stained by 4′, 6-diamidino-2-phenylindole (DAPI) and immunostained sections were examined using a fluorescence microscope for the staining intensity evaluation. Images were quantified using image j software.
Statistical analysis
GraphPad Prism (version 9) and Statistical Package for Social Sciences
(SPSS) version 22 were used to analysis the data. One way analysis of variance (ANOVA) followed by Tukey’s post-hoc were used for data analysis. The results were expressed as the mean ± SEM and the significance value was set at 0.05.