ATB 340 (A MODULATOR OF SULFITE OXIDASE ACTIVITY) REDUCES OXIDATIVE STRESS DURING HYPERGLYCEMIA AND IN STRESS EXPOSED GASTRIC MUCOSA IN OLD RATS ОРИГІНАЛЬНІ ДОСЛІДЖЕННЯ : ФУНДАМЕНТАЛЬНІ НАУКИ

(n=43) rats were divided into control groups with a standard diet (SD) and experimental groups with a 28-day high fructose level diet (HFD, V. Kozar, 2008) with and without acute stress induction (Takagi, 1964). During 19-28 day, HFD groups consumed per os: a) placebo (1.0 ml of saline per os); b) NaHS, 100 μ mol/kg/day; c) placebo and stress induction; d) NaHS, 100 μ mol/kg/day and stress induction; e) ASA, 10 mg/kg/ day and stress induction; f) conventional aspirin (ASA), 10 mg/kg/day and NaHS, 100 μ mol/kg/ day and stress induction; g) ATB-340, 17.5 mg/kg/day and stress induction. The integrity of the GM was analyzed based on the histological severity index, which was calculated with account for morphological studies of GM samples. The indicator of oxidative stress, malondialdehyde (MDA) and the activity of SO in the GM were assessed using the standard biochemical methods. Results. We have observed a different SO expression in the GM of adult vs old rats with SD and HFD with and without WIS. The effect of HFD resulted in an increase in the SO activity in old rats by 15%, whereas in adults this value was 14%. MDA content changes were similar. In animals that received NaHS, the changes were similar, whereas the SO activity in old rats was 23% higher. Stress induction reduced the SO activity by 35% in old animals and by 22% in adults. The difference in the MDA content amounted to 41%. The effect of exogenous NaHS in old rats under stress was the highest when combined with ASA, while the MDA content was the highest when classical aspirin was used, which also coincided with similar changes in histological severity index. Conclusion. Sul ﬁ te oxidase is the essential contributor to age-related oxidative stress in the GM under research. The use of ATB-340 (H 2 S-aspirin) increases the H 2 S content in the tissues, reduces symptoms of GM damage associated with a decrease in the SO activity and the content of MDA, which con ﬁ rms alleviation of oxidative stress and has anti-radical and anti-oxidant effects. Disclosures. No con ﬂ icts of interest, ﬁ nancial or otherwise, are declared by the authors. Author contribution. Yaroslav Pavlovskiy, Maksym Lutsyk drafted manuscript; Antonina Yashchenko edited and revised manuscript; Natalia Zaichko, John Wallace, Oksana Zayachkivska approved ﬁ nal version of the manuscript.


Introduction
The gastrointestinal (GI) tract shows a remarkable resilience to damage induced by the beverages and foods that we ingest, which can have a wide range of osmolarity, pH and temperature [20]. This resistance to tissue damage is collectively referred to as "mucosal defense" and many different chemical mediators participate in GM defense, inflammation and repair [9,18]. A new classifi cation of human diseases will become available soon, based on consensus in expert committees that decided to include new category "stress-related diseases" (SRD) [7]. It is widely accepted that SRD result from complex reciprocal interaction between epigenetic and genetic factors. One of the epigenetic factors is fructose-rich nutrition, which results in oxidative stress, however its prevention remains incompletely understood [3,14]. There are age-related features in gastric mucosa (GM) defense, inflammation and repair against cytolytic agents, including prostaglandin/cyclooxygenase activities [11]. The issue of preventing and correcting the cyto-aggressive action of nonsteroidal anti-infl ammatory drugs (NSAIDs), which widely used by elderly patients and most often causes GM damage to the elderly patients [1,2,6] and whatever age-related features in H 2 S mucosal defence against oxidative stress is related to that, is open.
Important bioregulators of prostaglandin-independent mechanisms of cytoprotection of the GM is sulfur compounds. Recent extensive research into their effects confi rmed as potent cyto-and vasoprotective effects, including against low-grade infl ammation [17]. It has been shown that Sulfhydryl (SH) groups play prominent role in gastroprotection by depleting glutathione (GSH) and induction oxidative stress [11,18]. Another important regulator is the gas transmitter H 2 S (hydrogen sulfi de), which is known for its vasodilator, antioxidant, antiapoptotic and pro-angiogenic properties, which provide cytoprotective and anti-infl ammatory effects [16,22]. A number of studies point out the unity of various ways of H 2 S synthesis: enzymatic and non-enzymatic (the transformation of thiols and thiol-containing compounds) [5,15,23]. The four most important enzymes involved in formation of hydrogen sulfi de are: cystathionine β-synthase -CBS, cystathionine γ-lyase -CSE, 3-mercaptopyruvate sulfurtransferase -3-MST, which cooperates with cysteine aminotrans- ferase -CAT [17]. It is known that these enzymes have specifi c tissue and cellular localization. We have shown that for the purpose of cytoprotection of the mucosal barrier of the digestive system (esophagus, stomach and intestines) and anti-infl ammatory action, the synthesis of H 2 S is carried out using CSE, as well as the maintenance of normal microfl ora in the cardiovascular system under physiological conditions [16,23]. It has been found that in activated neutrophils, H 2 S can be oxidized to thiosulfate (under the infl uence of sulfi te oxidase (SO, EC 1.8.3.1) oxidizes sulfi te to sulfate), and then converted to SO2 (by enzymes thiosulfate sulfutransferase or thiosulfate reductase). SO is one of the enzymes involved in the utilization of H 2 S in mitochondria too [5]. Both H 2 S pathways enzymatic and non-enzymatic act as crucial rheostat regulators of the redox state, affected by active forms of oxygen levels in cells, and have both been shown to combine together and perform key roles in cellular and subcellular survival and progression under extreme factors actions. However, the full concept explaining the role of SO in GM cytoprotection in the aspect of age-related changes is still unknown. Metabolic dysfunction induced by long-term fructose infl uence is not fi nally clarifi ed. Taking in account that of nonsteroidal anti-infl ammatory drugs (NSAIDs), potent cytolysis' agents for GM, are among the most commonly used medications, and their use is increasing with aging populations worldwide despite of the significant risk for pro-oxidative gastrointestinal (GI) effects, bleeding and ulceration, sometimes leading to death [6,10,12].

Aim.
To characterize the role of sulfi te oxidase (SO) and activity of oxidative stress in GM in older rats with fructose exposed hyperglycemia and to assess the therapeutic effect of the recently synthesized H 2 S donor -hybrid NSAID H 2 S-acetylsalicylic (H 2 S-ASA, ATB 340) adult vs old rat GM.

Materials and Methods
All experiments were carried out on adult and old rats (N=67, body weight 350±40g; n=5-6) in accordance with the norms of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientifi c Purposes (1986) and in accordance with the Committee on Bioethics of Danylo Halytsky Lviv National Medical University (protocol № 6 from 29.03.2017). Animals were maintained under a constant 12 h light and dark cycle and an ambient temperature of 21-23 0 C with 50±10% relative humidity. All animals were kept in raised mesh-bottom cages to prevent coprophagy. Animal from the control group were allowed free access to tap water and kept on standard diet (SD), experimental groups were on 28-days hypercaloric high-fructose diet (HFD by V. Kozar, 2008), with unrestricted access to 40% solution of fructose ad libitum within drinking-water [8].
Acute stress was induced by model of Takagi et al. that involves short-term exposure to water-immersion stress [20]. The rats were placed in restraint cages and immersed vertically to the level of the xiphoid process in a water bath of 23˚ for 3.5 h. The initial and fi nal body weights were recorded. Blood glucose concentrations were measured daily by glucometer (Achtung TD-4207, Germany) using a blood sample from the tail vein.
The rat stomach was removed immediately after euthanasia and cut with scissors in the longitudinal direction from the gastroesophageal junction to the pylorus. The gastric mucosal surface was gently washed with phosphate-buffered (pH 7.4) saline. Samples of the GM were fi xed in 10% formalin and embedded Visualization was performed using a microscope (Swift Instruments International, Japan) and a digital video camera (Echoo-Imager 5020200 Microscope Digital Eyepiece, China).
The activity of SO was determined in GM homogenates biochemically by the rate of oxidation of sulfate anion in the presence of potassium hexacyanoferrate by standart biochemical methods. GM was homogenized at 3000 rpm (Tefl on-glass) in a medium of 1.15% potassium chloride (ratio 1:3). The homogenates were centrifuged for 30 min. at 600g, the aliquots of the post-native supernatant were taken in the Eppendorf microtubules and stored at -20°C until the research was carried out [23].
The intensity of the oxidative component of oxidative stress was evaluated by changes in the rate of generation of an unstable free radical of oxygen -superoxide anion (O 2 -), hydroxyl radical (OH -) and by changes in the content of terminal products of lipid peroxidation (malonic dialdehyde -MDA).
The level of MDA was determined by reaction with thiobarbituric acid (TBA) by the diagnostic set TBK-Agate (Biokont, RF). With a thiobarbituric acid the lipid peroxidation products form a red stained complex, which is extracted with butanol, with a maximum light absorption at λ = 535 nm.
Statistical analysis of the results was carried out using programs «Excel» і «Statistica 7.0». The certainty of the changes was estimated by ANOVA Dunnett's test.
The relative difference between the physiological and biochemical parameters against control was calculated by formula: where Х і -value in the main group; Х n -value in the control group.

Results and discussion
Healthy adult rats on SD treated with vehicle exhibited normal gastric mucosa appearance, with histological scores of zero. The GM histological scores in old rats ranged between 1 and 2 (on a 0 to 12 scale) in HS. There was no detectable mucosal damage by HS of GM in adult rats under HFD vs old rats, however combination of WIS and HFD exposure caused sharply rise of HS (Fig. 1). The effects on the GM in old rats were with irregular subepithelial exudates, submucosal vascular dilation and mild leukocyte infi ltration in the gastric epithelium and submucosal clots ( Fig.  2A). Infl uence of treatment with NaHS and ATB 340 (donors of H 2 S synthesis) resulted in attenuated gastric injury induced by HFD, acute stress and ASA (Fig. 2B-2D).
We have observed a different expression of SO activity in GM in adult vs old rats with SD and HFD without and with WIS. In control groups that consumed SD, which we considered relative physiological norm, the SO activity in adult rats was 2,9 nmol/min·mg protein, in the old -4,1 nmol/min·mg protein, the effect of HFD caused an increase in SO activity in old animals by 15%, and in adult by 14%, as compared to control (Fig. 3).
Changes in MDA content had the same tendency. The use of exogenous NaHS, which increases the H 2 S content in the tissues of the body, reduced the activity of SO by 24% of adult rats, whereas in old rats it decreased by 23% compared to the groups without correction of the synthesis of H 2 S. The obtained data allows us to interpret the effect of H 2 S, on the background of HFD, as anti-oxidant and antiradical. Investigation of changes of MDA levels in all experimental groups are represented on Fig. 4.
Regarding the importance to test agerelated activity of H 2 S enzyme SO against GM injury caused by long-term high fructose diet (HFD), we combined exposure to HFD with WIS. Stress induction showed agerelated changes in the activity of SO. Older animals showed an increase in SO activity compared to adults by 18%, indicating better   [10]. The relative difference between experimental groups and control data are represented on Tab. 1.

Conclusions.
Long-term postprandial hyperglycemia induced by HFD is ones agent that decline mucosal defense in stomach. Sulfi te oxidase is the essential contributor to age-related oxidative stress in GM during exposure of HFD. Supplementation of NaHS and ATB-340 (H 2 S-aspirin), which increases the H 2 S content in the tissues, reduces the symptoms of GM damage associated with a decrease in the activity of SO and the content of MDA, indicating the alleviation of oxidative stress, showing an anti-radical and anti-oxidant effects.