Interactions between nitric oxide and hydrogen sulfide generating systems in gastric mucosa under condition of the combined action of stress and NDAIDs

Article history: Received on: 29/01/2017 Accepted on: 18/03/2017 Available online: 30/08/2017 The metabolic relationship between H2S and NO in gastric mucosa in norm and pathology is still poorly studied. Aim of this study was to determine mechanisms of interaction between NO and H2S generating systems under conditions of the combined actions of NSAIDs and stress. Water restraint stress (WRS) was used to induce peptic lessions in rats; naproxen and ATB-346 were administered prior to WRS. Nos2, Cbs and Ptgs2 gene expression level was determined by semiquantitative RT-PCR in gastric epitheliocytes. In the gastric mucosa were determined: alterations in H2S and NOx concentrations, changes in activity of myeloperoxidase. Both WRS and naproxen action prior to WRS cased a significant rise in myeloperoxidase activity. Administration of ATB346 resulted in a considerable decrease of myeloperoxidase activity. Naproxen action caused the downregulation of Nos2. The level of Cbs expression in group pretreated with naproxen was much higher than in group of WRS alone. We suppose that it increases as a result of Nos2 downregulation and the correspondent decrease of NO concentration. The relationship between NO and H2S in the gastric mucosa is likely mediated through the regulation of genes expression. As a result of the released H2S, ATB-346 administration decreased the severity of gastric mucosa lesions.


INTRODUCTION
The best characterized among numerous gaseous substance acting as signaling molecules are nitric oxide (NO) and hydrogen sulfide (H 2 S) (Wallace et al., 2015).Each has been shown to play important roles in many physiological and pathophysiological processes in the gastrointestinal system (Wallace, 2010).NO is produced from L-arginine by constitutive NO synthases (cNOS) and inducible (iNOS) isoenzyme (Lanas, 2008).cNOS are very important enzymes in synthesis of NO, which is involved in the control of the gastric blood flow and the regulation of gastric mucosal integrity (Brzozowski et al., 2008).iNOS (Ca 2+ -independent) isoenzyme is activated by proinflammatory stimuli such as cytokines and produces relatively large amounts of NO which contribute to injury and dysfunction of gastric mucosa (Nasadyuk and Sklyarov, 2013).The toxicity of NO has been attributed to the potent nitrating and oxidizing agent, peroxynitrite that affect proteins and DNA.Endogenous H 2 S is produced from L-cysteine either via pyridoxal-5-phosphate (P5P)dependent enzymes -cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), or the more recently described, cysteine aminotransferase (CAT; also P5P-dependent) and -3mercaptopyruvatesulfurtransferase (3-MST) pathway (Chan, Wallace, 2013).At physiological conditions H 2 S is produced by gastric mucosa and contributes to gastric ability to resist damage (Yan and Li, 2014), and like NO, it plays a role in modulating gastric inflammatory responses (Aboubakr et al., 2013).Both NO and H 2 S can exert pro-or anti-inflammatory effects depending on their concentrations; both are important mediators of gastric mucosal defense and as well contribute significantly to repair of damage and resolution of inflammation (Wallace et al., 2015).Moreover, there are close relationships between NO and H 2 S generating systems.
Nonsteroidal anti-inflammatory drugs (NSAIDs) and acute stress are considered to be the main risk factors for the development of gastrointestinal pathology.On the other hand, they can affect metabolism of NO and H 2 S. Adverse effects of NSAIDs are associated with the inhibition of cyclooxygenase (COX) and the deficiency of prostaglandins (PGs) (Takeuchi, 2012).Stress is well known to be associated with the formation of peptic ulcers.Ulcers frequently developed as a result of many stressful events including surgery, trauma, shock, sepsis and burns (Azlina et al., 2015).The pathological basis of stress is connected to the action of hormones such as epinephrine and glucocorticoids which result in oxidative damage, reduced gastric blood flow and inhibition of gastric mucosal prostaglandins synthesis (Fomenko et al., 2014).Both NSAIDs and stress increase the formation of reactive oxygen species (ROS), lipid peroxidation and cause a reduction of antioxidant status of the gastric mucosa (Konturek et al., 2011).Moreover, NSAID-or stress-induced damage causes an inhibition of cNOS and a deficiency of H 2 S that can result in disturbances of gastrointestinal motility, blood flow, secretion, etc (Wallace et al., 2012).
However, the combined action of NSAIDs and stress is still poorly studied, as well as mechanisms of the metabolic relationship between H 2 S and NO in the gastric mucosa.Thus, the purpose of this study was to determine the possible mechanisms of interaction between NO-and H 2 S-generating systems under conditions of the combined actions of NSAIDs and stress.

Animals
The structure of this study and the experimental procedures performed on the animals were approved by the Ethical Committee of L'viv National Medical University in accordance with the norms of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (1986).The experimental procedures were carried out in accordance with international guidelines for the use and care of laboratory animals.Male, outbred rats weighing 200-220 g were used.They were grouped and housed under conditions of controlled temperature (21-22 o C), humidity (65-75%) and light cycle and fed with standard rat chow and water ad libitum.

Study protocol
The rats were randomly divided into 4 groups (n=5-8 in each).Group 1 consisted of rats treated only with vehicle.In group 2, rats were exposed to 5 h of WRS to induce gastric lesions.For this reason, they were restrained in wire cages and immersed up to the depth of the xiphoid process in a water bath (23°C) for 5 hours to induce gastric mucosal lesions, as described by (Takagi et al. 1964).In group 3, naproxen, a non-selective cyclooxygenase (COX) inhibitor, (Sigma-Aldrich, Milwaukee) was administered prior to WRS.In group 4, an H 2 S-releasing derivative of naproxen, ATB-346 (Antibe Therapeutics Inc., Toronto, Canada), was administered.Before administration, naproxen and ATB-346 were dissolved in dimethylsulfoxide (DMSO)/1% carboxymethylcellulose (CMC); 5:95 ratio.Both naproxen and ATB-346 were administered intra-gastrically (via an orally introduced polyethylene tube) at a single dose (10 mgkg -1 ; volume of 1 ml) 30 min prior to WRS.This dose was selected because it produced significant and comparable anti-inflammatory activity in reducing swelling in experimental arthritis (Blackler et al., 2012).Rats were anesthetized with 1 mL of urethane at a dose of 1.1 mg/kg injected intraperitoneally and killed by cervical dislocation.Gastric mucosal samples from the whole mucosa were collected and homogenized in saline (1:4), centrifuged at 2,000 g and the supernatant was used for measurement of biochemical parameters.Mucosal specimens for RNA isolation were immediately snapfrozen in liquid nitrogen and stored at -80°C until analysis.

Histological Evaluation of Gastric Lesion
Specimens of the gastric walls from each rat were cut into pieces and fixed with 10% buffered formalin.The processed tissues were then infiltrated with paraffin to produce tissueparaffin embedding block.Sectioning of the stomach was accomplished by microtome at a thickness of 5 μm.The section was mounted on individual microscope slide.Then, tissues were stained with haematoxylin and eosin for histological evaluation.
Reproducibility of the amplification results was evaluated in parallel experiments by the repetition of the PCR reactions with all animals and each primer at least three times.Separation of PCR products was performed electrophoretically in 1,6 % agarose gel with 0,5 x TВЕ buffer following (Sambrook et al., 2000).For semi-quantitative analysis of amplicons expression based on densitometry the ImageJ 1.45s program was used.Indices of mRNA expression were calculated for each sample following (Konturek et al., 1998).

Measurement of myeloperoxidase (MPO) activity
Gastric mucosal MPO was assayed spectrophotometrically by the method of (Bradley et al., 1982) with some modifications.The MPO activity was analyzed spectrophotometrically as follows: 1 ml of homogenate was added to 2.9 ml of 0.1 M K 3 PO 4 buffer (pH 6.0) involving O-dianisidine dihydrochloride (0.167 mg/ml) and 0.005% hydrogen peroxide of the resection mixture was recorded at a wave length of 450 nm.One unit (U) of activity was defined as that degrading mole of peroxide/mg of protein.

Determination of nitrite/nitrate (NO 2 /NO 3 )
NOx (nitrite/nitrate) concentration in gastric mucosal tissues was assayed by the Griess reaction-dependent method of (Green et al., 1992)].In order to determine total (NO 2 /NO 3 ) concentration to deproteinized homogenates (1:100) of zinc for reduction of nitrate to nitrite or manganese sulfate for measurement of nitrate-anion where added.Naftil-ethylenediamine was used to perform Griess reaction.The absorbance was read in a Statfax at 520-560 (550) nm.Concentration of stable products of NO was expressed as nitrite+nitrate (mmol/g).

Determination of H 2 S concentration in gastric mucosa
1 ml of homogenates of gastric mucosa were incubated for 60 min at 37 o C with 1 ml of solution containing 4 mmol/l pyridoxal 5-phosphate, 20 mmol/l L-cysteine and Tris-HCL buffer (pH 8.5).Thereafter, 0.5 ml of 1 % zinc acetate, 0.5 ml of 20 mmol N,N-dimethyl-p-phenylenediamine in 7.2 M HCl were added and 0.4 ml of 0.4 mol FeCl3 were added and incubated for 20 min.Then, 0.5 ml of 20% trichloroacetic acid was added to precipitate any protein that might be present in the media and centrifugation (10,000 g) was performed.Absorbance (670 nm) of aliquots from the resulting supernatant was determined (Wilinski et al., 2011).The calibration curve of absorbance vs. H 2 S concentration was obtained by using NaHS solution.

Determination of proteins concentration in gastric mucosa
The concentration of proteins was determined by microbiuret method (Ruth et al., 1964).Benedict's reagent was added to the homogenates of gastric mucosa in an alkaline environment and the change of color from blue to purple was observed.

Statistical Analyses
Statistical processing of experimental data was performed using GraphPad Prism 4.03 ("GraphPad Software Inc.", USA).The normal Gaussian distribution of the data was verified by the Shapiro-Wilk normality test.Non-parametric Kruskal-Wallis test and Dunn's post test were performed on obtained data.Statistical significance was set at р ≤ 0.05.The data are expressed as means and standard deviations (SD).

RESULTS
The histological results showed that WRS induced mucosal congestion and disruption of surface epithelial cells with severe inflammatory cell infiltration at the base of mucosa (fig 1-B).Pre-treatment with naproxen potentiated the development of gastric lesions caused by WRS and showed a large hemorrhagic ulcerated area with significant leucocyte infiltration, edema, and disruption of deep mucosa (fig.1-C).The pre-treatment of rats with ATB-346 prior to WRS significantly reduced the disruption of surface epithelial cells (fig.1-D).The infiltration of the mucosa with leucocytes was significantly decreased proving the fact that H 2 S can reduce leucocyte migration to the sites of injury (Zanardo et al., 2006).COX is the enzyme responsible for PG production, and exists in two isozymes, the constitutively expressed COX-1 and the inducible one COX-2 (Kargman et al., 1996;Wallace, 2008).COX-2 plays an important role in the maintenance of mucosal defense in digestive tract, as well as in modulating mucosal inflammation (Wallace, 2008).There is considerable evidence for interactions between gasotransmitters and COX (Salvemini et al., 1993).In our research, WRS caused the activation of adaptive cytoprotection manifested by the upregulation by 2.1-fold (p≤0.001) of Ptgs2 gene expression (Fig. 2 -A), responsible for coding of COX-2 protein.Naproxen non-selectively inhibits activities of both and COX-2.When administered 30 min prior WRS, it significantly down-regulated Ptgs2 expression as compared to WRS alone, practically to the normal level.ATB-346 showed the similar effect on COX-2 gene expression to its parent drug.
Both WRS and nonselective COX inhibition by naproxen prior to WRS caused a significant rise in MPO activity (Fig. 3 -A), which indicates the enhancement of neutrophil migration in the gastric mucosa.As a result, neutrophils are involved in the damage process.Administration of ATB-346 prior to WRS resulted in a considerable decrease of MPO activity.This explains one of the aspects of H 2 S-releasing NSAID's reduced gastrotoxicity.
We previously reported that subjecting rats to WRS resulted in a considerable rise of iNOS activity, while the administration of naproxen prior to WRS resulted in its decrease in the gastric mucosa (Fomenko et al., 2014).In this research we have measured an expression of mRNA of Nos-2, the gene responsible for coding of iNOS protein.Nos2 expression was increased 3.7-fold (p≤0.001)under conditions of WRS.Application of naproxen caused the down-regulation of iNOS gene expression, which explains the decrease of iNOS activity.ATB-346 decreased Nos2 expression 1.7-fold (p≤0.001) more than naproxen.This may have been attributable to the actions of H S released from ATB-346, which can regulate iNOS gene expression.However, the concentration of stabile metabolite of NO was very similar in groups of rats pretreated with either of the NSAIDs (Fig. 3-B).It was previously shown that NO metabolites can regulate activity of CBS but not CSE (Taoka and Banerjee, 2001).In this research we estimated the level of Cbs mRNA expression, WRS caused a decline of Cbs expression, it was 2.6-fold lower than in the control group (p≤0.001),likely a consequence of the down-regulation of CBS gene expression, H 2 S concentration in gastric mucosa declined by 25% (p≤0.001).Unexpectedly, the level of Cbs expression in rats pretreated with naproxen prior to WRS was much higher than with WRS alone.It is possible that it increased as a result of Nos2 downregulation and the corresponding decrease of NO concentration.We speculate that NO can influence not only CBS activity but also Cbs expression.However, the H 2 S concentration in gastric mucosa of rats subjected to the combined action of WRS and naproxen was 25% lower (p≤0.01)than in control group.This suggests a key role of other enzymes involved in the generation of the intracellular H 2 S pool, such as CSE or pyridoxal-5-phosphate independent enzymes.
In the gastric mucosa of rats which were treated with ATB-346 prior to WRS, the H 2 S concentration was practically normal, buth there was still a down-regulation of Cbs expression similar to that observed in the group treated with WRS alone.

DISCUSSION
In this study we have examined the combined action of two types of NSAIDs: conventional (naproxen) and an H 2 Sreleasing derivative (ATB-346), as well as WRS, on the metabolism of gaseous mediators (NO and H 2 S) in the gastric mucosa.Both NO and H 2 S dose-dependently contribute to many physiological and pathological processes, including the maintenance of gastrointestinal integrity and the mechanism of gastroduodenal protection.Nevertheless, if the synthesis of NO and H 2 S is overwhelmed by injurious factors, such an acute stress or use of NSAIDs, a gastric mucosal lesion may develop (Wallace, 2010).
The exposure to stress is one of the most important risk factors in the pathogenesis of various diseases of gastrointestinal system (Huerta-Franco et al., 2013).Stress affects different functions of the digestive system including gastric secretion, gut motility, mucosal permeability, mucosal blood flow and the gastrointestinal microflora (Konturek et al., 2011).In our study, rats subjected to WRS developed ulcerative and hemorrhagic gastric lesions with corresponding histological changes.
As reported previously (Nie et al., 2004), up-regulation of the Ptgs2 and Nos2 genes, which are responsible for coding of pro-inflammatory enzymes COX-2 and iNOS, respectively, was detected in gastric mucosa of rats subjected to WRS.It was previously shown that there was an up-regulation of COX-2 mRNA post-stress which, may be important in the production of PGE 2 during the healing of ulcers (Nie et al., 2004).We had previously showed that considerable activation of iNOS takes place as a result of acute stress (Azlina et al., 2015).In the present study, we showed that the concentration of NO x increased as a result of Nos2 up-regulation and iNOS activation.Oxidative stress developed as a consequence of vasoconstriction in the WRS model.The rise of NOx concentration created grounds for the peroxynitrite generation, which is one of the most toxic derivatives of NO (Lundberg and Weitzberg, 2013).Thus, our research confirmed the suggestion that NO production from iNOS plays a key role in stress-induced gastric injuries (Azlina et al., 2015).
Inflammation of the gastric mucosa developed as a result of stress, which causes enhanced permeability of blood vessels, activation of neutrophils, and their excessive infiltration of the gastric mucosa (Azlina et al., 2015).As a result, we detected the considerable increase in the activity of MPO in the WRS group.Concentrations of another gaseous mediator, H 2 S, decreased under the stress conditions as a result of Cbs gene down-regulation.It created additional conditions for the development of oxidative stress, because the gastric mucosa loses the antioxidant potential of H 2 S.
A range of different NSAIDs are widely used for the treatment of different pathologies.Their use is often accompanied by acute or chronic stress.NSAID administration is associated with both topical and systemic effects leading to the formation of gastric lesions (Wallace, 2008).Endogenous H 2 S generation under these conditions is often inhibited and thus increases the susceptibility of the mucosa to damage induced by NSAIDs (Fomenko et al., 2014).In our study, e pretreatment of rats with a non-selective COX inhibitor (naproxen) under conditions of an acute stress considerably worsened the histological status of the gastric mucosa.The H 2 S-releasing derivative of naproxen (ATB-346) exhibited reduced gastro-toxicity as compared to its parent drug.This phenomenon was previously described and it was associated with the gastroprotective action of H 2 S (Fomenko et al., 2014).We previously reported that the reduced gastrotoxicity of ATB-346 under conditions of acute stress was not connected to the antioxidant role of H 2 S, because MDA concentrations in gastric mucosa were not different from those of rats subjected to simultaneous action of naproxen and stress (Fomenko et al., 2014).
In the present study we showed that ATB-346 considerably inhibited MPO activity in correspondence to the previously shown data (Palinkas et al., 2015).Thus, we can conclude that the main cytoprotective effect of H 2 S released from ATB-346 given at a single dose 10 mg•kg -1 is likely due to its inhibitory effect on infiltration of neutrophils to gastric mucosa.
Taking into account that both NO and H 2 S are actively involved in the mechanisms of gastroprotection and ulcerogenesis, and ATB-346 showed the reduced gastrotoxicity under condition of WRS as compared to its parent drug naproxen, the question of potent mechanisms of interaction between gasotransmitters is very important.It should be pointed out that stress caused a sharp upregulation in Nos2 expression and activity of iNOS while naproxen and ATB-346 administered prior the stress considerably decreased them.This was possibly mediated by the COX-1 inhibition by these NSAIDs and the interaction between COX and NOS (Mollace et al., 2005).The up-regulation of Nos2 under condition of stress was accompanied by a corresponding increase in NO x concentrations and led to Cbs down-regulation and a lack of H 2 S synthesis.Administration of either naproxen or ATB-346 prior the stress caused considerable up-regulation of Cbs mRNA.Still, the influence of ATB-346 on the level of Nos2 expression was more significant than that of naproxen, suggesting that H 2 S released from ATB-346 may decrease the generation of NO.Thus, the relationship between NO and H 2 S in the gastric mucosa under conditions of stress and NSAIDs is likely mediated through the regulation of genes expression.As a result of the released H 2 S, ATB-346 administration decreased the severity of gastric mucosa lesions.
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Fig. 2 :
Fig.2: Expression of mRNA for Ptgs2 (A), Nos2 (B) and Cbs (C) genes in gastric mucosa of vehicle-treated rats, rats subjected to WRS, pretreated with naproxen or ATB-346 prior to WRS.Mean ± SD. n=5 in each group of animals. -p0.05, -p0.001, in relation to vehicle-treated animals;  -p0.01, -p0.001, as compared to the rats subjected to WRS;  -p0.01, -p0.01, as compared to the animals pre-treated with naproxen prior to WRS.

Fig. 3 :
Fig.3: Activity of myeloperoxidase (MPO) (A), concentrations of stable products of NO (B) and H2S (C) in gastric mucosa of vehicle-treated rats, rats subjected to WRS, pretreated with naproxen orATB-346 prior to WRS.Mean ± SD, n=8 in each group of animals. -p0.05, -p0.001, in relation to vehicle-treated animals;  -p0.01, -p0.001, as compared to the rats subjected to WRS;  -p0.01, -p0.01, as compared to the animals pretreated with naproxen prior to WRS