Dapsone Attenuates the Severity of Gastric Erosion Induced by Ethanol; Stress; and Indomethacin in Rats: A Pharmacological Approach


 Background: Dapsone, as an antimicrobial compound, has shown strong anti-inflammatory properties in clinical and experimental studies. Inflammation in stomach mocusa produce gastric erosion. Several factors contribute to the development of gastric erosions, including corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs), alcohol, and stress. The aim of this study, therefore, was to evaluate the effects of dapsone against gastric erosions-induced by alcohol, stress, or indomethacin in rat.Methods: Gastric damage was induced in male rats in different three experimental models including ethanol (5 ml/kg, p.o.)-, water-immersion stress-, and indomethacin (30 mg/kg, p.o.)- induced gastric injury. Macroscopic lesion scores (the J- score), inflammatory cytokines levels, NF-κB protein expression, MPO activity, and histopathological evaluation were assessed. Results: Induction of erosion in gastric mocusal led to a significant increase in J-score as an index of macroscopic parameter, tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) as biochemical markers, NF-κB and myeloperoxidase (a marker of neutrophil activation, as well microscopic deterioration. Dapsone reduced the tissue injuries and erosion area, serum levels of inflammatory cytokines, the expression of NF-κB and tissue concentration of myeloperoxidase (a marker of neutrophil activation) in all three experimental groups. Furthermore, the histopathological studies revealed the reduction of damages in dapsone treated animals.Conclusions: To conclude, dapsone exhibits significant gastro-protective effects against the development of experimental gastric erosions in rats which seems to be related to its anti-inflammatory properties.


Introduction
Peptic ulcers is a major disorder in the digestive tract which is associated with high mortality and morbidity [1,2]. The major causes of gastric epithelial damage are H.pylori infection, stress, ethanol as well as medications such as corticosteroids and NSAIDs [3][4][5]. Different mechanisms contribute to the development of gastric erosions. For example inhibition of cyclooxygenase (COX), blockade of the production of prostaglandins, reduce the protective effects of prostaglandins on gastric mucosa, which causes submucosal erosion and gastrointestinal ulcer by NSAIDs [6,7]. Prostaglandins are also involved in protective and therapeutic effects of gastrointestinal hormones in the digestive system [8][9][10]. The essential role in the protection and healing of the mucous membrane plays the maintenance of adequate blood ow in the gut [11]. Mucosal ischemia following decreased gastric mucosal blood ow also plays a role in pathogenesis of gastric mucosal damage induced by stress and ethanol [12,13]; as well reduction of gastric mucosal blood and subsequent ischemia-reperfusion by stress [14]. In addition, in ammatory processes and oxidative stress plays a role in these processes. Disruption of gastric mucosal defense mechanisms such as mucus and bicarbonate secretion, anti-in ammatory and antioxidant agents are further factors leading to the development of peptic ulcers [15][16][17][18].
Dapsone is an antimicrobial compound of synthetic sulfone family used in the treatment of numerous systemic and dermatologic disorders. Similar to the sulphonamide drugs dapsone acts by competing with para-aminobenzoic acid for the active site of the enzyme dihydropteroate synthetase and via inhibiting the synthesis of dihydrofolic acid [19]. Dapsone has anti-in ammatory properties equivalent to the NSAIDs [20,21]. It appears that dapsone can reduce the level of in ammatory cytokines, adhesion molecules, lymphocyte activity and leukotrienes. Dapsone inhibits chemoattractant-induced signal transduction followed by suppression of neutrophil adherence in skin dermatitis [22]. Moreover, dapsone can inhibit the synthesis of both prostaglandins and leukotrienes by macrophages [23]. Studies have also shown that dapsone has antioxidant properties and is able to reduce reactive oxygen species (ROS) formation [24]. Besides, dapsone can reduce the amount myeloperoxidase enzymes leading to inhibition of HOCl formation [25].
Given anti-in ammatory effect of dapsone, this study was designed to investigate the protective effects of dapsone in gastric erosions induced from ethanol, water-immersion stress, and NSAIDs in rats.

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 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].
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 in ammatory factors such as TNF-α and IL1-betausingenzyme-linked immunosorbent assay (ELISA) method.
Finally, the gastric tissues were xed 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 xed in 10% of buffered formalin solution for 24 hours, embedded in para n 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.
In ammatory 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 para n-embedded tissue pieces were depara nized 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 uorescence microscope for the staining intensity evaluation. Images were quanti ed 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 signi cance value was set at 0.05.

Results
Effect of dapsone on macroscopic damage in ethanol induced peptic erosion As illustrated in table 1 hemorrhagic and necrotic area were visible in the gastric mucosa following ethanol administration in control groups (J-score = 61.5±3.90) which was signi cantly different with normal group (P<0.001). A remarkable reduction in gastric mucosal damage was observed in group that had received dapsone (10 mg/kg) (P<0.001). Pre-treatment with 1 mg/kg dapsone didn't show any signi cant changes in the gastric erosion area; but a remarkable decrease in J-score was observed in animals subjected to 3 and 10 mg/kg dapsone (J-score = 42.3±3.27, P<0.05; and J-score = 20.16±1.097 P<0.001, respectively). Table 1 Comparison of J-Score (macroscopic score) between control and dapsone treated groups in rats. Data are presented as Mean± SEM; Number of animals in each group: 8, ### P<0.001 compared to normal group, * P< 0.05; ** P< 0.01; *** P<0.001 compared to relative control group. Effect of dapsone on macroscopic damage in indomethacin-induced gastric damage

GROUPS
As demonstrated in table 1 indomethacin led to formation of multiple mucosal erosions in the control groups compare to normal group (P<0.001). On the contrary, According to Table1, the J-score in control rats exposed to indomethacin reached 43.1±4.99, which is markedly higher than rats that did not receive indomethacin. Pre-treatment with 10 mg/kg dapsone was protective against indomethacin-induced gastric damage (J-score= 15±2.06, P<0.01). Pre-treatment with dapsone at doses of 1 and 3 mg/kg did not show any signi cant protective effects on gastric lesions.
Histopathological assessment of gastric tissue Fig. 1a shows the histological features of the gastric mucosa in a normal rat. Histological examination of the gastric tissues in all control rats in all 3 models showed severe chronic active gastritis with mucosal erosion (Fig. 1b, 1d, and 1f) in comparison with normal group (P 0.01). The results showed that treatment with dapsone (10 mg/kg) improved mucosal congestion in the body of the stomach (as shown in Fig. 1c, 1g). Dapsone (10 mg/kg) treatment in ethanol and indomethacin-induced ulcerated rats showed mild submucosal erosions (about 1/3 and less than 1/4 of the mucosal thickness, respectively. In Fig. 1e, dapsone (10 mg/kg) treatment in stress-induced ulcerated rats showed normal histopathology without signi cant super cial mucosal epithelial degeneration.

Effect of dapsone on plasma in ammatory cytokines
In order to evaluation of in ammatory response, TNF-α and IL-1β, as the in ammatory factors, in serum of animals were detected by ELISA. There was a high serum level of TNF-α and IL-1β in the ethanol-, stress-, and indomethacin-induced gastric erosion groups as compared with normal group (P 0.001). Among the tested doses, high dose of dapsone (10 mg/kg, i.p.) showed a reduction on serum concentration of TNF-α and IL-1β in all three models.
As demonstrated in Fig. 2a and 2b, the increased TNF-α and IL-1β levels in ethanol-induced gastric erosion group was signi cantly reduced after administration of dapsone (10 mg/kg; P<0.001 and P<0.05; respectively). But with lower doses of dapsone (1, and 3 mg/kg), the levels of TNF-α and IL-1β did not change statistically.
In water immersion stress groups ( Fig. 3a and 3b), a statistically signi cant decrease in serum concentration of TNF-α was observed in both higher doses of dapsone (3 and 10 mg/kg) as compared to the control group (P<0.01 and P<0.001, respectively). With regard to serum IL-1β concentration, dapsone could only reduce it at the highest dose 10 mg/kg (P<0.01).

Effect of dapsone on NF-κB expression
We performed immunohistochemistry in order to determine the expression level of NF-κB protein in all three gastric erosion models (Fig. 6). In all control groups the NFκB protein expression was signi cantly raised in comparing to the normal groups. In ethanol-induced gastric erosion model based on results achieved from IHC staining method, the expression of this protein was dramatically decreased in the dapsone 10 mg/kg treated group in comparison with the control (ethanol-induced) group (P<0.001). In the same manner, pretreatment with 10 mg/kg dapsone revealed a signi cant decrease in NF-κB expression compared to the control group in stress-induced peptic erosion (Fig. 7) (P<0.001). According to (Fig. 8), similar to the previous two models, in the indomethacin-induced gastric erosion model remarkable reduction of NF-κB expression in dapsone 10 mg/kg treated group was detected (P<0.001).

Discussion
In the present study, the gastro-protective effects of dapsone in three different rat models of peptic erosion were demonstrated. Three different doses of dapsone (1, 3, and 10 mg/kg) were administered intraperitoneally before gastric erosion induction. According to the results of this research, dapsone at a dose of 1 mg/kg showed no signi cant protective effect in all three experimental models but at the doses of 3 mg/kg and specially 10 mg/kg, the effects of dapsone on reduction of mucosal erosions (J score), histopathological damages, as well as in ammatory factors were evident.
Studies have shown that ethanol can indirectly induce destructive effects in submucosal venules through leukotriene C4 (LTC4) over production [13]. Also it was reported that dapsone can inhibit the synthesis of leukotrienes [23], therefore it may suppress following in ammation. Various reports have proved that free radical formation and oxidative stress pathways play a critical role in the process of in ammation and can accelerate gastric injury following ethanol administration [30]. Reactive oxygen species (ROS), which are produced by in ammatory cells such as active neutrophils, enhance the severity of damages and cause concomitant increase in MPO activity which is considered as an indicator of neutrophil in ltration [31]. In agreement with previous reports, our study showed that the level of gastric MPO was increased remarkably after 1 hour of oral ethanol gavage in control group, but pretreatment with 10 mg/kg dapsone resulted in a signi cant decrease in MPO activity. In our previous study, reduction in the activity of myeloperoxidase due to dapsone administration have been exhibited in an animal model of renal ischemia reperfusion injury [32]. NF-κB is a transcription factor that facilitates essential in ammatory process in ethanol-induced gastric injury including the expression of several proin ammatory factors such as TNF-α and IL-8 [33]. NFκB contains of p65 and p50 subunits although NFκB-p65 subunit has been considered as an indicator for NFκB activation [34]. In recent studies, compounds that decreased the expression of NF-κB have been observed to be useful in the attenuation of gastric erosion [35]. Besides, ethanol administration can provoke a great number of proin ammatory cytokines generation such as IL-1β and TNF-α [36,37]. IL-1β induce macrophage in ltration and ICAM-1 over expression [38]. Moreover, TNF-α can suppresses gastric microcirculation and upregulate NF-κB expression [39], as well as adhesion molecules production [40].
We observed an increase amount of NF-κB protein expression as well as IL-1β, and TNF-α serum level following ethanol gavage in accordance with previous studies. Dehpour et al. have been shown previously that dapsone can decrease the level of IL-1β, and TNF-α in different animal in ammatory models [32,41,42]. Data of this study demonstrated that dapsone only at dose of 10 mg/kg showed a signi cant decrease in of these in ammatory markers in ethanol induced gastric erosion.
In the pathophysiology of stress induced ulcer, oxidative damage and excessive generation of ROS [43], production of proin ammatory cytokines (such as TNF-α, IL-1β and IL-6) and in ltration of neutrophils [44] are involved following local hypoxia and ischemia. Recent studies have shown that TNF-α, IL-1β can lead to delayed healing of gastric ulcers [45]. Most of these in ammatory cytokines are downstream mediators of NF-κB pathway that is reported p65 subunit of NF-κB plays a critical role in response to stress by stimulating the expression of speci c genes [46]. In this study water immersion restraint enhanced TNF-α, IL-1β, and NF-κB activity. In addition, it has been found that dapsone have antiin ammatory properties through down regulation of NF-kB pathway in a rat model of colitis [47]. The present study demonstrated that dapsone reduced mucosal injury of the stomach tissue and J score mean in a dose dependent manner in stress erosion. Although this should be taken into consideration that in microscopic study and J score evaluation, gastric damage resulted from stress showed lower intensity. In addition, we found that, dapsone attenuates stress induced erosion by suppressing of in ammatory cascades mediators. Our data are consistent with a study which report dapsone can inhibit human leukocyte enzyme MPO production, and this inhibition is depends on various factors such as dapsone concentration [48].
As mentioned in previous studies, NSAIDs have inhibitory effects on cyclooxygenase enzymes and subsequently prevent synthesis of protective mucosal prostaglandins. PGE2 could reduce TNF-α and IL-1 release from macrophages and leukotriene B4 [49]. Accumulated evidence show the ability of indomethacin to stimulate NF-κB expression and subsequently up regulation of its promoting downstream cytokines such as TNF-α and IL-1β [50][51][52]. Furthermore, an increase in gastric MPO level, indicates neutrophil in ltration during indomethacin-induced gastric injury [53]. Increased gastric mucosal permeability following disruption in mucosal barriers, leads to microbial invasion, neutrophil chemotaxis and in ammation [54]. Pretreatment with 10 mg/kg dapsone, resulted in a signi cant decrease in the level of MPO, TNF-α, and IL-1β as compared to control groups. In addition, dapsone prevented NF-κB expression and formation of gastric erosions in this model.
Since in ammatory pathways are highly involved in causing and advancing gastric ulcer [15], compounds with antioxidant and anti-in ammatory properties may represent protective roles in development of gastric injuries and erosions. A vast majority of antiin ammatory drugs such as corticosteroids or NSAIDs have been shown to breed gastric destruction [3,4] whereas dapsone showed gastro-protective effects via inhibition of in ammatory cascades, in this study.
This protection may be related to different anti-in ammatory properties of dapsone compared to mentioned drugs.

Conclusions
In summary, dapsone exhibits gastro-protective effects against 3 different causes of gastric mucosal damage namely ethanol, water-immersion stress, and indomethacin. According to present study, the effects of dapsone seems to be associated with a decrease in in ammatory markers (TNF-α, and IL-1β, and NF-κB) and gastric leukocyte in ltration which might be related to anti-in ammatory effects of dapsone. Nevertheless, further research is needed to distinguish the exact protective mechanisms of dapsone.

Ethics approval
The

Authors' contributions
Mohammad Sheibani: Co-designed the study, performed experiments, analysed data, co-wrote the paper. Sadaf Nezamoleslami: Co-designed the study, performed experiments, analysed data, co-wrote the paper.