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Vol. 295, Issue 2, 447-452, November 2000
Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine (W.-H.S., S.T., M.T., E.S.G., N.K., A.K., Y.K., M.Y., H.I., Y.O., Y.S., M.H.), and Department of Clinical Laboratory Science, School of Allied Health Sciences, Osaka University Faculty of Medicine, Yamadaoka, Suita, Japan (S.K.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Recent studies indicate an expression of mitogen-inducible cyclooxygenase (COX-2) in gastric mucosa. Rebamipide, a mucoprotective agent enhances prostaglandin (PG) synthesis. The present study was designed to clarify the mechanism for rebamipide-induced mucosal protection. Male Sprague-Dawley rats were administered 5, 15, or 50 mg/kg/day rebamipide for 14 days. The expression of constitutive cyclooxygenase (COX-1) and COX-2 in gastric mucosa was determined using Western blot analysis. Another series of rats was used to examine 1) the levels of PGE2 in stomach with and without pretreatment with a COX-2 inhibitor; 2) the protective action of rebamipide against gastric damage caused by 0.6 N HCl; and 3) the effects of a COX-2 inhibitor on rebamipide-induced gastric mucosal protection. COX-2 expression was enhanced, whereas COX-1 expression did not change significantly in the gastric mucosa of rats after treatment with rebamipide. The gastric mucosal PGE2 was higher in the rebamipide groups than in the vehicle-treated group. Rebamipide also suppressed gastric damage induced by HCl in a dose-dependent manner. A COX-2 inhibitor blocked the rebamipide-induced increase in mucosal PGE2, and mucosal protection induced by rebamipide. The results indicate that rebamipide induces COX-2 expression, increases PGE2 levels, and enhances gastric mucosal defense in a COX-2-dependent manner. Thus, COX-2 has an important role in the effects of rebamipide on gastric mucosal protection.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Prostaglandins
(PGs) such PGE2 have potent effects on gastric
mucosal protection (Robert, 1979
; Miller, 1983; Guth et al., 1984).
Endogenous PG synthesis also has an important role in gastric mucosal
defense (Robert et al., 1983; Arakawa et al., 1990). Synthesis of PGs
is governed by PG endoperoxide synthase, or cyclooxygenase (COX: EC
1.14.99.1), which consists of two isoforms (Masferrer et al., 1996).
The constitutive isoform (COX-1) is dominantly expressed in platelets,
prostate, and stomach. Expression of the mitogen-inducible isoform
(COX-2) is enhanced in gastric epithelial cells after growth
stimulation in vitro and in gastric epithelium after acid-induced
damage in vivo (Tsuji et al., 1996; Sawaoka et al., 1997; Sun et al.,
2000). Furthermore, COX-2-specific inhibitors delay healing of acetic
acid-induced gastric ulcers in mice (Mizuno et al., 1997; Sun et al.,
2000), suggesting an important role for this isozyme in peptic ulcer healing.
Rebamipide,
2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl]-propionic
acid, an antiulcer agent, prevents various acute experimental gastric
mucosal lesions and accelerates the healing of chronic ulcers (Yamasaki
et al., 1987, 1989; Kawano et al., 1991; Kleine et al., 1993; Suzuki et
al., 1994; Kim and Hong, 1995; Watanabe et al., 1996; Kokura et al.,
1997, 1998; Kim et al., 1998). In particular, rebamipide has
cytoprotective actions in humans and animals (Yamasaki et al., 1987;
Kawano et al., 1991). This compound increases PG levels in gastric
tissue and protects gastric mucosa from various insults (Kleine et al.,
1993). The mechanism of rebamipide-induced gastric mucosal protection,
however, is not known.
Therefore, the present study was designed to clarify the effects of rebamipide on expression of COX-1 and COX-2 in rat gastric mucosa. Because long-term effects of the agent on gastric mucosal protection would be clinically relevant, we focused our attention on the effects of 14-day administration of rebamipide on gastric mucosal expression of COX-1 and COX-2, on gastric mucosal levels of PGE2, and on gastric mucosal protection in rats.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals and Agents. Specific pathogen-free male Sprague-Dawley rats aged 7 weeks and weighing 200 to 230 g were fed with standard pellet chow and allowed free access to water, unless stated otherwise. All of the experiments were performed according to the guidelines of the Institutional Committee on Experimental Animals.
Rebamipide was a gift from Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan. A specific inhibitor of COX-2, NS-398, was purchased from Cayman Chemicals, Ann Arbor, MI. The agents were suspended in 0.5% carboxymethylcellulose (CMC; Sigma Chemical Co., St. Louis, MO). The other agents were purchased from Nacalai Tesque, Co., Kyoto, Japan, unless stated otherwise.Effects of Rebamipide on Expression of COX-1 and COX-2 in Rat Gastric Mucosa. The rats were randomly divided into four groups, six rats per group. To the control animals (group 1), only 0.5% CMC was given. Groups 2 through 4 rats were administered oral rebamipide at doses of 5, 15, and 50 mg/kg once a day. After 14 days of drug administration, these rats were anesthetized with sevoflurane, sacrificed, and immediately laparotomized. The stomach was harvested and opened along to the greater curvature. The oxyntic mucosa was scraped with glass slides, immediately frozen in liquid nitrogen, and stored for Western blotting analysis for COX-1 and COX-2.
The gastric mucosal samples were homogenized in phosphate-buffered saline containing 1% Nonidet-40, 0.5% sodium deoxycholate, and 0.1% SDS. Protein concentration of the homogenate was measured using a protein assay reagent (BCA kit; Pierce, Rockford, IL). The tissue homogenates, 100 µg of protein per lane, were electrophoresed in 10% SDS-polyacrylamide gel, and transferred onto polyvinylidene difluoride membranes (Immobilon; Millipore Corp., Bedford, MA) using a semidry transfer cell (Bio-Rad, Hercules, CA). The blots were pretreated in Tris-buffered saline containing 5% nonfat dry milk, 1% albumin, and 0.1% Tween 20, and incubated with antibodies for COX-1 and COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Filters were washed three times and incubated with a horseradish peroxidase-conjugated secondary antibody against goat IgG (Dako, Glostrup, Denmark), developed using a commercial enhanced chemiluminescence system (Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK), and exposed to films (Hyperfilm; Amersham Pharmacia Biotech Ltd). The expression of COX-1 and COX-2 was semiquantified using a densitometric scanner. For immunohistochemical analysis, gastric corpus tissue from rats treated with vehicle or 50 mg/kg rebamipide was fixed in 10% phosphate-buffered formalin. Four-micrometer-thick sections were cut from paraffin-embedded tissues and were deparaffinized. Sections were then microwaved in citrate buffer, pH 6.1, 95°C, for 10 min for antigen retrieval. Endogenous peroxidase activity was quenched by incubation in 3.0% H2O2 in methanol for 30 min. Nonspecific binding was blocked with 3% normal rabbit serum in phosphate-buffered saline, and sections were incubated in primary antibodies overnight at 4°C. Primary antibodies raised in goat against COX-1 or COX-2 were used alone (1:200 dilution) or after preincubation with 2 µg/ml pure blocking COX-1 or COX-2 peptide (Santa Cruz Biotechnology) as preabsorption controls. They were stained according to the avidin-biotin-peroxide complex (ABC) method using a commercial kit (Vecstatin kit; Vector Laboratories, Burlingame, CA) and visualized by 3,3'-diaminobenzidine (Vecstatin DAB kit; Vector Laboratories). Subsequently, the specimens were counterstained with hematoxylin.Effects of Rebamipide and NS-398 on PGE2 Levels in Rat Gastric Mucosa. Another series of 36 rats was randomized and divided to 6 groups, 6 animals per group. Group 1 animals received 0.5% CMC, whereas groups 2 through 4 were given rebamipide (5, 15, or 50 mg/kg) in 0.5% CMC for 14 days. These rats were fasted for 20 h but given 0.5% CMC 10 h after the final administration of rebamipide. Group 5 rats were given daily administration of rebamipide (50 mg/kg) for 14 days, and were treated with 30 mg/kg NS-398 10 h after the final administration of rebamipide. Group 6 animals were given 0.5% CMC for 14 days and then NS-398 (30 mg/kg). One hour later, the rats were anesthetized with sevoflurane, intragastrically administered with saline containing 100 µM indomethacin and 10 mM EDTA to block excess production of PG synthesis, and immediately laparotomized. The stomach was harvested and opened along to greater curvature. The gastric mucosa was scraped with glass slides, immediately frozen in liquid nitrogen. The tissue samples were weighed and homogenized in cold ethanol. The homogenate was acidified to pH 4 and centrifuged. PGE2 in the supernatant was purified using C18 solid phase extraction cartridges (Sep-Pak; Waters, Millford, MA), and eluted with ethyl acetate containing 1% methanol. PGE2 was solidified using a rotary evaporator, reconstituted in a buffer, and measured using enzyme immunoassay (Cayman Chemicals). The PGE2 level in the gastric mucosa was expressed as picograms of PGE2 per gram of tissue.
Effects of Rebamipide and NS-398 on Acid-Induced Gastric Mucosal Injury. Another series of 36 rats, 6 animals per group, was randomly assigned. Groups 1 through 4 were given either rebamipide (5, 15, or 50 mg/kg) or 0.5% CMC for 14 days. These groups were treated with 0.5% CMC 10 h after the 14-day administration of rebamipide or CMC. Group 5 rats were given 30 mg/kg NS-398 after daily administration of rebamipide (50 mg/kg) for 14 days. Group 6 animals were given 0.5% CMC as a vehicle and then NS-398 (30 mg/kg). One hour later, the rats were anesthetized and administered 1 ml of 0.6 N HCl through orogastric tubing (7 French feeding tube; Top, Tokyo, Japan). One hour later, the rats were sacrificed and laparotomized. The stomach was harvested, opened along the greater curvature, extended on a plastic board, and photographed. The area of the macroscopic hemorrhages and erosions was assessed using planimetry. The ulcer index was expressed as a percentage of the lesion area to the total gastric glandular area.
For histologic assessment, the gastric corpus wall was fixed in phosphate-buffered formalin, sectioned, and paraffin embedded. Semithin sections were deparaffinized, stained with H&E, and examined under a light microscope by a pathologist blind to the group to which the specimen belonged. The specimens were coded and assessed according to the criteria of Whittle et al. (1990). In brief, a 1-cm length of each
histologic section was assessed for epithelial cell damage (score = 1); glandular disruption, vasocongestion, or edema in the upper
mucosa (score = 2); hemorrhagic damage in the mid to lower mucosa
(score = 3); and deep necrosis and ulceration (score = 4).
Each section was evaluated on a cumulative basis to give the histologic
score, the maximum score thus being 10.
Statistical Analyses. Data were shown as mean ± S.E., and were analyzed using one-way ANOVA with Dunnett's multiple comparison test. A probability value less than .05 was considered statistically significant.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Rebamipide on Expression of COX-1 and COX-2 in Rat
Gastric Mucosa.
In the control rats treated with 0.5% CMC, the
gastric mucosa expressed COX-1. The mucosa also expressed small amounts
of COX-2. In the groups treated with rebamipide (5, 15, or 50 mg/kg) for 14 days, the expression of COX-1 did not change compared with that
of the control. Expression of COX-2 was enhanced in the animals after
daily administration of rebamipide for 14 days. The level of COX-2
immunoreactivity increased after administration of rebamipide in a
dose-dependent manner (Fig. 1). The
specificity of the COX-1 and COX-2 antibodies was confirmed by
neutralization study with the corresponding blocking peptides (data not
shown).
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Effects of Rebamipide and NS-398 on PGE2 Levels in Rat
Gastric Mucosa.
Gastric mucosal PGE2 levels
were higher in the rebamipide groups than in the CMC-treated control
group. Rebamipide increased gastric mucosal PGE2
levels in a dose-dependent manner. Treatment with NS-398 suppressed the
rebamipide-induced increase in gastric mucosal
PGE2 (Fig. 3).
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Effects of Rebamipide and NS-398 on Acid-Induced Gastric Mucosal
Injury.
In rats given 0.5% CMC, significant hemorrhagic streaks
and erosions developed mainly in the gastric corpus mucosa 1 h
after the intragastric administration with 0.6 N HCl. In the groups given rebamipide (5, 15, or 50 mg/kg), however, the ulcer index was
significantly smaller than in the control group treated with CMC. On
the other hand, there were no significant differences in the ulcer
index between the group given CMC and the group given CMC and NS-398.
Therefore, NS-398 did not aggravate the HCl-induced macroscopic injury
of the stomach. The ulcer index was significantly higher in the group
given rebamipide and NS-398 than the group given rebamipide and CMC
(Fig. 4).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PGs have long been implicated in protection of gastric mucosa from
various insults and acceleration of gastric wound healing (Miller,
1983). PGE2 is one of the major prostanoids in
gastric tissue. Rebamipide enhances endogenous production of PGs,
particularly that of PGE2 in the stomach, and
protects gastric mucosa from injury. The precise mechanism for
PG-synthesis stimulated by this mucoprotective agent is not yet known.
Because COX is the rate-limiting enzyme for production of PGs from
arachidonate, we investigated the influence of rebamipide on expression
of the two isoforms of COX. The present study demonstrated that
rebamipide, which was administered to the animals for 14 days, enhanced
expression of mitogen-inducible COX-2, but not that of constitutive
COX-1. Rebamipide also increased PGE2 in gastric
mucosa in a dose-dependent manner. The rebamipide-induced increase in
gastric mucosal PGE2 was blocked by a
COX-2-specific inhibitor. Therefore, 14-day administration with
rebamipide increases gastric mucosal PGE2 by
enhancing COX-2 expression.
After the discovery of the mitogen-inducible COX-2 (Fletcher et al.,
1992; Kujubu and Herschman, 1992), Seibert et al. (1994) examined mRNA
levels of COX-1 and COX-2 in normal rat tissue using RNase protection
assay. COX-1 mRNA was readily detectable in all normal tissue examined,
whereas levels of COX-2 mRNA were substantially lower with the
exception of brain. Our previous study also demonstrated that
unstimulated gastric mucosa expresses smaller amount of COX-2, whereas
gastric mucosal epithelium expresses this isoform after various stimuli
(Sawaoka et al., 1997; Sun et al., 2000). The present study confirmed
that gastric mucosa expresses both COX-1 and COX-2 in control rats
treated with vehicle. Our result is in accord with previous findings
demonstrating COX-2 expression in the stomach (Iseki, 1995; Zimmermann
et al., 1998).
On the other hand, daily administration of rebamipide for 14 days
enhanced COX-2 expression in gastric mucosa and significantly reduced
both macroscopic injury and histologic damage caused by HCl in a
dose-dependent manner. We did not examine the protective effects of
rebamipide against the other different types of gastric injury.
However, previous studies have shown that rebamipide reduces or
prevents experimental acute gastric mucosal injury caused by nonsteroidal anti-inflammatory drugs, restraint and
water-immersion stress, histamine (Yamasaki et al., 1989),
ischemia-reperfusion (Kim and Hong, 1995) and necrotizing agents such
as absolute ethanol and 0.2 N NaOH (Yamasaki et al., 1987). The present
study clearly shows that rebamipide-induced gastric mucosal protection
was associated with an increase in gastric mucosal
PGE2. NS-398 (30 mg/kg), a specific inhibitor of
COX-2, significantly blocked the rebamipide-induced gastric mucosal
protection against HCl, as well as the rebamipide-induced increase in
gastric mucosal PGE2. Several studies (Futaki et
al., 1993; Masferrer et al., 1994; Ogino et al., 1997) have confirmed that NS-398 inhibited COX-2 with an IC50 of 0.32 µM, but never affected COX-1 activity, even at 100 µM. Futaki et
al. (1993, 1997) reported that NS-398 even at 100 mg/kg did not
significantly suppress COX-1 activity in rats. In the previous studies
we confirmed that NS-398 specifically inhibits COX-2 in the doses
ranging from 0.4 to 40 mg/kg in rats (Sun et al., 2000) and from 10 to
100 mg/kg in mice (Sawaoka et al. 1998). Therefore, at the dose used in
this study, NS-398 is sufficient to inhibit COX-2 without significant influences to COX-1 activity. The protective effects of rebamipide appear to be mediated by PGs, such as PGE2, which
is synthesized from arachidonate by COX-2. A future study using a
COX-1-specific inhibitor, which was unavailable in the present study,
may support the idea that COX-2 is solely responsible for the
rebamipide-induced gastric mucosal protection.
Our present study demonstrated for the first time that daily
administration of rebamipide for 14 days induces COX-2 expression in
normal rat gastric mucosa. The precise mechanism for COX-2 induction by
this mucoprotective agent remains to be investigated. Tarnawski et al.
(1998) reported that rebamipide increases expression of epidermal
growth factor (EGF) and its receptor (EGF-R) both in normal and
ulcerated gastric mucosa in rats. Signaling through the EGF-R induces
COX-2 expression in some cell lines, including gastric epithelial cells
(DuBois et al., 1994; Sawaoka et al., 1999). We also found that
the expression of COX-2 mRNA and COX-2 protein was enhanced within
several hours after the ulcer induction. On the other hand, we found
that single administration of rebamipide did not induce COX-2
expression in stomach (our unpublished data).
The promoter region of COX-2 gene contains binding sites for a number
of important transcription factors, including cAMP response element,
nuclear factor for interleukin-6 expression, and nuclear factor-
B
(NF-B) (Reddy et al., 2000; Wadleigh et al., 2000). We did not
examine which transcription factor(s) contributes to the up-regulation
of COX-2 expression in the present study. However, a number of studies
have suggested that rebamipide is an oxygen radical scavenger and
thereby inhibits lipid peroxidation (Naito et al., 1995; Hahm et al.,
1997) and oxidant-mediated activation of NF-B (Kim et al., 2000).
Therefore, it is likely that transcriptional regulatory elements other
than NF-B play an important role in the rebamipide-induced
expression of COX-2. The precise mechanism for COX-2 induction caused
by long-term administration of rebamipide remains to be investigated in
future studies.
In conclusion, the present results clearly demonstrate that intragastric administration of rebamipide induces COX-2 expression in gastric mucosa, increases gastric mucosal PGE2 levels, and protects gastric mucosa from necrotizing agents in rats. Treatment with NS-398, a specific COX-2 inhibitor, abolishes the rebamipide-induced increase in gastric mucosal PGE2 and protection of gastric mucosa from acute injury. In this experimental ulcer model of ulcers in rats, the mucoprotective effects of rebamipide depend on COX-2 induced by the agent. Thus, rebamipide enhances gastric mucosal defense by increasing COX-2-dependent production of PGs in gastric mucosa.
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Footnotes |
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Accepted for publication July 5, 2000.
Received for publication March 27, 2000.
1 This study was supported by grants in aid from the Ministry of Health and Welfare, the Ministry of Education, Science, Sports and Culture, and the Smoking Research Foundation.
Send reprint requests to: Shingo Tsuji, M.D., Ph.D., Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, 565-0871, Japan. E-mail: tsuji{at}medone.med.osaka-u.ac.jp
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Abbreviations |
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PG, prostaglandin;
COX, cyclooxygenase;
CMC, carboxymethylcellulose;
EGF, epidermal growth factor;
EGF-R, epidermal
growth factor-receptor;
NF-B, nuclear factor-B.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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P < .01 versus the group treated
with rebamipide (50 mg/kg/day for 14 days) and then 0.5% CMC. Data are
shown as mean ± S.E., n = 6 per group, and
analyzed using ANOVA with Dunnett's multiple comparison test of the
means.
