Pharmacological Research, Vol. 46, No.

1, 2002
doi:10.1016/S1043-6618(02)00034-8, available online at http://www.idealibrary.com on

PROTECTIVE EFFECT OF OMEPRAZOLE AGAINST ACUTE GASTRIC

MUCOSAL LESIONS INDUCED BY COMPOUND 48/80,
A MAST CELL DEGRANULATOR, IN RATS
TAKASHI KOBAYASHI a , YOSHIJI OHTA b,∗ , KAZUO INUI a , JUNJI YOSHINO a and
SABURO NAKAZAWA a
a Department of Internal Medicine, Second Teaching Hospital, School of Medicine, Fujita Health University,
Nagoya, Aichi 454-0012, Japan, b Department of Chemistry, School of Medicine,
Fujita Health University, Toyoake, Aichi 470-1192, Japan

Accepted 12 March 2002

Omeprazole, a proton pump inhibitor is known to function not only as a proton pump inhibitor but
also as an anti-inflammatory agent, an antioxidant or a stimulator of gastric mucus secretion. We have
shown that the pathogenesis of acute gastric mucosal lesions induced by compound 48/80, a mast
cell degranulator, in rats involves neutrophil infiltration, lipid peroxidation, and mucin depletion,
but not acid secretion, in the gastric mucosal tissue. Therefore, we examined whether omeprazole
protects against acute gastric mucosal lesions induced by compound 48/80 in rats. Rats were injected
with omeprazole (10 or 50 mg kg−1 , i.p.) at 0.5 h before injection of compound 48/80 (0.75 mg kg−1 ,
i.p.). Omeprazole prevented gastric mucosal lesion development at 0.5 and 3 h after compound 48/80
injection. Omeprazole attenuated decreased nonprotein sulfhydryl content and increased myeloper-
oxidase and xanthine oxidase (XO) activities and lipid peroxide (LPO) content in the gastric mu-
cosa at 0.5 h after compound 48/80 injection and increased myeloperoxidase and XO activities and
LPO content, but not decreased hexosamine and adherent mucus contents, in the gastric mucosa at
3 h. These results indicate that omeprazole protects against compound 48/80-induced acute gastric
mucosal lesions in rats possibly through its anti-inflammatory and antioxidant actions.
© 2002 Elsevier Science Ltd. All rights reserved.

Key wo rds: omeprazole, gastric mucosal lesion (rat), compound 48/80, neutrophil infiltration, lipid
peroxidation.

INTRODUCTION thought to depend on its inhibitory action on gastric acid

Omeprazole, a substituted benzimidazole derivative, is a
potent inhibitor of gastric acid secretion both in humans
[1, 2] and in animals [3–5]. The antisecretory mecha-
nism of omeprazole depends on the blockage of the H+ ,
K+ -ATPase in parietal cells [6, 7]. This proton pump in-
hibitor is clinically used for the treatment of gastric ulcers
[8, 9]. Omeprazole is known to protect against gastric mu-
cosal injuries induced by a variety of ulcernogenic agents,
water immersion restraint stress, and hemorrhage shock
[4, 5, 10–15], although there are some reports showing that
this proton pump inhibitor has no protective effect against
indomethacin- and absolute ethanol-induced gastric mu-
cosal injuries [16, 17]. Many of these protective effects
of omeprazole against gastric mucosal injuries have been
∗ Correspondingauthor. Department of Chemistry, School of Medicine,
Fujita Health University, Toyoake, Aichi 470-1192, Japan. E-mail:
yohta@fujita-hu.ac.jp
secretion. However, Mattsson et al. [10] and Konturek
et al. [11] have shown that omeprazole given orally, but
not parenterally, protects against gastric mucosal injuries
induced by various necrotizing agents in rats by an un-
known mechanism not related to inhibition of gastric acid
secretion. Ohara et al. [12] have reported that omeprazole
given orally protects against acetylsalicylic acid-induced
gastric damage by preventing a decrease in gastric mucus
glycoprotein content in rats. Blandizzi et al. [13–15] have
also reported that omeprazole exerts a protective effect
against ethanol-HCl- and hemorrhagic shock-induced gas-
tric mucosal injuries by its intraduodenal and i.p. admin-
istrations, respectively, possibly through an enhancement
of gastric mucus secretion, in addition to inhibition of gas-
tric acid secretion, in rats. Ohtsuki et al. [18] have shown
that oral administration of omeprazole, but not of cime-
tidine, a histamine H2 -receptor antagonist, at effective
doses to inhibit gastric acid secretion twice daily prevents
gastric mucosal lesion development in rats with repeated

1043-6618/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
76
injection of compound 48/80, a mast cell degranulator. In
addition, omeprazole has been shown to inhibit neutrophil
chemotaxis, degranulation, and superoxide anion produc-
tion [19], to attenuate neutrophil–endothelial cell interac-
tion induced by the extract of Helicobacter pylori [20], to
be a potent inhibitor of leukocyte transmigration through
endothelial cells [21], and to attenuate the production of
oxygen free radicals in activated neutrophils by increasing
intralysosomal pH in the cells [22] in vitro. Omeprazole
has also been shown to scavenge hypochlorus acid (HOCl)
and to inhibit iron- and copper-derived oxidant damage
in vitro [23]. Thus, omeprazole functions not only as a
proton pump inhibitor but also as an anti-inflammatory
agent, an antioxidant or a stimulator of gastric mucus
secretion.
Recently, omeprazole in combination with antibiotics
has been often used to treat human gastritis and gastric
ulcers associated with H. pylori which induces severe
inflammation in gastric tissues [24–26]. Yamamoto et al.
[27] have found that H. pylori water extract induces the
degranulation of mast cells prepared from rat abdominal
cavity, and have suggested that H. pylori might be in-
volved in the gastric mucosal inflammation as a trigger of
mast cell degranulation for releasing chemical mediators.
Atuma et al. [28] have reported that H. pylori water ex-
tract reduces gastric mucosal blood flow in rats. The same
authors have indicated that the reduction in gastric mu-
cosal blood flow induced by H. pylori water extract in rats
is probably mediated through an acute inflammatory re-
sponse involving mast cell degranulation with consequent
platelet-activating factor secretion [28]. Thus, it has been
implicated that inflammation induced by mast cell de-
granulation is involved in the pathogenesis of gastritis and
gastric ulcers associated with H. pylori. We have shown in
rats with a single injection of compound 48/80 that acute
gastric mucosal lesions develop not only with neutrophil
infiltration but also with decreases in Se-glutathione
peroxidase activity and nonprotein sulfhydryl (NPSH),
vitamin E, and mucin contents and increases in xanthine
oxidase (XO) activity and lipid peroxide (LPO) content in
the gastric mucosal tissue and that gastric mucosal blood
flow decreases with gastric mucosal lesion formation and
the decreased blood flow recovers rapidly with the lesion
progression [29]. We have also shown that, in rats with a
single compound 48/80 injection, neutrophils infiltrating
into the gastric mucosal tissue participate in gastric mu-
cosal lesion formation, while the xanthine–XO system
in the gastric mucosal tissue participates in the lesion
progression rather than the lesion formation [30]. Fur-
thermore, we have shown in rats with a single compound
48/80 injection that degranulation of connective mast
cells contributes to the development of gastric mucosal le-
sions, that acutely released endogenous serotonin causes
gastric mucosal lesions with a decrease in gastric mucosal
blood flow, while released endogenous histamine mainly
contributes to the lesion development, and that pretreat-
ments with histamine H2 -receptor antagonists such as
cimetidine and famotidine at effective doses to inhibit
Pharmacological Research, Vol. 46, No. 1, 2002
gastric acid secretion have no effect on the development
of gastric mucosal lesions [29, 31]. These findings may
allow us to assume a possibility that omeprazole protects
against acute gastric mucosal lesions associated with
inflammation and ischemia-reperfusion in rats with a sin-
gle compound 48/80 injection by a mechanism related
to its anti-inflammatory, antioxidant, and/or gastric mu-
cus secretion-enhancing actions, but not to its inhibitory
action on gastric acid secretion.
In the present study, therefore, we examined the protec-
tive effect of omeprazole against acute gastric mucosal
lesions in rats with a single compound 48/80 injection.
Namely, we examined the effect of i.p. omeprazole pre-
treatment on gastric mucosal lesion development and
changes in the activities of gastric mucosal MPO, an in-
dex of tissue neutrophil infiltration [32], and XO and the
contents of gastric mucosal LPO, NPSH, hexosamine, an
index of gastric mucin [33], and adherent mucus with the
lesion development in compound 48/80-injected rats. In
addition, we examined the effect of pretreated omepra-
zole on changes in gastric mucosal blood flow and serum
serotonin and histamine concentrations with gastric mu-
cosal lesion development in the compound 48/80-injected
rats.
MATERIALS AND METHODS
Materials
Omeprazole was kindly provided by Fujisawa-Astra
Co. (Osaka, Japan). Compound 48/80, methyl serotonin,
3,3 ,5,5 -tetramethylbenzidine, and xanthine were pur-
chased from Sigma Chemical Co. (St. Louis, MO, USA);
alcian blue CX8, ethylenediaminetetraacetic acid (EDTA),
5,5 -dithibis(2-nitrobenzoic acid) (DTNB), reduced glu-
tathione (GSH), o-phthalaldehyde, 2-thiobarbituric acid,
and other chemicals from Wako Pure Chemicals Ind., Co.
(Osaka, Japan).
Animals
Male Wistar rats aged 6 weeks were obtained from
Nippon SLC Co. (Hamamatsu, Japan). The animals were
housed in cages in a ventilated animal room with con-
trolled temperature (23 ± 2 ◦ C) and relative humidity
(55 ± 15%) and with 12 h of light (7:00 to 19:00). They
were maintained on standard laboratory chow (Oriental
MF, Oriental Yeast Co., Tokyo, Japan) and tap water ad
libitum for 1 week. All animals received humane care in
compliance with the guideline of the Animal Care and
Use Committee of Fujita Health University.
Induction of gastric mucosal lesions by compound
48/80
Seven-week-old rats fasted for 24 h received a single
i.p. injection of compound 48/80 (0.75 mg kg−1 ), dis-
solved in distilled water, as described previously [29–31].
The control rats received an equal volume of distilled wa-
ter in the same manner. All animals were maintained with
Pharmacological Research, Vol. 46, No. 1, 2002
free access to water and without food during the experi-
ment. The animals were sacrificed under ether anesthesia
0.5 and 3 h after compound 48/80 injection. The stom-
achs were removed, inflated with 10 ml of 0.9% NaCl,
and put into 10% formalin for 10 min. The stomachs were
then opened along the greater curvature and examined
for lesions in the glandular part under a dissecting micro-
scope (10×). The severity of gastric mucosal lesions was
estimated using the index of the following eight grades
of lesions as described in our previous reports [29–31]:
grade 0, no lesion (normal); grade I, edema only; grade
II, damaged area of 1–10 mm2 ; grade III, damaged area
of 11–20 mm2 ; grade IV, damaged area of 21–30 mm2 ;
grade V, damaged area of 31–40 mm2 ; grade VI, dam-
aged area of 41–50 mm2 ; grade VII, damaged area
of >51 mm2 .
Treatments with omeprazole
Omeprazole was suspended in 0.5% carboxymethylcel-
lulose sodium salt solution containing 0.9% NaCl. Fasted
rats received a single i.p. injection of this drug (10 or
50 mg kg−1 ) at a constant dosing volume of 1 ml 100 g−1
at 0.5 h before compound 48/80 injection. Vehicle-treated
rats received an equal volume of the carboxymethylcellu-
lose sodium salt solution in the same manner at the same
time point.
Determinations of gastric mucosal enzymes and
compounds
Immediately after removal of the stomach, gastric mu-
cosal tissues were carefully collected on ice using a pair
of small scissors and then used for determinations of gas-
tric mucosal MPO, XO, LPO, NPSH, and hexosamine.
Gastric mucosal MPO was assayed by the method of
Suzuki et al. [34]. For this MPO assay, gastric mucosal
tissues were homogenized in 9 vol. of ice-cold 0.05 M
Tris–HCl buffer (pH 7.4). After sonication on ice for
20 s using a Handy Sonic model UR-20P (Tomy Seiko
Co., Tokyo, Japan), the homogenate was centrifuged at
4 ◦ C (10 000 g, 20 min), and the resultant supernatant
was dialyzed against 100 vol. of the same buffer at 4 ◦ C
for 24 h. MPO activity was assessed by measuring the
H2 O2 -dependent oxidation of tetramethylbenzidine at
37 ◦ C. One unit (U) of this enzyme is defined as the
amount of enzyme causing a change in absorbance of
1 min−1 at 655 nm. Gastric mucosal XO was assayed by
the method of Hashimoto [35]. For this XO assay, gastric
mucosal tissues were homogenized in 9 vol. of ice-cold
0.25 M sucrose. The homogenate was sonicated as de-
scribed above. The sonicated homogenate was centrifuged
at 4 ◦ C (10 000 g, 20 min), and the resultant supernatant
was dialyzed against 100 vol. of the same solution at
4 ◦ C for 24 h. XO activity was assessed by measuring the
increase in absorbance at 292 nm following the forma-
tion of uric acid at 30 ◦ C. One unit (U) of this enzyme
is defined as the amount of enzyme forming 1 µmol uric
acid min−1 . Gastric mucosal LPO was determined by the
thiobarbituric acid method of Ohkawa et al. [36] except
77
that 1 mM EDTA was added to the reaction medium. Gas-
tric mucosal NPSH was determined by the DTNB method
of Sedlak and Lindsay [37]. For both determinations,
gastric mucosal tissues were homogenized in 9 vol. of
ice-cold 20 mM EDTA. The amount of LPO is expressed
as that of MDA equivalents. The amount of NPSH is
expressed as that of GSH equivalents. Gastric mucosal
hexosamine was determined by the method of Oketani
et al. [38] using acetylacetone reagent and Ehrlich’s
reagent. Its amount is expressed as that of glucosamine
equivalents. Gastric mucosal adherent mucus was deter-
mined by the method of Kitagawa et al. [39] using alcian
blue staining. For this adherent mucus determination, the
glandular portion of the stomach was cut off in a circular
form (8 mm in diameter) with a razor and a template and
its wet weight was measured. The adherent mucus con-
tent is expressed as the amount of alcian blue that adhered
to the gastric mucosal surface (microgram per gram of
tissue).
Determinations of serum serotonin and histamine
For serum serotonin and histamine determinations,
blood was collected from the inferior vena cava of rats
upon sacrifice and then serum was obtained from the
collected blood by centrifugation. Serum samples were
deproteinized by adding perchloric acid at a final con-
centration of 3% and then centrifuged at 4 ◦ C for 10 min
(10 000 g). Serum serotonin was measured by the method
of Shibata et al. [40] using high-performance liquid
chromatography with electrochemical detection except
that 40 mM sodium dihydrogenphosphate used for the
mobile phase was replaced by 0.1 M citric acid/0.1 M
sodium acetate (0.7:1, v/v). Methyl serotonin was used as
an internal standard. Serum histamine was measured by
the methods of Lorenz et al. [41] and Shore et al. [42].
Histamine was reacted with o-phthalaldehyde and the in-
tensity of the resultant fluorescence was measured using
a spectrophotometer (the excitation wavelength, 360 nm;
the emission wavelength, 450 nm).
Measurement of gastric mucosal blood flow
Gastric mucosal blood flow was measured using a laser
Doppler flowmeter, Laser Flow BRL-100 (Bio Research
Center Co., Nagoya, Japan), as described in our previ-
ous reports [29–31]. Rats used for this measurement were
anesthetized with pentobarbital sodium 10 min before the
onset of the measurement and the abdomen was opened on
an operation mat. The mat was heated at 37 ◦ C during the
operation and blood flow measurement. The laser probe
was attached to the serosal side of the corpus mucosa by
aid of a cyanoacrylate-typed instantaneous adhesive, Aron
Alpha (Toha Gosei Co., Tokyo, Japan), and the blood flow
changes were monitored on a recorder for at least 5 min
after the onset of the measurement. Gastric mucosal blood
flow in compound 48/80-injected rats is expressed as a rel-
ative percentage toward the mean value of gastric mucosal
blood flow determined in control rats without compound
48/80 injection.
78 Pharmacological Research, Vol. 46, No. 1, 2002

Analysis of data RESULTS

Results obtained for gastric mucosal and serum com-
ponents and enzymes and gastric mucosal blood flow
are expressed as the means ± sd The results were an-
alyzed by computerized statistical package (StatView).
Each mean value was compared by one-way analysis of
variance (one-way ANOVA) and Fisher’s protected least
significant difference for multiple comparisons as the
post hoc test. Statistical analyses of the severity of mu-
cosal lesions were carried out using the Kruskal–Wallis
test. Values of significance were set at P < 0.05 for both
tests.
Effect of omeprazole on gastric mucosal lesions at
0.5 and 3 h after compound 48/80 injection
As shown in Table I apparent gastric mucosal lesions
were formed 0.5 h after a single injection of compound
48/80 (0.75 mg kg−1 ), at which time the ratios of the num-
bers of stomachs with grade II and III lesions to the total
number of stomachs studied were 30 and 70%, respec-
tively. Progressed gastric mucosal lesions occurred 3 h
after the compound 48/80 injection, at which time the ra-
tios of the numbers of stomachs with grade V, VI, and VII

Table I
Effect of omeprazole on gastric mucosal lesions in rats with a single compound 48/80 injection

Time after compound 48/80 Lesion index (%)

injection and groups
0 I II III IV V VI VII

0.5 h
Compound 48/80 0 0 30 70 0 0 0 0
+Omeprazole (10 mg kg−1 )∗ 0 40 60 0 0 0 0 0
+Omeprazole (50 mg kg−1 )∗ 0 70 30 0 0 0 0 0
3h
Compound 48/80 0 0 0 0 0 10 50 40
+Omeprazole (10 mg kg−1 )∗ 0 0 0 0 30 50 20 0
+Omeprazole (50 mg kg−1 )∗ 0 0 0 20 50 30 0 0

Rats received a single i.p. injection of omeprazole (10 or 50 mg kg−1 ) or vehicle at 0.5 h before i.p. injection of compound 48/80 (0.75 mg kg−1 ) and
the severity of gastric mucosal lesions was estimated 0.5 and 3 h after the compound 48/80 injection as described in MATERIALS AND METHODS.
The lesion index (%) represents the percentage of incidence of each lesion grade in each group. The number of rats used in each group is 10.
∗ P < 0.05 compared to the group treated with compound 48/80 alone at 0.5 or 3 h after compound 48/80 injection.

Fig. 1. Effect of omeprazole on serum serotonin (A) and histamine (B) concentrations and gastric mucosal blood flow (C) at 0.5 and 3 h after compound
48/80 injection. Rats were injected with omeprazole (10 or 50 mg kg−1 , i.p.) or vehicle at 0.5 h before injection of compound 48/80 (0.75 mg kg−1 , i.p.).
Control rats without any treatment received an equal volume of each vehicle in the same manner at the same time point. Serum serotonin and histamine
and gastric mucosal blood flow were determined 0.5 and 3 h after compound 48/80 injection as described in MATERIALS AND METHODS. Open
bars, control rats; closed bars, rats injected with compound 48/80 alone; crosshatched bars, compound 48/80-injected rats pretreated with 10 mg kg−1
of omeprazole; dotted bars, compound 48/80-injected rats pretreated with 50 mg kg−1 of omeprazole. Data represent the mean ± sd for 10 animals. An
asterisk (∗) denotes P < 0.05 versus control rats.
Pharmacological Research, Vol. 46, No. 1, 2002 79

lesions to the total number of stomachs studied were 10,

50, and 40%, respectively (Table I). The development of
gastric mucosal lesions found at 0.5 and 3 h after com-
pound 48/80 injection were significantly prevented by pre-
treatment with omeprazole at a dose of 10 or 50 mg kg−1 ,
and the preventive effect of 50 mg kg−1 of omeprazole was
stronger than that of 10 mg kg−1 of the drug (Table I).

Effect of omeprazole on serum serotonin and

histamine concentrations and gastric mucosal blood
flow at 0.5 and 3 h after compound 48/80 injection
Rats with a single compound 48/80 injection alone had
3.7- and 2.4-fold higher serum serotonin concentration
than control rats not injected with compound 48/80 at 0.5
and 3 h after the injection, respectively [Fig. 1(A)]. The
increased serum serotonin concentration at 0.5 or 3 h af-
ter compound 48/80 injection was not attenuated by pre-
treatment with omeprazole at a dose of 10 or 50 mg kg−1
[Fig. 1(A)]. The compound 48/80-injected group had 31.3-
and 3.5-fold higher serum histamine concentration than
the control group at 0.5 and 3 h after the injection, respec-
tively [Fig. 1(B)]. Pretreatment with omeprazole at a dose
of 10 or 50 mg kg−1 did not affect the increase in serum
histamine concentration at 0.5 or 3 h after compound 48/80
injection [Fig. 1(B)]. Gastric mucosal blood flow in the
compound 48/80-injected group was 25% of that in the
control group at 0.5 h after compound 48/80 injection, but
the decreased gastric mucosal blood flow returned up to
75% of the control level at 3 h [Fig. 1(C)]. Pretreatment
with omeprazole at a dose of 10 or 50 mg kg−1 did not af-
fect the reduction of gastric mucosal blood flow at 0.5 or
3 h after the compound 48/80 injection [Fig. 1(C)]
Effect of omeprazole on gastric mucosal MPO and
XO activities at 0.5 and 3 h after compound 48/80
injection
Rats with a single compound 48/80 injection alone had
2.5- and 2.8-fold higher gastric mucosal MPO activity
than control rats not injected with compound 48/80 at 0.5
and 3 h after the injection, respectively [Fig. 2(A)]. The
increase in gastric mucosal MPO activity at 0.5 or 3 h
after compound 48/80 injection was significantly attenu-
ated by pretreatment with omeprazole at a dose of 10 or
50 mg kg−1 ; gastric mucosal MPO activity in the com-
pound 48/80-injected group pretreated with 10 mg kg−1
of omeprazole was not significantly different from that
in the control group at 0.5 h after compound 48/80 injec-
tion and was 2-fold higher than that in the control group
at 3 h, while gastric mucosal MPO activity in the com-
pound 48/80-injected group pretreated with 50 mg kg−1
of omeprazole was almost equal to that in the control
group at 0.5 h after compound 48/80 injection and was
1.5-fold higher than that in the control group at 3 h [Fig.
2(A)]. The compound 48/80-injected group had 1.8- and
4.3-fold higher gastric mucosal XO activity than the con-
trol group at 0.5 and 3 h after compound 48/80 injection,
respectively [Fig. 2(B)]. Pretreatment with omeprazole
at a dose of 10 or 50 mg kg−1 significantly prevented
Fig. 2. Effect of omeprazole on gastric mucosal MPO (A) and XO (B)
activities at 0.5 and 3 h after compound 48/80 injection. Rats were in-
jected with omeprazole (10 or 50 mg kg−1 , i.p.) or vehicle at 0.5 h before
injection of compound 48/80 (0.75 mg kg−1 , i.p.). Control rats without
any treatment received an equal volume of each vehicle in the same
manner at the same time point. Gastric mucosal MPO and XO activities
were determined 0.5 and 3 h after compound 48/80 injection as described
in MATERIALS AND METHODS. Open bars, control rats; closed
bars, rats injected with compound 48/80 alone; crosshatched bars, com-
pound 48/80-injected rats pretreated with 10 mg kg−1 of omeprazole;
dotted bars, compound 48/80-injected rats pretreated with 50 mg kg−1
of omeprazole. Data represent the mean ± sd for 10 animals. An aster-
isk (∗) denotes P < 0.05 versus control rats; hash (#) denotes P < 0.05
versus rats injected with compound 48/80 alone.
the increase in gastric mucosal XO activity at 0.5 or 3 h
after compound 48/80 injection; gastric mucosal XO ac-
tivities in the compound 48/80-injected group pretreated
with 10 mg kg−1 of omeprazole at 0.5 and 3 h after com-
pound 48/80 injection were 1.5- and 3.6-fold, respec-
tively, higher than that in the control group, while gastric
mucosal XO activities in the compound 48/80-injected
group pretreated with 50 mg kg−1 of omeprazole at 0.5
and 3 h after compound 48/80 injection were 1.3- and
2.9-fold, respectively, higher than that in the control group
[Fig. 2(B)].
Effect of omeprazole on gastric mucosal LPO and
NPSH contents at 0.5 and 3 h after compound 48/80
injection
Rats with a single compound 48/80 injection alone had
1.5- and 2.1-fold higher gastric mucosal LPO content
than control rats not injected with compound 48/80 at
0.5 and 3 h after the injection, respectively [Fig. 3(A)].
The increase in gastric mucosal LPO content at 0.5 or
3 h after compound 48/80 injection was significantly pre-
vented by pretreatment with omeprazole at a dose of 10
80
Fig. 3. Effect of omeprazole on gastric mucosal LPO (A) and NPSH (B)
contents at 0.5 and 3 h after compound 48/80 injection. Rats were injected
with omeprazole (10 or 50 mg kg−1 , i.p.) or vehicle at 0.5 h before injec-
tion of compound 48/80 (0.75 mg kg−1 , i.p.). Control rats without any
treatment received an equal volume of each vehicle in the same manner
at the same time point. Gastric mucosal LPO and NPSH were determined
0.5 and 3 h after compound 48/80 injection as described in MATERIALS
AND METHODS. Open bars, control rats; closed bars, rats injected with
compound 48/80 alone; crosshatched bars, compound 48/80-injected
rats pretreated with 10 mg kg−1 body weight of omeprazole; dotted bars,
compound 48/80-injected rats pretreated with 50 mg kg−1 of omepra-
zole. Data represent the mean ± sd for 10 animals. An asterisk (∗) de-
notes P < 0.05 versus control rats; hash (#) denotes P < 0.05 versus
rats injected with compound 48/80 alone.
or 50 mg kg−1 ; gastric mucosal LPO contents in the com-
pound 48/80-injected group pretreated with 10 mg kg−1
of omeprazole at 0.5 and 3 h after compound 48/80 in-
jection were 1.2- and 1.8-fold, respectively, higher that
in the control group, while gastric mucosal LPO content
in the compound 48/80-injected group pretreated with
50 mg kg−1 of omeprazole was almost equal to that in
the control group at 0.5 h after compound 48/80 injection
and was 1.5-fold higher than that in the control group
at 3 h [Fig. 3(A)]. The compound 48/80-injected group
had 69% of gastric mucosal NPSH content in the control
group at 0.5 h after compound 48/80 injection, but the
reduced gastric mucosal NPSH content returned to the
control level at 3 h [Fig. 3(B)]. Pretreatment with omepra-
zole at a dose of 10 or 50 mg kg−1 significantly attenuated
the reduced gastric mucosal NPSH content at 0.5 h after
compound 48/80 but did not affect the gastric mucosal
NPSH content at 3 h; gastric mucosal NPSH contents in
the compound 48/80-injected group pretreated with 10
and 50 mg kg−1 of omeprazole were 77 and 90%, respec-
Pharmacological Research, Vol. 46, No. 1, 2002
Fig. 4. Effect of omeprazole on gastric mucosal hexosamine (A) and
adherent mucus (B) contents at 0.5 and 3 h after compound 48/80 in-
jection. Rats were injected with omeprazole (10 or 50 mg kg−1 , i.p.) or
vehicle at 0.5 h before injection of compound 48/80 (0.75 mg kg−1 , i.p.).
Control rats without any treatment received an equal volume of each
vehicle in the same manner at the same time point. Gastric mucosal hex-
osamine and adherent mucus were determined 0.5 and 3 h after com-
pound 48/80 injection as described in MATERIALS AND METHODS.
Open bars, control rats; closed bars, rats injected with compound 48/80
alone; crosshatched bars, compound 48/80-injected rats pretreated with
10 mg kg−1 of omeprazole; dotted bars, compound 48/80-injected rats
pretreated with 50 mg kg−1 of omeprazole. Data represent the mean ±sd
for 10 animals. An asterisk (∗) denotes P < 0.05 versus control rats.
tively, of that in the control group at 0.5 h after compound
48/80 injection [Fig. 3(B)].
Effect of omeprazole on gastric mucosal
hexosamine and adherent mucus contents at 0.5
and 3 h after compound 48/80 injection
Gastric mucosal hexosamine content in rats with a sin-
gle compound 48/80 injection alone was not different from
that in control rats not injected with compound 48/80 at
0.5 h after the injection but was 69% of that in control rats
at 3 h [Fig. 4(A)]. The reduction of gastric mucosal hex-
osamine content at 3 h after compound 48/80 injection was
not prevented by pretreatment with omeprazole at a dose
of 10 or 50 mg kg−1 [Fig. 4(A)]. Gastric mucosal adherent
mucus content in rats with a single compound 48/80 in-
jection alone was not different from that in control rats not
injected with compound 48/80 at 0.5 h after the injection
but was 75% of that in control rats at 3 h [Fig. 4(B)]. The
reduction of gastric mucosal adherent mucus content at 3 h
after compound 48/80 injection was not prevented by pre-
treatment with omeprazole at a dose of 10 or 50 mg kg−1
[Fig. 4(B)].
Pharmacological Research, Vol. 46, No. 1, 2002
DISCUSSION
The present study has clearly shown that omeprazole pro-
tects against acute gastric mucosal lesions associated with
inflammation and ischemia-reperfusion in rats with a sin-
gle injection of compound 48/80, a mast cell degranulator.
It has been reported that oral administration of omepra-
zole (30 or 100 mg kg−1 ), but not of cimetidine (100 or
300 mg kg−1 ), twice daily prevents gastric mucosal le-
sion development in rats injected with compound 48/80
(0.75 mg kg−1 ) once daily for 4 days, although both ad-
ministered omeprazole and cimetidine are effective in
inhibiting gastric acid secretion at doses used [18]. We
have reported that a single i.p. injection of a histamine
H2 -receptor antagonist, i.e. cimetidine (100 mg kg−1 ) or
famotidine (2 mg kg−1 ), each dose of which is effective in
inhibiting gastric acid secretion in rats, has no preventive
effect on gastric mucosal lesion development in rats with a
single injection of compound 48/80 (0.75 mg kg−1 ) [31].
In addition, it has been shown that a single i.p. injection
of compound 48/80 (3 or 5 mg kg−1 ) inhibits basal and
histamine-stimulated gastric acid secretion in rats [43].
These findings may allow us to think that omeprazole ex-
erts a protective effect against compound 48/80-induced
acute gastric mucosal lesions in rats without affecting
gastric acid secretion.
It has been shown that when rats are injected with
compound 48/80 (0.75 mg kg−1 ), marked increases in
serum serotonin and histamine concentrations occur 0.5 h
after the injection and the increases in serum serotonin
and histamine concentrations are partially reduced at 3 h
[30, 31, 44]. Takeuchi et al. [44] have reported that there
are little changes in serotonin and histamine contents
in the gastric mucosa of rats with a single injection
of compound 48/80 (0.75 mg kg−1 ) and that serotonin
and histamine are released from connective tissue mast
cells present in the extragastric tissues of the compound
48/80-injected rats. We have shown that, in rats with a
single compound 48/80 injection, endogenous serotonin
rather than histamine released from connective tissue mast
cells plays a major role in the formation of acute gastric
mucosal lesions [31]. In the present study, omeprazole
pretreated at doses of 10 and 50 mg kg−1 had no effect
on increases in serum serotonin and histamine concen-
trations at 0.5 and 3 h after compound 48/80 injection.
These results indicate that omeprazole protects against
compound 48/80-induced acute gastric mucosal lesions
in rats without affecting the compound 48/80-mediated
release of serotonin and histamine from connective tissue
mast cells.
We have reported that, in rats with a single compound
48/80 injection, gastric mucosal blood flow is reduced to
approximately 25% of the level of normal rats not injected
with compound 48/80 at 0.5 h after the injection and this
reduced gastric mucosal blood flow is recovered up to
approximately 75% of the level of normal rats at 3 h [30].
We have also shown that the reduction of gastric mucosal
blood flow in compound 48/80-injected rats is due to en-
81
dogenous serotonin released from connective tissue mast
cells [31]. Thus, ischemia-reperfusion occurs in the gastric
mucosa of rats with a single compound 48/80 injection. It
has been reported that a single i.v. injection of omeprazole
(5 µmol kg−1 ) causes a significant increase in gastric mu-
cosal blood flow in pentagastrin-treated rats, but not in nor-
mal rats, although topical application of the drug (2.5 mM)
to normal rats increases the blood flow by 15% [45]. It has
also been reported that omeprazole (6.25–50 mg) added
to the ex vivo chamber of normal rat stomachs increases
gastric mucosal blood flow in a dose-dependent manner,
which may play an important role in the protective effect
of the drug against ethanol-induced gastric mucosal le-
sions [46]. Besides, there is a report showing that a single
i.v. injection of omeprazole (2 or 20 mg kg−1 ) does not
affect gastric mucosal blood flow in normal rats [47] or
that a single i.v. injection of omeprazole (10 µmol kg−1 )
tends to increase blood flow in the corpus mucosal layer
of normal rats [48]. In the present study, i.p. pretreat-
ment with omeprazole (10 or 50 mg kg−1 ) did not affect
the change in gastric mucosal blood flow found at 0.5
and 3 h after compound 48/80 injection, indicating that
omeprazole protects against compound 48/80-induced
acute gastric mucosal lesions in rats without affecting
gastric mucosal blood flow. The observation that omepra-
zole has no enhancing effect on gastric mucosal blood
flow in compound 48/80-injected rats may result from
differences in experimental condition and administration
route between the present study and the above-described
studies in which the enhancing effect of omeprazole on
gastric mucosal blood flow is observed [45, 46].
We have shown that, in rats with a single compound
48/80 injection, neutrophils infiltrating into the gas-
tric mucosal tissue participate in gastric mucosal lesion
formation, while the xanthine–XO system in the gas-
tric mucosal tissue takes part in the lesion progression
rather than the lesion formation [30]. In addition, our
recent report has shown that pretreatment with ebselen,
a seleno-organic compound, which is known to pos-
sess glutathione peroxidase-like activity and antioxidant
and anti-inflammatory properties, prevents the devel-
opment of gastric mucosal lesions in rats with a single
compound 48/80 injection, and has suggested that this
preventive effect of ebselen could be due to its glu-
tathione peroxidase-like activity and antioxidant and
anti-inflammatory properties [49]. Our previous report
has also shown that cotreatment with the extract of
Oren-gedoku-to, a Chinese herbal medicine, which is
used for the therapy of gastric ulcer and gastritis in Japan
and possesses antioxidant and anti-inflammatory actions,
prevents the development of compound 48/80-induced
acute gastric mucosal lesions in rats possibly through its
antioxidant and anti-inflammatory actions [50]. Omepra-
zole has been reported to inhibit neutrophil chemotaxis
and degranulation [19], neutrophil–endothelial cell inter-
action induced by extracts of H. pylori [20] and leukocyte
transmigration through endothelial cells [21] in vitro.
In the present study, pretreated omeprazole attenuated
82
a compound 48/80-induced increase in the activity of
gastric mucosal MPO, an index of tissue neutrophil infil-
tration [32], at 0.5 and 3 h after compound 48/80 injection.
This result suggests that omeprazole could exert a protec-
tive effect against compound 48/80-induced acute gastric
mucosal lesions in rats through its anti-inflammatory ac-
tion. Pretreated omeprazole was also found to attenuate
a compound 48/80-induced increase in gastric mucosal
XO activity at 0.5 and 3 h after compound 48/80 injec-
tion. But, the mechanism by which pretreated ompera-
zole attenuates increased gastric mucosal XO activity in
compound 48/80-injected rats has not been clarified in
the present study. We have observed that omeprazole at
a concentration of 10 to 100 µg ml−1 has no effect on
XO activity in gastric mucosal tissue samples prepared
from compound 48/80-injected rats sacrificed at 3 h after
the injection (unpublished data). Our previous report has
shown that, in rats with a single compound 48/80 injec-
tion, pretreatment with antineutrophil antiserum or NPC
14686, an inhibitor of neutrophil recruitment, attenuates
increased gastric mucosal XO activity at 3 h, but not at
0.5 h, after the compound 48/80 injection [30]. Accord-
ingly, these findings may allow us to think that pretreated
omeprazole attenuates the increase in gastric mucosal XO
activity found at 3 h after compound 48/80 injection, at
least in part, through its anti-inflammatory action.
Our previous report has shown that, in rats with a single
compound 48/80 injection, gastric mucosal LPO con-
tent increases with a decrease in gastric mucosal NPSH
content at 0.5 h after the injection and further increases
despite return of the decreased gastric mucosal NPSH
content to the normal level at 3 h [29]. A large part of
NPSH present in rat gastric mucosal tissues is GSH [51].
GSH has been demonstrated to protect gastric mucosal
tissues against oxidative stress [52]. It has been reported
that omeprazole has significant antioxidant effects against
HOCl- or catalytic transition metal-mediated oxidative
injury in vitro [23]. It has been shown that acid degraded
omeprazole inhibits superoxide anion generation in ac-
tivated neutrophiles in vitro [19]. It has also been shown
that omeprazole attenuates the production of oxygen free
radicals in activated neutrophils by increasing intralyso-
somal pH in the cells in vitro [22]. In the present study,
pretreated omeprazole attenuated an increase in gastric
mucosal LPO content at 0.5 and 3 h after compound 48/80
injection and a decrease in gastric mucosal NPSH content
at 0.5 h. These results suggest that omeprazole could exert
a protective effect against compound 48/80-induced acute
gastric mucosal lesions in rats by inhibiting enhanced lipid
peroxidation and NPSH oxidation in the gastric mucosal
tissue through its antioxidant action. But, omeprazole has
been shown to have no scavenging effect on superoxide
anion generated in a cell free xanthine–XO system [19]
and no direct scavenging action of superoxide anion and
hydrogen peroxide [22]. In addition, it has been reported
that neutrophils mediate lipid peroxidation through the
production of superoxide anion via activated NADPH ox-
idoreductase in the cells [53, 54]. It has also been reported
Pharmacological Research, Vol. 46, No. 1, 2002
that MPO mediates lipid peroxidation in the presence of
hydrogen peroxide with halide ions [55, 56]. These find-
ings suggest that pretreated omeprazole could inhibit lipid
peroxidation mediated by infiltrated neutrophils rather
than the xanthine–XO system in the gastric mucosal tissue
of rats with a single compound 48/80 injection. However,
the possibility cannot be ruled out that pretreated omepra-
zole contributes to the reduction of enhanced gastric
mucosal lipid peroxidation and NPSH oxidation in com-
pound 48/80-injected rats by inhibiting neutrophil infil-
tration into the gastric mucosal tissue. It is known that the
conversion of xanthine dehydrogenase to XO in tissues
occurs through oxidation of essential sulfhydryl groups
in the xanthine dehydrogenase under hypoxic conditions
[57]. Accordingly, the prevention by omeprazole pretreat-
ment of gastric mucosal NPSH oxidation in compound
48/80-injected rats found at 0.5 h after compound 48/80
injection, at which time a marked reduction of blood flow,
i.e. hypoxia, occurred in the gastric mucosa as ascribed
above, may contribute to the aforementioned attenuating
effect of pretreated omeprazole on the increase in gastric
mucosal XO activity found at the same time point.
We have reported that the content of gastric mucosal
hexosamine, an index of gastric mucin, decreases with
the development of gastric mucosal lesions in rats with a
single compound 48/80 injection [29]. Ohara et al. [12]
have shown that a single oral administration of omepra-
zole (20 µmol kg−1 ) protects against acetylsalicylic
acid-induced gastric mucosal damage in rats by prevent-
ing a decrease in gastric mucus glycoprotein content in
the corpus. Blandizzi et al. [13–15] have also shown that
a single intraduodenal administration of omeprazole (15,
30 or 60 mg kg−1 ) protects against ethanol-HCl-induced
gastric mucosal lesions in rats with attenuation of de-
creased adherent gastric mucus content and that a single
i.p. administration of omeprazole (15, 30 or 60 mg kg−1 )
protects against hemorrhagic shock-induced gastric mu-
cosal lesions in rats with attenuation of decreased ad-
herent gastric mucus content. The same authors have
suggested that an enhancement of gastric mucus barrier
through stimulation of gastric mucus secretion, in addi-
tion to inhibition of gastric acid secretion, contributes to
the protective action of omeprazole against both gastric
mucosal injuries [13–15]. But, it has been shown that a
single oral administration of omeprazole (30 mg kg−1 ) to
normal rats does not increase hexosamine content in the
gastric lumen [58]. It has also been shown in an organ
culture system that omeprazole does not affect mucin
biosynthesis in the corpus region of rat stomachs [59].
In compound 48/80-injected rats, neither gastric mucosal
hexosamine and adherent mucus contents at 0.5 h after
the injection nor decreased gastric mucosal hexosamine
and adherent mucus contents at 3 h were altered by a sin-
gle i.p. pretreatment with omeprazole (10 or 50 mg kg−1 ).
These results indicate that omeprazole protects against
compound 48/80-induced acute gastric mucosal lesions
in rats without an enhancement of gastric mucosal barrier
due to secreted mucus. However, the reason why a single
Pharmacological Research, Vol. 46, No. 1, 2002
i.p. injection of omeprazole does not affect the secre-
tion of gastric mucus in compound 48/80-injected rats is
unclear at present.
In conclusion, the results obtained in the present study
indicate that omeprazole protects against acute gastric
mucosal lesions in rats with a single injection of com-
pound 48/80, and suggest that omeprazole could exert this
protective effect through its anti-inflammatory and antiox-
idant actions. Omeprazole in combination with antibiotics
has been often used to treat human gastritis and gastric
ulcers associated with H. pylori which induces severe
inflammation in gastric tissues [24–26]. It has been impli-
cated that inflammation induced by mast cell degranula-
tion is involved in the pathogenesis of gastritis and gastric
ulcers associated with H. pylori [27, 28]. Therefore, to fur-
ther investigate the precise mechanism for the protective
effect of omeprazole against compound 48/80-induced
acute gastric mucosal lesions in rats seems to be use-
ful for the elucidation of the pharmacological action of
omeprazole in gastritis and gastric ulcers associated with
H. pyroli.
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