1 Department of Applied Pharmacology and 2 Department of Biopharmaceutics, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We
investigated the role of nuclear factor-B (NF-
B) in
gastric ulcer healing in rats. NF-
B was activated in ulcerated
tissue but not in normal mucosa, and the level of the activation was decreased with ulcer healing. NF-
B activation was observed in fibroblasts, monocytes/macrophages, and neutrophils. Treatment of
gastric fibroblasts, isolated from the ulcer base, with
interleukin-1
activated NF-
B and the subsequently induced
cyclooxygenase-2 and cytokine-induced neutrophil
chemoattractant-1 (CINC-1) mRNA expression. Inhibition of activated
NF-
B action resulted in suppression of both their mRNA expression
and increases in PGE2 and CINC-1 levels induced by
interleukin-1
. Persistent prevention of NF-
B activation caused an
impairment of ulcer healing in rats. Gene expression of
interleukin-1
, CINC-1, cyclooxygenase-2, and inducible nitric oxide
synthase in ulcerated tissue had been inhibited before the delay
in ulcer healing became manifest. The increased levels of
cyclooxygenase-2 protein and PGE2 production were also
reduced. These results demonstrate that NF-
B, activated in ulcerated
tissue, might upregulate the expression of healing-promoting factors
responsible for gastric ulcer healing in rats.
interleukin-1; cytokine-induced neutrophil chemoattractant-1; cyclooxygenase-2; inducible nitric oxide synthase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GASTRIC ULCER HEALING
PROCEEDS through multiple steps, such as formation of granulation
tissue, angiogenesis, and epithelial regeneration. The healing response
is largely coordinated by a variety of inflammatory mediators,
cytokines, and growth factors that are produced locally in the
ulcerated portion. There has been accumulating evidence that
prostaglandin/cyclooxygenase (COX)-2, nitric oxide (NO)/inducible NO
synthase (iNOS), cytokine-induced neutrophil chemoattractants (CINCs),
interleukin (IL)-1, and hepatocyte growth factor are induced or
increased by gastric ulceration and might contribute to ulcer healing
(9, 10, 13, 21, 24, 25, 33). The genes of these factors
have nuclear factor-B (NF-
B) responsive elements in their
promoter regions, and their expression is regulated by NF-
B in many
cell types (2, 11, 22).
NF-B exists in the majority of cell types and consists of homo- or
heterodimers of structurally related proteins including p65 (Rel A),
c-Rel, and p50 (NF-
B1) (2, 11, 22). In resting cells,
NF-
B is coupled with inhibitor-
B (I-
B) and resides in the
cytosol as an inactive form. In response to inflammatory stimuli and
mitogens, I-
B is phosphorylated and dissociates from NF-
B. Subsequently, NF-
B translocates into the nucleus to function as an
active transcription factor. We speculated that NF-
B is activated in
ulcerated gastric tissue because gastric ulcers are associated with
inflammation. In the present study, we found that NF-
B is activated
only in ulcerated tissue and that its activation is sustained during
the healing of gastric ulcers in rats. Then we examined whether NF-
B
upregulates the expression of healing-promoting factors such as COX-2,
iNOS, CINC-1, and IL-1
in vitro (isolated gastric fibroblasts) and
in vivo (ulcerated gastric tissue in rats). In addition, we examined
the effect of pyrrolidine dithiocarbamate (PDTC, an inhibitor of
NF-
B activation) on ulcer healing in rats. Here we describe the
crucial role of NF-
B in gastric ulcer healing in rats.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evaluation of gastric ulcer healing in rats. Male Wistar ST rats (270-360 g; Nihon SLC, Hamamatsu, Japan) were anesthetized with pentobarbital (50 mg/kg ip), and the stomachs were exposed through an abdominal incision. Acetic acid (100%) was applied to the anterior serosal wall surface at the border between the antrum and the fundus through a plastic tube (15). Since deep and well-defined ulcers had been generated 3 days later, we defined the third day as day 0 (the onset of ulcer healing). The ulcerated area (mm2) was determined under a dissecting microscope (Olympus, Tokyo, Japan).
Histological examination of gastric ulcer healing was according to our previously reported method (21, 23). After histological sections were prepared from ulcerated tissues on day 14 and then stained with hematoxylin and eosin, the length of the regenerated mucosa on the ulcer base (mm), the thickness of the base (mm), and the density of neutrophils infiltrated in the base were determined under a light microscope (Olympus). Alternatively, the sections were immunostained with the antibody against von Willebrand factor (factor VIII-related endothelial antigen; DAKO, Glostrop, Denmark). The numbers of neutrophils and microvessels in the base were measured in three randomly chosen fields (0.0625 mm2), and their densities were expressed as the number of neutrophils and microvessels per square millimeter, respectively. The person evaluating ulcer healing was unaware of the treatment given the animals.Isolation of gastric fibroblasts.
Gastric fibroblasts were isolated from ulcerated tissue on day
7. Under ether anesthesia, rats were perfused via the left cardiac
ventricle with medium A [in mM: 25 HEPES-NaOH (pH 7.4), 132.4 NaCl, 5.4 KCl, 5 Na2HPO4, 1 NaH2PO4, 1.2 MgSO4, and 1 CaCl2 with 2 mg/ml glucose and 1 mg/ml BSA]. After
ulcerated tissues were finely minced in medium A, they were
incubated in medium A containing 0.05% collagenase (Wako
Pure Chemicals, Osaka, Japan) and 1 mM EDTA for 60 min at 37°C under
95% O2-5% CO2 and then filtrated through a
mesh (pore size 0.1 mm). The culture of the isolated cells was
maintained at 37°C under 5% CO2 in air in DMEM supplemented with 10% FBS (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 µg/ml amphotericin B
for >4 wk, after which contaminated immune and epithelial cells died
and cultures were >95% fibroblasts. The remaining cells were spindle-shaped and reactive to anti-vimentin antibody (DAKO) but not to
anti-smooth muscle -actin antibody (Nichirei, Tokyo, Japan). Their
growth was significantly stimulated by basic fibroblast growth factor.
For experiments, gastric fibroblasts were allowed to grow to confluence
in 60-mm dishes.
Effect of NF-B decoy oligonucleotide in
gastric fibroblasts.
Double-stranded NF-
B consensus motifs (decoys) were
synthesized as single-stranded oligonucleotides (NF-
B decoy,
5'-AGTTGAGGGGACTTTCCCAGGC-3' and 5'-GCCTGGGAAAGTCCCCTCAACT-3';
mutant decoy, 5'-AGTTGAGGCGACTTTCCCAGGC-3' and
5'-GCCTGGGAAAGTCCGCTCAACT-3') (Life Technologies) and annealed (17). The decoys (5 µg) were introduced to gastric
fibroblasts with LipofectAMINE PLUS Reagent (Life Technologies),
according to the instruction manual. Sixteen hours later, medium was
replaced with DMEM supplemented with 5% FBS, and then gastric
fibroblasts were stimulated with 10 ng/ml IL-1
. At the indicated
times, nuclear extracts and total RNAs were prepared, and then they
were subjected to gel shift assay and RT-PCR, respectively. In
addition, the medium was recovered 16 h after the addition of
IL-1
and subjected to measurement of PGE2 and CINC-1 by
enzyme immunoassay (PGE2 EIA kit, Cayman, Ann Arbor, MI;
GRO/CINC-1 EIA kit, Immuno Biological Laboratories, Fujioka, Japan).
NS-398 at 10 µM was added 5 min before IL-1
stimulation.
PGE2 and CINC-1 production were expressed as picograms per
milliliter per 16 h.
Preparation of nuclear extracts. Gastric tissues taken from normal and ulcerated stomachs were homogenized in buffer A [in mM: 10 HEPES-NaOH (pH 7.8), 10 KCl, 0.1 EDTA, 1 dithiothreitol (DTT), and 0.5 phenylmethylsulfonyl fluoride (PMSF) with 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1% Triton X-100] and then left on ice for 15 min. Gastric fibroblasts were scraped in PBS, followed by centrifugation at 800 g for 5 min. The cell pellets were suspended in buffer A, vortexed for 1 min, and then left on ice for 15 min. After the homogenates and cell lysates were centrifuged at 5,000 g for 5 min, the precipitates were suspended in 0.5 ml and 0.1 ml, respectively, of buffer B [in mM: 50 HEPES-NaOH (pH 7.8), 420 KCl, 0.1 EDTA, 5 MgCl2, 1 DTT, and 0.5 PMSF with 20% glycerol, 2 µg/ml aprotinin, and 2 µg/ml leupeptin] and then left on ice for 30 min with frequent agitation. Nuclear extracts were prepared by centrifugation at 15,000 g for 10 min. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA) with BSA as a standard.
Gel shift assay.
Nuclear extracts (5-10 µg) were incubated with
32P-labeled NF-B oligonucleotide probe (2 pmol, 50,000 cpm) in 20 µl of binding buffer [in mM: 10 HEPES-NaOH (pH 7.8), 50 KCl, 1 EDTA, 5 MgCl2, and 5 DTT with 100 µg/ml
poly(dI-dC)] at 30°C for 30 min. NF-
B consensus oligonucleotide
probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Madison, WI) was end
labeled with [
-32P]ATP (Amersham Pharmacia Biotech,
Little Chalfont, UK) and MEGALABEL (TaKaRa, Kyoto, Japan). The
specificity of the oligonucleotide probe-protein binding was determined
by competition reaction in which 200-fold excess of unlabeled NF-
B
probe or SP-1 probe (Promega) was added to the binding buffer.
Supershift assay was performed by adding anti-p65 subunit of NF-
B
antibody (Roche Diagnostics, Indianapolis, IN) or anti-activating
transcription factor (ATF)-2 antibody (New England Biolabs,
Beverly, MA). The DNA-protein complexes were subjected to nondenaturing
4% polyacrylamide gel electrophoresis. The gel was dried, and
detection of labeled complexes was carried out with an imaging analyzer
(BAS2000; Fuji Film, Tokyo, Japan).
Western blot analysis.
Gastric tissues taken from normal and ulcerated stomachs were
homogenized in 25 mM Tris · HCl (pH 8.0) buffer containing 250 mM sucrose, 1 mM EDTA, 0.5 mM PMSF, 2 µg/ml aprotinin, and 2 µg/ml leupeptin, and then COX proteins were partially purified as we previously described (26). Nuclear extracts and COX
proteins were subjected to SDS-PAGE (10%), and then the separated
proteins were electrophoretically transferred to Hybond P membranes
(Amersham Pharmacia Biotech) (28). The membranes were
incubated with anti-p65 subunit of NF-B antibody, anti-COX-1
antibody (Cayman), or anti-COX-2 antibody (Cayman) after nonspecific
binding sites had been blocked. Immunoreactive proteins were visualized
on X-ray films (Phototope-HRP Western Blot Detection kit; New England Biolabs).
Immunohistochemical analysis.
Histological sections of gastric tissues were incubated with anti-p65
subunit of NF-B antibody after deactivation of endogenous peroxidase
with 0.3% H2O2 and blocking of nonspecific
binding sites. This antibody only reacts to the activated NF-
B
because it binds to a p65 epitope that is exposed only after I-
B
dissociation (8). Activated NF-
B was visualized by the
avidin-biotin-peroxidase complex method (Vectastain ABC kit; Vector
Laboratories, Burlingame, CA), and then the sections were successively
stained with hematoxylin.
Determination of gastric acid secretion in rats. Gastric acid secretion on day 14 was determined by the pylorus ligation method. Two hours after the pylorus was ligated the gastric contents were collected, and then acidity was determined by titration of the contents against 100 mM NaOH to pH 7.0. Total acid output was calculated as volume × acidity and expressed as milliequivalents per hour.
Determination of PGE2 and CINC-1 production by gastric tissues of rats. PGE2 and CINC-1 production in the animals treated with PDTC for 7 days was determined. Normal and ulcerated gastric tissues were incubated at 37°C in 1 ml of DMEM supplemented with 5% FBS and the antibiotics for 1 h. The medium was subjected to enzyme immunoassay of PGE2. The levels of CINC-1 protein in the remaining tissues were also determined as we previously reported (33). PGE2 and CINC-1 production was expressed as picograms per milligram per hour.
RT-PCR. Total RNAs were extracted from gastric tissues with TRIzol Reagent (Life Technologies) (6). First-strand cDNAs were prepared from 1-3 µg of total RNAs with 150 units Moloney murine leukemia virus reverse transcriptase (Life Technologies) and 0.3 µg random hexamer oligonucleotides (TaKaRa). The DNAs were subjected to PCR with 0.625 units Taq DNA polymerase (Life Technologies). The primers were synthesized as we described previously (25), except for glyceraldehyde-3-phosphate dehydrogenase primers (5). After denaturation at 94°C for 5 min, PCR was performed for 22-25 cycles consisting of denaturation at 94°C for 45 s, annealing at 55-60°C for 45 s, and extension at 72°C for 30 s. The amplification was terminated by a 10-min final extension step at 72°C. The PCR products were subjected to 2% agarose gel electrophoresis and then visualized by ethidium bromide staining. The sequences of the PCR products were confirmed to be identical to known ones (with reference to the databases of GenBank and EMBL).
Materials.
Recombinant IL-1 (Otsuka Pharmaceutical, Tokushima, Japan) was
dissolved in DMEM. NS-398 (Calbiochem, La Jolla, CA) was dissolved in
dimethyl sulfoxide, followed by dilution with culture medium. The final
concentration of dimethyl sulfoxide was 0.5%, at which concentration
cell viability and PGE2 production were not affected. PDTC
(Nacalai Tesque, Kyoto, Japan) was dissolved in saline and administered
twice daily starting from day 0 for the indicated times. The
drug was administered orally in a volume of 5 ml/kg. Control animals
received saline alone. All other chemicals used here were of reagent grade.
Statistical analysis. The data are presented as means ± SE. Statistical differences in the dose-response studies were evaluated by Dunnett's multiple comparison test. Student's t-test was applied to comparisons between two groups. P values of <0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NF-B activation during gastric ulcer healing in
rats.
We examined whether NF-
B is activated in gastric tissues of rats.
There were round and deep ulcers in all animals on day 0,
and thereafter the ulcers healed spontaneously (Fig.
1A). Gel shift assay with
radiolabeled NF-
B-specific oligonucleotide probe revealed that
NF-
B activation was induced in ulcerated tissue (Fig.
1B). NF-
B activation was maintained during healing of
gastric ulcers, although the activation was reduced in a time-dependent manner. However, activated NF-
B was not detected in normal tissue. When the antibody against the p65 subunit of NF-
B was added to the
nuclear extract from ulcerated tissue on day 7, the
NF-
B-radiolabeled oligonucleotide probe complex was supershifted to
the upper band. On the other hand, anti-ATF-2 antibody (an
NF-
B-unrelated antibody) did not affect mobility of the
NF-
B-oligonucleotide probe complex (data not shown). In addition,
binding of the radiolabeled probe to NF-
B was abolished in the
presence of 200-fold molar excess of unlabeled probe. In contrast, the
complex formation was not affected by the same amount of an unrelated
oligonucleotide, SP-1 probe (data not shown). Activation of NF-
B was
also assessed by Western blotting (Fig. 1C). Similar to the
results with gel shift assay, activated NF-
B was detected only in
the nuclear extracts from ulcerated tissues, and the amount of
activated NF-
B decreased with ulcer healing.
|
|
NF-B-mediated expression of CINC-1
and COX-2 in response to IL-1
in rat
gastric fibroblasts.
We already found that endogenous IL-1 plays an important role in
gastric ulcer healing by upregulating the gene expression of
healing-promoting factors in rats (25). Therefore, we
isolated fibroblasts from ulcerated tissue and investigated the role of NF-
B in IL-1
-stimulated fibroblasts.
|
|
Prevention of gastric ulcer healing by persistent inhibition of
NF-B activation in rats.
We next examined the effect of PDTC on gastric ulcer healing in rats.
PDTC inhibits I-
B degradation, and the resultant NF-
B activation,
and has been widely used as an NF-
B inhibitor both in vitro and in
vivo (12, 19). PDTC (100 mg/kg) or vehicle was repeatedly
administered, and nuclear extracts from the ulcer bases were subjected
to gel shift assay (Fig. 5A).
The activation of NF-
B was potently inhibited by PDTC on day
7. The effect of PDTC on ulcer healing is shown in Fig.
5B. There was no difference of ulcerated area between
control and PDTC-treated groups on day 7. However, ulcer
healing was prevented by PDTC from day 10. On day
14, significant delay of ulcer healing caused by PDTC was observed
in a dose-dependent manner. In contrast, administration of 100 mg/kg
PDTC for 14 days did not cause any gastric lesions or ulcers in normal
rats.
|
|
Effect of NF-B inhibition on the
mRNA expression of CINC-1,
COX-2, and iNOS in ulcer healing.
We examined the effect of PDTC on the mRNA expression of CINC-1, COX-1,
COX-2, and iNOS in gastric ulcers by means of RT-PCR (Fig.
6A). Neither CINC-1, COX-2,
nor iNOS mRNAs were expressed in normal gastric mucosa, but their mRNA
expression was induced in ulcerated tissue on day 7.
Treatment with 100 mg/kg PDTC suppressed the induction of CINC-1,
COX-2, and iNOS mRNAs. COX-1 mRNA expression was observed in both
normal and ulcerated tissues, and the level was not affected by PDTC.
In addition, expression of COX-2 protein as well as its mRNA was also
induced in ulcerated tissue on day 7, whereas the expression
level of COX-1 protein in ulcerated tissue was similar to that in
normal mucosa (Fig. 6B). PGE2 production in
ulcerated tissue markedly increased by 3.4-fold compared with that in
normal mucosa (Fig. 6C). PDTC at 100 mg/kg inhibited the expression of COX-2 protein without affecting COX-1 protein level and
significantly reduced the increased PGE2 production. CINC-1 protein level was also significantly reduced by PDTC treatment (15.3 ± 3.4 pg · mg
1 · h
1 in the
control group and 6.7 ± 3.1 pg · mg
1 · h
1 in the 100 mg/kg PDTC group).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, NF-B is found to be activated in
ulcerated gastric tissue during a healing phase in rats. We had
confirmed that ulcer size reaches maximum and that granulation tissue
(ulcer base) is firmly formed until day 0 in the acetic
acid-induced gastric ulcer model. Thereafter, ulcer size decreases with
time. It is evident that NF-
B activation was evaluated during an
ulcer healing process (from day 0) but not during an ulcer
developing process (until day 0). Normal inflammatory
response to gastric ulceration is essential for mucosal repair and is
reduced with ulcer healing. There has been accumulating evidence that
inflammatory proteins, such as IL-1
, COX-2, iNOS, and CINCs, play
important roles in gastric ulcer healing (10, 13, 21, 25,
33). It is reasonable that NF-
B activation is maintained
until gastric ulcers are remarkably reduced.
The activation of NF-B was detected in fibroblasts,
monocytes/macrophages, and neutrophils in the ulcer base.
Immunoreactive NF-
B was observed both in the nucleus and the
cytosol. This is consistent with I-
B dissociation and NF-
B
activation in the cytosol and the subsequent nuclear translocation of
activated NF-
B (2, 11, 22). Gastric fibroblasts are a
major component of the ulcer base and produce PGE2,
cytokines, and growth factors, contributing to ulcer healing (3,
21, 23-27). The activation of NF-
B was confirmed in
IL-1
-stimulated cultured fibroblasts, because the activation
occurred in the fibroblasts in the ulcer base. Furthermore, our results
clearly indicate that, in response to IL-1
, NF-
B actually
functions as a positive regulator of CINC-1 and COX-2 gene expression
in gastric fibroblasts.
We found that NF-B might play a crucial role in gastric ulcer
healing in rats. Schmassmann et al. (18) and we
(21) reported that epithelial regeneration, maturation of
the ulcer base (reduction of the ulcer base size), angiogenesis, and
neutrophil infiltration are relevant events for efficient healing of
gastric ulcers. It is evident that NF-
B is implicated in all of the
above events. The mechanism of the NF-
B action in gastric ulcer
healing is discussed below. The present study revealed that NF-
B
might be involved in the gene expression of healing-promoting factors
such as COX-2, iNOS, and CINC-1 in gastric ulcers in rats as well as in
cultured gastric fibroblasts. Our group (21, 26) reported that COX-2 mRNA and protein are induced by gastric ulceration and that
the increased prostaglandins, derived from COX-2, have stimulatory
effects on epithelial regeneration, maturation of the base, and
angiogenesis. Konturek et al. (10) reported that NO might
maintain an increased blood flow at the ulcer margin and stimulate
angiogenesis in the ulcer base, contributing to gastric ulcer healing.
Most of the NOS activity in the ulcer base is due to iNOS activity
(25). These results strongly suggest that iNOS is also an
important factor for ulcer healing. CINC-1 exhibits potent neutrophil
chemotactic activity in vitro (20) and acts as a
functional chemoattractant for neutrophils in vivo (30,
31). Since CINCs belong to IL-8 chemokine family, and a rat
counterpart of human IL-8 has not been discovered to date, CINCs are
considered to play a predominant role in neutrophil infiltration in
rats (30, 31). The activation and infiltration of
neutrophils are essential for the elimination of wounded cells, cell
debris, and extracellular matrix proteins in tissue regeneration. It
was reported that CINC induces neovascularization in a rat corneal
pocket model and that NF-
B activation results in CINC production and
participates in the induction of retinal neovascularization in rats
(34). As we described previously (25, 33) and
here, CINC-1 mRNA and protein are induced in ulcerated gastric tissue. CINC-1 not only elicits neutrophil infiltration but also may enhance angiogenesis in the ulcer base. In addition, we recently reported that
IL-1 upregulates the gene expression of COX-2, iNOS, and CINC-1 in the
ulcer base, and the inhibition of IL-1 action causes an impairment of
ulcer healing in rats (25). This, together with in vitro
data, indicates that NF-
B might partly mediate IL-1 signaling to
induce these factors.
Administration of PDTC for more than 10 days prevented gastric ulcer
healing, although the drug did not affect the healing on day 7 when NF-B activation was inhibited. These findings are similar
to COX inhibitor-delayed healing (21). Neither
indomethacin nor NS-398 affect ulcer healing in early phase (by
day 7), but PGE2 production in ulcerated tissue
has been markedly inhibited since day 0. It is considered
that there is a lag period in which the delay of ulcer healing has not
yet become manifest, because the persistent inhibition of tissue repair
responses of cells results in visible healing impairment. In addition,
NF-
B inhibition suppresses new production of the healing-promoting
factors but has no influence on the levels of the preexisting ones. The
lag period might be attributable partly to the remaining factors.
NF-B decoy oligonucleotide and PDTC potently inhibited NF-
B
activation in IL-1
-stimulated gastric fibroblasts and ulcerated tissue, respectively, but their inhibitory effects on the expression of
healing-promoting factors were weaker than those on NF-
B activation. Since it is known that several transcription factors coordinately upregulate them, it is suggested that other pathways as well as the
NF-
B pathway might be important in their expression in gastric fibroblasts and ulcers. Actually, we have recently found that a
mitogen-activated protein kinase pathway is also involved in the
induction of these factors in gastric ulcers (Takahashi et al.,
unpublished data).
In contrast to the present results, it has been accepted that NF-B
plays a pivotal role in the pathogenesis of gastric mucosal inflammation and injury caused by Helicobacter pylori
infection. NF-
B mediates Helicobacter pylori-induced
IL-8 expression in gastric epithelial cells (8). Thus
NF-
B has both favorable and harmful actions in the gastric mucosa.
The bilateral actions are similarly observed in the case of IL-1. IL-1
provides protection of the gastric mucosa against various noxious
stimuli (16, 29) and might contribute to gastric ulcer
healing (25). On the other hand, IL-1 is responsible for
inflammation and injury in Helicobacter pylori-infected
mucosa (4, 7) and causes recurrence of healed gastric
ulcers (32). Factors controlling the roles of NF-
B and
IL-1 remain unclear, but it is likely that NF-
B and IL-1 exert
injurious effects on the gastric mucosa when severe inflammation is
sustained. In the case of ulcer healing delayed by indomethacin, severe
inflammation is persistently observed (1, 33). Sustained severe inflammation disrupts epithelial functions and therefore is
deleterious to the gastric mucosa (1, 4, 33). In
conclusion, NF-
B, activated in fibroblasts, monocytes/macrophages,
and neutrophils in the ulcer base, might upregulate the expression of
healing-promoting factors, contributing to gastric ulcer healing in rats.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Senye Takahashi (Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan) and Yoshimitsu Nakajima (Fujisawa Pharmaceutical, Tsukuba, Japan) for assisting with gel shift assays and histological studies, respectively.
![]() |
FOOTNOTES |
---|
This research was partly supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan [Grant-in-Aid for Scientific Research (B) #11470490].
Address for reprint requests and other correspondence: S. Takahashi, Dept. of Biopharmaceutics, Kyoto Pharmaceutical Univ., Misasagi, Yamashina, Kyoto 607-8414, Japan (E-mail: takahasi{at}mb.kyoto-phu.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 May 2000; accepted in final form 22 January 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arakawa, T,
Watanabe T,
Fukuda T,
Higuchi K,
Takaishi O,
Yamasaki K,
Kobayashi K,
and
Tarnawski A.
Indomethacin treatment during initial period of acetic acid-induced rat gastric ulcer healing promotes persistent polymorphonuclear cell-infiltration and increases future ulcer recurrence: possible mediation of prostaglandins.
Dig Dis Sci
41:
2055-2061,
1996[ISI][Medline].
2.
Baldwin, AS, Jr.
The NF-B and I-
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-681,
1996[ISI][Medline].
3.
Bamba, H,
Ota S,
Kato A,
and
Matsuzaki F.
Nonsteroidal anti-inflammatory drugs may delay the repair of gastric mucosa by suppressing prostaglandin-mediated increase of hepatocyte growth factor production.
Biochem Biophys Res Commun
245:
567-571,
1998[ISI][Medline].
4.
Blaser, MJ.
Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation.
Gastroenterology
102:
720-727,
1992[ISI][Medline].
5.
Borenstein, P,
Froissart M,
Laghmani K,
Bichara M,
and
Paillard M.
RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1224-F1228,
1995
6.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid-guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
7.
Crabtree, JE.
Role of cytokines in pathogenesis of Helicobacter pylori-induced mucosal damage.
Dig Dis Sci
43:
46S-55S,
1998[ISI][Medline].
8.
Keates, S,
Hitti YS,
Upton M,
and
Kelly CP.
Helicobacter pylori infection activates NF-B in gastric epithelial cells.
Gastroenterology
113:
1099-1109,
1997[ISI][Medline].
9.
Kinoshita, Y,
Nakata H,
Hassan S,
Asahara M,
Kawanami C,
Matsushima Y,
Naribayashi-Imoto Y,
Ping CY,
Min D,
Nakamura A,
and
Chiba T.
Gene expression of keratinocyte and hepatocyte growth factors during the healing of rat gastric mucosal lesions.
Gastroenterology
109:
1068-1077,
1995[ISI][Medline].
10.
Konturek, SJ,
Brzozowski T,
Majka J,
Pytko-Polonczyk J,
and
Stachura J.
Inhibition of nitric oxide synthase delays healing of chronic gastric ulcers.
Eur J Pharmacol
239:
215-217,
1993[ISI][Medline].
11.
Lenardo, MJ,
and
Baltimore D.
NF-B: a pleiotropic mediator of inducible and tissue-specific gene control.
Cell
58:
227-229,
1989[ISI][Medline].
12.
Liu, SF,
Ye X,
and
Malik AB.
Pyrrolidine dithiocarbamate prevents I-B degradation and reduces microvascular injury induced by lipopolysaccharide in multiple organs.
J Pharmacol Exp Ther
55:
658-667,
1999.
13.
Mizuno, H,
Sakamoto C,
Matsuda K,
Wada K,
Uchida T,
Noguchi H,
Akamatsu T,
and
Kasuga M.
Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice.
Gastroenterology
112:
387-397,
1997[ISI][Medline].
14.
Morishita, R,
Sugimoto T,
Aoki M,
Kida I,
Tomita N,
Moriguchi A,
Maeda K,
Sawa Y,
Kaneda Y,
Higaki J,
and
Ogihara T.
In vivo transfection of cis element "decoy" against nuclear factor-B binding site prevents myocardial infarction.
Nat Med
3:
894-899,
1997[ISI][Medline].
15.
Penney, AG,
Malcontenti-Wilson C,
O'Brien PE,
and
Andrews FJ.
NSAID-induced delay in gastric ulcer healing is not associated with decreased epithelial cell proliferation in rats.
Dig Dis Sci
40:
2684-2693,
1995[ISI][Medline].
16.
Robert, A,
Olafsson AS,
Lancaster C,
and
Zhang WR.
Interleukin-1 is cytoprotective, antisecretory, stimulates PGE2 synthesis by the stomach, and retards gastric emptying.
Life Sci
48:
123-134,
1991[ISI][Medline].
17.
Roshak, AK,
Jackson JR,
McGough K,
Chabot-Fletcher M,
Mochan E,
and
Marshall LA.
Manipulation of distinct NF-B proteins alters interleukin-1
-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.
J Biol Chem
271:
31496-31501,
1996
18.
Schmassmann, A,
Tarnawski A,
Peskar BM,
Varga L,
Flogerzi B,
and
Halter F.
Influence of acid and angiogenesis on kinetics of gastric ulcer healing in rats: interaction with indomethacin.
Am J Physiol Gastrointest Liver Physiol
268:
G276-G285,
1995
19.
Schreck, BR,
Meier B,
Männel DN,
Dröge W,
and
Baeuerle PA.
Dithiocarbamates as potent inhibitors of nuclear factor B activation in intact cells.
J Exp Med
175:
1181-1194,
1992[Abstract].
20.
Shibata, F,
Konishi K,
Kato H,
Komorita N,
Al-Mokdad M,
Fujioka M,
and
Nakagawa H.
Recombinant production and biological properties of rat cytokine-induced neutrophil chemoattractants, GRO/CINC-2, CINC-2
and CINC-3.
Eur J Biochem
231:
306-311,
1995[Abstract].
21.
Shigeta, J,
Takahashi S,
and
Okabe S.
Role of cyclooxygenase-2 in the healing of gastric ulcers in rats.
J Pharmacol Exp Ther
286:
1383-1390,
1998
22.
Siebenlist, U,
Franzoso G,
and
Brown K.
Structure, regulation and function of NF-B.
Annu Rev Cell Biol
10:
405-455,
1994[ISI].
23.
Suzuki, N,
Takahashi S,
and
Okabe S.
Relationship between vascular endothelial growth factor and angiogenesis in spontaneous and indomethacin-delayed healing of acetic acid-induced gastric ulcers in rats.
J Physiol Pharmacol
49:
515-527,
1998[ISI][Medline].
24.
Takahashi, M,
Ota S,
Hata Y,
Mikami Y,
Azuma N,
Nakamura T,
Terano A,
and
Omata M.
Hepatocyte growth factor as a key to modulate anti-ulcer action of prostaglandins in stomach.
J Clin Invest
98:
2604-2611,
1996
25.
Takahashi, S,
Kobayashi N,
and
Okabe S.
Regulation by endogenous interleukin-1 of mRNA expression of healing-related factors in gastric ulcers in rats.
J Pharmacol Exp Ther
291:
634-641,
1999
26.
Takahashi, S,
Shigeta J,
Inoue H,
Tanabe T,
and
Okabe S.
Localization of cyclooxygenase-2 and regulation of its mRNA expression in gastric ulcers in rats.
Am J Physiol Gastrointest Liver Physiol
275:
G1137-G1145,
1998
27.
Tominaga, K,
Arakawa T,
Kim S,
Iwao H,
and
Kobayashi K.
Increased expression of transforming growth factor-1 during gastric ulcer healing in rats.
Dig Dis Sci
42:
616-625,
1997[ISI][Medline].
28.
Towbin, H,
Staehelin T,
and
Gordon J.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354,
1979[Abstract].
29.
Uehara, A,
Okumura T,
Kitamori S,
Takasugi Y,
and
Namiki M.
Interleukin-1: a cytokine that has potent antisecretory and anti-ulcer actions via the central nervous system.
Biochem Biophys Res Commun
173:
585-590,
1990[ISI][Medline].
30.
Utsunomiya, I,
Ito M,
Watanabe K,
Tsurufuji S,
Matsushima K,
and
Oh-ishi S.
Infiltration of neutrophils by intrapleural injection of tumour necrosis factor, interleukin-1, and interleukin-8 in rats, and its modification by actinomycin D.
Br J Pharmacol
117:
611-614,
1996[Abstract].
31.
Watanabe, K,
Iida M,
Takaishi K,
Suzuki T,
Hamada Y,
Iizuka Y,
and
Tsurufuji S.
Chemoattractants for neutrophils in lipopolysaccharide-induced inflammatory exudate from rats are not interleukin-8 counterparts but gro-gene-product/melanoma-growth stimulating-activity-related factors.
Eur J Biochem
214:
267-270,
1993[Abstract].
32.
Watanabe, T,
Arakawa T,
Fukuda T,
Higuchi K,
and
Kobayashi K.
Role of neutrophils in a rat model of gastric ulcer recurrence caused by interleukin-1.
Am J Pathol
150:
971-979,
1997[Abstract].
33.
Yamada, H,
Takahashi S,
Fujita H,
Kobayashi N,
and
Okabe S.
Cytokine-induced neutrophil chemoattractants in healing of gastric ulcers in rats: expression of >40-kDa chemoattractant in delayed ulcer healing by indomethacin.
Dig Dis Sci
44:
889-895,
1999[ISI][Medline].
34.
Yoshida, A,
Yoshida S,
Hata Y,
Khalil AK,
Ishibashi T,
and
Inomata H.
The role of NF-B in retinal neovascularization in the rat: possible involvement of cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 family.
J Histochem Cytochem
46:
429-436,
1998