Egualen Na, (sodium 3-ethyl-7-isopropyl-1-azulemesulfonate
1/3 hydrate) (sodium), a more stable azulene derivative than sodium guaiazulene-3-sulfonate
(
Fig. 1), is known to exhibit antiulcer activity against a variety of
experimental gastric lesions in rats (1-3) and used as an antiulcer drug for
treatment of gastritis and gastric ulcers (4, 5). Although previous studies
suggested the mucosal protective action of this drug, which is due to an increase
in mucosal blood flow (3), the inhibition of endothelial injury (6), and thromboxane
(TX) A
2 antagonistic action (7), the actual
mechanisms involved remain unknown.
We recently found that gastric lesions generated by ischemia/reperfusion (I/R)
were prevented by drugs that inhibit TXA
2 production
or blockade TXA
2 TP receptors, suggesting the
pathogenic importance of this prostanoid in this model (8, 9). Because TXA
2
is known to potentiate the bradykinin-induced nociceptive response (10) and
may be a mediator of epigastric pain (11), TXA
2
antagonistic action is considered crucial to antiulcer drugs. We also set up
a model of gastric bleeding induced by double antiplatelet therapy with low-dose
aspirin (ASA) plus clopidogrel, a P2Y
12 receptor
antagonist (12), and found mucosal protective drugs to be effective in preventing
the bleeding (13, 14). Since recent studies have reported a risk of adverse
gastric reactions in patients taking antiplatelet drugs with nonsteroidal anti-inflammatory
drugs (NSAIDs) (15, 16), it is important to find drugs effective in this model.
Furthermore, clinical studies using capsule endoscopes or double-balloon endoscopes
have confirmed that NSAIDs damage the small intestine at a higher incidence
than previously thought (17-19). Since no satisfactory means for the prevention
and treatment of these lesions are currently available, except for the use of
PG analogs (19), the identification of effective therapies for the treatment
of NSAID-induced small intestinal damage remains an urgent priority. Thus, it
is critical to examine whether egualen has beneficial influences on these lesions
induced in the stomach and the small intestine, to provide further information
for clinical use of this drug in the treatment of gastrointestinal diseases.
In the present study, we examined the effects of egualen against gastric lesions produced by I/R as well as double antitplatelet therapy with ASA plus clopidogrel, and small intestinal lesions generated by loxoprofen in rats and investigated the possible mechanisms involved in the protective action of this drug.
MATERIALS AND METHODS
Animals
Male C57BL/6 mice (3 months old; SLC, Shizuoka, Japan) and Sprague-Dawley rats (200–260 g; Nippon Charles River, Shizuoka, Japan) were acclimated to standard laboratory conditions (12:12 h light-dark cycle, temperature 22±1°C). Experiments were carried out with or without fasting, using four to six animals in a conscious state, unless otherwise specified. All experimental procedures involving animals were approved by the Experimental Animal Research Committee of the Kyoto Pharmaceutical University.
Induction of ischemia and reperfusion-induced gastric lesions
Acute gastric mucosal lesions were produced by I/R in 18 h-fasted mice, according
to a recently published method (8). Briefly, under urethane anesthesia (1.25
g/kg i.p.), the celiac artery was clamped with a small clamp (disposable vascular
clip, holding force 40 g; BEAR Medical Corporation, Chiba, Japan), and 30 min
later reperfusion was achieved through removal of the clamp. Then, the animals
were killed 60 min after the onset of reperfusion following ischemia for 30
min, and the stomach was excised, inflated by injecting 0.4 ml of 2% formalin
for 10 min to fix the tissue walls, and opened along the greater curvature.
Egalen (30 and 60 mg/kg), ozagrel (TXA
2 synthase
inhibitor: 200 mg/kg), or seratrodast (TXA
2
antagonist: 3–30 mg/kg) was administered p.o. 60 min before ischemia. The area
(mm
2) of hemorrhagic lesions developed in the
stomach was measured under a dissecting microscope (Olympus, Tokyo, Japan) with
a square grid (x10). The person measuring the lesions did not know the treatments
given to the animals. In some cases, the gastric mucosa was examined with a
light microscope following I/R with or without egualen (60 mg/kg). The animals
were killed 60 min after the onset of reperfusion following ischemia for 30
min, and the stomach excised. The tissue samples were then immersed in 10% neutralized
formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin
and eosin (H&E).
Determination of myeloperoxidase (MPO) activity
MPO activity in the gastric mucosa was measured after I/R treatment in mice,
according to a modified version of the method of Krawisz
et al. (20).
At 60 min after I/R-treatment, the animals were sacrificed by the withdrawal
of blood from the heart through perfusion with saline, and the stomach was excised.
After rinsing of the tissue with cold saline, the mucosa was scraped with glass
slides, weighed, and homogenized in a 50 mM phosphate buffer containing 0.5%
hexadecyl-trimethyl-ammonium bromide (HTAB; pH 6.0; Sigma). The homogenized
samples were subjected to freezing and thawing three times, and centrifuged
at 2,000 g for 10 min at 4°C. MPO activity in the supernatant was determined
by adding 50 µl of the supernatant to 50 µl of 10 mM phosphate buffer (pH 6.0)
and 50 µl of 1.5 M o-dianisidine hydrochloride (Sigma) containing 0.0005% (w/v)
hydrogen peroxide. The changes in absorbance at 450 nm were recorded on a microplate
reader (VERSAmax; Molecular Device, OA). Sample protein content was estimated
by spectrophotometric assay (Pierce protein assay kit, IL), and the MPO activity
was obtained from the slope of the reaction curve, based on the following equation:
Specific activity (µmol H
2O
2/min/mg
protein) = (OD/min)/OD/µmol H
2O
2
x mg protein). Egalen (30 and 60 mg/kg), ozagrel (TXA
2
synthase inhibitor: 200 mg/kg) or seratrodast (TXA
2
antagonist: 3–30 mg/kg) was administered p.o. 60 min before ischemia.
Gastric bleeding and lesions caused by ASA plus clopidogrel
An experimental system for effectively evaluating gastric ulcerogenic and bleeding
responses to antiplatelet therapy was set up in 18 h-fasted rats under urethane
anesthesia, according to a previously published paper (13). Briefly, two catheters
were inserted into the rat stomach, one from an incision in the esophagus and
another through the pylorus
via an incision in the duodenum, and the
stomach was then perfused with saline at a rate of 0.4 ml/min using an infusion
pump, and the perfusate was collected every 5 min for determination of hemoglobin
concentrations. After an equilibration period with saline perfusion for 2 hours,
the stomach was perfused with 25 mM ASA plus 50 mM HCl for another 60 min. Clopidogrel
(30 mg/kg) was given p.o. 24 hours before the perfusion. The doses of ASA and
clopidogrel were determined based on our previous studies (13, 14). Gastric
bleeding was evaluated as the hemoglobin concentration in the perfusate. At
the end of the experiment (60 min after the onset of ASA+HCl perfusion) the
animals were killed for examination of the gastric mucosa. The stomach was excised,
treated with 2% formalin for fixation of the tissue walls, and then opened along
the greater curvature, and the mucosa was examined for damage under a dissecting
microscope (x10). The area (mm
2) of macroscopically
visible lesions was measured, summed for each tissue, and used as a lesion score.
The person measuring the lesions did not know the treatments given to the animals.
Egualen (10 and 30 mg/kg) or PGE
2 (1 mg/kg)
was given i.d. 60 min or i.v. 10 min before the ASA perfusion, respectively.
Determination of hemoglobin concentrations
Gastric bleeding was determined by an increase in the hemoglobin concentration of the gastric perfusate (13, 14). To this end the gastric perfusate was collected every 15 min during the experiment where the stomach was perfused with saline or 25 mM ASA in the presence of clopidogrel pretreatment (30 mg/kg). The concentration of hemoglobin was assessed on a Hitachi spectrophotometer (U-2000, Ibaraki, Japan), according to standard curves made by adding various amounts of rat hemoglobin (Sigma Chemicals, St. Louis, MO) to saline or 25 mM ASA solutions. When rat hemoglobin was dissolved in saline of pH 3.5, maximal absorption occurred at 386 nm (21). This wavelength was used for the estimation of hemoglobin in the gastric perfusates. Data were analyzed using the Short Softmax Program and the results were presented as micrograms per milliliter or micrograms per 90 min.
Induction of small intestinal damage by loxoprofen
Rats without fasting were administered loxoprofen (60 mg/kg) p.o. and killed
24 hours later under deep ether anesthesia (22). The small intestine was excised,
treated with 2% formalin for 10 min to fix the tissue walls, and opened along
the anti-mesenteric attachment. The area of macroscopically visible damage (mm
2)
was measured under a dissecting microscope with square grids (x10), summed per
tissue, and used as a lesion score. To highlight hemorrhagic lesions, a 1% Evans
blue solution was administered i.v. in a volume of 1 ml/animal 30 min before
killing. The person measuring the lesions did not know the treatment given to
the animals. Egualen was given p.o. twice daily (3–30 mg/kg: 9:30 AM and 8:30
PM) for 3 days before the administration of loxoprofen or acutely twice (10–100
mg/kg) 30 min before and 6 hours after loxoprofen. In some cases, the small
intestine was examined with a light microscope following the administration
of loxoprofen (60 mg/kg) with or without egualen (100 mg/kg). The animals were
killed 24 hours after the loxoprofen treatment, and the small intestine was
excised. The tissue samples were then immersed in 10% neutralized formalin,
embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin
(H&E).
Determination of mucus secretion
The amount of mucus secreted in the small intestine was determined by periodic acid-Schiff (PAS) staining. Three hours after the administration of loxoprofen (60 mg/kg, p.o.), the animals were killed under deep diethyl ether anesthesia, and the small intestines were removed. The removed tissues were fixed in Carnoy’s fluid (ethanol: acetic acid: chloroform = 6:1:3) for 24 hours, embedded in paraffin, and sectioned at a thickness of 8 µm. PAS staining was performed according to the conventional method. Egualen (100 mg/kg) was given p.o. with or without co-administration of loxoprofen. In the combined administration, this agent was given p.o. 30 min before the administration of loxoprofen.
Determination of enterobacterial counts
Enterobacteria were enumerated according to a method described by Deitch
et al. (23). Six hours after loxoprofen treatment (60 mg/kg, p.o.), the animals were killed under deep ether anesthesia, and the small intestines were removed. After each intestine was rinsed with sterile saline, the mucosa was scraped with glass slides, weighed, and homogenized in 1 ml of sterile phosphate-buffered saline (PBS) per 100 mg of wet tissue. Aliquots of the homogenate were placed on blood agar and Gifuco anaerobic medium agar (Nissui, Tokyo, Japan). Blood agar plates were incubated at 37°C for 24 hours under aerobic conditions (BBL Gas Pack Pouch Anaerobic System; BD Biosciences, San Jose, CA). Plates containing 10 to 300 colony-forming units (CFU) were examined for numbers of enterobacteria, and the data were expressed as log CFU per gram of tissue. Egualen (100 mg/kg) was given p.o. 30 min before the administration of loxoprofen.
Determination of iNOS mRNA expression
The expression of iNOS mRNA in the small intestinal mucosa was measured by reverse transcription-polymerase chain reaction (RT-PCR) (24). The animals were killed under deep ether anesthesia 6 hours after the administration of loxoprofen (60 mg/kg) p.o., and the small intestines were removed and stored at –80°C prior to use. Egualen (100 mg/kg) was given p.o. 30 min before the administration of loxoprofen. Total RNA was extracted from tissue samples using Sepasol RNA I (Nacalai Tesque, Kyoto, Japan). The total RNA was reverse-transcribed with a first strand cDNA synthesis kit (ReverTra Ace alpha, TOYOBO, Osaka, Japan). The sequences of the sense and antisense primers for rat iNOS and GAPDH and each product size are shown in Table 1. An aliquot of the RT reaction product served as a template with 30 s of denaturation at 95°C and 1 min of extension at 68°C using the Advantage 2 polymerase mixture (CLONTECH, Mountain View, CA) in a thermal cycler (PC-806, ASTEC, Fukuoka, Japan). A portion of the PCR mixture was electrophoresed on a 1.5% agarose gel in Tris-acetic acid-EDTA buffer (40 mM Tris, 20 mM acetic acid, and 2 mM EDTA; pH 8.1), and the gel was stained with ethidium bromide and photographed (BioDoc-It Imaging System, UVP, Upland, CA).
Table 1. Sequences
of sense and antisense primers for rat iNOS and GAPDH |
|
Preparation of drugs
The drugs used were urethane (Tokyo Kasei, Tokyo, Japan), egualen Na (Ajinomoto Pharm Co., Kawasaki, Japan), ozagrel, prostaglandin E
2 (Cayman Chemical, Ann Arbor, MI), seratrodast (LKT Laboratories, St. Paul, MN), clopidogrel (Sanofi-Aventis, Tokyo, Japan), and loxoprofen (Sigma Chemicals, St. Louis, MO). Loxoprofen was suspended in a hydroxypropylcellulose (HPC) solution (Wako Pure Chemicals, Osaka, Japan). Other agents were dissolved in saline. Each agent was prepared immediately before use and administered p.o. in a volume of 0.1 ml/20 g body weight or 1 ml/200 g body weight, respectively, in mice or rats, and i.d. or i.v. in a volume of 1 ml/200 g body weight or 0.1 ml/100 g body weight, respectively, in rats. Control animals received the vehicle in the same volume and
via the same route.
Statistical analysis
Data are presented as the mean±S.E. for 5–7 mice or 4–13 rats per group. Statistical analyses were performed using a one-way analysis of variance (ANOVA) and Student’s t-test or Dunnett’s multiple comparison test where appropriate, and values of P<0.05 were considered significant.
RESULTS
Effect of egualen on ischemia/reperfusion-induced gastric damage in mice
Laparotomy without clamping of the gastric artery (sham operation) did not produce
any damage in the mouse gastric mucosa. In the animals subjected to I/R treatment
(30 min of ischemia followed by reperfusion for 60 min), however, multiple hemorrhagic
lesions were observed in the gastric mucosa, the lesion score being 9.6.2±1.9~10.3±1.1
mm
2 (
Fig. 2). Pretreatment of the animals
with ozagrel (200 mg/kg, p.o.), a TXA
2 synthase
inhibitor, significantly prevented the I/R-induced development of gastric lesions,
the inhibition being 57.1%. Likewise, the severity of these lesions was dose-dependently
reduced by prior administration of seratrodust (3–30 mg/kg), a TXA
2
antagonist, and the effect was significant at 10 mg/kg or greater. Egualen (30
and 60 mg/kg) also dose-dependently and significantly mitigated the severity
of the I/R-induced gastric damage, and the effect at 60 mg/kg was as potent
as that of seratrodast at 30 mg/kg, the inhibition being 64.1%. In sham-operated
animals without I/R treatment, no damage was detected even by histological observation
(
Fig. 3A). By contrast, severe damage was observed histologically in
the stomach after I/R treatment; most of the damage was restricted to the surface
epithelium, but some damage occurred deep in the mucosa (
Fig. 3B). When
the animals were pretreated with egualen (60 mg/kg, p.o.), the severity of the
histological damage was markedly reduced, and only slight damage was observed
in the surface epithelium (
Fig. 3C).
|
Fig. 1. Chemical structure
of egualen Na (sodium 3-ethyl-7-isopropyl- 1-azulemesulfonate 1/3 hydrate). |
|
Fig. 2. Effects of egualen,
ozagrel, and seratrodast on gastric lesions induced by I/R in mice. Under
urethane anesthesia, the celiac artery was clamped. Reperfusion followed
30 min later with removal of the clamp, and then, the stomach was excised
60 min later. Egualen (3 and 60 mg/kg), ozagrel (200 mg/kg), or seratrodast
(330 mg/kg) was given p.o. 30 min before the onset of ischemia.
Data are presented as the mean±S.E. for 5~7 mice. *Significant
difference from control, at P<0.05. |
|
Fig. 3. Histological observations
of I/R-induced gastric damage. Under urethane anesthesia, the celiac artery
was clamped for 30 min. Reperfusion followed with removal of the clamp,
and the stomach was excised after 60 min of reperfusion. Egualen (60 mg/kg)
was given p.o. 30 min before the onset of ischemia. Fig. 3A shows:
sham: Fig. 3B: control (I/R+vehicle); Fig. 3C: I/R plus
egualen. |
Myeloperoxidase (MPO) activity
Gastric mucosal MPO activity in sham-operated mice was less than 0.02 µmol H
2O
2/min/mg
protein. The MPO activity was markedly increased after I/R, the values being
0.044±0.007 µmol H
2O
2/min/mg
protein. This response was significantly abrogated by the prior administration
of egualen (60 mg/kg, p.o.), the inhibition being 51.5%. Both ozagrel (200 mg/kg,
p.o.) and seratorodast (30 mg/kg) also suppressed the increase of MPO activity
induced by I/R, the inhibition being 66.7% and 66.9%, respectively.
Effect of egualen on gastric bleeding and ulcerogenic responses to acidified ASA with or without clopidogrel pretreatment
Perfusion of the rat stomach with acidified ASA (25 mM in 50 mM HCl) produced
few lesions, but the ulcerogenic response to acidified ASA was aggravated by
pretreatment with clopidogrel (30 mg/kg). These treatments caused a time-dependent
increase in the hemoglobin concentration, resulting in many hemorrhagic lesions
in the stomach (
Figs. 4 and
5A). PGE
2
(1 mg/kg, i.v.) markedly reduced gastric bleeding and hemorrhagic damage induced
by acidified ASA in the presence of clopidogel; the total hemoglobin output
and hemorrhagic lesions were both reduced to about 20% of that in the vehicle-treated
animals. Egualen (10 and 30 mg/kg, i.d.) dose-dependently reduced gastric bleeding
and ulcerogenic responses to acidified ASA in the presence of clopidogel, and
the effects were significant at 30 mg/kg. Notably, prior administration of egualen
at 30 mg/kg also decreased the severity of lesions under such conditions almost
as effectively as PGE
2 at 1 mg/kg, the inhibition
being 62.1%. As shown in
Fig. 5B and
Fig. 5C, the severity of
gastric lesions produced by 25 mM ASA plus clopidogrel was markedly reduced
by pretreatment with egualen (30 mg/kg), converting the lesions from hemorrhagic
to non-hemorrhagic.
|
Fig.
4. Effects of egualen and PGE2 on
gastric bleeding induced by luminal perfusion of acidified ASA in the
presence of clopidogrel in urethane-anesthetized rats. The stomach was
perfused by 25 mM ASA plus 50 mM HCl for 60 min. Clopidogrel (30 mg/kg)
was given p.o. 24 hours before ASA. Egualen (30 and 60 mg/kg) or PGE2
(1 mg/kg) was given i.d. 60 min or i.v. 10 min, respectively, before the
onset of ASA perfusion. Fig. A shows the time-course of change
in the hemoglobin concentration in the luminal perfusate, and the data
are presented as the mean±S.E. of values determined every 5 min
from 68 rats. Fig. B shows total hemoglobin output in the
perfusate for the last 60 min, and the data are presented as the mean±S.E.
for 68 rats. Significant difference at P<0.05; * from control
(without clopidogrel); # from vehicle. |
|
Fig.
5. Effects of egualen and PGE2 on
gastric hemorrhagic lesions produced by luminal perfusion of acidified
ASA in the presence of clopidogrel in urethane-anesthetized rats. The
stomach was perfused by 25 mM ASA plus 50 mM HCl for 60 min. Clopidogrel
(30 mg/kg) was given p.o. 24 hours before ASA. Egualen (30 and 60 mg/kg)
or PGE2 (1 mg/kg) was given i.d. 60 min
or i.v. 10 min, respectively, before the onset of ASA perfusion. Fig.
A shows hemorrhagic lesions, and the data are presented as the mean±S.E.
for 68 rats. Significant difference at P<0.05; * from control
(without clopidogrel); # from vehicle. Right panel shows macroscopic
appearances of hemorrhagic gastric lesions induced by luminal perfusion
with 25mM ASA plus 50mM HCl in the presence of clopidogrel (30 mg/kg),
in rats without (B) or with pretreatment of egualen (10 mg/kg)
(C). Significant difference at P<0.05; * from control (without
clopidogrel); # from vehicle. |
Effect of egualen on loxoprofen-induced small intestinal damage
Orally administered loxoprofen (60 mg/kg) in normally fed rats produced multiple
hemorrhagic lesions in the small intestine, the lesion score being 223.2±25.1
mm
2 (
Fig. 6). When the animals were pretreated
with egualen (3–30 mg/kg) given p.o. twice daily for 3 days prior to loxoprofen
treatment, the development of these intestinal lesions was prevented in a dose-dependent
manner, and a significant effect was observed at 30 mg/kg, the inhibition being
64.3%. Likewise, this drug exhibited a prophylatic effect when administered
acutely twice 30 min before and 6 hours after the administration of loxoprofen.
In this case, egualen (10–100 mg/kg) also dose-dependently reduced the severity
of the small intestinal lesions generated by loxoprofen, although the effective
dose was slightly higher than that of the repeated administrations for 3 days.
In particular, this drug at 100 mg/kg significantly suppressed the occurrence
of these lesions in response to loxoprofen; the inhibition being 68.7%. Histologically,
the lesions produced by loxoprofen were deep in the mucosa, almost reaching
the muscularis mucosae (
Fig. 7), while the severity of the damage was
markedly lessened in rats pretreated with egualen (100 mg/kg).
|
Fig. 6. Effect of egualen
on loxoprofen-induced small intestinal damage in rats. Animals were given
loxoprofen (60 mg/kg) p.o. and killed 24 hours later. Egualen (10100
mg/kg) was given p.o. twice 30 min before and 6 hours after the administration
of loxoprofen or twice daily (9:30 AM and 8:30 pM) for 3 days prior to
loxoprofen treatment. Data are presented as the mean±S.E. for 413
rats. *Significant difference from control, at P<0.05. |
|
Fig. 7. Microscopical observations
of small intestinal lesions induced by loxoprofen in rats, with or without
pretreatment with egualen. Animals were given loxoprofen (60 mg/kg) p.o.
and killed 24 hours later. Egualen (100 mg/kg) was given p.o. twice 30
min before and 6 hours after loxoprofen. Figures show: (A) normal;
(B) loxoprofen alone; and (C) egualen plus loxoprofen. Note
that loxoprofen caused deep damage in the mucosa, but the severity of
damage was apparently lessened by prior administration of egualen. |
Effect of egualen on various events induced in the small intestinal mucosa by loxoprofen treatment
It was found that egualen prevented the occurrence of damage in the small intestine after loxoprofen treatment when the animals were even acutely pretreated 30 min before loxoprofen treatment, and this effect at 100 mg/kg was significant and reproducible. To further investigate the functional mechanism responsible for the prophylactic action of egualen, we examined the effects of egualen at 100 mg/kg on various events that are considered critical in the pathogenesis of NSAID-induced enteropathy (24).
Enterobacterial invasion
Aerobic and anaerobic bacterial counts in the normal intestinal mucosa were 6.11±0.30 and 6.49±0.34 log CFU/g tissue, respectively. Those following the administration of loxoprofen (60 mg/kg) were about 70–90 times greater after 6 hours, being 8.02±0.47 and 8.04±0.40 log CFU/g tissue, respectively. Pretreatment with egualen (100 mg/kg) given p.o. 30 min before loxoprofen significantly suppressed the enhanced invasion of enterobacteria following loxoprofen treatment, the number of bacteria being 6.76±0.26 and 6.88±0.22 log CFU/g tissue, respectively.
Mucosal expression of iNOS mRNA
Expression of iNOS mRNA was not detected in the normal intestine, yet markedly
upregulated in the mucosa when examined 6 hours after the administration of
loxoprofen (60 mg/kg) (
Fig. 8). The upregulation of iNOS expression caused
by loxoprofen was mitigated by prior administration of egualen (100 mg/kg).
|
Fig. 8. Effect of egualen
on the expression of iNOS mRNA in the rat small intestine after the administration
of loxoprofen. The animals were given loxoprofen (60 mg/kg) p.o., and
the expression of iNOS mRNAs was examined 6 hours later by RT-PCR. Egualen
(100 mg/kg) was given p.o. 30 min before the administration of loxoprofen.
M: Marker. Note that loxoprofen upregulated iNOS expression, but this
response was apparently mitigated by prior administration of egualen. |
Mucus secretion
In the normal intestinal mucosa, PAS-positive substances were clearly observed
over the surface epithelial cells and along the glands (
Fig. 9A). Loxoprofen
(60 mg/kg) apparently reduced the amount of PAS-positive substances on the epithelial
cells as well as in the glands (
Fig. 9B). However, when the animals were
pretreated with egualen (100 mg/kg, p.o.) before the loxoprofen treatment, the
decrease in PAS staining was prevented and the amount of PAS-positive materials
in the mucosa was largely restored (
Fig. 9C). In addition, egualen alone
also increased the amount of PAS-positive substances in the mucosa when compared
to the control mucosa (
Fig. 9D).
|
Fig. 9. Effect of loxoprofen
on mucus secretion (PAS staining) in the rat small intestine, with or
without loxoprofen treatment. The animals were given loxoprofen (60 mg/kg)
or egualen (100 mg/kg) p.o. and killed 3 hours later for examination of
PAS staining. In the combined administration, egualen was given p.o. 30
min before loxoprofen. Figures show: (A) normal; (B)
loxoprofen alone; (C) egualen plus loxoprofen; (D) egualen
alone. (PAS; x100). Note that loxoprofen markedly decreased PAS-positive
materials in the mucosa, but this response was apparently prevented by
prior administration of egualen. |
|
Fig.
10. Factors involved in the development of loxoprofen-induced small
intestinal damage, and the influences of egualen on these processes. Loxoprofen
causes functional changes such as an increase in intestinal motility and
a decrease in mucus secretion, followed by enterobacterial invasion in
the mucosa. Endotoxin released from enterobacteria upregulates iNOS expression
and NO production as well as inflammation, which results in damage to
the small intestine. Egualen increases mucus secretion and hampers the
mucosal invasion of enterobacteria, and by so doing suppresses the upregulation
of iNOS expression, eventually leading to protection against damage in
the small intestine. |
DISCUSSION
Egualen, a stable azulene derivative, has been used to treat gastritis and gastric ulcers (4, 5). Although previous studies have showed a prophylactic effect of this drug in various models of damage in the stomach and duodenum (1-3, 6), no study had evaluated the effectiveness of egualen on small intestinal damage. In the present study, we examined the effect of egualen on NSAID-induced enteropathy as well as gastric injury induced by I/R or dual antiplatelet therapy, and found that the drug effectively suppressed the occurrence of damage.
First, we examined the effect of egualen on the development of gastric damage
under I/R conditions. Previous study showed that I/R-induced gastric damage
was worsened by pretreatment with indomethacin and rofecoxib (a selective cyclooxygenase
(COX)-2 inhibitor) but not SC-560 (a selective COX-1 inhibitor), and this aggravation
was abrogated by co-administration of iloprost (a prostacyclin (PGI
2)
analogue), suggesting a role for COX-2/PGI
2
in mucosal defense under such conditions (8, 26). Furthermore, we have also
shown that the severity of these lesions was reduced by pretreatment with ozagrel
or seratorodast, similar to iloprost, suggesting the involvement of COX-1/TXA
2
in the pathogenesis of I/R-induced gastric damage, in addition to COX-2/PGI
2
(9). As expected, the present study showed that egualen significantly prevented
the development of gastric damage under I/R conditions, similar to ozagrel and
seratorodast. We also confirmed the neutrophil infiltration in the stomach during
I/R-treatment, as represented by a marked increase of MPO activity (27), and
further observed that egualen attenuated the increase of MPO activity following
I/R treatment. Because egualen is known to have a TXA
2
antagonistic action (7), the present results further supported the pathogenic
importance of TXA
2 in I/R-induced gastric damage.
Recent clinical studies showed that the risk of gastric bleeding is increased
by the concomitant use of antiplatelet drugs with NSAIDs or low-dose ASA in
the presence of exogenous or endogenous acid in the rat stomach (15, 16, 28).
Clopidogrel is an antiplatelet drug that specifically and irreversibly inhibits
the P2Y
12 subtype of the adenosine diphosphate
receptor, which is important to the aggregation of platelets and cross-linking
by the protein fibrin (29). The present study confirmed in rats that pretreatment
with clopidogrel aggravated gastric bleeding in response to acidified ASA, and
eventually increased the severity of the acidified ASA-induced hemorrhagic damage
in the stomach. Ritchie
et al. (30) proposed three factors essential
for the generation of gastric lesions; 1) gastric barrier disruption, 2) luminal
acid, and 3) gastric mucosal ischemia. ASA is known to disrupt the gastric mucosal
barrier, damage the stomach in the presence of acid, and attenuate the gastric
hyperemic response to acid back-diffusion following barrier disruption by suppressing
PG production (31). Thus, the model we used fulfills the conditions necessary
to generate damage in the stomach. In the present study, egualen dose-dependently
and significantly mitigated gastric bleeding and ulcerogenic responses to acidified
ASA plus clopidogrel and the effect was almost equivalent to that of PGE
2.
Similar effects have been observed with several other agents used as mucosal
protective drugs in the treatment of gastric ulcers (13, 14, 28). The mechanism
by which egualen prevented gastric bleeding and damage under these conditions
remains unknown. However, because egualen binds to the gastric mucosa
via
a non-specific hydrophobic interaction to form a complex that is less vulnerable
to ASA irritation and because this drug exhibits gastric hyperemic and anti-inflammatory
effects (1-3, 6, 7), it is assumed that egualen prevents gastric bleeding and
lesions under such conditions, probably by its local mucosal protective action
and anti-inflammatory action. At present, the clinical effectiveness of mucosal
protective drugs against gastric bleeding associated with dual antiplatelet
therapy remains unproven. Further animal and clinical studies are needed to
clarify these points.
Most important in the present study is the finding that egualen, given acutely or subacutely for 3 days before, prevented the development of small intestinal damage following the administration of loxoprofen, a NSAID frequently used in Asian countries. NSAIDs cause intestinal ulceration in humans and laboratory animals after short-term and long-term administration (32, 33). In particular, recent clinical studies using capsule endoscopes or double-balloon endoscopes confirmed that NSAIDs damage the small intestine at a higher incidence than previously thought (34). Several factors have been implicated in the pathogenesis of NSAID-induced small intestinal ulceration, including bacterial flora, bile acid, and hypermotility, in addition to PG deficiency (24, 25, 35). Since egualen did not affect the decreased PGE
2 content in the presence of loxoprofen (data not shown), it is assumed to act downstream of the events resulting from the PG deficiency caused by loxoprofen and eventually prevent the development of small intestinal lesions.
NSAID-induced intestinal damage is prevented by pretreatment with antibiotics such as ampicillin (24, 25), suggesting a key pathogenic role for enterobacteria in this model. The importance of bacterial was also demonstrated in the pathogenesis of not only the small intestinal damage but also colonic damage induced by NSAIDs (36). Boughton-Smith
et al. (37) reported that bacterial endotoxin enhanced intestinal permeability through up-regulation of iNOS expression and overproduction of NO in the mucosa. This was further supported by the findings that indomethacin increased iNOS activity and NO production, preceeding the onset of intestinal damage, and that aminoguanidine prevented the intestinal ulcerogenic response by suppressing NO production due to iNOS (24). As expected, we observed that loxoprofen caused bacterial invasion in the mucosa, followed by the up-regulation of iNOS expression and MPO activity, and these responses were suppressed by egualen. These findings suggest that the protective effect of egualen against loxoprofen- induced intestinal damage is functionally associated with the down-regulation of iNOS expression resulting from the suppression of bacterial invasion. The mechanism by which enterobacteria invade the mucosa remains unknown, yet previous studies suggest that a decrease in mucus secretion and an increase of mucosal permeability may contribute to this process after indomethacin treatment (22, 38, 39). Since mucus plays a crucial role in innate host defenses against intestinal pathogens and irritants, it is possible that a decrease in mucus secretion weakens the intestinal barrier, resulting in bacterial invasion.
In the present study, we found that the amount of PAS-positive materials in
the small intestine was markedly reduced after loxoprofen treatment, but this
response was restored by prior administration of egualen. Furthermore, egualen
by itself apparently increased the amount of PAS-positive substances in the
mucosa. It is assumed that egualen stimulates mucus secretion, thereby increasing
the mucus gel’s thickness and hampering bacterial invasion following the administration
of loxoprofen. Thus, the present results together suggest that egualen protects
the small intestine against loxoprofen-induced damage, and this effect may be
functionally associated with an increase in the secretion of mucus, resulting
in suppression of bacterial invasion and iNOS expression, the major pathogenic
events in NSAID-induced small intestinal ulceration (
Fig. 10).
Given the findings of the present study, we conclude that egualen has a prophylactic effect against gastric damage induced by I/R and gastric bleeding induced by double antiplatelet therapy with ASA plus clopidogrel as well as small intestinal damage generated by loxoprofen, probably through its characteristic pharmacological properties, such as TXA
2 antagonistic action, local mucosal protection, and increase in mucosal blood flow as well as mucus secretion (2, 3, 7).
Acknowledgements:
This work in its preliminary from has been presented during 7th
International Symposium on Cell/Tissue Injury and Cytoprotection Organoprotection.
September 9-11 2012, Honolulu, Hawaii organized by Prof. K. Takeuchi (Kyoto,
Japan) and Prof. H. Matsui (Tsukuba, Japan).
Conflict of interests: None declared.
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