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Protective Effect of Rebamipide against Hydrogen Peroxide-Induced Hemorrhagic Mucosal Lesions in Rat Stomach
Protective
Effect
of Rebamipide
Hemorrhagic
Mucosal
Kazushi Department
of Pharmacology
against
and Experimental 463-10
Received
Lesions
Sakurai
and
Therapeutics,
Kagasuno, August
Hydrogen
Katsuya
Tokushima
Kawauchi-cho, 9, 1993
in Rat
Stomach
Yamasaki Research Institute,
Tokushima
Accepted
Peroxide-Induced
771-01,
December
Otsuka Pharmaceutical
Co., Ltd.,
Japan
30, 1993
ABSTRACT-Intragastric administration of 6°1oH202 induces gastric mucosal hemorrhagic lesions in rats. Intraperitoneal administration of rebamipide at 10 to 100 mg/kg dose-dependently prevented the H202 induced mucosal lesions. The fact that cimetidine, ranitidine and omeprazole could not prevent the gastric mucosa from developing H202-induced lesions indicates that acid secretion might not be the main cause of these lesions. The protective effect of rebamipide was partially reduced by indomethacin and completely blocked by diethyl maleate, a glutathione depressor. Gastric mucosal glutathione level and glutathione peroxidase and superoxide dismutase (SOD) activities were significantly decreased by 6% H202 instillation. Rebamipide at 100 mg/kg significantly inhibited the decreases in gastric mucosal glutathione level and SOD activity. These results suggest that H202-induced gastric mucosal lesions might partially involve a decrease in defense activities against reactive oxygen species and that the protective effect of rebamipide might be related to its ability to improve oxidative stress in gastric mucosa. Keywords:
Rebamipide,
H2O2i Gastric
mucosal
lesion,
Reactive oxygen species are known to be involved in the pathogenesis of mucosal lesions in the gastrointestinal tract by ischemia-reperfusion and necrotizing agents such as ethanol and aspirin (1 -4). Due to their high reactivity and short life, it has still not been elucidated which free radicals, i.e., the superoxide or hydroxyl radical, play an important role in these mucosal lesions (1-5). Several investigations (6, 7) have been performed using H2O2 as the necrotizing agent. It is well known that H202 can be metabolized to the hydroxyl radical, which is one of the most potently damaging free radicals (8). Rebamipide has been reported to prevent various acute experimental gastric mucosal lesions and to accelerate the heeling of chronic gastric ulcers (9, 10). Ogino et al. (11) showed that the antiulcer effect of rebamipide might be related to the inhibition of the production of reactive oxy gen species by activated neutrophils in a diethyldithio carbamate-induced antral ulcer model in rats. Recently, it was indicated that rebamipide was one of the most potent hydroxyl radical scavengers studied by electron spin resonance (12). However, there is still no direct evidence that rebamipide protects gastric mucosa from the damage induced by reactive oxygen species. The present study was undertaken to examine the protective effect of rebamipide
Glutathione,
Reactive
against H202-induced not its effect might
oxygen
lesions and to elucidate whether or be connected to gastric mucosal
glutathione level, glutathione SOD activity.
MATERIALS
peroxidase
activity
and/or
AND METHODS
Animals Male Wistar/ST strain rats (SLC, Shizuoka) weighing 200 300 g were housed in wire-bottom cages to prevent coprophagy. Food and water were respectively withheld from 24 hr and 20 hr prior to the experiment. Chemicals Rebamipide, ranitidine and omeprazole were synthe sized by Otsuka Pharmaceutical Co., Ltd. (Tokushima). Cimetidine and diethyl maleate were purchased from Nacalai Tesque (Osaka). Catalase (19,900 U/mg pro tein), superoxide dismutase (SOD, 4,200 U/mg protein), indomethacin and glutathione were obtained from Sigma Chemical Co. (St. Louis, MO, USA). H2O2 was supplied by Wako Pure Chemicals (Osaka). Rebamipide, cimeti dine, ranitidine and omeprazole were prepared by suspen sion in 0.5% carboxymethyl cellulose (CMC). Catalase
and SOD were dissolved in saline. Solutions were freshly prepared each day. H202-induced gastric mucosal necrosis Rebamipide at 10, 30 and 100 mg/kg was administered i.p. 30 min prior to intragastric administration of 6% H2O2. Cimetidine, ranitidine or omeprazole at 100 mg/kg was administered i.p. 1 hr prior to H202. Catalase at 500,000 U/kg and/or SOD at 30,000 U/kg were admin istered s.c. 30 min before H2O2 instillation. Control animals received 0.5% CMC or saline instead of drugs. Parenteral administration was used to avoid possible inter ference from the production of mucosal lesions by adap tive cytoprotection (13). In separate experiments, indo methacin at 5 mg/kg or diethyl maleate at 1 ml/kg was given s.c. 1 hr prior to the 6% H202 instillation. Follow ing these procedures, 6% H2O2 in a volume of 1 ml/body was administered i.g. through an oral gastric tube. Thirty minutes after H202 administration, the rats were sacrificed by dislocation of the cervical vertebrate and the stomach was excised. After light fixation with 10 ml of 3% buffered formalin, the stomach was opened along the great curvature and washed with saline. The total glandu lar and hemorrhagic mucosal lesion area were measured by an image analyzer (Luzex-IID; Nikon, Tokyo). Determination of GSH level, GSHpx and SOD activities Rebamipide at 10, 30 and 100 mg/kg was administered i.p. 30 min prior to intragastric administration of 6% H2O2. Control animals received i.p. 0.5% CMC. The animals were sacrificed by cervical dislocation 30 min af ter H2O2 instillation. In a separate experiment, rebami
Statistical analyses The data was expressed
as the mean ±S.E.
The statisti
cal difference was determined by Student's two-tailed t-test or one-way analysis of variance followed by two tailed Dunnett's was considered
or Scheffe's test. A difference statistically significant.
at P < 0.05
RESULTS Effect of rebamipide, antisecretory agents and reactive oxygen metabolite scavenging enzymes on H202-induced gastric mucosal lesions Intragastric administration of 6% H202 produced hemorrhagic, necrotic mucosal lesions in the fundic area. Intraperitoneal administration of rebamipide at 10 to 100 mg/kg prevented the mucosal lesions in a dose-dependent manner (Fig. 1). The protective effect of rebamipide was significant at 30 and 100 mg/kg. Pretreatment with cimeti dine, ranitidine or omeprazole at a dose of 100 mg/kg, i.p. 1 hr before H202 was not able to prevent the gastric mucosa from developing H202-induced lesions (Fig. 2). Catalase at 500,000 U/kg, s.c. and/or SOD at 30,000 U/kg, s.c. also failed to protect the gastric mucosa from H202-induced hemorrhagic mucosal lesions (Fig. 2). Effect of rebamipide with indomethacin or diethyl male ate on H202-induced gastric mucosal lesions Hemorrhagic mucosal lesions induced by 6% H202 were not aggravated by pretreatment with indomethacin
pide at 10, 30 and 100 mg/kg was administered i.p. to nor mal rats, which were sacrificed 1 hr after administration. The stomach was immediately excised and opened along the great curvature. After rinsing with ice-cooled saline, the entire gastric mucosa was scraped with a slide glass and weighed. Ten percent of the mucosal homogenates was prepared in 50 mM potassium phosphate buffer pH 7.0 using a Polytron homogenizer (Kinematica, Lucerne, Switzerland; output 5 to 6). The homogenates were stored at -80V until the determination of total glutathione (GSH) level and glutathione peroxidase (GSHpx) and su peroxide dismutase (SOD) activities. GSH level was deter mined according to the method of Sies and Akerboom (14) and expressed as nmol/mg protein. Protein was deter mined by the method of Lowry et al. (15). GSHpx activity was spectrophotometrically measured by the method of Paglia and Valentaine (16) and expressed as units (U)/mg protein. SOD activity was determined by the nitrite method (17) and expressed as nitrite units (NU)/mg pro tein.
Fig.
1.
lesions
Inhibitory induced
Rebamipide H202. dular rats.
Lesion stomach. **P<0.01
effect by
of
intragastric
at 10 to 100 mg/kg,
rebamipide administration i.p.
on
hemorrhagic of
was administered
6070 H202
mucosal in rats.
30 min before
area was expressed as a percentage of the total glan Each column represents the mean±S.E. of 9 or 10 vs. the control
group.
Fig. 2. Effect of antisecretory drugs, SOD and catalase on hemor rhagic mucosal lesions induced by intragastric administration of 6076 H202 in the rats. (A) Cimetidine, ranitidine and omeprazole at 100 mg/kg, i.p. were administered 1 hr before H2O2. (B) SOD at 30,000 U/kg and/or catalase at 500,000 U/kg, s.c. were administered 30 min before H2O2. Each column represents the mean ±S.E. of 6 to 8 rats.
at 5 mg/kg, s.c. 30 min before H202 instillation. The pro tective effect of rebamipide was partially reversed by pretreatment with indomethacin (Fig. 3). Pretreatment with diethyl maleate at 1 ml/kg, s.c. did not change the magnitude of the H202-induced gastric mucosal lesions, but completely abolished the protective effect of rebami
Fig. 3. Influence of pretreatment with indomethacin or diethyl maleate on the protective effect of rebamipide against H202-induced hemorrhagic mucosal lesions in the rats. (A) Indomethacin at 5 mg/kg, s.c. or (B) diethyl maleate at 1 ml/kg, s.c. was administered 30 min before rebamipide at 100 mg/kg, i.p. and 1 hr prior to 6% H2O2. Each column represents the mean±S.E. of 8 rats. **P<0.01 vs. the left column group.
pide.
H202 (Table 1). Rebamipide at 10 to 100 mg/kg, i.p. 30 min
Effect of H202 and rebamipide on gastric mucosal total GSH level and GSHpx and SOD activities in gastric mu cosa Gastric mucosal total GSH was significantly decreased (41010) 30 min after intragastric administration of 6%
prior to H202 instillation dose-dependently inhibited the decrease in mucosal GSH, and the inhibiting effect was sig nificant at 100 mg/kg. GSHpx and SOD activities in gas tric mucosa were also significantly reduced by 6010H202 administration to 19% and 41010of those of normal rats, respectively. Rebamipide at 10 to 100 mg/kg, i.p. showed
Table 1. Effects of intragastric administration of 6% H202 and pretreatment with rebamipide on total glutathione level (GSH) and glutathione peroxidase (GSHpx) and superoxide dismutase (SOD) activities in the gastric mucosa
a tendency
to inhibit
the reduction
of GSHpx
activity
in
a dose-dependent manner. Rebamipide at 100 mg/kg, i.p. significantly inhibited the reduction of gastric mucosal SOD activity. On the other hand, in normal rats, intra peritoneal rebamipide at 10 to 100 mg/kg did not signifi cantly affect gastric mucosal GSH level or GSHpx and SOD activities.
DISCUSSION It has been reported that several necrotizing agents, i.e., ethanol and aspirin, induce gastric mucosal hemor rhagic lesions by promoting the generation of reactive oxy gen metabolites (3, 4). Furthermore, it has been indicated that compounds and enzymes that are capable of scaveng ing or inhibiting the generation of reactive oxygen species are able to protect the gastric mucosa from such hemor rhagic lesions (18, 19). In this study, intragastric adminis tration of 6% H202 induced gastric mucosal hemorrhagic lesions in rats. H202 seems to be a suitable agent for inves tigating reactive oxygen metabolite-induced gastric mu cosal lesions because H2O2 itself is a reactive oxygen metabolite. Rebamipide is a protective antiulcer agent with activ ities to increase prostaglandin level in gastric mucosa and stimulate mucus secretion (9, 20, 21). In this study, re bamipide at 30 and 100 mg/kg, i.p. significantly reduced gastric mucosal hemorrhagic lesions induced by intragas tric H202. It has also been reported that rebamipide can prevent gastric mucosa developing from acute gastric mu cosal lesions induced by ethanol and aspirin (9, 10), which are thought to generate reactive oxygen metabolites (3, 4). Recently, Yoshikawa et al. (12) indicated that rebamipide has a potent activity to scavenge the hydroxyl radical,
which is thought to be one of the most toxic free radicals. These results suggest that hydroxyl radical generated from H2O2 in gastric mucosa might be related to these le sions and that rebamipide's scavenging of the hydroxyl radical may play some roles in its protective effect. Pretreatment with indomethacin partially diminished the protective effect of rebamipide against 6% H202 induced lesions. Kleine et al. recently reported that in traperitoneal administration of rebamipide stimulated prostaglandin synthesis in the rat stomach (22). Further more, 16,16-dimethyl PGE2 was previously reported to protect gastric mucosa from 6% H202-induced injury (7). This data suggests that the elevation of gastric prostaglan dins might, in part, be due to the protective effect of rebamipide. Pretreatment with diethyl maleate, which is a depressor of GSH, completely blocked the protective effect of rebamipide. These findings suggest that the pro tective effect of rebamipide on gastric mucosal gluta thione level may play a more important role in the detoxi cation of H202 and H202-derived oxygen metabolites than the stimulation of prostaglandin synthesis in gastric mucosa. On the other hand, neither cimetidine, ranitidine nor omeprazole was able to protect the gastric mucosa from 6% H202-induced lesions. Aggravation rather than no effect by antisecretory agents was reported by Laudanno et al. (6). The same phenomena were indicated in ethanol induced mucosal lesions (23). These findings suggest that the gastric mucosa could not be protected by the inhibi tion of acid secretion from 6% H202-induced lesions. Although there have been reports of the free radical scavenging enzymes catalase and/or SOD being able to protect the gastric mucosa from injuries caused by reac tive oxygen metabolites (3), they failed to protect against
6%%o H202 lesions in this study. It seems reasonable to as sume that SOD scavenges superoxide and generates H202, but the ineffectiveness of catalase can not be explained in this context. The fact that the intravenous administration of catalase at 100,000 to 1,000,000 U/kg also failed to pro tect the gastric mucosa (data not shown) might suggest that exogenous catalase was not able to reach the site to fully exhibit its scavenging activity against intragastric H202 because of its large molecular size (about MW 200,000). In this study, gastric mucosal GSH level and GSHpx and SOD activities were significantly decreased by instilla tion of 6% H2O2. It has been reported that sulfhydryl compounds mediate gastric cytoprotection (24). Oxygena tion of proteins by H202 and H202-metabolites, which is partly caused by GSH depression, might be related to the decrease of GSHpx and SOD activities (18). Rebamipide at 10 to 100 mg/kg dose-dependently inhibited the decrease of gastric mucosal GSH, and this inhibition was significant at 100 mg/kg. These findings are in accord with the result that pretreatment with diethyl maleate abolished the protection provided by rebamipide. It was thought that the mechanism of the inhibition of H202 induced GSH depletion by rebamipide in gastric mucosa was due to (1) the stimulation of GSH synthesis, (2) the result of the inhibition of gastric mucosal lesion, which was not related to GSH, or (3) the inhibition of GSH con sumption in gastric mucosa. In normal rats, rebamipide did not significantly affect gastric mucosal total GSH level or GSHpx and SOD activities, suggesting that the inhibi tion of GSH-depletion by rebamipide might be related to its inhibitory effect on the consumption of GSH during H202 attack. The H202-induced decreases in both GSHpx and SOD activities were also inhibited by pretreatment with rebamipide. Because Cu,Zn-SOD activity has been reported to be inhibited by the hydroxyl radical (25), the scavenging effect of rebamipide on the hydroxyl radical may be related to the protection of SOD activity. In conclusion, our results suggest that rebamipide pro tects gastric mucosa from H202-induced mucosal lesions and that its protective effect might be involved in improv ing oxidative stress in gastric mucosa through inhibition of the decrease in gastric mucosal GSH level and GSHpx and SOD activities.
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REFERENCES
1 Parks DA, Bulkley GB and Granger DN: Ischemic injury in the cat small intestine: role of superoxide radicals. Gastroenter ology 82, 9-15 (1982) 2 Ito M and Guth PH: Role of oxygen-derived free radicals in hemorrhagic shock-induced gastric lesions in the rat. Gastro enterology 88, 1162-1167 (1985) 3 Terano A, Hiraishi H, Ota S, Shiga J and Sugimoto T: Role of
20
21
superoxide and hydroxyl radicals in rat gastric mucosal injury induced by ethanol. Gastroenterol Jpn 24, 488-493 (1989) Pihan G, Regillo C and Szabo S: Free radicals and lipid peroxi dation in ethanol or aspirin-induced gastric mucosal injury. Dig Dis Sci 32, 1395-1401 (1987) Perry MA, Wadhwa S and Parks DA: Role of oxygen radicals in ischemia-induced lesions in cat stomach. Gastroenterology 90, 362 367 (1986) Laudanno OM, Bedini OA, Cesolari JA and Miguel PS: Evi dence of anti-oxidant role of sucralfate in gastric mucosal pro tection. Ital J Gastroenterol 22, 19-21 (1990) Schurer-Maly CC, Haussner V and Halter F: Effect of 16.16 dimethyl prostaglandin E2, n-acetyl-cysteine and the proton pump inhibitor BY 831-78 on hydrogen peroxide-induced mu cosal damage in the rat stomach. Digestion 46, 97-106 (1990) Chance B, Sies H and Boveris A: Hydroperoxide metabolism in mammalian organs. Physiol Rev 59, 527-604 (1979) Yamasaki K, Ishiyama H, Imaizumi T, Kanbe T and Yabuuchi Y: Effect of OPC-12759, a novel antiulcer agent, on chronic and acute experimental gastric ulcer and gastric secretion in rats. Jpn J Pharmacol 49, 441-448 (1989) Yamasaki K, Kanbe T, Chijiwa T, Ishiyama H and Morita S: Gastric mucosal protection by OPC-12759, a novel antiulcer compound, in the rat. Eur J Pharmacol 142, 23 30 (1987) Ogino K, Hobara T, Ishiyama H, Yamasaki K, Kobayashi H, Izumi Y and Oka S: Antiulcer mechanism of action of rebami pide, a novel antiulcer compound, on diethyldithiocarbamate induced antral gastric ulcers in rats. Eur J Pharmacol 212, 9-13 (1992) Yoshikawa T, Naito Y and Kondo M: Free radical scavenging activity of the novel anti-ulcer agent rebamipide studied by elec tron spin resonance. Arzneimittelforschung 43, 363-366 (1993) Robert A: Cytoprotection by prostaglandin. Gastroenterology 77, 761-767 (1979) Sies H and Akerboom TPM: Glutathione disulfide (GSSG) efflux from cells and tissues. Methods Enzymol 105, 445-451 (1984) Lowry OH, Rosebrough NJ, Farr AL and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-275 (1951) Paglia DE and Valentaine WN: Studies on the glutathione and glutathione characterization of erythrocyte glutathione peroxi dase. J Lab Clin Med 70, 158 -169 (1967) Ohyanagi Y: Establishment of nitrite-kit for SOD activity deter mination. Jpn J Inflam 4, 63 -73 (Abstr in English) Cho CH, Pfeiffer CJ and Misra HP: Ulcerogenic mechanism of ethanol and the action of sulphanilyl fluoride on the rat stomach in-vivo. J Pharm Pharmacol 43, 495-498 (1991) Ito M, Shii D, Segami T, Kojima R and Suzuki Y: Preventive actions of n-(3-aminopropionyl)-1-histidinato zinc (Z-103) through increases in the activities of oxygen-derived free radical scavenging enzymes in the gastric mucosa on ethanol-induced gastric mucosal damage in rats. Jpn J Pharmacol 59, 267-274 (1992) Kawano S, Sato N, Kamada T, Yamasaki K, Imaizumi T and Komemushi S: Protective effect of rebamipide (OPC-12759) on the gastric mucosa in rats and humans. Folia Pharmacol Jpn 97, 371-380 (1990) (Abstr in English) Ishihara K, Komuro Y, Nishiyama N, Yamasaki K and Hotta K: Effect of rebamipide on mucus secretion by endogenous
22
23
prostaglandin-independent mechanism in rat gastric mucosa. Arzneimittelforschung 42, 1462-1466 (1992) Kleine A, Kluge S and Peskar BM: Stimulation of prostaglan din biosynthesis mediates gastroprotective effect of rebamipide in rats. Dig Dis Sci 38, 1441-1449 (1993) Tarnawski A, Hollander D and Gergely H: Comparison of anti acid sucralfate, cimetidine and ranitidine in protection of the
24 25
gastric mucosa against ethanol injury. Am J Med 79, 19-23 (1985) Szabo S, Trier JS and Frankel PW: Sulfhydryl compounds may mediate gastric cytoprotection. Science 214, 200-202 (1981) Sinet PM and Garber P: Inactivation of the human Cu, Zn superoxide dismutase during exposure to 02 and H202. Arch Biochem Biophys 212, 411-416 (1981)
Effect
of Rebamipide
Hemorrhagic
Mucosal
Kazushi Department
of Pharmacology
against
and Experimental 463-10
Received
Lesions
Sakurai
and
Therapeutics,
Kagasuno, August
Hydrogen
Katsuya
Tokushima
Kawauchi-cho, 9, 1993
in Rat
Stomach
Yamasaki Research Institute,
Tokushima
Accepted
Peroxide-Induced
771-01,
December
Otsuka Pharmaceutical
Co., Ltd.,
Japan
30, 1993
ABSTRACT-Intragastric administration of 6°1oH202 induces gastric mucosal hemorrhagic lesions in rats. Intraperitoneal administration of rebamipide at 10 to 100 mg/kg dose-dependently prevented the H202 induced mucosal lesions. The fact that cimetidine, ranitidine and omeprazole could not prevent the gastric mucosa from developing H202-induced lesions indicates that acid secretion might not be the main cause of these lesions. The protective effect of rebamipide was partially reduced by indomethacin and completely blocked by diethyl maleate, a glutathione depressor. Gastric mucosal glutathione level and glutathione peroxidase and superoxide dismutase (SOD) activities were significantly decreased by 6% H202 instillation. Rebamipide at 100 mg/kg significantly inhibited the decreases in gastric mucosal glutathione level and SOD activity. These results suggest that H202-induced gastric mucosal lesions might partially involve a decrease in defense activities against reactive oxygen species and that the protective effect of rebamipide might be related to its ability to improve oxidative stress in gastric mucosa. Keywords:
Rebamipide,
H2O2i Gastric
mucosal
lesion,
Reactive oxygen species are known to be involved in the pathogenesis of mucosal lesions in the gastrointestinal tract by ischemia-reperfusion and necrotizing agents such as ethanol and aspirin (1 -4). Due to their high reactivity and short life, it has still not been elucidated which free radicals, i.e., the superoxide or hydroxyl radical, play an important role in these mucosal lesions (1-5). Several investigations (6, 7) have been performed using H2O2 as the necrotizing agent. It is well known that H202 can be metabolized to the hydroxyl radical, which is one of the most potently damaging free radicals (8). Rebamipide has been reported to prevent various acute experimental gastric mucosal lesions and to accelerate the heeling of chronic gastric ulcers (9, 10). Ogino et al. (11) showed that the antiulcer effect of rebamipide might be related to the inhibition of the production of reactive oxy gen species by activated neutrophils in a diethyldithio carbamate-induced antral ulcer model in rats. Recently, it was indicated that rebamipide was one of the most potent hydroxyl radical scavengers studied by electron spin resonance (12). However, there is still no direct evidence that rebamipide protects gastric mucosa from the damage induced by reactive oxygen species. The present study was undertaken to examine the protective effect of rebamipide
Glutathione,
Reactive
against H202-induced not its effect might
oxygen
lesions and to elucidate whether or be connected to gastric mucosal
glutathione level, glutathione SOD activity.
MATERIALS
peroxidase
activity
and/or
AND METHODS
Animals Male Wistar/ST strain rats (SLC, Shizuoka) weighing 200 300 g were housed in wire-bottom cages to prevent coprophagy. Food and water were respectively withheld from 24 hr and 20 hr prior to the experiment. Chemicals Rebamipide, ranitidine and omeprazole were synthe sized by Otsuka Pharmaceutical Co., Ltd. (Tokushima). Cimetidine and diethyl maleate were purchased from Nacalai Tesque (Osaka). Catalase (19,900 U/mg pro tein), superoxide dismutase (SOD, 4,200 U/mg protein), indomethacin and glutathione were obtained from Sigma Chemical Co. (St. Louis, MO, USA). H2O2 was supplied by Wako Pure Chemicals (Osaka). Rebamipide, cimeti dine, ranitidine and omeprazole were prepared by suspen sion in 0.5% carboxymethyl cellulose (CMC). Catalase
and SOD were dissolved in saline. Solutions were freshly prepared each day. H202-induced gastric mucosal necrosis Rebamipide at 10, 30 and 100 mg/kg was administered i.p. 30 min prior to intragastric administration of 6% H2O2. Cimetidine, ranitidine or omeprazole at 100 mg/kg was administered i.p. 1 hr prior to H202. Catalase at 500,000 U/kg and/or SOD at 30,000 U/kg were admin istered s.c. 30 min before H2O2 instillation. Control animals received 0.5% CMC or saline instead of drugs. Parenteral administration was used to avoid possible inter ference from the production of mucosal lesions by adap tive cytoprotection (13). In separate experiments, indo methacin at 5 mg/kg or diethyl maleate at 1 ml/kg was given s.c. 1 hr prior to the 6% H202 instillation. Follow ing these procedures, 6% H2O2 in a volume of 1 ml/body was administered i.g. through an oral gastric tube. Thirty minutes after H202 administration, the rats were sacrificed by dislocation of the cervical vertebrate and the stomach was excised. After light fixation with 10 ml of 3% buffered formalin, the stomach was opened along the great curvature and washed with saline. The total glandu lar and hemorrhagic mucosal lesion area were measured by an image analyzer (Luzex-IID; Nikon, Tokyo). Determination of GSH level, GSHpx and SOD activities Rebamipide at 10, 30 and 100 mg/kg was administered i.p. 30 min prior to intragastric administration of 6% H2O2. Control animals received i.p. 0.5% CMC. The animals were sacrificed by cervical dislocation 30 min af ter H2O2 instillation. In a separate experiment, rebami
Statistical analyses The data was expressed
as the mean ±S.E.
The statisti
cal difference was determined by Student's two-tailed t-test or one-way analysis of variance followed by two tailed Dunnett's was considered
or Scheffe's test. A difference statistically significant.
at P < 0.05
RESULTS Effect of rebamipide, antisecretory agents and reactive oxygen metabolite scavenging enzymes on H202-induced gastric mucosal lesions Intragastric administration of 6% H202 produced hemorrhagic, necrotic mucosal lesions in the fundic area. Intraperitoneal administration of rebamipide at 10 to 100 mg/kg prevented the mucosal lesions in a dose-dependent manner (Fig. 1). The protective effect of rebamipide was significant at 30 and 100 mg/kg. Pretreatment with cimeti dine, ranitidine or omeprazole at a dose of 100 mg/kg, i.p. 1 hr before H202 was not able to prevent the gastric mucosa from developing H202-induced lesions (Fig. 2). Catalase at 500,000 U/kg, s.c. and/or SOD at 30,000 U/kg, s.c. also failed to protect the gastric mucosa from H202-induced hemorrhagic mucosal lesions (Fig. 2). Effect of rebamipide with indomethacin or diethyl male ate on H202-induced gastric mucosal lesions Hemorrhagic mucosal lesions induced by 6% H202 were not aggravated by pretreatment with indomethacin
pide at 10, 30 and 100 mg/kg was administered i.p. to nor mal rats, which were sacrificed 1 hr after administration. The stomach was immediately excised and opened along the great curvature. After rinsing with ice-cooled saline, the entire gastric mucosa was scraped with a slide glass and weighed. Ten percent of the mucosal homogenates was prepared in 50 mM potassium phosphate buffer pH 7.0 using a Polytron homogenizer (Kinematica, Lucerne, Switzerland; output 5 to 6). The homogenates were stored at -80V until the determination of total glutathione (GSH) level and glutathione peroxidase (GSHpx) and su peroxide dismutase (SOD) activities. GSH level was deter mined according to the method of Sies and Akerboom (14) and expressed as nmol/mg protein. Protein was deter mined by the method of Lowry et al. (15). GSHpx activity was spectrophotometrically measured by the method of Paglia and Valentaine (16) and expressed as units (U)/mg protein. SOD activity was determined by the nitrite method (17) and expressed as nitrite units (NU)/mg pro tein.
Fig.
1.
lesions
Inhibitory induced
Rebamipide H202. dular rats.
Lesion stomach. **P<0.01
effect by
of
intragastric
at 10 to 100 mg/kg,
rebamipide administration i.p.
on
hemorrhagic of
was administered
6070 H202
mucosal in rats.
30 min before
area was expressed as a percentage of the total glan Each column represents the mean±S.E. of 9 or 10 vs. the control
group.
Fig. 2. Effect of antisecretory drugs, SOD and catalase on hemor rhagic mucosal lesions induced by intragastric administration of 6076 H202 in the rats. (A) Cimetidine, ranitidine and omeprazole at 100 mg/kg, i.p. were administered 1 hr before H2O2. (B) SOD at 30,000 U/kg and/or catalase at 500,000 U/kg, s.c. were administered 30 min before H2O2. Each column represents the mean ±S.E. of 6 to 8 rats.
at 5 mg/kg, s.c. 30 min before H202 instillation. The pro tective effect of rebamipide was partially reversed by pretreatment with indomethacin (Fig. 3). Pretreatment with diethyl maleate at 1 ml/kg, s.c. did not change the magnitude of the H202-induced gastric mucosal lesions, but completely abolished the protective effect of rebami
Fig. 3. Influence of pretreatment with indomethacin or diethyl maleate on the protective effect of rebamipide against H202-induced hemorrhagic mucosal lesions in the rats. (A) Indomethacin at 5 mg/kg, s.c. or (B) diethyl maleate at 1 ml/kg, s.c. was administered 30 min before rebamipide at 100 mg/kg, i.p. and 1 hr prior to 6% H2O2. Each column represents the mean±S.E. of 8 rats. **P<0.01 vs. the left column group.
pide.
H202 (Table 1). Rebamipide at 10 to 100 mg/kg, i.p. 30 min
Effect of H202 and rebamipide on gastric mucosal total GSH level and GSHpx and SOD activities in gastric mu cosa Gastric mucosal total GSH was significantly decreased (41010) 30 min after intragastric administration of 6%
prior to H202 instillation dose-dependently inhibited the decrease in mucosal GSH, and the inhibiting effect was sig nificant at 100 mg/kg. GSHpx and SOD activities in gas tric mucosa were also significantly reduced by 6010H202 administration to 19% and 41010of those of normal rats, respectively. Rebamipide at 10 to 100 mg/kg, i.p. showed
Table 1. Effects of intragastric administration of 6% H202 and pretreatment with rebamipide on total glutathione level (GSH) and glutathione peroxidase (GSHpx) and superoxide dismutase (SOD) activities in the gastric mucosa
a tendency
to inhibit
the reduction
of GSHpx
activity
in
a dose-dependent manner. Rebamipide at 100 mg/kg, i.p. significantly inhibited the reduction of gastric mucosal SOD activity. On the other hand, in normal rats, intra peritoneal rebamipide at 10 to 100 mg/kg did not signifi cantly affect gastric mucosal GSH level or GSHpx and SOD activities.
DISCUSSION It has been reported that several necrotizing agents, i.e., ethanol and aspirin, induce gastric mucosal hemor rhagic lesions by promoting the generation of reactive oxy gen metabolites (3, 4). Furthermore, it has been indicated that compounds and enzymes that are capable of scaveng ing or inhibiting the generation of reactive oxygen species are able to protect the gastric mucosa from such hemor rhagic lesions (18, 19). In this study, intragastric adminis tration of 6% H202 induced gastric mucosal hemorrhagic lesions in rats. H202 seems to be a suitable agent for inves tigating reactive oxygen metabolite-induced gastric mu cosal lesions because H2O2 itself is a reactive oxygen metabolite. Rebamipide is a protective antiulcer agent with activ ities to increase prostaglandin level in gastric mucosa and stimulate mucus secretion (9, 20, 21). In this study, re bamipide at 30 and 100 mg/kg, i.p. significantly reduced gastric mucosal hemorrhagic lesions induced by intragas tric H202. It has also been reported that rebamipide can prevent gastric mucosa developing from acute gastric mu cosal lesions induced by ethanol and aspirin (9, 10), which are thought to generate reactive oxygen metabolites (3, 4). Recently, Yoshikawa et al. (12) indicated that rebamipide has a potent activity to scavenge the hydroxyl radical,
which is thought to be one of the most toxic free radicals. These results suggest that hydroxyl radical generated from H2O2 in gastric mucosa might be related to these le sions and that rebamipide's scavenging of the hydroxyl radical may play some roles in its protective effect. Pretreatment with indomethacin partially diminished the protective effect of rebamipide against 6% H202 induced lesions. Kleine et al. recently reported that in traperitoneal administration of rebamipide stimulated prostaglandin synthesis in the rat stomach (22). Further more, 16,16-dimethyl PGE2 was previously reported to protect gastric mucosa from 6% H202-induced injury (7). This data suggests that the elevation of gastric prostaglan dins might, in part, be due to the protective effect of rebamipide. Pretreatment with diethyl maleate, which is a depressor of GSH, completely blocked the protective effect of rebamipide. These findings suggest that the pro tective effect of rebamipide on gastric mucosal gluta thione level may play a more important role in the detoxi cation of H202 and H202-derived oxygen metabolites than the stimulation of prostaglandin synthesis in gastric mucosa. On the other hand, neither cimetidine, ranitidine nor omeprazole was able to protect the gastric mucosa from 6% H202-induced lesions. Aggravation rather than no effect by antisecretory agents was reported by Laudanno et al. (6). The same phenomena were indicated in ethanol induced mucosal lesions (23). These findings suggest that the gastric mucosa could not be protected by the inhibi tion of acid secretion from 6% H202-induced lesions. Although there have been reports of the free radical scavenging enzymes catalase and/or SOD being able to protect the gastric mucosa from injuries caused by reac tive oxygen metabolites (3), they failed to protect against
6%%o H202 lesions in this study. It seems reasonable to as sume that SOD scavenges superoxide and generates H202, but the ineffectiveness of catalase can not be explained in this context. The fact that the intravenous administration of catalase at 100,000 to 1,000,000 U/kg also failed to pro tect the gastric mucosa (data not shown) might suggest that exogenous catalase was not able to reach the site to fully exhibit its scavenging activity against intragastric H202 because of its large molecular size (about MW 200,000). In this study, gastric mucosal GSH level and GSHpx and SOD activities were significantly decreased by instilla tion of 6% H2O2. It has been reported that sulfhydryl compounds mediate gastric cytoprotection (24). Oxygena tion of proteins by H202 and H202-metabolites, which is partly caused by GSH depression, might be related to the decrease of GSHpx and SOD activities (18). Rebamipide at 10 to 100 mg/kg dose-dependently inhibited the decrease of gastric mucosal GSH, and this inhibition was significant at 100 mg/kg. These findings are in accord with the result that pretreatment with diethyl maleate abolished the protection provided by rebamipide. It was thought that the mechanism of the inhibition of H202 induced GSH depletion by rebamipide in gastric mucosa was due to (1) the stimulation of GSH synthesis, (2) the result of the inhibition of gastric mucosal lesion, which was not related to GSH, or (3) the inhibition of GSH con sumption in gastric mucosa. In normal rats, rebamipide did not significantly affect gastric mucosal total GSH level or GSHpx and SOD activities, suggesting that the inhibi tion of GSH-depletion by rebamipide might be related to its inhibitory effect on the consumption of GSH during H202 attack. The H202-induced decreases in both GSHpx and SOD activities were also inhibited by pretreatment with rebamipide. Because Cu,Zn-SOD activity has been reported to be inhibited by the hydroxyl radical (25), the scavenging effect of rebamipide on the hydroxyl radical may be related to the protection of SOD activity. In conclusion, our results suggest that rebamipide pro tects gastric mucosa from H202-induced mucosal lesions and that its protective effect might be involved in improv ing oxidative stress in gastric mucosa through inhibition of the decrease in gastric mucosal GSH level and GSHpx and SOD activities.
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