Protective effect ofN,N’-dimethylthiourea against stress-induced gastric mucosal lesions in rats

In the present study, we examined the protective effect of N,N’‐dimethylthiourea (DMTU), a scavenger of hydroxyl radical (·OH), against water‐immersion restraint stress (WIRS)‐induced gastric mucosal lesions in rats. When male Wistar rats fasted for 24 h were exposed to WIRS for 3 h, gastric mucosal lesions occurred with increases in the levels of gastric mucosal myeloperoxidase (MPO), an index of tissue neutrophil infiltration, pro‐inflammatory cytokines (tumor necrosis factor alpha and interleukin 1beta), lipid peroxide (LPO), and nitrite/nitrate (NOx), an index of nitric oxide synthesis, and decreases in the levels of gastric mucosal nonprotein SH and vitamin C and gastric adherent mucus. DMTU (1, 2.5, or 5 mmol/kg) administered orally at 0.5 h before the onset of WIRS reduced the severity of gastric mucosal lesions with attenuation of the changes in the levels of gastric mucosal MPO, pro‐inflammatory cytokines, LPO, NOx, nonprotein SH, and vitamin C and gastric adherent mucus found at 3 h after the onset of WIRS in a dose‐dependent manner. Serum levels of corticosterone and glucose, which are indices of stress responses, increased in rats exposed to WIRS for 3 h, but DMTU pre‐administered at any dose had no effect on these increases. These results indicate that DMTU protects against WIRS‐induced gastric mucosal lesions in rats by exerting its antioxidant action including ·OH scavenging and its anti‐inflammatory action without affecting the stress response.

gastric mucosal lesions; inflammation; N; N’‐dimethylthiourea; oxidative stress; water‐immersion restraint stress (rats)

Abbreviations

BMC‐AML bromocriptine and amlodipine co‐administration.

BMC bromocriptine

CCBs calcium channel blockers

CPZ chlorpromazine

n.s. not significant

NAs neurobehavioural abnormalities

NDs neurodegenerative disorders

Acute gastric mucosal lesions (AGML) induced by stress are often encountered in clinical status, and the management of stress‐induced AGML is of primary importance. Salim [1] has shown in patients with stress‐induced AGML that treatment with allopurinol, an inhibitor of xanthine oxidase (XO) which generates reactive oxygen species (ROS), or dimethyl sulfoxide, a scavenger of hydroxyl radical (∙OH), prevents the development of AGML when the severity of AGML is observed endoscopically. The author has suggested that ROS are directly implicated in stress‐induced AGML in humans and that removing ROS protects against stress‐induced AGML and its complications [1] .

Water‐immersion restraint stress (WIRS) is widely used as an experimental model of stress‐induced AGML, because this model has the reliable reproducibility on gastric mucosal lesions without surgical and anesthetic procedures [2] . Oxidative stress associated with ROS generated by infiltrated neutrophils and the xanthine‐XO system, excessive nitric oxide (∙NO) generated by inducible nitric oxide synthase (iNOS), disrupted antioxidant defense systems, and lipid peroxidation in gastric mucosal tissues are involved in the development of WIRS‐induced gastric mucosal lesions in rats [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] . Inflammation associated with neutrophil infiltration, pro‐inflammatory cytokines such as interleukin 1beta (ΙL‐1β) and tumor necrosis factor alpha (TNF‐α), and iNOS is also involved in the development of WIRS‐induced gastric mucosal lesions in rats [11] , [12] , [13] , [14] . Yasukawa et al. [7] have demonstrated using the in vivo ESR/nitroxyl probe technique for noninvasive analysis of ROS that ROS generated from the xanthine‐XO system induce neutrophil infiltration into the gastric mucosa of WIRS‐exposed rats, resulting in ·OH production by infiltrated neutrophils in the tissue and have suggested that this ∙OH generated by infiltrated neutrophils induces gastric mucosal lesion formation in WIRS‐exposed rats. Asano et al. [15] have demonstrated using the histochemical technique that the staining levels of redox‐active non‐heme iron and hydrogen peroxide (H2O2) increase in the oncotic parietal cells located at the erosive lesion in the gastric mucosa of WIRS‐exposed rats and have suggested that ·OH generated through the iron‐catalyzed Haber‐Weiss reaction is involved in the development of WIRS‐induced gastric mucosal lesions. Thus, it has been implicated that ∙OH generated in the gastric mucosa of rats with WIRS contributes to the development of gastric mucosal lesions.

Fox [16] has shown that N,N’‐dimethylthiourea (DMTU), a powerful scavenger of ∙OH, blocks ·OH production by activated neutrophils in vitro without interfering other neutrophil functions, such as the production of superoxide radical (O2−∙), myeloperoxidase (MPO) activity, chemotaxis, degranulation, or aggregation. DMTU scavenges hypochlorous acid (HOCl) which is generated by MPO in the presence of H2O2 and chloride ion, thereby protecting against neutrophil‐mediated tissue damage [17] . DMTU inhibits tissue damage due to peroxynitrite (ONOO−) which is generated by the interaction between O2−∙ and ∙NO[18] . It has been reported that DMTU protects against gastric mucosal injury induced by ethanol, indomethacin, and ∙NO donors in rats possibly by scavenging ∙OH in gastric mucosal tissues [19] , [20] , [21] . At present, however, there is no available information on whether DMTU protects against WIRS‐induced gastric mucosal lesions in experimental animals.

In the present study, therefore, we examined the protective effect of DMTU against gastric mucosal lesions in rats exposed to WIRS.

3,3′,5,5′‐Tetramethylbenzidine (TMB), dioctyl sodium sulfosuccinate (DSS), and xanthine were purchased from Sigma‐Aldrich Japan (Tokyo, Japan). DMTU, VC (l‐ascorbic acid), reduced glutathione (GSH), α,α’‐dipyridyl, 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB), N,N‐dimethylformamide, alcian blue, ethylenediaminetetraacetic acid (EDTA), corticosterone (CORT), thiobarbituric acid, trichloroacetic acid (TCA), and other chemicals were obtained from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). All chemicals used were of reagent grade and were not further purified.

Male Wistar rats aged 6 weeks were purchased from Nippon SLC Co. (Hamamatsu, Japan). The animals were housed in cages a ventilated animal room with controlled temperature (23 ± 2 °C) and relative humidity (55 ± 5%) with 12 h of light (7:00 to 19:00). The animals were maintained with free access to rat chow, Oriental MF (Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum for 1 week. All animals received humane care in compliance with the Guidelines of the Management of Laboratory Animals in Fujita Health University. The animal experiment was approved by the Institutional Animal Care and Use Committee and its approved protocol number was M1461.

Rats aged 7 weeks were starved for 24 h before experiment, but were allowed free access to tap water. Rats were restrained in wire cages and immersed up to the depth of the xiphoid process in a 23 °C water bath to induce WIRS‐induced gastric mucosal lesions, as described by Takagi and Okabe [2] . Rats with and without WIRS were killed under pentobarbital anesthesia at 3 h after the onset of WIRS. The observation of gastric mucosal lesions was conducted as follows: 10 mL of 0.9% NaCl was infused into the stomachs of rats with and without WIRS through the duodenum after ligation of the esophagus at 5 mm proximal to the gastroesophageal junction and then the duodenum was ligated at 10 mm distal to the pylorus. The stomachs filled with 0.9% NaCl were removed, fixed with 10% formaldehyde for 10 min, and cut open along with the greater curvature. The gastric mucosa was carefully examined for lesions recognized as linear breaks (erosions) at the mucosal surface of the glandular part under a stereoscopic microscope (10×). The extent of the lesion (lesion index) is expressed as the sum of the length of these breaks per stomach.

DMTU dissolved in 0.9% NaCl was orally administered to WIRS‐exposed rats at a volume of 1 mL/100 g body weight (BW), that is, at a dose of 1, 2.5, or 5 mmol/kg BW at 0.5 h before the onset of WIRS. WIRS‐exposed and unexposed rats without DMTU administration were orally administered with an equal volume of 0.9% NaCl at the same time point.

All rats used for the assays of serum components and gastric mucosal components and enzymes were killed under pentobarbital anesthesia at which time blood was collected from the inferior vena cava. Serum was obtained from the collected blood by centrifugation. Immediately after killing, stomachs were isolated and cut open along the grater curvature. The gastric mucosa was removed using a pair of small scissors. The collected serum and gastric mucosa were stored at −80 °C until use.

Serum CORT was fluorometrically assayed by the method of Guillemin et al. [22] using authentic CORT as a standard. Serum glucose was assayed using a kit of Glucose CII‐Test Wako (Wako Pure Chemicals Ind., Litd.).

Gastric mucosal tissues were homogenized using a Physcotron handy microhomogenizer (Microtec Co., Funabashi, Japan). Gastric mucosal tissues were homogenized in nine volumes of ice‐cold 50 mm Tris‐HCl buffer (pH 7.4) containing 1 mm EDTA. The prepared homogenate was used for the assays of VC, nonprotein SH (NSPH), and LPO. VC was assayed by the α,α’‐dipyridyl method of Zannoni et al. [23] using authentic l‐ascorbic acid as a standard. NPSH was assayed by the DTNB method of Sedlak and Lindsay [24] using authentic GSH as a standard. LPO was assayed by the thiobarbituric acid method of Ohkawa et al. [25] using tetramethoxypropane as a standard except that 1 mm EDTA was added to the reaction mixture. The amount of gastric mucosal LPO is expressed as that of malondialdehyde (MDA) equivalents. Nitrite/nitrate (NOx), an index of ∙NO synthesis, in gastric mucosal tissues was assayed by the Griss reaction‐dependent method of Green et al. [26] . NOx in the sample was determined using a nitric oxide assay kit (Roche‐diagnostics Co., Tokyo, Japan). Gastric adherent mucus was assayed by the method of Kitagawa et al. [27] . The glandular portion of the removed stomach (50 mm2, i.e., ~8 mm in diameter) was excised with a scalpel, and the excised part was weighed. The excised stomach was soaked in 2 mL of 0.1% alcian blue dissolved in 0.16 m sucrose buffered with 50 mm sodium acetate (pH 5.8) for 2 h. After removing dye not complexed by two successive washes in 2 mL of 0.25 m sucrose for 15 and 45 min, dye complex with mucus was extracted with 30% DSS for 2 h and then centrifuged (700 g, 10 min). The optical density of the resultant supernatant was read at 620 nm, and the concentration of the extracted alcian blue was calculated in comparison with calibration curve obtained from known concentrations of alcian blue solution. The concentration of gastric mucosa adherent mucus is expressed as that of alcian blue adhered to the gastric mucosal surface (mg/g tissue).

Gastric mucosal tissues were homogenized in nine volumes of ice‐cold 50 mm Tris‐HCl buffer (pH 7.4) and ice‐cold 0.25 m sucrose for the assays of gastric mucosal MPO and XO, respectively. Gastric mucosal MPO was assayed by the method of Suzuki et al. [28] . This enzyme activity was assessed by measuring the H2O2‐dependent oxidation of TMB dissolved in N,N‐dimethylformamide at 37 °C. One unit (U) of this enzyme is defined as the amount of enzyme causing a change in absorbance of 1.0 per min at 655 nm. Gastric mucosal XO was assayed by the method of Hashimoto [29] using xanthine as a substrate. This enzyme activity was assessed by measuring the increase in absorbance at 292 nm following the formation of uric acid at 30 °C. One unit (U) of this enzyme is defined as the amount of enzyme forming 1 μmol uric acid per min.

Protein in gastric mucosal tissue samples was assayed using a commercial Rapid Protein Assay kit (Wako Pure Chemical Ind. Ltd.). Bovine serum albumin was used as a standard in this protein assay.

Gastric mucosal TNF‐α and IL‐1β were assayed using the ELISA kits of Quantikine Rat TNF‐α and Quantikine Rat IL‐1β (R & D Systems, Inc., Minneapolis, MN, USA), respectively. The assays of TNF‐α and IL‐1β in gastric mucosal tissues were performed according to the manufacture's recommendation.

All results obtained are expressed as the mean ± SD of at least triplicate determinations. The statistical analyses of the results were performed using a computerized statistical package (StatView II; Abacus Concepts Inc., Barkley, CA, USA). Each mean value was compared by the one‐way analysis of variance (anova) and Bonferroni/Dunn for multiple comparisons. The significance level was set at P < 0.05.

When rats with 3 h of WIRS were administered with DMTU at a dose of 1, 2.5, or 5 mmol/kg BW at 0.5 h before the onset of WIRS, the index for gastric mucosal lesions (lesion index) was significantly lower in DMTU pre‐administered rats with WIRS than in rats with WIRS alone (Figure[NaN] ). Pre‐administered DMTU attenuated the lesion index in a dose‐dependent manner and DMTU (5 mmol/kg BW) reduced the lesion index to approximately one‐third of that in rats with WIRS alone (Figure[NaN] ).

The typical gross features of stomachs in WIRS‐exposed rats with and without DMTU (5 mmol/kg BW) pre‐administration are shown in Figure[NaN] . The stomach of DMTU pre‐administered rats with WIRS showed much less erosion than that of rats with WIRS alone, although control rats showed no lesion in the stomach; the lesion index in rats with WIRS and DMTU (5 mmol/kg BW) pre‐administration was approximately one‐third of that in rats with WIRS alone (Figure[NaN] ).

The levels of gastric mucosal VC and NPSH and gastric adherent mucus were significantly lower in WIRS‐exposed rats than in unexposed control rats (Figure[NaN] ). DMTU pre‐administered to WIRS‐exposed rats at a dose of 1, 2.5, or 5 mmol/kg BW significantly attenuated the decreased gastric mucosal VC and NPSH and gastric adherent mucus levels in a dose‐dependent manner (Figure[NaN] ).

Gastric mucosal LPO level was significantly higher in WIRS‐exposed rats than in unexposed control rats, while there was no significant difference in gastric mucosal XO activity between both groups (Figure[NaN] ). DMTU pre‐administered to WIRS‐exposed rats at a dose of 1, 2.5, or 5 mmol/kg BW significantly attenuated the increased gastric mucosal LPO level in a dose‐dependent manner (Figure[NaN] a,b). DMTU pre‐administered to WIRS‐exposed rats did not affect gastric mucosal XO activity at any dose (Figure[NaN] c).

Gastric mucosal MPO activity and TNF‐α, IL‐1β, and NOx levels were significantly higher in WIRS‐exposed rats than in unexposed control rats (Figure[NaN] ). DMTU pre‐administered to WIRS‐exposed rats at a dose of 1, 2.5, or 5 mmol/kg BW significantly attenuated the increased gastric mucosal MPO activity and TNF‐α, IL‐1β, and NOx levels in a dose‐dependent manner (Figure[NaN] ).

Serum CORT and glucose levels were significantly higher in WIRS‐exposed rats than in unexposed control rats, but any dose of DMTU pre‐administered to WIRS‐exposed rats had no effect on these increased levels (Figure[NaN] ).

The present study has clearly shown that DMTU, a potent of ∙OH scavenger, protects against WIRS‐induced gastric mucosal lesions in rats through its antioxidant and anti‐inflammatory actions. DMTU administered orally to rats at a dose of 1, 2.5, or 5 mmol/kg BW at 0.5 h before the onset of WIRS attenuated WIRS‐induced gastric mucosal lesions in a dose‐dependent manner. Upon the observation of the gross feature of stomachs, the extent of erosion in the stomach was much less in rats with WIRS and DMTU (5 mmol/kg) pre‐administration than in rats with WIRS alone.

Oxidative stress associated with increased lipid peroxidation and ROS generation, and decreased antioxidant defense systems in gastric mucosal tissues is involved in the development of WIRS‐induced gastric mucosal lesions in rats [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] . It has been implicated that ∙OH generated by infiltrated neutrophils and through the iron‐catalyzed Haber‐Weiss reaction in gastric mucosal tissues is involved in the development of WIRS‐induced gastric mucosal lesions in rats [7] , [15] . GSH and VC are consumed by reacting with ∙OH[30] , [31] . The majority of NPSH in stomachs is GSH [32] . Gastric mucus plays a critical role in the primary defense of the gastric epithelium [33] . Gastric mucus level is known to decrease with the development of WIRS‐induced gastric mucosal lesions in rats [27] , [34] . Gastric mucin reacts with ROS, especially ∙OH[35] . In the present study, pre‐administered DMTU attenuated increased gastric mucosal LPO and decreased gastric mucosal NPSH and VC and gastric adherent mucus levels in rats with 3 h of WIRS in a dose‐dependent manner. These findings indicate that DMTU exerts a protective effect against WIRS‐induced gastric mucosal lesions in rats through the maintenance of gastric mucosal non‐enzymatic antioxidant defense system and gastric barrier possibly by scavenging ∙OH generated in the gastric mucosa. In the present study, the activity of XO, an enzyme to generate ROS, in the gastric mucosa of rats with 3 h of WIRS did not changed, as shown in our previous reports [3] , [36] . Any dose of DMTU pre‐administered to rats with 3 h of WIRS did not affect gastric mucosal XO activity. Yasukawa et al. [7] have shown in WIRS‐exposed rats that ∙OH generation by neutrophils infiltrated into the gastric mucosa depends on the infiltration of neutrophils into the gastric mucosa which is mediated by ROS generated from the hypoxanthine‐XO system. However, the authors have not clarified whether the generation of ROS by the hypoxanthine‐XO system occurs within the gastric mucosa of WIRS‐exposed rats. We have shown in rats with 3 h of WIRS that ROS generated by XO increasing in the blood are involved in neutrophil infiltration into the gastric mucosa [36] .

Inflammation associated with neutrophil infiltration, pro‐inflammatory cytokines such as TNF‐α and IL‐1β, and iNOS plays a critical role in the development of WIRS‐induced gastric mucosal lesions in rats [11] , [12] , [13] , [14] . It has been demonstrated that the activity of iNOS derived from infiltrated neutrophils increases in the gastric mucosa of rats with WIRS, resulting in an excessive generation of ∙NO in the tissue [11] , [37] . In the present study, pre‐administered DMTU reduced increases in the activity of MPO, an index of tissue neutrophil infiltration, and the levels of TNF‐α, IL‐1β, and NOx, an index of ∙NO production, in the gastric mucosa of rats with 3 h of WIRS in a dose‐dependent manner. These results indicate that orally administered DMTU exerts a protective effect against gastric mucosal lesions in rats with WIRS by attenuating inflammation in the gastric mucosa.

N,N’‐Dimethylthiourea blocks ∙OH production by activated neutrophils without interfering MPO activity in vitro [16] and protects against neutrophil‐mediated tissue damage by scavenging HOCl generated by MPO in the presence of H2O2 and chloride ion in vitro [17] . These findings suggest that DMTU administered to WIRS‐exposed rats protects against gastric mucosal lesions by blocking ∙OH production by infiltrated neutrophils and by scavenging HOCl generated by MPO in infiltrated neutrophils without affecting MPO activity in gastric mucosal tissues. Activated neutrophils mediate lipid peroxidation in liposomes via the respiratory burst in vitro [38] , [39] . MPO induces lipid peroxidation in liposomes in the presence of H2O2 and chloride ion in vitro [40] . Besides, MPO functions as a major enzymatic catalyst for the initiation of lipid peroxidation at sites of inflammation in vitro and in vivo [41] . Therefore, it can be assumed that administered DMTU exerts a protective effect against WIRS‐induced gastric mucosal lesions in rats by suppressing lipid peroxidation induced by infiltrated neutrophils and/or MPO present in infiltrated neutrophils through inhibition of neutrophil infiltration in the gastric mucosa. Furthermore, we have suggested that ONOO− is generated by the reaction of ·NO produced by increased iNOS with O2−∙ in the gastric mucosa of WIRS‐exposed rats [4] and that such a generated ONOO− contributes to WIRs‐induced gastric mucosal lesions by enhancing gastric mucosal lipid peroxidation and NPSH oxidation [5] . DMTU is known to inhibit tissue damage due to ONOO−[19] . Accordingly, it is conceivable that DMTU protects against ONOO−‐mediated gastric mucosal lesions in WIRS‐exposed rats.

Plasma or serum adrenocorticotropic hormone, CORT, adrenaline, noradrenalin, and glucose levels increase in response to WIRS in rats [42] , [43] . It has been reported that attenuation of the stress response leads to protection against WIRS‐induced gastric mucosal lesions in rats [44] , [45] , [46] , [47] . In the present study, any dose of DMTU pre‐administered to rats with 3 h of WIRS did not affect increases in serum CORT and glucose levels. Accordingly, it can be thought that orally administered DMTU protects against WIRS‐induced gastric mucosal lesions in rats without affecting the stress response.

In conclusion, the results obtained from the present study indicate that DMTU protects against WIRS‐induced gastric mucosal lesions in rats through its antioxidant action including ·OH scavenging and its anti‐inflammatory action without affecting the stress response. However, further studies are needed to clarify the exact mechanism by which DMTU protects against WIRS‐induced gastric mucosal lesions in rats and whether DMTU is able to exert a therapeutic effect on WIRS‐induced gastric mucosal lesions in rats.

This study was partially supported by a grant from the Research Foundation of Fujita Health University.

The authors declare that they have no conflicts of interest concerning this article.

Graph: Effect of pre‐administered N , N’ ‐dimethylthiourea ( DMTU ) on gastric mucosal lesions in rats with 3 h of water‐immersion restraint stress ( WIRS ). DMTU (1, 2.5, or 5 mmol/kg body weight) was orally administered at 0.5 h before the onset of WIRS. Gastric mucosal lesions were determined using the lesion index as described in. Each value is a mean ± SD of 10 animals in each group. #P < 0.05, compared with rats with WIRS alone.

Graph: Typical gross features of stomachs in water‐immersion restraint stress ( WIRS )‐exposed rats with and without N , N’ ‐dimethylthiourea ( DMTU ) pre‐administration. (a) control rats without WIRS ; (b) rats with 3 h of WIRS alone; (c) rats with WIRS and DMTU (5 mmol/kg body weight) pre‐administration.

Graph: Effect of pre‐administered N , N’ ‐dimethylthiourea ( DMTU ) on gastric mucosal VC (a) and NPSH (b) and gastric adherent mucus (c) levels in rats with 3 h of water‐immersion restraint stress ( WIRS ). DMTU (1, 2.5, or 5 mmol/kg body weight) was orally administered at 0.5 h before the onset of WIRS. Gastric mucosal VC and NPSH and gastric adherent mucus were assayed as described in. Each value is a mean ± SD of eight animals in each group. * P < 0.05, compared with control rats without WIRS alone; #P < 0.05, compared with rats with WIRS alone.

Graph: Effect of pre‐administered N , N’ ‐dimethylthiourea ( DMTU ) on gastric mucosal lipid peroxide ( LPO ) level (a) and xanthine oxidase ( XO ) activity (b) in rats with 3 h of water‐immersion restraint stress ( WIRS ). DMTU (1, 2.5, or 5 mmol/kg body weight) was orally administered at 0.5 h before the onset of WIRS. Gastric mucosal LPO and XO were assayed as described in. Each value is a mean ± SD of eight animals in each group. * P < 0.05, compared with control rats without WIRS alone; #P < 0.05, compared with rats with WIRS alone.

Graph: Effect of pre‐administered N , N’ ‐dimethylthiourea ( DMTU ) on gastric mucosal myeloperoxidase ( MPO ) activity (a) and tumor necrosis factor alpha ( TNF ‐α) (b), IL ‐1β (c), and nitrite/nitrate ( NO x) (d) levels in rats with 3 h of water‐immersion restraint stress ( WIRS ). DMTU (1, 2.5, or 5 mmol/kg body weight) was orally administered at 0.5 h before the onset of WIRS. Gastric mucosal MPO , TNF ‐α, IL ‐1β, and NO x were assayed as described in. Each value is a mean ± SD of eight animals in each group. * P < 0.05, compared with control rats without WIRS alone; #P < 0.05, compared with rats with WIRS alone.

Graph: Effect of pre‐administered N , N’ ‐dimethylthiourea ( DMTU ) on serum corticosterone ( CORT ) (a) and glucose (b) levels in rats with 3 h of water‐immersion restraint stress ( WIRS ). DMTU (1, 2.5 or 5 mmol/kg body weight) was orally administered at 0.5 h before the onset of WIRS. Serum CORT and glucose were assayed as described in. Each value is a mean ± SD of eight animals in each group. * P < 0.05, compared with control rats without WIRS alone.