JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2011, 62, 1, 75-86
www.jpp.krakow.pl
M. PAWLIK, R. PAJDO, S. KWIECIEN, A. PTAK-BELOWSKA, Z. SLIWOWSKI, M. MAZURKIEWICZ-JANIK,
S.J. KONTUREK, W.W. PAWLIK, T. BRZOZOWSKI
NITRIC OXIDE (NO)-RELEASING ASPIRIN EXHIBITS A POTENT ESOPHAGOPROTECTION
IN EXPERIMENTAL MODEL OF ACUTE REFLUX ESOPHAGITIS. ROLE OF NITRIC OXIDE
AND PROINFLAMMATORY CYTOKINES
Department of Physiology Jagiellonian University Medical College, Cracow, Poland
The purpose of this study was to develop an acute animal model of reflux esophagitis, which would be suitable to induce
the esophageal damage caused by gastric acid reflux, thus mimicking the esophageal injury of human gastroesophageal
reflux disease (GERD). Global research indicates that GERD is rapidly increasing among the world's population. NSAIDs
are known to induce gastrointestinal damage and low doses of aspirin (ASA) have been shown to increase the incidences
of GERD in humans. Gastric acid and pepsin secretion and enhanced COX-2 expression were implicated in the
pathogenesis of reflux esophagitis, but the effect of selective COX-2 inhibitors against lesions induced by the reflux of
gastric acid content into esophagus has not been thoroughly studied. Here, we compared the effect of aspirin (ASA) and so
called "safe" nitric oxide (NO) derivative of ASA with those of non-selective and selective cyclooxygenase (COX)-1 and
COX-2 in rat model of reflux esophagitis. Reflux esophagitis was induced in anesthetized rats by ligating the pylorus and
limiting ridge transitional region between the forestomach and the corpus of stomach. Subsequently, the total gastric
reservoir to store gastric juice was greatly diminished, resulting in the reflux of this juice into the esophagus. Rats with
esophagitis received intragastric (i.g.) pretreatment either with: 1) vehicle (saline), 2) ASA or NO-ASA (100 mg/kg); 3) the
non-selective COX inhibitor, indomethacin (5 mg/kg); 4) the selective COX-1 inhibitor, SC-560 (10 mg/kg), and 5) the
selective COX-2 inhibitor, celecoxib (5 mg/kg). In a separate series of rats with reflux oesophagitis, the efficacy of ASA
combined with a donor of NO, glyceryl trinitrate (GTN; 10 mg/kg i.g.) to prevent esophageal mucosal injury was
investigated. Four hours after induction of esophagitis the gross mucosal damage was graded with a macroscopic lesion
index (LI) from 0-6. The esophageal blood flow (EBF) was determined by H2-gas clearance technique, the oesophageal
mucosal and blood samples were collected for histology and analysis of the RT-PCR expression and release of
proinflammatory cytokines IL-1β, TNF-α and IL-6 using specific ELISA. The exposure of the esophagus to reflux of
gastric acid time-dependently increased the esophageal LI and morphologic damage, and decreased EBF with the most
significant changes observed at 4 hrs after the ligation procedure. The pretreatment with native ASA in the dose that
suppressed the generation of mucosal PGE2, enhanced gross and histologic esophageal damage and produced a significant
fall in EBF. NO-ASA or ASA coupled with GTN counteracted the aggravation of the damage and accompanying fall in
EBF when compared with native ASA applied alone to rats with esophagitis. The proinflammatory cytokines IL-1β and
TNF-α were overexpressed in rats with esophagitis and those pretreated with ASA but this effect was significantly
attenuated by NO-ASA. Plasma IL-1β, TNF-α and IL-6 were negligible in the intact rats but significantly increased in those
with esophagitis, with this effect being further enhanced by non-selective (indomethacin) and selective (SC-560, celecoxib)
COX-1 and COX-2 inhibitors. We conclude that conventional NSAID such as aspirin augments esophagitis, while NOASA exerts the beneficial protective effect against reflux esophagitis via the enhancement of esophageal microcirculation
due to NO release and an inhibitory effect on expression and release of pro-inflammatory cytokines.
K e y w o r d s : aspirin, celecoxib, esophageal blood flow, glyceryl trinitrate, interleukin-1β, nitric oxide-releasing aspirin, reflux
esophagitis, tumor necrosis factor-α
INTRODUCTION
Gastroesophageal reflux disease (GERD) is one of to the
most common diseases affecting about 40% of the world's
population (1-3). The major mechanism by which GERD
develops depends upon the tension of the lower esophageal
sphincter (LES). When there is a transient decrease in the tension
of the LES, gastric contents may leak into the esophagus (4-7).
This disease is considered to be a failure of the physiological
anti-reflux barrier to protect against frequent and abnormal
amounts of gastroesophageal reflux (8, 9). The pathogenesis of
GERD is complex, resulting from an imbalance between
defensive factors protecting the esophagus such as; the antireflux barrier, esophageal acid clearance, esophageal tissue
resistance and the aggressive factors from the stomach and
duodenum such as gastric acid, pepsin and duodenal contents
76
which can damage the esophagus (10-13). The esophageal
damage is developed in response to the prolonged exposure of
the esophagus to acid and digestive enzymes found in the gastric
fluid (pepsin), or duodenal contents containing for instance, bile
salts regurgitated into the stomach. Gastric acid and alkaline
contents may induce and promote irritation and result in
symptoms and morphological changes to the esophageal mucosa
(9, 10, 14).
The esophageal defense against acid damage consists of the
anti-reflux mechanisms, including the mucosal barrier, which is
created by special physiological properties of the
gastroesophageal junction (4). The resistance to damage of the
stratified squamous epithelium of the normal esophagus is
governed by several factors. Besides this structural component,
the functional component of mucosal resistance includes the
ability of the esophageal epithelium to buffer and extrude
hydrogen ions. The intraepithelial buffering capacity is
accomplished by proteins, as well as bicarbonates (12, 14, 15).
Other functional defense mechanisms include the removal of the
reflux material from the esophagus and the restoration of
physiological pH of the esophageal mucosa after exposure to
acid (6-9). Circulating blood delivers oxygen, nutrients and
bicarbonate and removes hydrogen ion, CO2 and other
metabolites, thereby maintaining the proper mucosal acid-base
balance. Previous studies documented that blood flow to the
esophageal mucosa increases in response to an increase of
mucosal secretory activity and the stress-induced increase of the
luminal acid (11).
Among the endogenous aggressive factors of gastric origin,
the reflux of gastric acid and pepsin play a major role. It has been
demonstrated that acid alone induces minor injury with a pH of
less than 3 (13, 16). However, when acid is combined with even
small amounts of pepsin, the damage to the mucosal barrier
occurs due to increased hydrogen ion permeability which causes
local hemorrhage (16). In addition to gastric and pepsin, the
alkaline duodenal content refluxed into esophagus could cause
injury to the esophageal mucosa (17). Persistent reflux of gastric
contents into the esophagus can induce breaks, disruptions and
erosions of the mucosal cell layer (17, 18), as particular lines of
the esophageal mucosal defense system becomes unable to
withhold and repair local damage. Experimental studies
demonstrated that bile acids induced esophageal damage is
exaggerated in the presence of acid and pepsin (17-19).
Ingestion of various drugs could also contribute to the
imbalance between offensive and defensive factors and the
pathogenesis of reflux esophagitis. It is well established that the
use of commonly used medications such as nonsteroidal antiinflammatory drugs (NSAIDs), may increase the vulnerability of
gastrointestinal mucosa to the development of peptic lesions,
such as erosions and even ulceration as well as serious ulcer
complications such as bleeding and perforation (20), however,
the influence of NSAIDs on gastroesophageal reflux has not
been fully explored. NSAIDs such as aspirin, naproxen,
indomethacin and ibuprofen have been identified as risk factor
for development of erosive esophagitis (21-24). It has also been
demonstrated that aspirin makes the esophageal mucosa more
sensitive to the injurious action of acid and pepsin (25)
suggesting that this NSAID can act synergistically with
gastroduodenal factors responsible for the damage of esophageal
mucosa.
Since NSAIDs, aside from Helicobacter pylori (26), are
considered as potential independent risk factor of
gastrointestinal bleedings and peptic ulcer disease, the new
strategy against adverse effect of classic NSAIDs includes the
novel nitric oxide (NO)-releasing NSAIDs that were developed
by the coupling of nitric oxide ( NO)-releasing moiety to
standard NSAIDs (27-32). In contrast to classic NSAIDs, the
new NO-releasing derivatives of flurbiprofen, ketoprofen,
diclofenac and NO-releasing aspirin have been shown to exhibit
lower gastrointestinal toxicity than parent compounds, and they
spare the gastrointestinal tract even when administered
repeatedly for several weeks (33-35). The NO-NSAID were
shown to inhibit non specifically both cyclooxygenases (COX)1 and COX-2, with a comparable potency to parent NSAID but
in contrast, they have shown a beneficial effect on the gastric
mucosa, because they fail to induce neutrophil adherence and to
produce gastric ulceration (36, 37).
After carefully assessing previous research on the matter, it
is our belief that the effects on esophageal mucosal damage in
the model of gastric reflux taking into account the new
derivatives of ASA, particularly the NO releasing ASA have not
been fully explored. Therefore, using a modified method of
gastric acid reflux as proposed by Omura et al. (38) and selecting
the optimal time of reflux esophagitis to cause reproducible
mucosal damage, we attempted to determine the effect of
pretreatment with NO-ASA in comparison with conventional
ASA on the esophageal mucosal injury and esophageal blood
flow (EBF) in rodent model of acute acid reflux esophagitis in
rats. The involvement of endogenously generated prostaglandins
and the contribution of cyclooxygenase (COX)-1 and COX-2 to
the development of esophageal damage by acid reflux with and
without NO-ASA or ASA was determined by the employment of
non-selective and highly selective COX-1 and COX-2 inhibitors
administered to animals with reflux esophagitis in the doses that
were previously reported to inhibit mucosal PG biosynthesis in
vivo (39). Moreover, an attempt was made to determine not only
the gross but also morphology of esophageal mucosa in rats with
reflux esophagitis administered with COX inhibitors and NO
releasing ASA. Furthermore, the effect of concurrent
administration of NO donor combined with ASA was
determined in order to check whether addition of exogenous NO
could influence the esophageal damage and accompanying
changes in the EBF comparable with those evoked by NO-ASA.
In addition, we attempted to assess the effect of the treatment
with NO-ASA, ASA and non-selective and selective COX-1 and
COX-2 inhibitors on the esophageal expression of
proinflammatory cytokines IL-1β and TNF-α and plasma levels
of IL-1β and TNF-α and IL-6 in animals with gastric reflux.
MATERIAL AND METHODS
Male Wistar rats weighting 190-250 g and fasted for 24
hours before the experiment were used in all studies on the
development of acute esophagitis and esophago-protection. The
experimental procedures of this study were approved by the
Institutional Animal Care and use Committee of Jagiellonian
University Medical College in Cracow. All experimentations
were followed in accordance with statements of the Helsinki
Declaration regarding handling of experimental animals.
Development of reflux esophagitis and experimental groups
The model of reflux esophagitis utilized in our present study
with slight modification has been described previously in detail
by Omura et al. (38). Briefly, under ether anesthesia in about 140
rats that underwent overnight fasting, a midline laparotomy was
performed to expose the stomach, and then both the pylorus and
the transitional junction between the fore-stomach and the
corpus were first exposed and later ligated with 2-0 silk thread
but without pyloric ring with a piece of 18 Fr Nelaton catheter as
originally proposed by Omura et al. (38) (see Fig. 1). After the
surgical preparation was completed, the EBF was measured to
confirm that esophageal blood vessels were not ligated and then
77
the abdomen cavity was temporarily closed. In a separate series
of experiments, the time-course relationship between changes in
EBF and the degree of esophageal mucosal lesions and duration
of development of acute esophagitis were investigated. In these
series of experiments animals were treated with vehicle (saline)
only. The rats were then anesthetized second time at 2 hrs, 4 hrs,
8 hrs, 12 hrs, and 24 hrs after the onset of the reflux esophagitis
induction. Then, a laparotomy was performed and the EBF was
measured. After EBF determination was completed the animals
were killed with an overdose of pentobarbital (50 mg/kg i.p.) at
2 hrs, 4 hrs, 8 hrs, 12 hrs and 24 hrs, and the esophagus with the
proximal portion of stomach was gently dissected, then removed
and pinned open for the assessment of macroscopic lesions. The
esophageal biopsy samples with and without mucosal lesions
present were gently rinsed with saline, excised and the fixed in
10% buffered formalin. Samples were embedded in paraffin, and
3 µm sections were prepared and stained with H&E for
microscopic assessment.
After these series of experiments were completed, we
selected the duration model of acute esophagitis based on the
time of reflux being 4 hrs from the time of the procedure of
ligation. With this particular time of 4 hrs, the oesophageal
lesions were fully developed and the experiments were the
most reproducible with a given satisfactory index of the lesions
in control animals. Animals with reflux esophagitis were
randomized into the six experimental groups (A-F), consisting
of 6-10 rats per group and they received i.g. the following
pretreatment with: A) vehicle (saline), B) non-selective COX1 and COX-2 inhibitor, ASA (100 mg/kg) or indomethacin (5
mg/kg), C) COX-1 selective inhibitor, SC-560 (5 mg/kg), D)
selective COX-2 inhibitor, celecoxib (10 mg/kg), E) NO-ASA
(NicOx S.A., Sophia Antipolis, France; 100 mg/kg), and F)
GTN (10 mg/kg i.g.), a NO donor combined with ASA (100
mg/kg, i.g.) (17). In separate group of animals with reflux
esophagitis, the pretreatment with ASA applied i.g. in graded
doses ranging from 12.5 mg/kg up to 100 mg/kg were
determined.
Determination of mucosal generation of PGE2 and plasma
proinflammatory cytokines
Measurement of esophageal blood flow (EBF) and assessment
of esophageal injury
The mRNAs for interleukin-1β and TNF-α were determined
by reverse transcriptase-polymerase chain reaction in the
esophageal mucosa of the intact rats or those exposed to reflux
esophagitis with or without pretreatment with a non-selective
and selective COX-1 and COX-2 inhibitors. Samples of the
gastric oxyntic mucosa (about 200 mg) were scraped off on ice
using a glass slide and then immediately snapped frozen in liquid
nitrogen, and stored at -80°C. Total RNA was isolated from the
esophageal mucosa according to Chomczynski and Sacchi
(1987) (41) using a rapid guanidinum isothiocyanate/phenol
chloroform single step extraction kit from Stratagene (Stratagene
GmbH, Heidelberg, Germany).
First strand cDNA was synthesized from total cellular RNA
(5 µg) using 200 U Strata Script TM reverse transcriptase and
oligo (dt) primers (Stratagene GmbH, Heidelberg, Germany).
The primers for the rat β-actin and IL-1β were synthesized by
Biometra (Gottingen, Germany). The IL-1β primer sequences
were designed according to the published cDNA sequence for
primer sequences were as follows: up-stream, 5' GCT ACC TAT
GTC TTG CCC GT; downstream, 3' GAC CAT TGC TGT TTC
CTA GG. The expected length product was 546 bp. The TNF-α
primer sequences were as follows: up-stream, 5'TAC TGA ACT
TCG GGG TGA TTG GTC C; downstream, 3' CAG CCT TGT
CCC TTG AAG AGA ACC. The expected length product was
296 bp. Concomitantly, amplification of control rat β-actin was
performed on the same samples to verify the RNA integrity.
DNA amplification was carried out under the following
conditions; denaturation at 94°C for 1 min, annealing at 60°C for
45 s and extension at 72°C for 45 s. Each PCR-product (8 µl)
Upon the termination of gastric acid reflux in most
experiments at the standard time of 4 hrs after the ligation as
described above, the animals were anesthetized with
pentobarbital, their abdomens were opened by midline incision,
and the abdominal part of the esophagus was exposed for
measurement of EBF by means of the H2-gas clearance
technique as described previously (19, 34). For this purpose
double electrode of an electrolytic regional blood flowmeter
(Biotechnical Science, Model RBF-2, Osaka, Japan) was
inserted into esophageal mucosa. The measurements were made
in three areas of the mucosa and the mean values of the EBF was
calculated and expressed as a percent changes of those compared
in the control vehicle-treated animals. The animals were
euthanized by pentobarbital overdose and the esophagus was
removed. Immediately after its removal it was opened
longitudinally, rinsed with saline and pinned open for
macroscopic examination. The lesion index score was calculated
(macroscopic degree of injury 0-6) after gross inspection of the
esophagus under a dissecting microscope (19) by a researcher
blind to the experimental grouping. The total area (mm2) of
lesions that had developed in the esophagus was determined
under a dissecting microscope (10x) with a square grid, and
graded with the lesion index (LI), as follows: 0) no visible
lesions; 1) a few erosions and bleedings; 2) total area of lesions
<15 mm2; 3) total area of lesions <30 mm2; 4) total area of
lesions <40 mm2; 5) total area of lesions 45 mm2; 6) perforation.
In groups of rats exposed to acid reflux without or with
pretreatment with non-selective COX-1 and COX-2 inhibitor
ASA, the oesophageal mucosal samples from were taken by
biopsy (about 200 mg) from grossly unchanged oesophageal
mucosa without mucosal lesions immediately after the animals
were sacrificed to determine the mucosal generation of
prostaglandin E2 by radioimmunoassay (RIA) as described
previously (39). The mucosal samples were placed in
preweighed Eppendorf vial and 1 ml of Tris buffer (50 mm, pH
9.5) was added to each vial. The samples were finely minced
(during 15 s) with scissors, washed and centrifuged for 10 s, the
pellet being resuspended again in 1 ml of Tris. Then, each
sample was incubated on a Vortex mixer for 1 min and
centrifuged for 15 s. The pellet was weighed and the supernatant
was transferred to a second Eppendorf vial containing
indomethacin (10 mM) and kept at -20°C until the
radioimmunoassay. The capability of the oesophageal mucosa to
generate prostaglandin E2 was expressed in nanograms of wet
tissue weight (39, 40).
At the termination of the experiments, immediately after
EBF measurements, a venous blood sample was withdrawn from
the vena cava into EDTA containing vials and used for the
determination of plasma TNF-α, IL-1β and IL-6 by a solid phase
sandwich ELISA (Biosource International Inc. Camarillo, CA,
USA) according to the manufacturer's instructions. Briefly, each
sample (50 µl) was incubated with biotinylated antibodies
specific for rat TNF-α, IL-1β and IL-6, was washed three times
with an assay buffer and finally conjugated with streptavidin
peroxidase to form a complex with stabilized chromogen as
described in our previous study (20, 39, 40).
Expression of IL-1β and TNF-α mRNA transcripts in the
esophageal mucosa determined by reverse transcriptasepolymerase chain reaction (RT-PCR)
78
was electrophoresed on 1.5% agarose gel stained with ethidium
bromide, and then visualized under UV light. The location of the
predicted PCR product was confirmed by using a 100-base pair
ladder (Gibco BRL/Life Technologies, Eggenstein, Germany) as
a standard marker. A comparisons between different treatment
groups were made by determining the IL-1β and TNF-α/β-actin
ratio of the immunoreactive area by densitometry.
Statistical analysis
Results are expressed as means ±S.E.M. Statistical analysis
was done using analysis of variance and two way ANOVA test
with Tukey post hoc test where appropriate. Differences of
p<0.05 were considered significant.
RESULTS
Time sequence of changes in esophageal injury and esophageal
blood flow (EBF) during development of reflux esophagitis
Fig. 2 shows the effects of duration of reflux esophagitis on
the alterations in EBF and the degree of macroscopic esophageal
mucosal injury as determined by the area of esophageal mucosa
occupied by the damage and expressed by a lesion index. As
shown in Fig. 2, the significant fall in the EBF and a significant
increase in the mucosal injury index were observed already at 2
hrs after the onset of reflux of gastric juice. Mucosal injury at 2
hrs was characterized by the appearance of local mucosal edema
with a small number of local hemorrhagic erosions, however, the
average damage score was low. Four hours after induction of
RE, further developments of esophageal mucosal lesions were
observed which increased the lesion score to the mean value of
3.5±0.5 and further significant fall in EBF by about 15% when
compared to that observed at 2 hrs after the end of the ligation
procedure to induce reflux. With a prolongation of the time of
acid exposure to the esophageal mucosa up to 8 hrs, 12 hrs and
24 hrs, the EBF was decreased by about 40% from control and
the lesion score rose significantly to the values of 4.1±0.6,
4.2±0.8 and 4.2±0.4, respectively, comparing to those obtained
in control animals at 2 hrs (Fig. 2). The increase in lesion index
observed at 8 hrs, 12 hrs and 24 hrs of the mucosal lesion score
was not significantly higher than this observed at 4 hrs of reflux.
On the basis of this experiment, the decision was made to choose
the time of 4 hrs as the standard time of observation for further
experimentations. This time after initiation of reflux was also
employed for the evaluation of ability of various NSAIDs
including ASA, indomethacin, celecoxib, NO-ASA, and GTN
combined with ASA to influence the esophageal mucosal injury
induced by gastric reflux and the accompanied changes in EBF.
Effect of native ASA and non-selective and selective COX-1
and COX-2 inhibitors on esophageal lesion index, gross and
microscopic injury and alterations in EBF
Fig. 3 shows the data on the administration of ASA applied
i.g, in graded doses ranging from 12.5 mg/kg up to 100 mg/kg
on the mean lesion score and accompanying changes in the
EBF and the content of PGE2 in the esophageal mucosa of rats
with esophagitis. The mean value of EBF in the intact
esophagus without reflux reached the value of 72.6±2.5
ml/min/100g of tissue, and this was considered to be the
control. The lesion score at 4 hrs of duration of reflux was
similar to that presented in Fig. 2. The pretreatment with ASA
administered i.g. at the dose of 12.5 mg/kg failed to affect both
the lesion index and the EBF as compared to vehicle. However,
when ASA was administered in graded doses starting from 25
mg/kg up to 100 mg/kg, the dose-dependent increase in the
lesion score was observed accompanied by the progressive fall
in the EBF. The quantitative data on the effect of non-selective
and selective COX-1 and COX-2 inhibitors on the lesion score
index and the accompanying changes in EBF are presented in
Fig. 4. The pretreatment with ASA (100 mg/kg i.g.) or
indomethacin (5 mg/kg i.g), resulted in a significant rise in the
lesion index followed by the significant fall in the EBF in rats
with acute esophagitis induced by gastric reflux. The
pretreatment with SC-560 (5 mg/kg i.g.), the selective COX-1
inhibitor, also significantly increased the lesion index and
reduced the EBF as it was observed in the case of ASA and
indomethacin (Fig. 4). In contrast, the pretreatment with
Fig. 1. The scheme of methods
employed in this study to
induce esophagitis. Both, the
duodenum and the transitional
between the forestomach and
the glandular portion was
ligated with 2-0 silk thread to
enhance reflux of gastric acid
into the esophagus.
79
Fig. 2. Esophageal mucosa lesion index
and the alterations in the esophageal
blood flow (EBF) at 2, 4, 8, 12 and 24
hours after induction of experimental
reflux esophagitis. Mean±S.E.M. of 6-8
rats. Asterisk indicates significant change
(p<0.02) as compared to intact rats. Cross
indicates a significant difference as
compared to the value of lesion index at 2
hours after the initiation of gastric acid
reflux.
Fig. 3. Esophageal mucosal lesion index,
the esophageal blood flow (EBF) and
mucosal generation of PGE2 in rats at 4 h
after induction acute esophagitis without
and with intragastric (i.g.) pretreatment
with vehicle or aspirin (ASA)
administered at graded doses starting
from 12.5 mg/kg up to 100.0 mg/kg.
Results are mean±S.E.M. of 6-8 rats.
Asterisk indicates significant change
(p<0.02) as compared to the value
recorded in vehicle-saline pretreated
animals.
celecoxib (10 mg/kg i.g), which produced a smaller fall in the
EBF, caused significantly less mucosal damage as compared
with those observed in indomethacin- or SC-560-pretreated
rats at 4 hrs of gastric reflux. At the same experimental
conditions, the pretreatment with NO-ASA significantly
attenuated the lesion index and significantly raised the EBF as
compared to respective values in vehicle-control and those
recorded in ASA-, indomethacin-, SC-560- and celecoxibpretreated animals.
The macroscopic appearance of the intact esophagus and
that exposed to vehicle (saline) control, ASA, indomethacin,
SC-560, celecoxib and NO-releasing ASA is presented in Fig.
5A-G. As shown in Fig. 5A, the intact esophageal mucosa
showed the normal appearance with no lesions present. In
contrast, the exposure of esophagus for 4 hrs to the reflux of
gastric content resulted in the formation of mucosal damage
(Fig. 5B), and these lesions were significantly potentiated by
the pretreatment with ASA (100 mg/kg i.g.), indomethacin (5
mg/kg i.g) or SC-560 (5 mg/kg i.g.) (Figs. 5C, 5D and 5E).
The pretreatment with celecoxib (10 mg/kg i.g.) also increased
the area of esophageal lesions and significantly decreased the
EBF but the lesion index was significantly smaller and the
values of EBF were significantly higher in celecoxib group
than those recorded in ASA- and indomethacin-pretreated rats
exposed to gastric reflux (Fig. 5F). In contrast, the
pretreatment with NO-ASA (100 mg/kg i.g.) markedly
reduced the area of esophageal damage when compared with
that treated with conventional ASA, indomethacin, SC-560
and celecoxib (Fig. 5G). The extent of injury was significantly
smaller in NO-ASA pretreated animals than those pretreated
with vehicle Figs. 5B and 5G).
The histological determination of esophageal mucosa
stained with H&E documented that no microscopic mucosal
changes were observed in intact animals (Fig. 6A). The normal
esophagus exhibited a thin epithelial layer with squamous
cells and only few inflammatory cells were found in
submucosal layers. In contrast, at 4hrs after induction of
reflux esophagitis, the mucosal damage and hyperemia of
epithelial layers and edema in mucosa and submucosa was
observed in vehicle (saline)-control animals (Fig. 6B). In
ASA- and SC-560 -treated rats (Figs. 6C and 6D), a marked
mucosal thickening accompanied by further exaggeration of
80
Fig. 4. Esophageal mucosal lesion index
and esophageal blood flow (EBF) in
acute esophagitis without or with
intragastric (i.g.) pretreatment with
aspirin (ASA) (100 mg/kg i.g.) or NOaspirin (NO-ASA) (100 mg/kg i.g.),
indomethacin (5 mg/kg i.g.), SC-560 (5
mg/kg i.g.), and celecoxib (10 mg/kg i.g).
Results are mean±S.E.M. of 6-8 rats.
Asterisk indicates significant change
(p<0.02) as compared to the value
recorded in vehicle-saline pretreated rats.
Cross indicates a significant change
(p<0.02) as compared to the value in
ASA-, indomethacin-, SC-560- and
celecoxib-pretreated rats. Double crosses
indicate a significant change (p<0.05) as
compared to the value recorded in ASAand indomethacin-pretreated rats.
Fig. 5. Macroscopic appearance of esophageal
mucosa in intact rats (A) and those pretreated i.g.
30 min before initiation of reflux esophagitis
with vehicle (B), ASA (100 mg/kg) (C),
indomethacin (5 mg/kg) (D), SC-560 (5 mg/kg)
(E), celecoxib (10 mg/kg) (F) and NO-releasing
ASA (100 mg/kg) (G). Note, the formation of
esophageal lesions in vehicle-control rat exposed
to gastric reflux that is significantly exaggerated
in rat treated with ASA (B vs. C), indomethacin
(B vs. D) and SC-560 (B vs. E). In contrast to
ASA- and indomethacin-pretreated rat, the NOreleasing ASA significantly attenuated the
damage induced by reflux esophagitis.
the damage, edema and heavy inflammatory cell infiltration
were observed. In celecoxib group (Fig. 6E), the esophageal
damage, edema and neutrophil infiltration were still observed,
however these changes were significantly less pronounced as
in case of group pretreated with ASA and SC-560. As shown
in Fig. 6F, the mucosal damage, neutrophil infiltration and
edema in the mucosa and submucosa were significantly
reduced in rats pretreated with NO-ASA and then exposed to
acid reflux.
Effect of pretreatment with ASA alone, NO-ASA and GTN
combined with ASA on the lesion index and accompanying
changes in EBF
Table 1 shows the macroscopic lesion index of esophageal
injury and the alterations in EBF following reflux esophagitis in
rats pretreated with vehicle, ASA alone and ASA combined with
GTN, a donor of NO. Treatment with ASA (100 mg/kg i.g.)
significantly increased the index of mucosal lesions, and
81
Fig. 6. Microscopic appearance of histological findings for the esophagus. The esophageal mucosa in intact rats (A) and those pretreated
i.g. 30 min before the initiation of reflux esophagitis with vehicle (B), ASA (100 mg/kg) (C), SC-560 (5 mg/kg) (D), celecoxib (10 mg/kg)
(E) and NO-releasing ASA (100 mg/kg ) (F). Note, the intact rat shows the thin epithelial layer with squamous cells and few inflammatory
cells reflecting the normal appearance of the esophageal mucosa (A). Vehicle-pretreated rats exposed to reflux esophagitis showed the
mucosal thickening, edema, mucosal damage, basal hyperplasia and leukocyte (neutrophil and eosinophils) infiltration (B). This damage
to esophageal structure was exaggerated in rat with reflux esophagitis pretreated with ASA (B vs. C) and SC-560 (B vs. D). In celecoxibtreated group, the mucosal damage and neutrophil infiltration were less pronounced, however the mucosal thickening and edema were
present. In contrast to ASA- and SC-560-pretreated rat, the pretreatment with NO-releasing ASA significantly attenuated the damage
induced by reflux esophagitis as reflected by partial preservation of the mucosal architecture (C and D vs. F).
Table 1. Esophageal mucosal lesion index and esophageal blood
flow ( EBF) in rats with acute reflux esophagitis without or with
intragastric pretreatment with aspirin (ASA) (100 mg/kg i.g) or
NO-aspirin (NO-ASA) (100 mg/kg i.g) or glyceryl trinitrate (GTN)
(10 mg/kg i.g) in combination with ASA (100 mg/kg i.g). Results
are mean±S.E.M. of 6-8 rats. Asterisk indicates significant change
(p<0.05) as compared to the value recorded in vehicle-saline
pretreated rats. Cross indicates significant change (p<0.02) as
compared to the value in ASA-pretreated rats.
2
$+
4
"#$
&'"
5!1)
.!,)
!6)
!,)
3
!6
!,7
!58
!.8
.,)+
6,)57
9,).8
9+)68
decreased the EBF when compared to those in the vehicletreated animals. In contrast, pretreatment with NO-ASA given in
similar dose as conventional ASA, significantly reduced the
esophageal lesions index score and this effect was accompanied
by a significant increase in EBF. Concurrent treatment with GTN
(10 mg/kg i.g) in combination with ASA reversed the fall in the
EBF and increase in mucosal lesion index caused by native ASA
alone. This reduction in the lesion index and the rise in the EBF
is almost identical to these evoked by NO-ASA.
Mucosal expression of IL-1β and TNF-α by RT-PCR and
plasma levels of IL-1β, TNF-α and IL-6 in the rats with
esophagitis treated with NO-ASA and COX-inhibitors
As shown in Fig. 7, the plasma levels of IL-1β, TNF-α and
IL-6 reached significantly higher values after 4 hrs of RE in
vehicle-treated rats when compared to those recorded in the
intact animals. The control concentration of IL-1β in the intact
animals were 15±2 pg/ml and a significant increase in IL-1β
level was observed averaging about 75 pg/ml in vehicle
pretreated rats with gastric reflux. Pretreatment with ASA (100
mg/kg i.g.) prior to onset of reflux esophagitis significantly
raised the plasma level of IL-1β when compared to that
measured in the vehicle pretreated animals with reflux. The
treatment with NO-ASA (100 mg/kg i.g.) restored the plasma IL1β to the level observed in vehicle pretreated rats, exposed to
RE. Treatment with indomethacin (5 mg/kg i.g.) and celecoxib
(10 mg/kg i.g.) significantly raised the plasma level of IL-1β in
comparison to the level recorded in vehicle-treated rats though
the increase in this cytokine level in the animals pretreated with
celecoxib were significantly less pronounced when compared to
that in ASA, indomethacin and SC-560 treated rats. Similarly,
the concentration of TNF-α and IL-6 remained at low levels
(0.5±0.01 pg/ml and 1.2±0.2 pg/ml) in intact animals but
following 4 hrs of reflux esophagitis, there was a remarkable rise
in TNF-α and IL-6 concentration, reaching the value of about
4.0±0.7 pg/ml and 8.1±1.7 pg/ml, respectively (Fig. 7).
Pretreatment with ASA or indomethacin resulted in a significant
increase in plasma TNF-α and IL-6 level when compared with
those measured in the vehicle-treated rats subjected to 4 hrs of
reflux esophagitis. In celecoxib-pretreated rats, a significantly
82
Fig. 7. Plasma levels of interleukin-1 beta (IL-1β), tumor
necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in
intact rats and animals with acute esophagitis without or
with intragastric (i.g.) pretreatment with vehicle (saline),
aspirin (ASA) (100 mg/kg), NO-aspirin (NO-ASA) (100
mg/kg), indomethacin (5 mg/kg), SC-560 (5 mg/kg) and
celecoxib (10 mg/kg). Results are mean±S.E.M. of 6-8 rats.
Asterisk indicates significant change (p<0.02) as compared
to intact rats. Cross indicates significant change (p<0.05) as
compared to the vehicle. Asterisk and cross indicate a
significant change (p<0.02) as compared with the values
obtained in rats pretreated with ASA, indomethacin and SC560. Double crosses indicate a significant change (p<0.05)
as compared to the value recorded in ASA-, indomethacinand SC-560-pretreated rats.
smaller increase in the plasma TNF-α and IL-6 were observed as
compared to the levels of these cytokines in ASA or
indomethacin treated rats. The pretreatment of animals with NOASA significantly decreased the plasma concentrations of TNFα and IL-6 below the value recorded in vehicle-treated animals
exposed to gastric reflux (Fig. 7).
Fig. 8 (left panel A) shows the expression of IL-1β mRNA
in the intact esophageal mucosa and in that exposed to acid
reflux with or without the pretreatment with NO-ASA or native
ASA. The weak signal for IL-1β mRNA was detected in intact
esophageal mucosa and this was significantly increased with
acid reflux, the effect that was further enhanced in the
esophageal mucosa exposed to ASA. In NO-ASA pretreated rats,
the signal for IL-1β mRNA expression was significantly
inhibited. The ratio of IL-1β mRNA to β-actin confirmed that
mRNA for IL-1β was significantly up-regulated in rats exposed
to acid reflux and those pretreated with ASA when compared to
intact gastric mucosa while NO-ASA inhibited the expression of
mRNA for IL-1β (Fig. 8, right panel). As shown in Fig. 8 (left
panel B), TNF-α mRNA was detected in the gastric mucosa of
all rats including control animals and those exposed to acid
reflux with and without ASA and NO-ASA administration.
Signal intensity for TNF-α mRNA was weakly detectable in
intact animals but it was increased in reflux esophagitis and
further significantly augmented in rats pretreated with ASA. The
pretreatment with NO-ASA inhibited the mRNA for TNF-α in
the esophageal mucosa of rats with acid reflux. The ratio of
TNF-α mRNA to β-actin mRNA confirmed that expression of
mRNA for TNF-α was increased in rats with reflux esophagitis
and further overexpressed in those pretreated with ASA as
compared to intact rats. The ratio of TNF-α to β-actin mRNA
confirmed that TNF-α mRNA was significantly inhibited in NOASA-pretreated animals and exposed 30 min later to acid reflux
as compared to that in rats exposed to acid reflux with native
ASA administration (Fig. 8, right panel).
DISCUSSION
Gastric esophageal reflux disease (GERD) is one of the most
important gastrointestinal disorders of our population nowadays
and the mechanisms responsible for this disease should be
extensively examined (42, 43). In this study we attempted to
develop the experimental model of esophagitis that serves as a
83
Fig. 8. Determination of IL-1β (A) and
TNF-α (B) and β-actin (C) mRNA
expression by RT-PCR (left panel) and
the ratio of IL-1β and TNF-α mRNA
over β-actin mRNA (right panel) in the
intact esophageal mucosa (lane 1) and in
those pretreated i.g. with vehicle (saline)
(lane 2), and NO-ASA (100 mg/kg) (lane
3) or ASA (100 mg/kg ) (lane 4) and
exposed to reflux esophagitis for 4 hrs; M
- DNA size marker. Mean±S.E.M. of 4-6
rats. Asterisk indicates a significant
change (p<0.05) as compared with
vehicle control gastric mucosa. Cross
indicates a significant change (p<0.05) as
compared with vehicle and ASA
treatment. Double crosses indicate a
significant change (p<0.05) as compared
to the value obtained in vehicle-control
and NO-ASA groups.
complementary tool for a clinical investigation offering insight
into the mechanism of damage to the mucosa of the esophagus
by aggressive factors and COX inhibitors. The model of
gastroesophageal reflux described in this paper seems to be
especially suitable for examination of the early mechanisms of
acute esophageal injury caused by gastric reflux and does not
entirely reflect the changes associated with chronic esophagitis
or long term GERD. In this experimental model, the gastric acid
secreted by parietal cells and pepsin activity in the stomach but
not intestinal alkaline content, are the most important pathogenic
factors of reflux esophagitis. The maintenance of integrity of the
epithelial barrier of the esophageal mucosa against aggressive
factors involves several mechanisms, mainly undisturbed
mucosal microcirculation which is potentially responsible for
maintaining normal mucosal acid-base balance. An increase in
the esophageal blood flow takes place in response to an
activation of the esophageal secretory activity and the presence
of luminal refluxate from the stomach and duodenum (8, 13). An
imbalance between defensive and offensive factors is proposed
as a major mechanism in the development of reflux esophagitis
(4-8). Recently, we reported that the inhibition of COX/PG and
NOS/NO systems and the deactivation of sensory nerves
rendering the esophageal mucosa highly susceptible to damage
by the exposure to exogenously administered acid-pepsin or to
the combination of acid, pepsin and bile solution (19).
The major observation of our present study is the evidence
accumulated for the first time, that NO-ASA, in contrast to
native NSAIDs such as conventional ASA or indomethacin,
protects esophageal mucosa against mucosal injury induced by
gastroesophageal reflux. Similar protective activity is
demonstrated by NO-ASA in the stomach of experimental
animals and humans (27, 29-32). Mechanism of this protection
by NO-ASA against the damage induced reflux esophagitis may
involve excessive release of NO from this NO derivative of ASA
as it was shown to result in gastroprotection induced by those
agents (27-35). Moreover, the NO-ASA-induced esophageal
protection and the subsequent increase in the EBF was
accompanied by the inhibition of COX-1 and the suppression of
the PG generation and did not differ from the inhibitory effect of
this agent and classic NSAIDs on gastric PG as described earlier
(31). It is of interest that these esophago-protective actions of the
NO-ASA was accompanied by a profound attenuation of the
expression and release of proinflammatory cytokines such as IL1β and TNF-α. Furthermore, the plasma level of
proinflammatory cytokine IL-6 was also markedly diminished in
animals pretreated with NO-ASA. On contrary, the expression of
mRNA for IL-1β and TNF-α and their plasma levels were
markedly increased in rats with acute gastric acid reflux and this
effect was further potentiated in those treated with non-selective
and selective COX-1 inhibitors. This supports the notion that
inhibition of COX-1 stimulates pro-inflammatory factors
resulting in the aggravation of mucosal injury and the
impairment of mucosal recovery from the damage (44, 46).
The esophagoprotective activity of NO-ASA shown in this
paper is consistent with previous data from our and others
laboratories that NO accelerated experimental ulcer healing,
whereas inhibition of endogenous NO generation by NOsynthase inhibitor impaired healing of chronic gastric ulcers (34,
35, 37). The mechanism through which NO released from ASA
induces inhibition of esophageal mucosa lesion is complex and
not entirely clear but could be attributed to its well established
potent vasodilatatory properties and an ability to increase the
mucosal blood flow (31, 34, 35). NO was shown to suppress the
release of pro-inflammatory cytokines and contribute to the
enhanced generation of mucosal esophageal PGE2 (18, 19).
Similar protective activity of NO-ASA has been recently
demonstrated in rodent stomach (27-29, 31). Our results
emphasize the protective role of NO released from NO-ASA in
the action of new ASA derivative on esophageal mucosa against
acute reflux esophagitis. The present investigations attempted to
determine the involvement of NO in the mediation of the
esophageal protective action of NO-ASA and for this purpose
the exogenous donor of NO, GTN was employed to mimic the
effect of NO releasing ASA. The combination of native ASA
with GTN induced the same degree of esophageal protection as
NO-ASA and enhanced significantly the EBF to the level
observed after application of NO-ASA. These protective and
microcirculatory effects of GTN combined with ASA could be
attributed to a very well recognized circulatory and the antiinflammatory activity of NO, which was apparently released
from GTN (31). Further studies are needed to assess which
isoform of COX, is involved in esophageal protection and
84
whether selective blockade of COX-1, or COX-2 or both (47),
might be responsible for the enhancement of esophageal damage
induced by gastric reflux.
Our study possesses evident limitation related to the duration
of inflammatory response since we have shown the ability of
NO-releasing derivative of commonly used native ASA to
protect the esophageal mucosa during early but not prolonged
chronic stage of esophagitis. However, experimental surgical
method of induction of reflux esophagitis via gastro-esophageal
reflux of acid caused visible mucosal damage to the esophageal
structure detectable as early as 2 hrs following surgical ligation.
The method of lesion induction employed in our present study
after original technique by Omura et al. (38), does not require
the animal to be under anesthesia for a long time during
experimentation as is the case of the method using esophageal
perfusion of acid and bile (19) and therefore we postulate that
this procedure most likely mimics the human scenario of acute
esophagitis. Moreover, in our present study, the damage was
achieved by endogenously secreted gastric acid but not
exogenous artificial acid as in our previous report (19).
The ingestion of NSAIDs has been associated with serious
complications and adverse effects such as an increase in the
vulnerability of the gastrointestinal and esophageal mucosa in
the development of peptic lesions including inflammation,
erosions and bleeding ulcerations (20, 25, 47). These destructive
effects of NSAIDs are related to inhibition of constitutive
enzyme COX-1, mainly responsible for PG generation and
esophageal protection. The COX-2 is induced through the
mechanism involving cytokines, growth factors and endotoxins
(40). Selective COX-2 inhibitors such as rofecoxib and
celecoxib, were specifically designed to preferentially inhibit the
COX-2-induced production of inflammatory eicosanoids by
sparing the endogenous protective prostanoids generated via
COX-1 activity in the GI-tract (39, 40, 47). The mechanisms by
which PG exhibit protection of GI-tract mucosa include mucosal
hyperemia, stimulation of mucus and bicarbonate secretion and
strengthening of epithelial component of the mucosal barrier.
Among these mechanisms, the maintenance of the mucosal
microcirculation is thought to be the most important mechanism
of the protective effects of PG in the GI-tract (34).
The adverse effects of traditional NSAID are related to
topical mucosal injury which usually occurs after the ingestion
of aspirin and other NSAIDs, especially under acidic conditions.
The apparent reduction in gastrointestinal blood flow induced by
NSAIDs such as ASA occurs also as a result of enhanced
adherence of neutrophils to vascular endothelium in
microvasculature via increased expression of adhesion
molecules on endothelial cells (43, 44). In our present study
native ASA dose-dependently augmented the damage induced
by acid reflux and this effect was accompanied by potent
suppression of PGE2 generation and a significant fall in the EBF.
It has been documented that PG such as PGI2 are very potent
vasodilators responsible for control of blood flow through
resistance vessels and microcirculation and these eicosanoids are
involved in the mediation of the vascular auto-regulatory
responses including functional hyperemia in the gastrointestinal
tract including the esophagus. Thus, it is likely that the
mechanism of the esophageal lesions induced by ASA could be
attributed to PG inhibition and vasoconstriction, ischemia and a
final consequence tissue hypoxia leading to the impairment of
the mucosal barrier. These damaging effects of ischemia on
esophageal mucosa are potentiated by the presence of hydrogen
ions and pepsin which alone can induce mucosal damage.
We have shown that at this acute stage of both COX-1 and
COX-2 activity could be involved in the mechanism of acute
esophagitis. This is supported by the fact that both non-selective
and selective COX-1 inhibitors aggravated the esophageal
damage and decreased the esophageal blood flow. An attempt
has been made to determine the involvement of COX-2 in the
mechanism of acute esophagitis by the use of celecoxib, the
selective COX-2 inhibitor. While COX-1 inhibition is achieved
by pretreatment with ASA, indomethacin and the selective
inhibitor SC-560 potentiated the damage induced by gastric
reflux, celecoxib was found to be superior to both non-selective
and selective inhibitors as documented by the weaker
aggravatory effect on esophagitis of celecoxib comparing to
COX-1 inhibitors. Similarly, the degree of reduction of EBF
observed after pretreatment with celecoxib in rats with gastric
reflux was significantly smaller than that observed after
pretreatment with selective COX-1 inhibitor, SC-560. Although
there are many differences between COX-1 and COX-2, one
notable distinction is that COX-2 is believed to be the principal
COX isoform, which is involved in inflammation. It is also
debated wither COX-2 activity could also be detected in the noninflamed stomach (47). Our data with celecoxib is only in partial
agreement with study by Hayakawa et al. (48) who found that
COX-2 inhibitors such as celecoxib and nimesulide significantly
reduced the severity of chronic esophagitis (on day 21) but failed
to affect acute oesophageal lesions (on day 3). They
demonstrated (48) that mRNA and protein expression of COX-2
and mPGES-1 was markedly increased in rat acid reflux
esophagitis compared with normal esophagus, while only
modest changes in expression of COX-1, cPGES, and mPGES2 were observed. Similar as in our present study, the PGE2 levels
were significantly elevated in esophagitis comparing to baseline
and in addition, the increased expression of COX-2 and mPGES1 on both days 3 and 21 were observed by these authors (48).
Because celecoxib significantly reduced the reflux-induced
increase in PGE2 synthetic activity on both days 3 and 21, they
concluded (48) that PGE2 derived from COX-2 and mPGES-1
play a significant role in the development of chronic esophagitis
but to lesser degree in acute esophageal lesions. The discrepancy
between our present results and those reported by Hayakawa et
al. (48) might be attributed to different method of esophagitis
employed and short time of acute esophagitis selected (up to 4
and 24 hours) in our experiments comparing with longer time (3
and 21 days) of observation in their study.
The esophagus is the first anatomical part of GI-tract which
undergoes exposure to NSAIDs. Esophageal ulcers are thought
to be initiated by disruption of mucosal barrier by refluxate thus
allowing the diffusion of hydrogen ion. Previous studies
revealed that under experimental conditions and in humans, the
ulceration of the esophageal mucosa can be induced after a few
oral doses of a NSAID or by low dose of ASA (20). Indeed, in
our study, the native ASA when applied i.g. induced a dosedependent potentiation of damage induced by gastric reflux and
this effect was accompanied by a parallel fall in EBF. These
effects could be explained by the ability of ASA to inhibit COX1 and the suppression of the generation of esophageal PG. In this
light, the most important finding of this study is the discovery
that NO-derivative of ASA protects esophageal mucosa against
mucosal injury induced by acid refluxed from the stomach to the
esophagus. Both, the NO-ASA-induced esophageal protection
and accompanying increase in the EBF take place in the face of
inhibition of COX-1 and suppressing of endogenous PG
generation. Further studies are needed to determine whether NOASA could exert inhibitory action in progression of chronic
esophagitis into Barrett's adenocarcinoma under experimental
conditions described by our group recently (49).
In summary, we have shown that pretreatment of animals
with NO-releasing ASA derivative or concomitant pretreatment
by native ASA with an NO donor (glyceryl trinitrate) has
beneficial effect on esophageal mucosa in reflux esophagitis.
The mechanisms through which NO induces inhibition of
85
esophageal mucosa lesion may involve an increase in the
esophageal blood flow. NO can also reduce the generation of
proinflammatory factors, e.g. reactive oxygen metabolites (50)
and contribute to an increased generation of esophageal mucosal
PGE2. Our present study implies that the beneficial effect of NOreleasing derivative of commonly used ASA to protect the
esophageal mucosa during early stage of gastro-esophageal
reflux depends upon the enhancement of the esophageal
microcirculation, the inhibition of proinflammatory cytokines
IL-1β, TNF-α and IL-6 induced by acidic refluxate and the
replacement by vasodilatory NO as a compensatory mechanism
to the decrease in endogenous PG induced by conventional ASA.
Conflict of interests: None declared.
REFERENCES
1. Dent J, El-Serag HB, Wallander MA, Johansson S.
Epidemiology of gastro-oesophageal reflux disease: a
systemic review. Gut 2005 ; 54: 710-717.
2. Fox M, Forgacs I. Gastro-oesophageal reflux disease. Br
Med J 2006; 332: 88-93.
3. El-Searag HB. Time trends of gastroesophageal reflux
disease: a systemic review. Clin Gastroenterol Hepatol
2007; 5: 17-26.
4. Kahrilas PJ, Lee TJ. Pathophysiology of gastroesophageal
reflux disease. Thorac Surg Clin 2005; 15: 323-333.
5. Sifrim D, Mittal R, Fass R, et al. Acidity and volume of the
refluxate in the genesis of gastro-oesophageal reflux disease
symptoms. Aliment Pharmacol Ther 2007; 25: 1003-1015.
6. Lanas A, Royo Y, Ortego J, Molina M, Sainz R.
Experimental esophagitis induced by acid and pepsin in
rabbits
mimicking
human
reflux
esophagitis.
Gastroenterology 1999; 116: 997-1107.
7. Vaezi MF, Richter JE. Role of acid and
duodenogastroesophageal reflux in gastroesophageal reflux
disease. Gastroenterology 1996; 111: 1192-1199.
8. Oh DS, Hagen JA, Fein M. The impact of reflux
composition on mucosal injury and esophageal function. J
Gastrointest Surg 2006; 10: 787-797.
9. Orlando RC, Bryson JC, Powell DW. Mechanisms of H+
injury in rabbit esophageal epithelium. Am J Physiol 1984;
246: G718-G724.
10. Tobey NA, Carson JL, Alkiek RA, Orlando RC. Dilated
intercellular spaces: a morphological feature of acid refluxdamaged human esophageal epithelium. Gastroenterology
1996; 111: 1200-1205.
11. Szentpali K, Kaszaki J, Eros G, et al. Changes in
microcirculation of esophageal mucosa during acute
experimental reflux. Pathogenic role of the biliary
component. Magy Seb 2003; 56: 61-67.
12. Abdulnour-Nakhoul S, Nakhoul NL, Wang P, Swenson ER,
Orlando RC. HCO3- secretion in the esophageal submucosal
glands. Am J Physiol Gastrointest Liver Physiol 2006; 288:
G736-G744.
13. Nagahama K, Yamoto M, Nishio H, Takeuchi K. Essential
role of pepsin in pathogenesis of acid reflux esophagitis in
rats. Dig Dis Sci 2006; 51: 303-309.
14. Tobey NA, Hossein SS, Argote CM., et al. Dilated
intercellular spaces and shunt permeability in nonerosive
acid-damaged esophageal epithelium. Am J Gastroenterol
2004; 9: 13-22.
15. Fujiwara Y, Higuchi K, Takashima, et al. Roles of epidermal
growth factor and Na+/H+ exchanger-1 in esophageal
epitheial defense against acid-induced injury. Am J Physiol
2006; 290: G665-G673.
16. Naito Y, Uchiyama K, Kuroda M, et al. Role of pancreatic
trypsin in chronic esophagitis induced by gastroduodenal
reflux in rats. J Gastroenterol 2006; 41: 198-208.
17. Lillemoe KD, Johnson LF, Harmon JW. Alkaline
esophagitis: a comparison of the ability of components of the
gastroduodenal contents to injure the rabbit esophagus.
Gastroenterology 1983; 85: 621-628.
18. Poplawski C, Sosnowski D, Szaflarska-Poplawska A,
Sarosiek J, McCallum R, Bartuzi Z. Role of bile acids,
prostaglandins and COX inhibitors in chronic esophagitis in
a mouse model. World J Gastroenterol 2006; 12: 1739-1742.
19. Konturek SJ, Zayachkivska O, Havryluk XO, et al.
Protective influence of melatonin against acute esophageal
lesions involves prostaglandins, nitric oxide and sensory
nerves. J Physiol Pharmacol 2007; 58: 371-387.
20. Armstrong HP, Blower AL. Non-steroidal anti-inflammatory
drugs and life-threatening complications of peptic
ulceration. Gut 1987; 28: 527-532.
21. Kikendall JW, Friedman AC, Oyewole MA, Fleischer D,
Johnson LF. Pill-induced esophageal injury: case reports
and review of the medical literature. Dig Dis Sci 1983; 28:
174-181.
22. Heller SR, Fellows IW, Ogilvie AL, Atkinson M. Nonsteroidal antiinflammatory drugs and benign oesophageal
stricture. Br Med J 1982; 285: 167-168.
23. Safaie-Shirazi S, Zike WL, Brubacher M, DenBensten L.
Effect of aspirin, alcohol, and pepsin on mucosal
permeability of esophageal mucosa. Surg Forum 1974; 25:
335-337.
24. Carlsby B, Densert O. Esophageal lesions caused by orally
administered drugs: an experimental study in the cat. Eur
Surg Res 1980; 12: 270-282.
25. Lanas A, Hirschowitz BI. Significant role of aspirin use in
patients with esophagitis. J Clin Gastroenterol 1991; 13:
622-627.
26. Konturek PC, Konturek SJ, Brzozowski T. Helicobacter
pylori infection in gastric cancerogensesis. J Physiol
Pharmacol 2009; 60: 3-21.
27. Wallace JL, Reuter B, Cicala C, McKnight W, Grishman M,
Cirino G. A diclofenac derivative without ulcerogenic
properties. Eur J Pharmacol 1994; 257: 249-255.
28. Elliott SN, McKnight W, Cirino G, Wallace JL. A nitric
oxide-releasing non steroidal anti-inflammatory drug
accelerates gastric ulcer healing in rats. Gastroenterology
1995; 109: 524-530.
29. Davies NM, Rosenth AG, Appleyard CB, et al. NO-naproxen
vs. naproxen: ulcerogeniic, analgesic and anti-inflammatory
effects. Aliment Pharmacol Ther 1997; 11: 69-79.
30. Tashima K, Fujita A, Umeda M, Takeuchi K. Lack of gastric
toxicity of nitric oxide-releasing aspirin, NCX-4026, in the
stomach of diabetic rats. Life Sci 2000; 67: 1639-1652.
31. Brzozowski T, Konturek PC, Konturek SJ, Sliwowski Z,
Drozdowicz D., Kwiecien S. Gastroprotective and ulcer
healing effects of nitric oxide-releasing non-steroidal antiinflammatory drugs. Dig Liver Dis 2000; 32: 583-594.
32. Fiorucci S, Santucci C, Gresele P, Del Soldato P, Morelli A.
Gastrointestinal safety of NO-aspirin (NCX-4016) in healthy
human volunteers; a proof of concept endoscopic study.
Gastroenterology 2003; 124: 600-607.
33. Burgaud JL, Ongini E, Del Soldato P. Nitric oxide-releasing
drugs: a novel class of effective and safe therapeutic agents.
Ann NY Acad Sci 2002; 962: 360-371.
34. Brzozowski T, Konturek PC, Konturek SJ. Nitric oxidereleasing agents-a new generation of drugs for gastrointestinal
diseases. Front Gastrointest Res 2002; 25: 158-165.
35. Brzozowska I, Targosz A, Sliwowski Z, et al. Healing of
chronic gastric ulcers in diabetic rats treated with native
86
36.
37.
38.
39.
40.
41.
42.
43.
44.
aspirin, nitric oxide (NO)-derivative of aspirin and
cyclooxygenase (COX)-2 inhibitor. J Physiol Pharmacol
2004; 55: 773-790.
Lee M, Aldred K, Lee E, Feldman M. Aspirin-induced acute
gastric mucosal injury is a neutrophil-dependent process in
rats. Am J Physiol 1992; 263: G920-G926.
Elliott SN, McKnight W, Cirino G, Wallace JL. A nitric
oxide-releasing non steroidal anti-inflammatory drug
accelerates gastric ulcer healing in rats. Gastroenterology
1995; 109: 524-530.
Omura N, Kashiwagi H, Chen G, Suzuki Y, Yano F, Aoki T.
Establishment of surgically induced chronic acid reflux
esophagitis in rats. Scand J Gastroenterol 1999; 34: 948-953.
Brzozowski T, Konturek PC, Konturek SJ, et al. Role of
prostaglandins generated by cyclooxygenase-1 and
cyclooxygenase-2 in healing of ischemia-reperfusioninduced gastric lesions. Eur J Pharmacol 1999; 385: 47-61.
Konturek SJ, Konturek PC, Brzozowski T. Role of
prostaglandins in ulcer healing. J Physiol Pharmacol 2005;
56: 33-56.
Chomczynski P, Sacchi N. Single-step method of RNA
isolation by acid guanidinum thiocyanate-phenolcholroform extraction. Anal Biochem 1987; 162: 156-159.
Essen G. The epidemiology of gastroesophageal reflux
disease: what we know and what we need to know. Am J
Gastroenterol 2001; 96 (Suppl. 8): S16-S18.
El-Searag HB. Time trends of gastroesophageal reflux
disease: a systemic review. Clin Gastroenterol Hepatol
2007; 5: 17-26.
Wallace JL, McKnight W, Miyasaka M, et al. Role of
endothelial adhesion molecules in NSAID-induced gastric
mucosal injury. Am J Physiol 1993; 265: G993-G998.
45. Hamaguchi M, Fujiwara Y, Takashima T, et al. Increased
expression of cytokines and adhesion molecules in rat
chronic esophagitis. Digestion 2003; 68: 189-197.
46. Yamaguchi T, Yoshida N, Tomatsuri N, et al. Cytokineinduced neutrophil accumulation in the pathogenesis of acute
reflux esophagitis in rats. Int J Mol Med 2005; 16: 71-77.
47. Wallace JL, McKnight W, Reuter B, Vergnolle N. NSAIDinduced gastric damage in rats: Requirement for inhibition
of both cyclooxygenase 1 and 2. Gastroenterology 2000;
119: 706-714.
48. Hayakawa T, Fujiwara Y, Hamaguchi M, et al. Roles of
cyclooxygenase 2 and microsomal prostaglandin E synthase
1 in rat acid reflux oesophagitis. Gut 2006; 55: 450-456.
49. Majka J, Rembiasz K, Migaczewski M, et al. Cyclooxygenase2 (COX-2) is the key event in pathophysiology of Barrett's
esophagus. Lesson from experimental animal model and
human subjects. J Physiol Pharmacol 2010; 61: 409-418.
50. Brzozowski T, Konturek PC, Konturek SJ, et al.
Implications of reactive oxygen species and cytokines in
gastroprotection against stress-induced gastric damage by
nitric oxide releasing aspirin. Int J Colorectal Dis 2003; 18:
320-329.
R e c e i v e d : November 22, 2010
A c c e p t e d : February 24, 2011
Author's address: Prof. Tomasz Brzozowski, Chair of
Department of Physiology, Jagiellonian University Medical
College, 16 Grzegorzecka Street, 31-531 Cracow, Poland;
Phone: (+4812) 421-10-06; Fax: (+4812) 422-20-14;
E-mail: [email protected]