1 Departments of Internal Medicine and of Integrative Biology and Pharmacology, 2 Department of Surgery, and 3 Trauma Research Center, The University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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Mesenteric
ischemia-reperfusion injury is a serious complication of shock.
Because activation of nuclear factor-B (NF-
B) has been implicated
in this process, we treated rats with vehicle or the I
B-
inhibitor BAY 11-7085 (25 mg/kg ip) 1 h before mesenteric ischemia-reperfusion (45 min of ischemia followed by
reperfusion at 30 min or 6 h) and examined the ileal injury
response. Vehicle-treated rats subjected to
ischemia-reperfusion exhibited severe mucosal injury, increased
myeloperoxidase (MPO) activity, increased expression of interleukin-6
and intercellular adhesion molecule 1 protein, and a biphasic peak of
NF-
B DNA-binding activity during the 30-min and 6-h reperfusion
courses. In contrast, BAY 11-7085-pretreated rats subjected to
ischemia-reperfusion exhibited less histological injury and
less interleukin-6 and intercellular adhesion molecule 1 protein
expression at 30 min of reperfusion but more histological injury at
6 h of reperfusion than vehicle-treated rats subjected to
ischemia-reperfusion. Studies with phosphorylation
site-specific antibodies demonstrated that I
B-
phosphorylation at
Ser32,Ser36 was induced at 30 min of
reperfusion, whereas tyrosine phosphorylation of I
B-
was induced
at 6 h of reperfusion. BAY 11-7085 inhibited the former, but not
the latter, phosphorylation pathway, whereas
-melanocyte-stimulating
hormone, which is effective in limiting late
ischemia-reperfusion injury to the intestine, inhibited
tyrosine phosphorylation of I
B-
. Thus NF-
B appears to play an
important role in the generation and resolution of intestinal
ischemia-reperfusion injury through different activation pathways.
ileum; inflammation; ischemic bowel; transcription factor; ileus
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INTRODUCTION |
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MESENTERIC
ISCHEMIA-REPERFUSION is an often-fatal clinical problem
that complicates septic, cardiogenic, and hemorrhagic shock, vascular
surgery, and small bowel transplantation. Mesenteric ischemia-reperfusion causes histological evidence of mucosal
injury and gut dysfunction characterized by increased intestinal
epithelial permeability and impaired motility (12).
Ischemia-reperfusion injury evokes a molecular and cellular
inflammatory response within the intestine, including activation of the
transcription factors nuclear factor-B (NF-
B) and signal
transducer and activator of transcription 3, induction of granulocyte
colony-stimulating factor and interleukin (IL)-6, and recruitment of
neutrophils and monocytes into the intestinal muscularis (12, 15,
19, 31, 34, 38). Several molecular mediators have been
implicated in mesenteric ischemia-reperfusion injury, including
cytokines (6), reactive oxygen species (10),
nitric oxide (33), arachidonic acid derivatives
(26), and cell adhesion molecules (27).
NF-B is known to be activated in the gut by a number of
proinflammatory stimuli, including sepsis (7), cytokines
(29), and oxidative stress (3). NF-
B
activation leads to the coordinated expression of many genes that
encode proteins involved in mediator synthesis and the further
amplification and perpetuation of the inflammatory response, including
proinflammatory cytokines, adhesion molecules, chemokines, growth
factors, and inducible enzymes such as cyclooxygenase-2 and inducible
nitric oxide synthase (9). Consequently, NF-
B is an
obvious target for anti-inflammatory treatment. Pharmacological agents
that inhibit NF-
B include glucocorticoids (1),
antioxidants (37), certain cyclooxygenase inhibitors (35), proteosome and calpain inhibitors (23),
and inhibitors of I
B-
phosphorylation (28).
NF-B is usually kept inactive in the cytoplasm through association
with a member of the I
B family. The most well-characterized pathway
of NF-
B activation involves phosphorylation of I
B-
on
Ser32,Ser36 by the I
B kinase complex. This
phosphorylation event targets I
B-
for ubiquitination and
degradation by the ubiquitin-26S proteasome pathway, allowing nuclear
transmigration of NF-
B, where the transcription factor binds to
specific DNA sequences located in the promoter regions of many
proinflammatory genes (9, 18, 30, 36). A wide array of
stimuli, including cytokines, endotoxin, and phorbol esters, activates
this I
B-
phosphorylation-ubiquitination-degradation pathway. Two
additional activation pathways have been reported. One results from
oxidant stress (21, 25, 32) or pervanadate treatment
(17) and appears to require phosphorylation of I
B-
at Tyr42. The dissociation of tyrosine-phosphorylated
I
B-
from NF-
B is not the result of degradation by the 26S
proteasome (17). This pathway appears to be specific for
I
B-
, because Tyr42 is not conserved in other I
B
family members. The exact protein tyrosine kinase(s) or phosphatase(s)
involved in this pathway remains unknown, but members of the Src family
have been implicated. Moreover, this activation pathway is well
characterized only in lymphocytes, and there are no data for the ileum
in vivo. The second atypical scheme for NF-
B activation occurs in
cells exposed to short-wavelength ultraviolet radiation and involves
induction of I
B-
degradation by the 26S proteasome, without
phosphorylation of Ser32,Ser36 or
Tyr42 (4, 24). The mechanisms for this
alternative pathway of I
B-
degradation are unknown. Both of these
alternative pathways yield lower levels of NF-
B activation than have
been observed in response to classical activators such as cytokines or endotoxin.
Work from our laboratory (13) and others (15,
38) in experimental animal models has established that NF-B
is activated in the small intestine after mesenteric
ischemia-reperfusion. We showed that mesenteric
ischemia-reperfusion results in a biphasic activation pattern
of NF-
B DNA-binding activity in the postischemic rat ileum
during reperfusion. After 45 min of mesenteric ischemia, the first peak of NF-
B DNA-binding activity occurred at 30 min of
reperfusion, followed by lower levels of activity and a second peak after 6 h of reperfusion. We further demonstrated that
exogenous administration of the anti-inflammatory neuropeptide
-melanocyte-stimulating hormone (
-MSH) before mesenteric
ischemia-reperfusion protected intestinal transit and
morphology and reduced ischemia-reperfusion-induced activation
of intestinal NF-
B after prolonged (6 h) reperfusion (13). This association of reduced late NF-
B activity
and protection against mesenteric ischemia-reperfusion injury
suggested that targeted inhibition of NF-
B at the onset of
mesenteric ischemia-reperfusion might prove protective.
Accordingly, in the present report, we tested the in vivo effects of
[(E)3-(4-t-butylphenylsulfonyl)-2-propenenitrile] (BAY 11-7085), a compound that inhibits NF-
B activation by blocking inducible I
B-
phosphorylation at
Ser32,Ser36 (28), on mesenteric
ischemia-reperfusion injury, NF-
B activation, MPO activity
as an index of neutrophil influx, and expression of cytokine and
adhesion molecule genes known to be upregulated in intestinal
ischemia-reperfusion injury. This compound has been shown to inhibit
NF-
B activation and to exert anti-inflammatory activity in vivo in
several models of inflammation (20, 22, 28). Our results
indicate a dual effect of the NF-
B inhibitor to abrogate early
intestinal ischemia-reperfusion injury but exacerbate ischemia-reperfusion injury at later stages of reperfusion. These distinct phases coincide with NF-
B activation through different I
B-
phosphorylation pathways. The data suggest that NF-
B may play a role not only in the initiation of mesenteric
ischemia-reperfusion injury but also in a late reparative
process that at least partly depends on the specific mechanisms of
I
B-
phosphorylation.
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MATERIALS AND METHODS |
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Animal model.
Male Sprague-Dawley rats (Harlan Labs, Houston, TX; 250-350 g body
wt) were cared for in accordance with the guidelines of The University
of Texas Medical School at Houston Animal Welfare Committee. The rats
were fed standard rat chow and water ad libitum. Operative
procedures were performed using standard sterile technique under
general anesthesia with inhaled isoflurane. The animals were fasted for
18 h before the operative procedures. The procedures for superior
mesenteric artery (SMA) occlusion (45 min) and sham surgery were as
previously described for our laboratory (13). BAY 11-7085 (25 mg/kg; Biomol) or an equal volume of vehicle (polyethylene glycol
400 diluted 1:5 in 5% bovine serum albumin/H2O) was
administered intraperitoneally before the SMA was clamped. This
concentration of BAY 11-7085 has been shown to be effective in other in
vivo models of inflammation (20, 22, 28). The rats were
killed 30 min or 6 h after release of the SMA clamp or sham
surgery. Histology, NF-B DNA-binding activity, ICAM-1 and IL-6
expression, MPO activity, and immunoblots or immunoprecipitations for
phosphorylated I
B-
at Ser32,Ser36 or
tyrosine residues were then determined. Thus the following groups of
animals were studied: sham surgery with ischemia followed by
reperfusion at 30 min [sham + I/R (30 min)], vehicle with
ischemia followed by reperfusion at 30 min [Veh + I/R (30 min)], BAY 11-7085 treatment with ischemia followed by
reperfusion at 30 min [BAY + I/R (30 min)], vehicle with
ischemia followed by reperfusion at 6 h [Veh + I/R
(6 h)], and BAY 11-7085 treatment with ischemia followed by
reperfusion at 6 h [BAY + I/R (6 h)].
Histological assessment of injury. In a separate set of experiments, biopsies of the distal ileum were taken from sham-operated animals and animals subjected to mesenteric ischemia-reperfusion treated with vehicle or BAY 11-7085 after 45 min of SMA occlusion and 30 min or 6 h of reperfusion. Tissues were fixed with 10% neutral-buffered formalin and processed by routine techniques before they were embedded in paraffin wax. Sections (4 µm) were stained with hematoxylin and eosin and examined under a light microscope. For histological assessment of intestinal injury, we graded the injury on a scale of 0-4, where 0 is normal histology, 1 is slight disruption of the surface epithelium, 2 is epithelial cell loss injury at villus tip, 3 is mucosal vasocongestion, hemorrhage, and focal necrosis with loss of less than one-half of villi, and 4 is damage extending to more than one-half of villi. All processed tissues were examined under light microscopy by two blinded, experienced observers, and the scores of the observers were averaged.
Electrophoretic mobility shift and supershift assays.
Nuclear and cytoplasmic extracts from full-thickness ileal tissue were
prepared by the method of Deryckere and Gannon (8) as
described previously (13). The DNA-binding activity of
NF-B in ileal nuclear extracts was determined by electrophoretic
mobility shift assay (EMSA). The NF-
B consensus oligonucleotide
5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Promega, Madison, WI) was end
labeled with [
-32P]ATP using T4 polynucleotide kinase.
Nuclear extract (10 µg) was then incubated for 20 min with gel shift
binding buffer [10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 1 µg poly(dI-dC)] and 1 µl of labeled
probe. For competition assays, a 100-fold molar excess of unlabeled
NF-
B oligonucleotide was added to the binding reaction. For
supershift assays, 2 µl of antibody to NF-
B subunit p50 or p65
(Santa Cruz Biotechnology, Santa Cruz, CA) were added before
addition of the labeled probe. Gel loading buffer was added to the
mixture, and the samples were electrophoresed on a nondenaturing 5%
polyacrylamide gel. The gels were then dried and analyzed by autoradiography.
Western blotting and immunoprecipitation.
For Western blots, cytoplasmic extracts were directly resuspended in
SDS sample buffer, boiled for 5 min, and analyzed on 7.5% or 10%
polyacrylamide gels as described previously. The proteins were
electrophoretically transferred to polyvinylidene difluoride membranes
(Hybond ECL, Amersham) and probed with antibodies against IL-6, ICAM-1,
IB-
, or Ser32,Ser36-phosphorylated
I
B-
(all from Santa Cruz Biotechnology) overnight at 4°C. The
blots were washed extensively with a solution containing 50 mM Tris, pH
8.0, 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20. The antigen-antibody
complexes were detected by the enhanced chemiluminescence protocol
using horseradish peroxidase-conjugated IgG secondary antibody.
Tyrosine-phosphorylated proteins were immunoprecipitated from
cytoplasmic extracts with A/G agarose together with an
antiphosphotyrosine antibody (Santa Cruz Biotechnology) by methods
routinely used in our laboratory (39). Protein blots of
the immunoprecipitates were prepared, probed with anti-I
B-
antibodies, and processed for enhanced chemiluminescence detection as
described above.
MPO assay. Cytoplasmic extracts from full-thickness ileal tissue were diluted 1:5 in buffer A (0.6% Nonidet P-40, 150 mM NaCl, 10 mM HEPES, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 30 µl/ml protease inhibitor cocktail). Ten microliters of each sample were then added to wells of 96-well plates and incubated with 100 µl of tetramethylbenzidine peroxidase substrate (KPL, Gaithersburg, MD) at room temperature for 20 min. The reaction was stopped with 100 µl of 0.18 M sulfuric acid. Optical density was measured at 450 nm with an ELISA plate reader. Assays were performed in duplicate, and the results were normalized for protein content.
Statistical analysis. Quantitative data are expressed as means ± SE and were analyzed with one-way analysis of variance (ANOVA) or t-test as appropriate. P < 0.05 was considered significant.
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RESULTS |
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BAY 11-7085 protects against early, but exacerbates late,
mesenteric ischemia-reperfusion injury in ileum.
Representative hematoxylin-and-eosin-stained sections of ileum
from Veh + I/R (30 min), Veh + I/R (6 h), BAY + I/R
(30 min), and BAY + I/R (6 h) rats are depicted in Fig.
1A. The degree of ischemia-reperfusion-induced mucosal damage expressed as a
microscopic damage score is shown in Fig. 1B. In the
sham-operated group, microscopic assessment of the tissue revealed no
significant abnormalities. In agreement with our earlier report
(13), mesenteric ischemia-reperfusion caused
significant epithelial abnormalities as well as infiltration of
inflammatory cells into the villi (Fig. 1). Pretreatment with BAY
11-7085 compared with vehicle + mesenteric
ischemia-reperfusion reduced the
ischemia-reperfusion-associated increase in the microscopic damage score at 30 min of reperfusion (Fig. 1). At 6 h of
reperfusion, vehicle-treated mesenteric rats subjected to
ischemia-reperfusion exhibited more prominent neutrophil
infiltration and capillary congestion, but injury was significantly
less than in Veh + I/R (30 min) rats (Fig. 1). In BAY + I/R
(6 h) rats, these signs of tissue injury and inflammation were much
more severe (Fig. 1). Thus NF-B inhibition by BAY 11-7085 was
beneficial in the initial response to ischemia-reperfusion
injury but exacerbated injury at later phases of reperfusion.
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BAY 11-7085 inhibits early IL-6 and ICAM-1 expression in ileum
after mesenteric ischemia-reperfusion.
IL-6 is known to be induced in the gut early in the
postischemic phase and to participate in the inflammatory
response (15). ICAM-1 is an adhesion molecule that is
induced by various inflammatory mediators and plays an important role
in gut ischemia-reperfusion-induced mucosal dysfunction and
neutrophil infiltration (5, 34). Both of these proteins
are regulated in part by NF-B. To determine whether BAY 11-7085 inhibition of NF-
B modulated the expression of these molecules,
immunoblot analysis of IL-6 and ICAM-1 expression in cytoplasmic
extracts from full-thickness ileal tissues was performed in the various
groups of animals. IL-6 was induced nearly twofold after mesenteric
ischemia and 30 min of reperfusion but returned to its low
basal level by 6 h of reperfusion (Fig.
2,A and B). BAY
11-7085 pretreatment resulted in IL-6 expression levels approximating
that of the sham controls at 30 min and 6 h of reperfusion (Fig.
2, A and B). ICAM-1 protein was induced in the
Veh + I/R (30 min) rats to levels ~30% greater than in sham
controls and remained elevated at 6 h of reperfusion compared with
sham (Fig. 2, C and D). BAY 11-7085 completely
inhibited the ischemia-reperfusion-induced expression of ICAM-1
at 30 min, but not at 6 h, of reperfusion (Fig. 2, C
and D).
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BAY 11-7085 inhibits early, but not late, induction of ileal
MPO activity after mesenteric ischemia-reperfusion.
Neutrophil infiltration occurs early in the course of mesenteric
ischemia-reperfusion injury. To assess neutrophil infiltration, MPO activity in ileal homogenates was measured. Our previous study showed that MPO activity in ileal samples harvested from rats subjected
to mesenteric ischemia and 6 h of reperfusion was
significantly increased compared with that in samples from sham
controls (13). BAY 11-7085-pretreated rats subjected to
ischemia-reperfusion exhibited lower levels of MPO activity at
30 min, but not at 6 h, of reperfusion than vehicle-treated rats
subjected to ischemia-reperfusion at the same time points (Fig.
3). There was a trend toward greater MPO
activity in BAY + I/R (6 h) than in Veh + I/R (6 h) animals, but this did not achieve statistical significance. These MPO data are
consistent with the effects of BAY 11-7085 on ICAM-1 expression after
mesenteric ischemia-reperfusion (Fig. 2).
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Differential responses of BAY 11-7085 and -MSH in I
B-
phosphorylation pathways.
On the basis of our findings in the present report that BAY 11-7085 inhibited I
B-
loss and NF-
B activation at 30 min, but not at
6 h, of reperfusion and our earlier report that
-MSH inhibited NF-
B activity induced at 6 h of reperfusion (13),
we hypothesized that the two agents might be acting on different
signaling cascades that converge on NF-
B activation. Accordingly,
using cytosolic extracts of ileum from rats subjected to
ischemia-reperfusion, we studied I
B-
phosphorylation at
Ser32,Ser36 by Western blot analysis with
antibodies raised against I
B-
phosphorylated at these residues,
analyzed tyrosine phosphorylation of I
B-
by immunoprecipitation
with an antiphosphotyrosine antibody, and analyzed the
immunoprecipitated proteins with anti-I
B-
by Western blot.
I
B-
phosphorylation at Ser32,Ser36 was
strongly induced at 30 min, but not at 6 h, of reperfusion (Fig.
6A), consistent with our data
concerning I
B-
degradation and NF-
B activation. In contrast,
rats pretreated with BAY 11-7085 exhibited no increase in
immunoreactivity for I
B-
phosphorylation at
Ser32,Ser36 induction at either time point
(Fig. 6A). Immunoreactivity for tyrosine-phosphorylated
I
B-
was detected principally at 6 h of reperfusion; little
immunoreactivity was detected at 30 min of reperfusion (Fig.
6B). BAY 11-7085 pretreatment had no inhibitory effect on
tyrosine phosphorylation of I
B-
, as would be expected from the
mechanism of action of the drug, at either time point (Fig.
6B). We also detected expression of tyrosine-phosphorylated I
B-
in ileum of sham animals, suggesting that this pathway may in
part be responsible for the constitutive expression of NF-
B that has
been observed by us and others.
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DISCUSSION |
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We used BAY 11-7085, an irreversible inhibitor of IB-
phosphorylation on Ser32,Ser36, to test in vivo
the hypotheses that signaling through NF-
B mediates, at least in
part, mesenteric ischemia-reperfusion injury in the rat and
that blockade of NF-
B activation would abrogate postischemic
gut injury. In the aggregate, our data suggest that NF-
B activation
by the classical pathway of serine phosphorylation of I
B-
plays
an important role in the injury response of the ileum during the early
reperfusion period following mesenteric ischemia. In addition,
induction of this transcription factor via tyrosine phosphorylation of
I
B-
appears to play a role in sustaining or aggravating injury
cascades and/or blocking inflammatory resolution at later
postischemic time points, because inhibition of this pathway by
-MSH (Fig. 6) also protected against late postischemic
injury (13). Thus NF-
B activation, mediated by different phosphorylation pathways, is associated with the onset and
aggravation or perpetuation of postischemic injury in this model. These results contrast with the reciprocal roles of NF-
B postulated in other in vivo studies of the effects of NF-
B
inhibition on inflammation. In rat carrageenin pleurisy and mouse
carrageenin air pouch models, NF-
B activation was associated with
the onset and resolution of inflammation (22). During
resolution, however, NF-
B activity was not associated with the
release of proinflammatory mediators but rather with the expression of
endogenous anti-inflammatory pathways and leukocyte apoptosis.
Inhibition of NF-
B during the resolution of inflammation protracted
the inflammatory response, prevented clearance of leukocytes, and
aggravated injury (22). The mechanistic basis for the
different phases of NF-
B activation and, specifically, of I
B-
phosphorylation pathways in these models, however, was not established.
Our data suggest that differential signaling via distinct
phosphorylation events to NF-
B may underlie specific phases of
postischemic injury and repair in the intestine.
Other factors may be responsible for the fact that IB-
loss was
much less at the later stage of reperfusion. It may be that ischemia-reperfusion in the later phase (6 h) of reperfusion
disrupts the ATP-dependent ubiquitin-dependent proteosome pathway,
which degrades phosphorylated I
B-
, so that any I
B-
that
escapes inhibition by BAY 11-7085 and is phosphorylated would tend to accumulate. Indeed, ATP-dependent reassociation of the 20S catalytic and PA700 regulatory subunits to form the active 26S proteasome is
severely, specifically, and irreversibly impaired in the hippocampus after transient forebrain ischemia (2). These
possibilities and others require further investigation.
The detrimental effects of BAY 11-7085 treatment on
postischemic injury at 6 h of reperfusion contrast with
the protective effect of -MSH on mesenteric
ischemia-reperfusion injury at this time point of reperfusion
we previously reported (13). Although BAY 11-7085 inhibited NF-
B activation more significantly early than late in the
reperfusion period,
-MSH did the opposite, inhibiting late, but not
early, NF-
B activation. Similarly, BAY 11-7085 inhibited early, but
not late, MPO induction, whereas
-MSH inhibited late MPO induction
(early time points were not assessed in that study). The fact that
-MSH blocked only late tyrosine phosphorylation of I
B-
,
whereas BAY 11-7085 inhibited only early
Ser32,Ser36 phosphorylation of I
B-
,
provides a mechanistic basis for these discrepant results. However,
because
-MSH exerts many other effects though signaling pathways
other than NF-
B, other mechanisms could be operative. Our results
showing that
-MSH did not alter I
B-
degradation rates are in
agreement with studies in two cutaneous melanoma cell lines where the
neuropeptide was found to inhibit NF-
B activation without affecting
I
B-
protein levels (14). However, work in glioma
cells and pulmonary epithelial cells (16) suggested that
-MSH inhibited I
B-
degradation and, consequently, NF-
B
activation. Indeed, we previously demonstrated cell type-specific mechanisms by which
-MSH inhibits transcriptional activation of
proinflammatory genes (11).
As in most cases of pharmacological inhibition, NF-B inhibitors may
have some nonspecific effects independent of the NF-
B pathway.
However, the use of inhibitors allows modulation of NF-
B at specific
stages of the inflammatory response in vivo, which is central to the
findings reported here. Unfortunately, however, available molecular
tools do not allow modulation of the NF-
B pathway at different
stages of the inflammatory response in the gut in vivo. The present
study indicates that the course of mesenteric ischemia-reperfusion injury in this model can be modulated by pharmacological interference with NF-
B activation and that the therapeutic impact of such inhibition on the gut critically depends on
the duration of reperfusion and the specific signaling cascades to
NF-
B activation. These results also lead to the prediction that
combined treatment with BAY 11-7085 and
-MSH would block both phases
of NF-
B activation and provide maximal protection from
ischemia-reperfusion injury to the ileum.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Tri Phan and the graphic assistance of Max Turk are gratefully acknowledged.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants RO1-DK-50745 and P50-GM-38529 and Department of Defense Disaster Relief and Emergency Medical Services grant to B. C. Kone.
Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, 6431 Fannin, MSB 4.138, Houston, TX 77030 (E-mail: Bruce.C.Kone{at}uth.tmc.edu).
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.
First published December 4, 2002;10.1152/ajpgi.00431.2002
Received 10 October 2002; accepted in final form 2 December 2002.
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