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Effects of various mucosal protective drugs on diethyldithiocarbamate-induced antral ulcer in rats
European Journal of Pharmacology, 197 (1991) 99-102 0 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991003602
99
EJP 51840
Effects sf various mucosall protective drugs on diethyldithiocarb antral ulcer in rats Shinji Oka *, Keiki Ogino 2, Tatsuya Hobara 2, Shunji Yoshimura Tadayosi Takemoto 5 and Yozo Iida 1
3, Yukinori Okazaki 4,
’ Kokura Memorial Hospital. Kitakyurhu, ’ Department of Public Health, Yamaguchi University, School of Medicine, Ube, Japan, ’ Nagato General Hospital, Nagato. Japan, ’ Shuto General Hospital Yanai. Japan and Tokyo Women’s Medical College, Tokyo, Japan
Received 14 January 1991, accepted 5 February 1991
This study was designed to determine the effects of exogenous Cu,Zn-superoxide dismutase (SOD) and various mucosal protective agents against antral ulcer induced by diethyldithiocarbamate (DDC), inhibitor of Cu,Zn-SOD. Exogenous Cu,Zn-SOD reduced ulcer formation and prevented a decrease in SOD activity in gastric mucosa. This result indicates that maintenance of mucosal SOD activity is essential to prevent the ulcerogenicity of DDC. Many mucosal protective drugs that increase blood flow, mucus secretion, and endogenous prostaglandin failed to prevent ulcer formation and decrease of mucosal SOD activity. Rebamipide, however, significantly reduced ulcerogenesis and maintained mucosal SOD activity. This suggests that rebamipide has new protective effects on gastric mucosa. Diethyldithiocarbamate-induced
antral ulcer; Cu,Zn-superoxide dismutase; Gastric mucosa; Mucosal protective drugs
1. Introduction
Recent studies have indicated that active oxygen species play an important role in the pathogenesis of various gastric mucosal injuries and that some scavengers show protective effects against mucosal injury induced by active oxygen species (Perry et al., 1986; Pihan et al., 1987; Szelenyi and Bnme, 1988). Among the various scavengers, we studied the role and significance of superoxide dismutase (SOD) in the gastric mucosa because exogenous SOD has been reported to prevent mucosal injury induced by active oxygen species (Parks et al., 1982; Itoh and Guth, 1985; Perry et al., 1986; Pihan et al., 1987). There are at least three different enzymes of SOD in mammalian tissue, which include cytosolic copper,zinc-SOD (Cu,Zn-SOD), mitochondrial manganese-SOD and extracellular SOD. In the process of studying SOD, we found that diethyldithiocarbamate (DDC), an inhibitor of Cu,Zn-SOD, showed ulcerogenicity (Ogino et al., 1987; 1988). Previous reports have indicated that active oxygen species are involved in the pathogenesis of this unique experimental ulcer (Ishiyama et al., 1989; Ogino et al., 1990; Oka et al., 1990).
Correspondence to: S. Oka, Kokura Memorial Hospital, tyou. Kokura Kitaku, 802 Kitakyushu. Japan.
l-l Kifune-
Many anti-ulcer drugs are used clinically in Japan, especially so-called mucosal protective drugs, the antiulcer drugs that do not affect acid secretion and increase mucosal protective power. Several reports have shown that these drugs prevent various experimental ulcers without inhibiting acid secretion (Takagi and Yano, 1972; Suzuki et al., 1976; Murakami et al., 1981; 1982; Saziki et al., 1984; Ysmasaki et al., 1989). This study concerned the effects of exogenous Cu,Zn-SOD and various other mucosal protective drugs on DDC-induced antral ulcer.
2. Materials and methods 2. I. Animals Male Wistar rats weighing 190-230 g were used. The animals were kept in cages with wide mesh wire bottoms to prevent coprophagia. The rats were fasted and allowed access to water ad libitum for 24 h prior to the experiment. 2.2. Chemicals The following chemicals were purchased from the sources indicated: diethyldithiocarbamate (Wako Pure
Chemiot\ls), sulfonilic acid (Nakarai). hypoxanthine ~Nakarai), hydroxylamine-o-sulfate (Merck). xanthine ringer h~~~~eirn for that used in the oxidase t ~~e~s~~~~n~ of SOD activity on gastric mucosa. Sigma for that used in the study of the effect of Rebamipide on the SOD assay system), N-l-naphthylethylene-diamine (Tokyo Kasei), Cu.&t-SOD (Sigma), Cetraxate ~Daii~~ Pharmaceutical}, Gefatnate (Sumitimo), ~falcone (T&ho Ph~aceutical}, Teprenone (E&i), Rebamipide (Otsuka Pharmaceutical). 2.3. Production cf DDC-induced antral ulcer (Ishiyama et al.. 1989) The rats were anesthetized with ether and the pylorus was ligated. After pylorus ligation, DDC resolved in saline was injected S.C.in a dose of 800 mg/kg. Next, 1 ml of 0.1 N NC1 was administered orally using a metal orogastric tube. The rats were killed 7 h later. The stomach was removed and filled with 10 ml of 10% form&n. The ulcer index was measured under a dissecting microscope with a square grid eyescope and was expressed as the area of the antral ulcer (mm’). 2.3. I. Effeert of C&Zn-SOD
Cu.Zn-SOD was dissolved in saline and administered S.C.30 min before the experiment. In the control group, saline was injected S.C.
TABLE 1 Effect of exogenous Cu.&t-SOD on SOD activity in the an&al mucosa. The data show the mean + SE. Group
Number of rats
SOD activity (NU/mg protein)
Normal antrum mucosa DDC + saline DDC + Cu,Zn-SOD (60~ U/kg)
8 8
15.9*0.7 6.6 + 0.5 h
8
12.4+1.7’”
8 P c 0.05. h P c 0.01 when compared with normal antrum mucosa. ’ P c 0.01 when compared with the DDC + saline group.
concentration of the samples was measured method of Lowry (1951).
by the
2.3. Effect of Rebamipide on the SOD assay system Rebamipide was dissolved in 2% NaHC4, and the solution was prepared to a final concentration of lo-‘, 10M5, IOe4 mol/l. The data are expressed as absorbance values at 550 nm. 2.6. Statistical analysis Statistical significance of the difference between two groups was determined by Student’s t-test and by oneway analysis of variance for multiple groups. A P < 0.05 was considered significant.
2.32. Effects of various mucosai p~orectiue drugs
Each drug was suspended in 0.5% carboxymethylcellulose (CMC) and administered i.p. 30 min before the experiment. Each drug was used in a dosage that would prevent the various experimental ulcers (Takagi and Yano, 1972; Suzuki et al., 1976; Murakami et al., 1981; 1982; Saziki et al., 1984; Konturek et al., 1986; Yamasaki et al., 1989; Kuroiwa et al., 1990). The effects of Rebamipide were studied at three different doses (7.5. 15. 30 mg/kg). 2-d. ~e~ure?~len~ of SOD activity in antraf mucosa
Because SOD activity in antral mucosa reaches its lowest value in 1 h, the samples were obtained within I h (Ishiyama et al., 1989). The rats were killed I h after pylorus ligation, DDC treatment and 0.1 N HCI administration. The stomach was removed and the antral mucosa was scraped. The mucosa was homogenized with 50 mM Tris-HCl buffer, pH 7.4 and sonicated under cooling with ice. The homogenate was centrifuged at 2Oooc) x g for 20 min. The supematant was used to measure SOD activity in the antral mucosa. SOD activity was measured as described by Oyanagui (1984) and expressed in nitrite units (NU)/mg protein. The protein
3. Result 3.1. Effect of exogenous Cu,Zn-SOD The ulcer index in the control group (saline) was 6.45 f 1.01. Exogenous Ct.&&-SOD ~60000 U/kg) significantly (P c 0.01) reduced the ulcer index to 2.20 + 0.50. SOD activity was significantly reduced in the DDC plus saline group (table 1). Cu,Zn-SOD administration significantly prevented a decrease in SOD activity compared with the DDC plus saline group.
TABLE 2 Effects of various mucosal protective drugs on DDC-induced ulcer. The data show the mean i S.E. Treatment Saline Gefarnate Cetraxate Teprenone Sofalcone Rebamipide
Dose f mg/kg)
Number of rats
Ulcer index 0nm2)
I~bition (W
500 300 200 100 30
9 7 8 7 8 7
6.45 It 0.72 6.36 f 0.93 6.19&0.45 5.71*0.78 5.50*0.53 3,00&0.64h
1.4 4.0 11.5 14.7 53.5
’ P <: 0.01 when compared with the saline treatment group.
antral
101 TABLE 3 Effects of Rebamipide on DDC-induced the mean f SE. Treatment Control Rebamipide
antral ulcer. The data show
Dose (mg/kg!
Number of rats
Ulcer index (mm2)
7.5 15 30
10 a 10 10
5.85 kO.71 5.19+ 1.06 3.10*0.67” 2.75 + 0.75 ’
a P -z 0.05, ’ P c 0.01 when compared with the control group.
3.2. Effects of various mucosal protective drugs Table 2 shows the inhibition rates for Gefamate, Cetraxate, Teprenone and Sofalcone. The differences between these drug treatment groups and the saline group were not significant. Only Rebamipide treatment significantly reduced the ulcer index; the effect was dose-dependent (table 3). SOD activity was significantly reduced in the saline, Gefarnate, Cetraxate, Teprenone, and Sofalcone treatment groups, but Rebamipide treatment prevented the reduction of SOD activity as seen from the ulcer index (table 4). 3.3. Effect of Rebamipide on the SOD assay system The following values were found for absorbance of the control, 10m6, 10e5 and 10m4 mol/l Rebapimide: 0.127, 0.136,0.136 and 0.126. Absorbance did not show a difference according to drug concentration. Rebamipide had no effect on the assay system for SOD activity.
4. Discussion DDC-induced antral ulcer has two features. One is that the ulcerogenicity of DDC depends on a decrease in protection against active oxygen species in gastric mucosa, because some scavengers inhibit ulcer formation, and because increases in lipid peroxide are found
TABLE 4 Effects of various mucosal protective drugs on SOD activity in the antral mucosa. The data show the mean& SE. Group
Number of rats
SOD activity (NU/mg protein)
Normal antrum mucosa DDC + saline DDC + Gefarnate DDC + Cetraxate DDC + Teprenone DDC + Sofalcone DDC + Rebamipide
7 7 7 7 7 7 7
17.2kO.2 7.5 f 0.7 h 8.1 f 0.5 h 8.6 f 0.6 ’ 7.3 f0.9b 7.9*0.7b 12.3f0.9’.=
’ P < 0.01 when compared with normal antrum mucosa. when compared with DDC + saline.
-
P < 0.01
in the gastric mucosa before the appearance of mucosal injury (IShiYaIkXt et al., 1989; Oka et al., 1990). The other feature is that this experimental ulcer is similar to the human gastric ulcer and is useful for elucidating the etiology of the human gastric ulcer. In this model, 0.1 N I-ICI was administered immediately after pylorus &ation. HCl administration is sometimes used to aggravate the mucosa! injury in experimental ulcers because exogenous HCl decreases mucosal blood flow and intramural pH caused by back diffusion of luminal H’ (Kivilaakso et al., 1978; Hirose et al., 1985; Morishita and Guth, 1987). AS DDC inhibits acid secretion (Ogino et al., 1990), additional HCl was useful to decrease the luminal pH by increasing back diffusion. A previous study had shown that DDC itself was toxic to gastric mucosa, but DDC treatment without HCI administration failed to induce a deep ulcer in some rats (Oka et al., 1989). HCl administration was considered to help the ulcerogenicity of DDC by decreasing luminal pH. As deep ulcer is the specific feature, HCI administration was useful for this model. The effect of exogenous Cu,Zn-SOD on this ulcer model was studied first. Cu,Zn-SOD pretreatment reduced the ulcer index and prevented the decrease in SOD activity in the gastric mucosa. As a previous study had indicated that DDC decreases only Cu,Zn-SOD activity (Ishiyama et al., 1989) an increase of mucosal SOD activity after Cu.Zn-SOD administration was considered to depend on exogenous Cu,Zn-SOD. These results indicate that reduction in Cu,Zn-SOD activity plays an important role in the pathogenesis of the DDC-induced antral ulcer. Maintenance of mucosal SOD activity, especially of Cu,Zn-SOD, is essential for preventing the ulcerogenicity of DDC. The effects of various mucosal protective drugs on the ulcer index and mucosal SOD activity were studied next. There were two reasons why we used i.p. administration with this model. One is that intragastric administration may induce adaptive cytoprotection. Adaptive cytoprotection is- caused by the direct stimulation of gastric mucosa by the drugs, and when drugs are administered intragastrically, they may act just like a mild irritant (Robert et al., 1983). This is not the pharmacological effect of the drugs. Another is that this model makes use of HCl administration. In order to prevent the effect of drugs on HCl, we chose i.p. administration. Each mucosal protective drug used in this experiment has been shown to increase mucosal blood flow (Cetraxate) (Kuroiwa et al., 1990) mucus secretion (Gefamate and Teprenone) (Takagi and Yano, 1972; Murakami et al., 1982) or endogenous prostaglandin (Gefarnate and Sofalcone) (Kobayashi et al., 1983; Konturek et al., 1986). These drugs have been reported to prevent vatious experimental ulcers according to the above effects. A dose high enough to prevent other experimental ulcers was used, but Gefarnate, Cetraxate, Teprenone, and
to prevent ulcer formation. At the same s failed to maintain SOD activrty in the From these results. it is suggested that er than the already known ones (mucosal ow. mucus secretion. and prostaglandin) plays a in this model. The new mucosal protective drug, showed dose-dependent effectiveness against DDC-induced antral ulcer and prevented sig~~ifica~tly reduction of mucosal SOD activity. We examined whether tke Rebamipide contained in the sample affected the SOD assay system, but found that amipide had no effect on the assay system and that it did not havre SOD-like activity. Therefore, the antiulcer effect on DDC-induced astral ulcer may depend on the maintenance of SOD activity in the gastric mucosa. As various drugs that increase already known mucosal protective factors failed to prevent this experimental ulcer. it is suggested that Rebamipide has a new effect on the gastric mucosa. This new effect is considered to be an effect on SOD activity in the gastric mucosa. It remains to be studied how Rebamipide maintains SOD activity and which types of SOD are increased by Rebamipide treatment.
References Hirose. H.. T. Mizui. N. Shimono and D. Doteuchi. 1985, Gastric antral ulcers induced by a combination of acid, indomethacin and ischemia in rats. Jap. J. Pharmacol. 38, 223. lsbiyama. H.. K. Yamasaki. T. Imaizumi, T. Kanbe, K. Ogino. S. Oka, Y. Okazaki and T. Takemoto, 1989, Gastric antral ulcer produced by dietbyldithiocarbamate in rats, J. Clin. B&hem. Nutr. 5. 155. Itoh. M. and P.H. Gut!), 1985. Role of oxygen derived free radicals in hemorrhagic shock-induced gastric lesions in the rat, Gastroenterology 88. 1162. Kivilaakso, E.. D. Fromm and W. Silen. 1978, Relationship between ulceration and intramural pH of gastric mucosa during hemorrhagic shock. Surgery 84,70. Kobayashi.K..T. Arakawa, H. Nakamura, S. Chono, H. Yamada. H. Satoh and T. Ono. 1983, Effect of gefamate on endogenous prostacyclin, prostaglandin E, and thromboxane in water-immersed rats, Tohoku J. Exp. Med. 140, 181. Konturek. S.J.. T. Radecki, T. Brzozowski, D. Drozdowicz, I. Piastucki, M. Muramatsu. M. Tanaka and H. Aihara, 1986. Antiulcer and gastroprotective effects of solon, a synthetic flavonoid derivative of sopharadin. Role of endogenous prostaglandins, European J. Pharmacol. 125, 185. Kuroiwa M., S. Sugiyama, H. Goto, Y. Tsukamoto, S. Makazawa and T. Ozawa. 199% The role of mucosal prostaglandin levels in healing of water immersion-induced gastric ulcer in rats, Stand. J. Gastroenterol. 25, 59. Low% 0-H.. M.H. Rosebrough. A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265.
Morishita. T. and P.H. Guth. 1987. Effect of exogenous acid on the rat gastric mucosal microcirculation in hemorrhagic shock. Gastroenterology 92, 1958. Murakami, M.. K. Oketani. H. Fujisaki. T. Wakabayashi and T. Ohgo, 1981, Antiulcer effect of geranylgeranylacetone, a new acyclic polisoprenoid on experimentally induced gastric and duodenal ulcer in rats. Atzneim. Forsch. 31, 799. Murakami, M., K. Oketani. H. Fujisaki. T. Wakabayashi and T. Ohgo. 1982, Effects of antiulcer drug geranylgeranylacetone on aspirin-induced gastric ulcers in rats, Jap. J. Pharmacol. 32, 299. Ogino, K., T. Hobara. T. Kawamoto. H. Kobayashi, S. Iwamoto. S. Oka, Y. Okazaki. 1990, Mechanism of diethyldithiocarbamate induced gastric ulcer formation in the rat. Pharmacol. Toxicol. 66, 133. Ogino, K.. S. Oka. S. Matsuura. I. Sakaida, S. Yoshimura, K. Matsuda, K. Sasaki, K. Yamamoto. T. Yoshikawa. Y. Okazaki and T. Takemoto, 1987. Ulcer formation in rat stomach wnh diethyldithiocarbamate, J. Clin. B&hem. Nutr. 3, 189. Ogino. K.. S. Oka, Y. Okazaki and T. Takemoto, 1988, Gastric mucosal protectton and superoxide dismutase. J. Clin. Gastroenterol. 10 (Suppl.). S129. Oka, S.. K. Ogino, T. Hobara, S. Yoshimura, H. Yanai, Y. Okazaki, T. Takemoto, H. Ishiyama, T. Imaizumi, K. Yamasaki and T. Kanbe. 1990, Role of active oxygen species in diethyldithiocarbamate-induced gastric ulcer in the rats, Experientia 46, 281. Oyanagui. Y.. 1984, Reeva!uation of assay methods and establishment of kit for superoxide dismutase activity, Anal. B&hem. 142, 290. Parks. D., G. Bulkley. D. Granger, S. Hamilton and J. McCord, 1982, lschemic injury in cat small intestine: Role of superoxide radicals, Gastroenterology 82, 9. Perry, M.A., S. Wadhwa. D.A. Parks. W. Pickard and D.N. Granger, 1986, Role of oxygen radicals in ischemic-induced lesions in the cat stomach. Gastroenterology 90, 362. Pihan, G., C. Regillo and S. Szabo, 1987, Free radical and lipid peroxidation in ethanol- or aspirin-induced gastric mucosal injury, Dig. Dis. Sci. 32. 1393. Robert, A., J.E. Nezamis, C. Lancaster, J.P. Davis, S.O. Field and A.J. Hanchar. 1983, Mild irritants prevent gastric necrosis through adaptive cytoprotection mediated by prostaglandin. Am. J. Physiol. 245. G113. Saziki, R., 1. Arai. Y. Isobe, H. Hirose and H. Aihara. 1984, Effects of sofalcone on necrotizing agents-induced gastric lesions and on endogenous prostaglandins in rats stomachs, J. Pharm. Dyn. 7, 791. Suzuki, Y., M. Hayashi, M. Ito and I. Yamagami, 1976, Anti-ulcer effects of 4’-(2-carboxyethyl)phenyl trans 4-aminomethyl cyclohexane carboxylate hydrochloride (cetraxate) on various experimental gastric ulcer in rats, Jap. J. Pharmacol. 26. 471. Szelenyi. I. and K. Brune. 1988, Possible role of oxygen free radicals in ethanol-induced gastric mucosal damage in rats, Dig. Dis. Sci. 33, 865. Takagi. K. and S. Yano, 1972, Effect of anti-ulcer drugs on gastric mucous hexosamine in rats subjected to several ulcerogenic conditions. Chem. Pharm. Bull. 20, 1170. Yamasaki, K.. H. Ishiyama, T. Imaizumi, T. Kanbe and Y. Yabuuchi, 1989, Effects of OPC-12759, a novel antiulcer agent, on chronic and acute experimental gastric ulcer and gastric secretion in rats, Jap. J. Pharmacol. 49, 441.
99
EJP 51840
Effects sf various mucosall protective drugs on diethyldithiocarb antral ulcer in rats Shinji Oka *, Keiki Ogino 2, Tatsuya Hobara 2, Shunji Yoshimura Tadayosi Takemoto 5 and Yozo Iida 1
3, Yukinori Okazaki 4,
’ Kokura Memorial Hospital. Kitakyurhu, ’ Department of Public Health, Yamaguchi University, School of Medicine, Ube, Japan, ’ Nagato General Hospital, Nagato. Japan, ’ Shuto General Hospital Yanai. Japan and Tokyo Women’s Medical College, Tokyo, Japan
Received 14 January 1991, accepted 5 February 1991
This study was designed to determine the effects of exogenous Cu,Zn-superoxide dismutase (SOD) and various mucosal protective agents against antral ulcer induced by diethyldithiocarbamate (DDC), inhibitor of Cu,Zn-SOD. Exogenous Cu,Zn-SOD reduced ulcer formation and prevented a decrease in SOD activity in gastric mucosa. This result indicates that maintenance of mucosal SOD activity is essential to prevent the ulcerogenicity of DDC. Many mucosal protective drugs that increase blood flow, mucus secretion, and endogenous prostaglandin failed to prevent ulcer formation and decrease of mucosal SOD activity. Rebamipide, however, significantly reduced ulcerogenesis and maintained mucosal SOD activity. This suggests that rebamipide has new protective effects on gastric mucosa. Diethyldithiocarbamate-induced
antral ulcer; Cu,Zn-superoxide dismutase; Gastric mucosa; Mucosal protective drugs
1. Introduction
Recent studies have indicated that active oxygen species play an important role in the pathogenesis of various gastric mucosal injuries and that some scavengers show protective effects against mucosal injury induced by active oxygen species (Perry et al., 1986; Pihan et al., 1987; Szelenyi and Bnme, 1988). Among the various scavengers, we studied the role and significance of superoxide dismutase (SOD) in the gastric mucosa because exogenous SOD has been reported to prevent mucosal injury induced by active oxygen species (Parks et al., 1982; Itoh and Guth, 1985; Perry et al., 1986; Pihan et al., 1987). There are at least three different enzymes of SOD in mammalian tissue, which include cytosolic copper,zinc-SOD (Cu,Zn-SOD), mitochondrial manganese-SOD and extracellular SOD. In the process of studying SOD, we found that diethyldithiocarbamate (DDC), an inhibitor of Cu,Zn-SOD, showed ulcerogenicity (Ogino et al., 1987; 1988). Previous reports have indicated that active oxygen species are involved in the pathogenesis of this unique experimental ulcer (Ishiyama et al., 1989; Ogino et al., 1990; Oka et al., 1990).
Correspondence to: S. Oka, Kokura Memorial Hospital, tyou. Kokura Kitaku, 802 Kitakyushu. Japan.
l-l Kifune-
Many anti-ulcer drugs are used clinically in Japan, especially so-called mucosal protective drugs, the antiulcer drugs that do not affect acid secretion and increase mucosal protective power. Several reports have shown that these drugs prevent various experimental ulcers without inhibiting acid secretion (Takagi and Yano, 1972; Suzuki et al., 1976; Murakami et al., 1981; 1982; Saziki et al., 1984; Ysmasaki et al., 1989). This study concerned the effects of exogenous Cu,Zn-SOD and various other mucosal protective drugs on DDC-induced antral ulcer.
2. Materials and methods 2. I. Animals Male Wistar rats weighing 190-230 g were used. The animals were kept in cages with wide mesh wire bottoms to prevent coprophagia. The rats were fasted and allowed access to water ad libitum for 24 h prior to the experiment. 2.2. Chemicals The following chemicals were purchased from the sources indicated: diethyldithiocarbamate (Wako Pure
Chemiot\ls), sulfonilic acid (Nakarai). hypoxanthine ~Nakarai), hydroxylamine-o-sulfate (Merck). xanthine ringer h~~~~eirn for that used in the oxidase t ~~e~s~~~~n~ of SOD activity on gastric mucosa. Sigma for that used in the study of the effect of Rebamipide on the SOD assay system), N-l-naphthylethylene-diamine (Tokyo Kasei), Cu.&t-SOD (Sigma), Cetraxate ~Daii~~ Pharmaceutical}, Gefatnate (Sumitimo), ~falcone (T&ho Ph~aceutical}, Teprenone (E&i), Rebamipide (Otsuka Pharmaceutical). 2.3. Production cf DDC-induced antral ulcer (Ishiyama et al.. 1989) The rats were anesthetized with ether and the pylorus was ligated. After pylorus ligation, DDC resolved in saline was injected S.C.in a dose of 800 mg/kg. Next, 1 ml of 0.1 N NC1 was administered orally using a metal orogastric tube. The rats were killed 7 h later. The stomach was removed and filled with 10 ml of 10% form&n. The ulcer index was measured under a dissecting microscope with a square grid eyescope and was expressed as the area of the antral ulcer (mm’). 2.3. I. Effeert of C&Zn-SOD
Cu.Zn-SOD was dissolved in saline and administered S.C.30 min before the experiment. In the control group, saline was injected S.C.
TABLE 1 Effect of exogenous Cu.&t-SOD on SOD activity in the an&al mucosa. The data show the mean + SE. Group
Number of rats
SOD activity (NU/mg protein)
Normal antrum mucosa DDC + saline DDC + Cu,Zn-SOD (60~ U/kg)
8 8
15.9*0.7 6.6 + 0.5 h
8
12.4+1.7’”
8 P c 0.05. h P c 0.01 when compared with normal antrum mucosa. ’ P c 0.01 when compared with the DDC + saline group.
concentration of the samples was measured method of Lowry (1951).
by the
2.3. Effect of Rebamipide on the SOD assay system Rebamipide was dissolved in 2% NaHC4, and the solution was prepared to a final concentration of lo-‘, 10M5, IOe4 mol/l. The data are expressed as absorbance values at 550 nm. 2.6. Statistical analysis Statistical significance of the difference between two groups was determined by Student’s t-test and by oneway analysis of variance for multiple groups. A P < 0.05 was considered significant.
2.32. Effects of various mucosai p~orectiue drugs
Each drug was suspended in 0.5% carboxymethylcellulose (CMC) and administered i.p. 30 min before the experiment. Each drug was used in a dosage that would prevent the various experimental ulcers (Takagi and Yano, 1972; Suzuki et al., 1976; Murakami et al., 1981; 1982; Saziki et al., 1984; Konturek et al., 1986; Yamasaki et al., 1989; Kuroiwa et al., 1990). The effects of Rebamipide were studied at three different doses (7.5. 15. 30 mg/kg). 2-d. ~e~ure?~len~ of SOD activity in antraf mucosa
Because SOD activity in antral mucosa reaches its lowest value in 1 h, the samples were obtained within I h (Ishiyama et al., 1989). The rats were killed I h after pylorus ligation, DDC treatment and 0.1 N HCI administration. The stomach was removed and the antral mucosa was scraped. The mucosa was homogenized with 50 mM Tris-HCl buffer, pH 7.4 and sonicated under cooling with ice. The homogenate was centrifuged at 2Oooc) x g for 20 min. The supematant was used to measure SOD activity in the antral mucosa. SOD activity was measured as described by Oyanagui (1984) and expressed in nitrite units (NU)/mg protein. The protein
3. Result 3.1. Effect of exogenous Cu,Zn-SOD The ulcer index in the control group (saline) was 6.45 f 1.01. Exogenous Ct.&&-SOD ~60000 U/kg) significantly (P c 0.01) reduced the ulcer index to 2.20 + 0.50. SOD activity was significantly reduced in the DDC plus saline group (table 1). Cu,Zn-SOD administration significantly prevented a decrease in SOD activity compared with the DDC plus saline group.
TABLE 2 Effects of various mucosal protective drugs on DDC-induced ulcer. The data show the mean i S.E. Treatment Saline Gefarnate Cetraxate Teprenone Sofalcone Rebamipide
Dose f mg/kg)
Number of rats
Ulcer index 0nm2)
I~bition (W
500 300 200 100 30
9 7 8 7 8 7
6.45 It 0.72 6.36 f 0.93 6.19&0.45 5.71*0.78 5.50*0.53 3,00&0.64h
1.4 4.0 11.5 14.7 53.5
’ P <: 0.01 when compared with the saline treatment group.
antral
101 TABLE 3 Effects of Rebamipide on DDC-induced the mean f SE. Treatment Control Rebamipide
antral ulcer. The data show
Dose (mg/kg!
Number of rats
Ulcer index (mm2)
7.5 15 30
10 a 10 10
5.85 kO.71 5.19+ 1.06 3.10*0.67” 2.75 + 0.75 ’
a P -z 0.05, ’ P c 0.01 when compared with the control group.
3.2. Effects of various mucosal protective drugs Table 2 shows the inhibition rates for Gefamate, Cetraxate, Teprenone and Sofalcone. The differences between these drug treatment groups and the saline group were not significant. Only Rebamipide treatment significantly reduced the ulcer index; the effect was dose-dependent (table 3). SOD activity was significantly reduced in the saline, Gefarnate, Cetraxate, Teprenone, and Sofalcone treatment groups, but Rebamipide treatment prevented the reduction of SOD activity as seen from the ulcer index (table 4). 3.3. Effect of Rebamipide on the SOD assay system The following values were found for absorbance of the control, 10m6, 10e5 and 10m4 mol/l Rebapimide: 0.127, 0.136,0.136 and 0.126. Absorbance did not show a difference according to drug concentration. Rebamipide had no effect on the assay system for SOD activity.
4. Discussion DDC-induced antral ulcer has two features. One is that the ulcerogenicity of DDC depends on a decrease in protection against active oxygen species in gastric mucosa, because some scavengers inhibit ulcer formation, and because increases in lipid peroxide are found
TABLE 4 Effects of various mucosal protective drugs on SOD activity in the antral mucosa. The data show the mean& SE. Group
Number of rats
SOD activity (NU/mg protein)
Normal antrum mucosa DDC + saline DDC + Gefarnate DDC + Cetraxate DDC + Teprenone DDC + Sofalcone DDC + Rebamipide
7 7 7 7 7 7 7
17.2kO.2 7.5 f 0.7 h 8.1 f 0.5 h 8.6 f 0.6 ’ 7.3 f0.9b 7.9*0.7b 12.3f0.9’.=
’ P < 0.01 when compared with normal antrum mucosa. when compared with DDC + saline.
-
P < 0.01
in the gastric mucosa before the appearance of mucosal injury (IShiYaIkXt et al., 1989; Oka et al., 1990). The other feature is that this experimental ulcer is similar to the human gastric ulcer and is useful for elucidating the etiology of the human gastric ulcer. In this model, 0.1 N I-ICI was administered immediately after pylorus &ation. HCl administration is sometimes used to aggravate the mucosa! injury in experimental ulcers because exogenous HCl decreases mucosal blood flow and intramural pH caused by back diffusion of luminal H’ (Kivilaakso et al., 1978; Hirose et al., 1985; Morishita and Guth, 1987). AS DDC inhibits acid secretion (Ogino et al., 1990), additional HCl was useful to decrease the luminal pH by increasing back diffusion. A previous study had shown that DDC itself was toxic to gastric mucosa, but DDC treatment without HCI administration failed to induce a deep ulcer in some rats (Oka et al., 1989). HCl administration was considered to help the ulcerogenicity of DDC by decreasing luminal pH. As deep ulcer is the specific feature, HCI administration was useful for this model. The effect of exogenous Cu,Zn-SOD on this ulcer model was studied first. Cu,Zn-SOD pretreatment reduced the ulcer index and prevented the decrease in SOD activity in the gastric mucosa. As a previous study had indicated that DDC decreases only Cu,Zn-SOD activity (Ishiyama et al., 1989) an increase of mucosal SOD activity after Cu.Zn-SOD administration was considered to depend on exogenous Cu,Zn-SOD. These results indicate that reduction in Cu,Zn-SOD activity plays an important role in the pathogenesis of the DDC-induced antral ulcer. Maintenance of mucosal SOD activity, especially of Cu,Zn-SOD, is essential for preventing the ulcerogenicity of DDC. The effects of various mucosal protective drugs on the ulcer index and mucosal SOD activity were studied next. There were two reasons why we used i.p. administration with this model. One is that intragastric administration may induce adaptive cytoprotection. Adaptive cytoprotection is- caused by the direct stimulation of gastric mucosa by the drugs, and when drugs are administered intragastrically, they may act just like a mild irritant (Robert et al., 1983). This is not the pharmacological effect of the drugs. Another is that this model makes use of HCl administration. In order to prevent the effect of drugs on HCl, we chose i.p. administration. Each mucosal protective drug used in this experiment has been shown to increase mucosal blood flow (Cetraxate) (Kuroiwa et al., 1990) mucus secretion (Gefamate and Teprenone) (Takagi and Yano, 1972; Murakami et al., 1982) or endogenous prostaglandin (Gefarnate and Sofalcone) (Kobayashi et al., 1983; Konturek et al., 1986). These drugs have been reported to prevent vatious experimental ulcers according to the above effects. A dose high enough to prevent other experimental ulcers was used, but Gefarnate, Cetraxate, Teprenone, and
to prevent ulcer formation. At the same s failed to maintain SOD activrty in the From these results. it is suggested that er than the already known ones (mucosal ow. mucus secretion. and prostaglandin) plays a in this model. The new mucosal protective drug, showed dose-dependent effectiveness against DDC-induced antral ulcer and prevented sig~~ifica~tly reduction of mucosal SOD activity. We examined whether tke Rebamipide contained in the sample affected the SOD assay system, but found that amipide had no effect on the assay system and that it did not havre SOD-like activity. Therefore, the antiulcer effect on DDC-induced astral ulcer may depend on the maintenance of SOD activity in the gastric mucosa. As various drugs that increase already known mucosal protective factors failed to prevent this experimental ulcer. it is suggested that Rebamipide has a new effect on the gastric mucosa. This new effect is considered to be an effect on SOD activity in the gastric mucosa. It remains to be studied how Rebamipide maintains SOD activity and which types of SOD are increased by Rebamipide treatment.
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