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Antimicrobial Agents and Chemotherapy, August 1998, p. 1895-1899, Vol. 42, No. 8
Department of Microbiology,
Received 20 October 1997/Returned for modification 6 February
1998/Accepted 8 May 1998
Helicobacter pylori is a major etiological agent in
gastroduodenal disorders. The adhesion of H. pylori to
human gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is
thus a therapeutic target in the prevention of H. pylori
infection. Experiments were performed to evaluate the effect of
rebamipide, a novel antiulcer agent, on H. pylori adhesion
to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from
human gastric carcinomas, were used as target cells. Ten H. pylori strains isolated from patients with chronic gastritis and
gastric ulcer were used in the study. We evaluated the effect of
rebamipide on H. pylori adhesion to MKN-28 and MKN-45 cells quantitatively using our previously established enzyme-linked immunosorbent assay. The adhesion of H. pylori to MKN-28
and MKN-45 cells was significantly inhibited by pretreatment of these
cells with 100 µg of rebamipide per ml. However, the adhesion was not affected by the pretreatment of H. pylori with rebamipide.
On the other hand, the viabilities of H. pylori, MKN-28
cells, and MKN-45 cells were not affected by rebamipide. Our studies
suggest that rebamipide inhibits the adhesion of H. pylori
to gastric epithelial cells.
In humans, Helicobacter
pylori plays a causal role in histologic gastritis (21)
and peptic ulcers (4) and is a cofactor in the occurrence of
gastric cancer (3). H. pylori infection occurs in
the gastric mucosa (20). The adhesion of H. pylori to human gastric epithelial cells is the initial step of
H. pylori infection. Inhibition of the adhesion would be the
ideal target for the prevention of H. pylori colonization.
Accordingly, we have developed an enzyme-linked immunosorbent assay
(ELISA) to quantitatively evaluate H. pylori adhesion to
gastric epithelial cells (6).
We investigated the effect of rebamipide, a novel antiulcer agent that
has antioxidant and free-radical scavenging activities (5, 12,
18), on H. pylori adhesion to gastric epithelial cells
using our established ELISA.
Target cells.
MKN-28 and MKN-45 cells, derived from human
gastric carcinomas, were used for the analysis of H. pylori
adhesion (11). The cells were suspended at a concentration
of 3 × 105 cells/ml in RPMI 1640 medium (ICN
Biomedicals, Costa Mesa, Calif.) containing 10% fetal calf serum,
penicillin G (100 U/ml), and streptomycin (0.1 mg/ml). For the assay
described here, 100 µl of cell suspension was placed in each well of
a flat-bottom 96-well tissue culture plate (Falcon 3072; Becton
Dickinson, Lincoln Park, N.J.), and the plate was incubated at 37°C
under 8% CO2 for 2 days.
Bacteria.
In this study, 10 H. pylori strains,
obtained from five patients with chronic gastritis and five patients
with gastric ulcer, were used for the evaluation of rebamipide.
Following primary isolation, these strains were passaged one to three
times and were frozen at Anti-H. pylori antibody.
Polyclonal antibody
against H. pylori was prepared from a male
specific-pathogen-free New Zealand White rabbit (weight, 3.5 kg). The
rabbit was immunized by the following schedule. Three basal
immunizations with a mixture of three different H. pylori clinical isolates (1.6 × 108 bacteria) were given
subcutaneously at 7-day intervals. After 1 week, four booster
injections of the same immunogen were given intravenously at 7-day
intervals. The antibody was purified by using a protein A Cellulofine
column (Chisso, Tokyo, Japan). The specificity of this antibody was
tested by Western blotting.
Rebamipide and related compounds.
Rebamipide and 11 related
compounds, OPC-12763, OPC-12804, OPC-12823, OPC-12853, OPC-12924,
OPC-12963, OPC-12994, OPC-22016, OPC-12758, OPC-12822, and DM-1212,
were synthesized at Otsuka Pharmaceutical Co. (Tokushima, Japan) (Fig.
1) (19). Bovine serum albumin
(BSA) was used as the control agent.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Rebamipide, a Novel Antiulcer Agent, on
Helicobacter pylori Adhesion to Gastric Epithelial
Cells
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
80°C in brain heart infusion broth (Difco,
Detroit, Mich.) supplemented with 15% glycerol. Subsequent analyses
were performed with strains derived from the frozen stocks. H. pylori was inoculated onto brain heart infusion agar (Difco)
containing 8% horse blood, and the plates were incubated at 37°C
under 8% CO2 for 5 days (15). The organisms
were washed with 10 mM phosphate-buffered saline (PBS; pH 7.4) and were
suspended in RPMI 1640 without fetal calf serum and antibiotics at a
concentration of 109 bacteria/ml.
View larger version (20K):
[in a new window]
FIG. 1.
Structures of rebamipide and 11 related compounds.
ELISA. After the MKN-28 or MKN-45 cells had formed confluent monolayers, the medium was decanted from the microplates. The plates were then washed three times with PBS, 100 µl of H. pylori suspension (109 bacteria/ml) was added to each well, and the plates were incubated at 37°C under 8% CO2 for 90 min. The plates were then washed three times to remove the unadhered H. pylori, 100 µl of 8% paraformaldehyde was added to each well, and adherent H. pylori and cells were fixed at 4°C for 60 min. After the plates were washed, 100 µl of 1% H2O2 in methanol was added to each well and the plates were incubated at room temperature for 10 min, inactivating the endogenous peroxidase. After the plates were washed, 100 µl of rabbit anti-H. pylori polyclonal antibody (10 µg/ml) was added to each well and the plates were incubated for 2 h at 37°C. After the plates were washed, 100 µl of peroxidase-conjugated goat anti-rabbit immunoglobulins (Wako Chemicals, Osaka, Japan) diluted 1:1,000 in PBS was added to each well and the plates were incubated for 2 h at 37°C. After the final wash, 100 µl of o-phenylenediamine (0.4 mg/ml) in 100 mM citrate-phosphate buffer (pH 5.0) containing 0.02% H2O2 was added to each well and the plates were incubated at room temperature for 15 min. The reaction was terminated by adding 50 µl of 2 M H2SO4. The optical density (OD) of the reaction was measured at 490 nm with a microplate reader (model 3550 EIA Reader; Bio-Rad, Richmond, Calif.). The OD represents the amount of H. pylori adhering to the target cells (6).
Effect of rebamipide on MKN-28 and MKN-45 cells. Before the assay of H. pylori adhesion to MKN-28 or MKN-45 cells by ELISA, 100 µl of RPMI 1640 medium containing rebamipide or BSA (25 to 100 µg/ml) was added to each well containing MKN-28 or MKN-45 cells, and the plates were incubated at 37°C under 8% CO2 for 30 to 120 min. After the cells were washed and the rebamipide or BSA was removed, 100 µl of H. pylori suspension (109 bacteria/ml) was added to each well, and the plates were incubated at 37°C under 8% CO2 for 90 min. After the cells were washed, the amount of H. pylori adhering to the target cells was quantified by ELISA.
The viabilities of MKN-28 and MKN-45 cells were assessed by using a Cell Counting Kit (Dojindo, Kumamoto, Japan) (7, 8) before and after the treatment with rebamipide. With this kit, the viability of the cells was represented by the OD at 450 nm.Effect of rebamipide on H. pylori. Before the assay of H. pylori adhesion to MKN-28 cells, H. pylori was suspended in RPMI 1640 medium containing rebamipide (25 to 100 µg/ml), and the plates were incubated at 37°C under 8% CO2 for 90 min. The H. pylori treated with rebamipide was washed with PBS and was resuspended in RPMI 1640 medium at a concentration of 109 bacteria/ml. Subsequently, 100 µl of this H. pylori suspension was added to each well containing MKN-28 cells, and the plates were incubated at 37°C under 8% CO2 for 90 min. After the cells were washed, the amount of adherent H. pylori was quantified by ELISA.
The viability of H. pylori was assessed by measuring the numbers of CFU of the H. pylori suspension before and after the treatment with rebamipide. The MICs of rebamipide for the H. pylori were determined by the agar dilution method. The H. pylori strains were inoculated onto brain heart infusion blood agar plates containing rebamipide (25 to 1,600 µg/ml), and the plates were incubated at 37°C under 8% CO2 for 3 days. The MICs were the lowest concentrations of rebamipide that visibly inhibited bacterial growth.Effect of rebamipide on E. coli adhesion to MKN-28 cells. Before the assay of E. coli adhesion to MKN-28 cells, the cells were pretreated with rebamipide or BSA (25 to 100 µg/ml) for 90 min. After washing for the removal of rebamipide or BSA, 100 µl of the E. coli suspension (109 bacteria/ml) was added to each well, and the plates were incubated at 37°C under 8% CO2 for 60 min. After the cells were washed, the amount of E. coli adhering to MKN-28 cells was quantified by ELISA with rabbit anti-E. coli polyclonal antibody (Biogenesis, Poole, United Kingdom) in place of anti-H. pylori antibody.
Assay of rebamipide binding to MKN-28 cells. For the assay of rebamipide binding to MKN-28 cells, 5 × 105 cells were added to each well of a flat-bottom, 24-well tissue culture plate (Falcon 3047; Becton Dickinson) and the plates were incubated at 37°C under 8% CO2 for 24 h. After the MKN-28 cells had formed confluent monolayers, 500 µl of RPMI 1640 medium containing 25 to 100 µg of rebamipide per ml, which consisted of a 1:100 mixture of 14C-labeled (114 mCi/mmol) and nonlabeled rebamipide, was added to each well and the plates were incubated at 37°C under 8% CO2 for 60 min. After the MKN-28 cells were washed twice, the cells were lysed at 0°C with 1 M NaOH for 10 min and neutralized with 1 M HCl. After a scintillator (Aquasol-2; Packard, Meriden, Conn.) was added to the cell lysate, the radioactivity was measured with a liquid scintillation counter (Liquid Scintillation System LS5000CE; Beckman, Fullerton, Calif.) and the amount of rebamipide binding to MKN-28 cells was calculated.
Effects of related compounds. Before the adhesion assay, 100 µl of RPMI 1640 medium containing one of the rebamipide-related compounds (100 µg/ml) was added to each well containing MKN-28 cells and the plates were incubated at 37°C under 8% CO2 for 90 min. After the cells were washed, 100 µl of the H. pylori suspension (109 bacteria/ml) was added to each well and the plates were incubated at 37°C under 8% CO2 for 90 min. After the cells were washed, the amount of adherent H. pylori was quantified by ELISA.
Statistics. Data are presented as means ± standard deviations (SDs). The difference between rebamipide and the control was evaluated by paired Student's t test. The correlation between the effect of rebamipide and the concentration of rebamipide was evaluated by Spearman's rank correlation. A two-tailed P value of less than 0.05 was considered statistically significant.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of rebamipide on MKN-28 and MKN-45 cells.
The amount of
H. pylori adhering to MKN-28 cells was reduced by
pretreating MKN-28 with rebamipide and was dependent on the incubation
time (Fig. 2). The inhibitory activity
reached a plateau after 90 min of incubation, at which point the
experiments were carried out. The amount of H. pylori
adhering to MKN-28 cells decreased in a dose-dependent manner
(r = 0.963; P < 0.05 by Spearman's
rank correlation) with the concentration of rebamipide (Fig.
3). Furthermore, the amount of H. pylori adhering to MKN-45 cells also decreased in a dose-dependent
manner (r = 0.974; P < 0.05 by
Spearman's rank correlation; Fig. 4).
However, there was a difference in the reproducibility of the results
between MKN-28 and MKN-45 cells. MKN-28 cells showed more consistent
results than MKN-45 cells, which indicates that MKN-28 cells are more suitable for the analysis of H. pylori adhesion than MKN-45
cells.
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) or BSA (
) for 30 to
120 min. The amount of adherent H. pylori is expressed as
the percentage of the amount of H. pylori adhering to
untreated target cells. Each value represents the mean ± SD for
10 strains tested in this study. The difference between rebamipide and
BSA was evaluated by two-tailed paired Student's t test.
*, P < 0.01.
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Effect of rebamipide on H. pylori. The adhesion activity of H. pylori to MKN-28 cells was not affected by the pretreatment of H. pylori with rebamipide (Table 1). The viability of H. pylori was not affected by the treatment with rebamipide (Table 1). The MICs of rebamipide for all H. pylori strains tested in this study were >1,600 µg/ml, indicating that rebamipide has no activity against H. pylori in vitro.
Effect of rebamipide on E. coli adhesion to MKN-28
cells.
The results for E. coli were similar to those
for H. pylori. The amount of E. coli adhering to
MKN-28 cells was reduced by the pretreatment of MKN-28 cells with
rebamipide (Fig. 5) and was dependent on
the dose of rebamipide (r = 0.971; P < 0.05 by Spearman's rank correlation).
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Binding of rebamipide to MKN-28 cells. In order to examine the direct binding of rebamipide to target cells, a binding assay was carried out. The amount of rebamipide that bound to MKN-28 cells increased in a dose-dependent manner (r = 0.992; P < 0.01 by Spearman's rank correlation) (Fig. 6).
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Effects of related compounds. The effects of rebamipide-related compounds on H. pylori adhesion to MKN-28 cells are indicated in Table 2. The compounds, each of which has a p-chlorophenyl group, reduced the level of adhesion. On the other hand, the compounds which do not have this group did not.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study, MKN-28 and MKN-45 cells, derived from human gastric carcinomas (11), were used as target cells. The manners of adhesion may differ in these cells and normal human gastric mucosal cells. However, normal human gastric mucosal cells are not available for laboratory adhesion assays and such cells, obtained from biopsied or surgical specimens, may show heterogeneous characteristics. Therefore, we adopted these cell lines as target cells to obtain reproducible results.
The adhesion of H. pylori to MKN-28 and MKN-45 cells was significantly inhibited by the pretreatment of these cells with 100 µg of rebamipide per ml for 90 min compared with the level of adhesion inhibition for the controls. This concentration can be achieved in the gastric mucous layer with the recommended clinical dose of rebamipide (13). Furthermore, rebamipide did not affect the viabilities of MKN-28 and MKN-45 cells at this concentration. These results suggest that rebamipide can inhibit H. pylori adhesion to gastric epithelial cells without affecting the viability of the cells. On the other hand, H. pylori adhesion to MKN-28 cells was not affected by the pretreatment of H. pylori with the same concentration of rebamipide. This indicates that rebamipide directly affects the gastric epithelial cells and does not act on H. pylori.
Rebamipide bound to MKN-28 cells and reduced the level of H. pylori adhesion in a dose-dependent manner. These results suggest that target molecules of rebamipide exist on or within MKN-28 cells. These target molecules could be responsible for the adhesion of H. pylori to MKN-28 cells. Several adhesins of H. pylori have been identified (1, 2, 9, 10). One possible mechanism is that rebamipide has some structural similarity to those adhesins and competitively inhibits H. pylori adhesion. To investigate the precise antiadhesion mechanism of rebamipide, the target molecules must be identified. On the other hand, rebamipide and related compounds which showed antiadhesion activities had the p-chlorophenyl group, without exception. Thus, this group may play an important role in the antiadhesion mechanism of rebamipide.
However, rebamipide did not completely inhibit H. pylori adhesion. The adhesion of H. pylori to gastric epithelial cells will be due to some combinations of H. pylori adhesins and their receptors. Rebamipide may only partially inhibit the combinations. On the other hand, rebamipide also inhibited E. coli adhesion to MKN-28 cells, which indicates that the antiadhesion effect of rebamipide is not specific for H. pylori. The target molecules of rebamipide may be common receptors for bacterial adhesion to alimentary tract epithelial cells.
Rebamipide itself did not directly affect the viability of H. pylori in vitro. However, our studies suggest that rebamipide has potential as an agent for the prevention of H. pylori adhesion. Furthermore, from preliminary data, triple therapy with omeprazole, amoxicillin, and rebamipide combined showed a strong eradication effect in a human clinical trial (14). In the gastric mucosa, H. pylori localizes on the surface of the epithelial cells as well as in the mucous layer (16). H. pylori colonized on epithelial cells can induce mucosal injury by direct and indirect mechanisms (17). On the other hand, H. pylori in the mucous layer can survive after insufficient eradication therapy, and the organisms adhere again to gastric epithelial cells and recolonize. Thus, the antiadhesion effect of rebamipide can contribute to the prevention of H. pylori recolonization, provided that the clinical dose of rebamipide completely overlays the surface of the gastric epithelial cells. As a result, prolonged use of rebamipide combined with antibiotics may enhance the eradication rate (14).
In conclusion, rebamipide may have potential as a new therapeutic agent against H. pylori infection.
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ACKNOWLEDGMENTS |
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This study was supported in part by a Grant-in-Aid for Scientific Research (grant 09770188) from the Japanese Ministry of Education, Science, Sports and Culture, a grant (grant 8-14) from the Japanese Ministry of Health and Welfare, and a grant from the Sapporo Medical University Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Tochigi-ken 329-0498, Japan. Phone: 81-285-58-7332. Fax: 81-285-44-1175. E-mail: shunhaya{at}jichi.ac.jp.
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Antimicrobial Agents and Chemotherapy, August 1998, p. 1895-1899, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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