Departments of 1 Surgery, 2 Internal Medicine, and 3 Integrative Biology and Pharmacology, and 4 Trauma Research Center, University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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Mesenteric
ischemia-reperfusion (I/R) injury to the intestine is a common
and often devastating clinical occurrence for which there are few
therapeutic options. -Melanocyte-stimulating hormone (
-MSH) is a
tridecapeptide released by the pituitary gland and immunocompetent
cells that exerts anti-inflammatory actions and abrogates
postischemic injury to the kidneys and brainstem of rodents. To
test the hypothesis that
-MSH would afford similar protection in the
postischemic small intestine, we analyzed the effects of this
peptide on intestinal transit, histology, myeloperoxidase activity, and
nuclear factor-
B (NF-
B) activation after 45 min of superior
mesenteric artery occlusion and
6 h of reperfusion. Rats subjected to
I/R exhibited markedly depressed intestinal transit, histological
evidence of severe injury to the ileum, increased myeloperoxidase
activity in ileal cytoplasmic extracts, and biphasic activation of
NF-
B in ileal nuclear extracts. In contrast, rats treated with
-MSH before I/R exhibited intestinal transit and histological injury
scores comparable to those of sham-operated controls. In addition, the
-MSH-treated rats demonstrated less I/R-induced activation of
intestinal NF-
B and myeloperoxidase activity after prolonged (6 h)
reperfusion. We conclude that
-MSH significantly limits
postischemic injury to the rat small intestine.
transcription factor; nuclear factor-B; ileus; small
intestine
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INTRODUCTION |
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MESENTERIC
ISCHEMIA-REPERFUSION (I/R) is a common clinical problem
in the settings of shock, sepsis, vascular surgery, and small bowel
transplantation. It is associated with considerable morbidity and
mortality for which there are virtually no therapeutic options.
Mesenteric I/R causes gut dysfunction characterized by histological
evidence of mucosal injury, increased intestinal epithelial
permeability, and impaired motility (16). The molecular details underlying the mechanisms of acute intestinal injury and repair
after I/R are incompletely characterized. Numerous mediators have been
implicated in mesenteric I/R injury including cytokines (8), reactive oxygen species (13), nitric
oxide (42), arachidonic acid derivatives
(32), and cell adhesion molecules (37). The expression of these inflammatory mediator genes is controlled, at least
in part, by nuclear factor-B (NF-
B) in most cell types.
NF-B is a well-known redox-sensitive and cytokine-inducible
cytoplasmic transcriptional factor that belongs to the Rel family of
inducible transcriptional factors. NF-
B is activated in the gut by a
number of proinflammatory stimuli including sepsis (10), cytokines (38), and oxidative stress (1).
Recent reports have demonstrated activation of NF-
B in
postischemic rat intestine (44). Under basal
conditions, NF-
B is sequestered in the cytoplasm as a ternary
complex tethered with a family of inhibitory proteins known as I
Bs,
whose expression varies in different cell types. On activation, a large
multiprotein signalsome, which contains NF-
B-inducing kinase
(homologous I
B kinases), phosphorylates I
B
, targeting it for
ubiquitination and subsequent proteosome-dependent degradation. This
rapidly frees NF-
B to translocate to the nucleus, where it binds to
specific DNA sequences located in the promoter regions of a number of
proinflammatory genes.
-Melanocyte-stimulating hormone (MSH) is a
proopiomelanocortin-derived tridecapeptide
(1SYSMQHFRWGKPV13) released by the pituitary
gland and immunocompetent cells that exerts broad anti-inflammatory
actions in mammals (31). The actions of
-MSH are
transmitted via a five-member family of specific melanocortin G protein
receptors that activate adenylyl cyclase and elevate cAMP. Of these
receptors, the melanocortin-3 and -4 receptors have been found in the
gut (12).
-MSH has been tested in several models of
sepsis and inflammatory organ failure, and it has specifically been
shown to protect against liver damage and mortality in endotoxemia
(4) and against renal injury after renal I/R in mice and
rats (5). Most recently, systemically administered
-MSH
was shown to inhibit NF-
B activation in a model of
lipopolysaccharide (LPS)-induced brain inflammation (20). The beneficial effects of
-MSH in these experimental models appear to result from its ability to limit induction of genes encoding proinflammatory cytokines, chemokines, cell adhesion molecules, and
inducible nitric oxide synthase (4, 5). By limiting these
injury pathways, neutrophil infiltration, capillary congestion, and
exposure of cell constituents to damaging reactive oxygen and nitrogen
intermediates is reduced. In this study, we examined the effects of
-MSH on intestinal injury after mesenteric I/R. We hypothesized that
-MSH would limit NF-
B activation and protect the gut from
I/R-mediated injury.
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MATERIALS AND METHODS |
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Animal model. Male Sprague-Dawley rats (Harlan, Houston, TX) weighing 250-350 g were cared for in accordance with the guidelines of University of Texas Medical School at Houston Animal Welfare Committee. Rats were housed individually at room temperature (25°C) with alternating 12:12-h light-dark cycles and fed standard rat chow and water ad libitum during a minimum stabilization period of 5 days. 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.
Rats received eitherIntestinal transit. Small intestinal transit in unanesthetized, unrestrained animals was measured by using a technique previously described (34). Briefly, 0.1 ml of nonabsorbable FITC-dextran (9,400 molecular wt; Sigma, St. Louis, MO) was injected into the catheter and flushed into the duodenum with 0.2 ml normal saline. Thirty minutes later, the animals were killed under anesthesia and the entire small intestine was removed and divided into 10 equal segments. The distal end of each segment was clamped and placed over a glass tube, and the contents were flushed with 3 ml of 5 mM Tris buffer (pH 10.3). The FITC-dextran concentration in each segment was measured by using an optical scanner (STORM model 860; Molecular Dynamics), expressed as a fraction of total tracer recovered and presented as the mean geometric center of distribution.
Histological studies.
In a separate set of experiments, biopsies of the distal ileum (~10
cm from the ileocecal valve) were taken from sham, mesenteric I/R, and
mesenteric I/R + -MSH treated animals after 45 min of SMA
occlusion and 0.5-, 1-, 2-, and 6-h reperfusion. The tissues were
immersed in 10% formalin for at least 24 h and then imbedded in
paraffin, cut into 5-µm sections, and stained with hematoxylin and
eosin. All processed tissues (n = 4 in each group) were
examined under light microscopy by a blinded, experienced observer and scored using the following grading scale (7): Grade
0, normal mucosa; Grade 1, development of subepithelial
space at the apex of the villus ± capillary congestion;
Grade 2, extension of the subepithelial space with moderate
lifting of epithelial layer from the lamina propria; Grade
3, massive epithelial lifting down the sides of villi and a few
tips may be denuded; Grade 4, denuded villi with lamina
propria and dilated capillaries exposed; Grade 5, digestion
and disintegration of lamina propria, and hemorrhage and ulceration.
Preparation of nuclear extracts.
Nuclear extracts from full-thickness ileal tissue were prepared by the
method of Deryckere and Gannon (11). Frozen tissue (~250
mg) was ground with a mortar in liquid nitrogen and transferred to a
Dounce tissue homogenizer. Tissue powder was then homogenized (~10
strokes) in 3 ml of buffer A (in mM): 150 NaCl, 10 HEPES (pH
7.9), 1 EDTA, 0.5% phenylmethylsulfonyl fluoride, 0.6% Nonidet P-40,
and 30 µl/ml protease inhibitor cocktail (Sigma) and centrifuged at
2,000 rpm for 30 s to pellet tissue debris. The supernatant was
incubated on ice for 5 min and then centrifuged at 5,000 rpm for 5 min.
The resulting supernatant containing the cytoplasmic extracts was
collected and stored at 80°C. Pelleted nuclei were then resuspended
in a small volume (50-100 µl) of buffer B (in mM): 20 HEPES (pH 7.9), 420 NaCl, 1.2 MgCl2, 0.2 EDTA, 0.5 dithiothreitol, and 0.5 phenylmethylsulfonyl fluoride with 25%
glycerol and 30 µl/ml of a protease inhibitor cocktail and incubated
on ice for 20 min. The lysed nuclei were then transferred to a
microcentrifuge tube, centrifuged at 12,000 rpm for 10 min, and the
supernatant containing the nuclear extracts were collected and stored
at
80°C. Protein contents of the extracts were assayed with the
BCA-protein estimation kit (Pierce, Rockwood, IL).
Electrophoretic mobility shift and supershift assays.
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 (in mM): 10 Tris (pH 7.5), 50 NaCl, 1 dithiothreitol, 1 EDTA, and 5% glycerol, 1 µg of
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 subunits p50, p52, c-Rel, RelB, or p65 (Santa Cruz
Biotechnology, Santa Cruz, CA) was added before the addition of the
labeled probe. Gel loading buffer was then added to the mixture, and
the samples were electrophoresed on a nondenaturing 5% polyacrylamide
gel. The gels were then dried and analyzed by autoradiography.
Deconvolution indirect immunofluorescence microscopy.
For indirect immunofluorescence microscopy, biopsies of ileum were
obtained from sham, mesenteric I/R (6-h reperfusion), and mesenteric
I/R (6-h reperfusion) + -MSH treated animals and cut into
6-µm sections using a Minotone Cryostat (International Equipment). Sections were quickly dried onto 18-mm coverslips coated with poly-L-lysine and rinsed in cold PBS before 5-min fixation
in 3.7% formaldehyde. The sections were then stained by using a
protocol previously described (2). Briefly, coverslips
were inverted onto ~50 µl of blocking solution (10% goat serum in
PBS), supported by a piece of parafilm (American Can, Greenwich, CT)
and incubated for 30 min in a humidified cell incubator at 37°C.
After being blocked, the sections were incubated for 30 min with rabbit
polyclonal anti-p50 or -p65 antibodies (Santa Cruz Biotechnologies)
diluted 1:100 in blocking solution. As a negative control, primary
antibody was omitted from the immunostaining procedure. After three
washes in PBS containing 0.05% Tween 20, the sections were incubated with Cy5-congugated goat anti-rabbit IgG (Molecular Probes) diluted 1:500 in a PBS containing 10% normal goat serum and 0.05% Tween 20. After final washes in PBS containing 0.05% Tween, the cells were
postlabeled with FITC-phalloidin (to identify F-actin) and 4',6-diamidine-2-phenylindole (DAPI) (to identify nuclei) and mounted in the antifade reagent Elvanol (DuPont, Wilmington, DE). Sections were then imaged by using an Olympus IX70 inverted
epifluorescence microscope. Data sets were acquired by using a mercury
short-arc lamp and stored in digital format by using a cooled
charge-coupled device camera (Applied Precision, Delta Vision System).
Data sets were then transferred to a Silicon Graphics workstation for
deconvolution and three-dimensional reconstruction (33).
Delta Vision System SoftWoRx (Applied Precision, Isasaquah, WA) was
used to deconvolve 0.1-µm optical sections before reconstruction.
Data sets were then imported into Imaris 3 (Bitplane, Zurich,
Switzerland) for digital image restoration and shadowing.
MPO assay. Cytoplasmic extracts from full-thickness ileal tissue were diluted 1:5 in buffer A. Ten microliters of each sample were then added to wells of 96-well plates and incubated with 100 µl tetramethylbenzidine Microwell peroxidase substrate (KPL, Gaithersburg, MD) at room temperature for 20 min. The reaction was stopped with 100-µl 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. Band intensities on autoradiograms were scanned by using image analysis software (Optimas 6.1). Quantitative data are expressed as means ± SE and were analyzed with one-way ANOVA. Individual group means were then compared with a Tukey multiple comparison test. P values <0.05 were considered significant.
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RESULTS |
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-MSH preserves small intestinal transit after SMA I/R.
We (27) previously reported that the mean geometric center
of distribution for unmanipulated rats is 6.4 ± 0.2. The mean geometric center of distribution of fluorescent tracer in the small
intestine for sham-operated animals (4.9 ± 0.2) was comparable to
that of
-MSH-treated animals (4.7 ± 0.3), whereas the mean geometric center of distribution for the mesenteric I/R (6-h
reperfusion) animals was 45% lower (2.7 ± 0.2, P < 0.01). In contrast, transit in rats treated with
-MSH during
mesenteric I/R (6-h reperfusion) was not significantly different from
sham-operated controls (4.2 ± 0.5). Figure
1 presents a histogram of the transit
data. The apparent reduction in intestinal transit of the sham-operated rats, compared with the historical, unmanipulated control rats, likely
reflects the well-documented depressive effects of anesthesia and
surgical gut manipulation on intestinal motility (23,
25).
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-MSH limits histologic evidence of injury of the ileum after SMA
I/R.
Representative hematoxylin- and eosin-stained sections of ileum from
sham, mesenteric I/R (6-h reperfusion), and mesenteric I/R (6-h
reperfusion) +
-MSH-treated animals are depicted in Fig.
2. Whereas the ileum of sham animals
exhibited normal mucosal architecture with intact villi, the mesenteric
I/R animals exhibited significant histological injury to the ileal
mucosa within 30 min of reperfusion (data not shown). By 6 h of
reperfusion, denuded villi, disintegration of the lamina propria, and
exposed capillaries were apparent (Fig. 2). The mesenteric I/R (6-h
reperfusion) +
-MSH-treated animals exhibited only capillary
congestion and mild epithelial lifting from the lamina propria (Fig.
2). The injury score for the mesenteric I/R (6-h reperfusion) animals (Grade 4.3 ± 0.9) was significantly (P < 0.05)
greater than both the sham-operated (Grade 0.0 ± 0.0) and
mesenteric I/R (6-h reperfusion) +
-MSH-treated (Grade 0.8 ± 0.3) animals.
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-MSH limits late NF-
B p50/p50 activation in ileum after SMA
I/R.
Time-course experiments after various durations of reperfusion for
NF-
B DNA binding activity in nuclear extracts harvested from sham,
mesenteric I/R, and mesenteric I/R +
-MSH treated animals were
performed. NF-
B DNA binding activities on EMSAs were quantified by
scanning densitometry. As seen in Fig.
3A, NF-
B DNA binding
activity exhibited a biphasic response with progressive durations of
reperfusion. Animals subjected to 45 min ischemia and 1 h
of reperfusion exhibited NF-
B DNA binding activity levels 40%
greater than sham-operated controls. In contrast, NF-
B DNA-binding
activity was only 60% of sham-operated controls in I/R rats subjected
to 2-h reperfusion. After 6-h reperfusion, NF-
B DNA binding activity
again increased and was significantly greater than sham-operated
animals or animals subjected to ischemia and shorter period of
reperfusion. The mesenteric I/R +
-MSH treated rats exhibited a
pattern of NF-
B DNA binding activity over time similar to that of
the mesenteric I/R animals, except at the 6-h reperfusion time point at
which mesenteric I/R +
-MSH treated animals exhibited ~30%
lower levels of NF-
B DNA binding activity (Fig. 3A). A
2-h reperfusion time point was not obtained for I/R +
-MSH
treated animals, because downregulation of NF-
B was not a focus of
the study.
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-MSH inhibits induction of MPO activity after SMA I/R.
MPO is a component of neutrophil granules and, as such, its activity
serves as a quantitative measure of neutrophil infiltration. MPO
activity was determined in ileal samples harvested from sham, mesenteric I/R (6-h reperfusion), and mesenteric I/R (6-h
reperfusion) +
-MSH treated animals (Fig.
5). MPO activity was approximately threefold greater in the I/R compared with the sham rats. In contrast, the I/R +
-MSH-treated rats exhibited MPO activity that was not different from sham or
-MSH-treated sham controls.
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DISCUSSION |
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In this report, we demonstrated in a rat model of SMA occlusion
that mesenteric I/R promotes severe mucosal injury and a reproducible decrement in intestinal transit. This injury was temporally associated with activation of nuclear DNA binding activity for NF-B p50/p50 homodimers in ileal extracts, nuclear localization of NF-
B p50 in
enterocytes and cells of the lamina propria, and an increase in ileal
MPO activity. We further show that the administration of intravenous
-MSH during mesenteric I/R protected the ileum from morphologic and
functional injury, as judged by histology and intestinal transit. In
addition,
-MSH limited the late activation of NF-
B in the mucosa
of the postischemic ileum and prevented the increase in ileal
MPO activity after I/R injury. Thus the anti-inflammatory effects of
this neuroimmunomodulator, noted in multiple other inflammatory models,
appear to extend to this well-established model of intestinal injury.
Clinically, ileus occurs after sepsis and shock-induced mesenteric I/R.
We and others (18, 19, 43) have shown that, in animal
models, mesenteric I/R causes an ileus characterized by depressed small
intestinal smooth muscle contractility in vitro as well as impaired
intestinal transit in vivo. Whereas the mechanisms involved in
postinjury ileus are not clearly understood, inflammatory processes
within the gut seem to play a significant role. Recent studies suggest
that prolonged postinjury ileus is due to leukocyte-mediated inflammation (23, 25) within the intestinal muscularis. In agreement with this work, we observed a dramatic increase in MPO activity, a quantitative marker of neutrophil infiltration, after mesenteric ischemia and 6-h reperfusion. This response was
completely abolished by treatment with -MSH.
-MSH is known to
have inhibitory effects on neutrophil migration, and neutrophils
express mRNA for the melanocortin-1 MSH receptor (31). The
peptide inhibited production of potent neutrophil chemokine IL-8 in the
renal I/R model (6) and inhibited in vitro neutrophil
migration in an IL-8 gradient (31). The specific
mechanisms by which
-MSH reduces induction of ileal MPO activity in
the postischemic intestine merit further study.
Recent work (22, 24, 39) has also implicated iNOS and
COX-2, genes whose transcription is controlled, at least in part, by
NF-B in postinjury ileus. The ability of
-MSH to suppress late
activation of NF-
B in intestinal mucosa and to ameliorate villous
injury in response to I/R suggests local protective mechanisms. However,
-MSH partially ameliorated I/R-induced inhibition of intestinal transit although there was no suggestion of an I/R-induced activation of NF-
B p50 in intestinal muscle cells. This suggests that transit may be influenced by events taking place in the mucosa. This would not be surprising, because nervous reflexes between muscle
and mucosa have been demonstrated (14). On the other hand,
a few cells in the muscle layer did show NF-
B p50 activation, perhaps enough to affect transit. It remains possible that NF-
B activation is merely associated with, and not causal for, impaired motility. Finally, neutrophil infiltration is known to be associated with impaired motility in the postischemic bowel
(19), so that the ability of
-MSH to dramatically
suppress neutrophil activity in the postischemic ileum might
account for much of the protective effects on transit we observed. The
specific mechanisms by which
-MSH preserves intestinal transit after
mesenteric I/R needs further investigation.
Relatively little is known about the role of NF-B in the
postischemic intestine. In a similar model of mesenteric I/R
injury, Yeh et al. (44) recently demonstrated activation
of NF-
B in the jejunum. They found that NF-
B activity was
increased by 1 h and remained elevated between 6 and 12 h
after reperfusion. In contrast to their study in jejunum, however, we
found that NF-
B activity was less at 2 h compared with its
initial increase and then was greater again at 6 h in the
postischemic ileum. The reasons for the biphasic course of
NF-
B activation in our model are unclear. Decline in NF-
B
activity after its early activation might represent cellular attempts
to attenuate the activation of proinflammatory and potentially
injurious genes. The second wave of increased NF-
B activity at 6-h
reperfusion suggests that new stimuli promoted activation of this
transcription factor. For example, in a recent study of
experimental inflammation, NF-
B activity was found to peak early
during the proinflammatory response, but also during the resolution of
inflammation where it induced the expression of endogenous
anti-inflammatory pathways and leukocyte apoptosis
(29). Similarly, in a model of I/R injury to rat skeletal muscle NF-
B activation displayed a biphasic pattern, showing peak
activities from 30 min to 3 h postperfusion and 6 to 16 h postperfusion, with a decline to baseline binding activity levels between 3 and 6 h (30). In a glial model, a biphasic
response was also evident (26). Early increase in NF-
B
activity was due to rapid degradation of I
B
, whereas after 1 h, I
B
was resynthesized to levels exceeding the amounts present
in unstimulated cells leading to low levels of nuclear NF-
B binding
activity. Degradation of both I
B
and I
B
contributed to the
late phase of induction. Further studies will be needed to elucidate
the complex regulation of NF-
B in the postischemic intestine.
In addition to I/R, both platelet activating factor and bacterial LPS
have been shown to activate intestinal NF-B p50/p50 homodimers
(9, 10). In the case of LPS administration, both NF-
B
p50/p50 and p50/p65 complexes were observed (10). Platelet activating factor is secreted by the gut mucosa after experimental I/R
injury (28) and may contribute to both the activation of NF-
B p50/p50 and p50/p65 complexes and the pathogenesis of I/R injury.
Our observation in the postischemic ileum, coupled with those
reported for the jejunum, suggests selective activation of NF-B p50/p50 homodimers in response to I/R in this tissue. Whereas DNA
binding activity of NF-
B containing p65 was present constitutively in the ileum, it was not influenced by I/R. These collective findings contrast with the responses of other tissues, such as heart, liver, and
kidney, in which p50/p65 dimers are activated after I/R (3, 35,
45). Siebenlist et al. (41) showed that p50 lacks a transactivation domain, and in some in vitro assays, the p50/p50 complex fails to recruit several coactivators required to transactivate target genes bearing
B sites (40). Accordingly, in most
promoter contexts, p50 homodimers compete for binding of p50/p65
complexes and repress gene expression. In other examples, higher order
complexes of p50 homodimers with other transcription factors have been
shown to activate gene transcription. For example, complexes of p50 homodimers and oncoprotein Bcl-3 transactivate the P-selectin gene in
bovine aortic endothelial cells (36). The specific role of
NF-
B p50 homodimer induction plays in the pathogenesis of gut I/R
injury will require additional studies for elucidation. Other
redox-sensitive and cytokine-inducible transcriptional factors such as
AP-1 are activated in the postischemic intestine
(44) and are likely involved in regulating proinflammatory
gene expression after intestinal I/R injury. The role of these and
other transcriptional factors needs further investigation.
Interestingly,
-MSH was recently found to rapidly inhibit peroxide
generation and glutathione peroxidase activation in keratinocytes and
melanocytes subjected to oxidative stress, an effect that preceded the
inhibitory actions of the peptide on activation of NF-
B in these
cells (17). If similar events occur in the
postischemic intestine, protection against oxidative damage
could be central to the protective effects of
-MSH in this setting.
Chiao et al. (5) showed that -MSH reduces renal injury
after renal I/R in mice and rats. They demonstrated that this effect was associated with a reduction in renal IL-8 and ICAM-1 mRNA, NOS II
protein, and nitration of kidney proteins. Interestingly, they found
that
-MSH was effective even when administered
6 h after the
ischemic insult. In a follow-up study (6), these authors demonstrated that
-MSH reduced renal I/R injury in ICAM-1 knockout mice that had 75% less neutrophil infiltration than
background mice after ischemia, suggesting that
-MSH
inhibits neutrophil-independent pathways of injury as well. In a recent
study, Ichiyama et al. (20) demonstrated that systemically
administered
-MSH reduced the activation of NF-
B in brain tissue
after an intracerebroventricular injection of LPS. Activation of
NF-
B requires phosphorylation, ubiquitination, and proteasomal
degradation of the I
B subunit. I
B release allows the NF-
B
complex to migrate to the nucleus and induce transcription. In vitro
studies suggested that
-MSH prevents the degradation of I
B
and
thereby blocks activation of NF-
B (21). Our study lends
further support to the concept that
-MSH exerts its broad
anti-inflammatory properties upstream in inflammatory cascades by
limiting the activation of inducible transcriptional factors such as
NF-
B and CCAAT/enhancer binding protein-
(15) that
are known to transactivate a variety of proinflammatory genes. Clearly,
however, the rather small effects (30% inhibition) of
-MSH on
NF-
B induction at 6 h of reperfusion may not explain the
peptide's dramatic protective effects on postischemic ileal
injury, as evaluated by transit, histology, and neutrophil activation
observed in the present study.
We observed nuclear expression of NF-B p50 after I/R in both the
epithelial cells and some of the cells of the lamina propria of the
ileum. NF-
B p65 has been shown to immunolocalize in a similar
distribution in the rat ileum after LPS treatment (10). Interestingly, whereas the
-MSH + I/R rats demonstrated no
appreciable immunoreactivity for NF-
B p50 in the epithelial cells,
they exhibited comparable immunofluorescence in the lamina propria
cells to that of the I/R rats. Whether this differential response
reflects differences between the enterocytes and lamina propria cells
in melanocortin receptor distribution or in the
-MSH-responsive
signaling machinery leading to activation of NF-
B remains open to investigation.
In summary, I/R results in activation of NF-B, mucosal injury, MPO
activity, and functional compromise of the rat ileum.
-MSH partially
prevents these events. In addition,
-MSH selectively blocks
I/R-induced activation of NF-
B p50/p50 in the enterocytes of the
ileum. These data suggest therapeutic potential for
-MSH, an
endogenously produced and safe molecule, in clinical settings associated with mesenteric I/R injury.
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Tri Phan and Mark Snuggs is gratefully acknowledged.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-50745 (to B. C. Kone) and National Institute of General Medical Sciences Grant P50-GM-20529 (to B. C. Kone, F. A. Moore, and N. W. Weisbrodt), and the Department of Defense "DREAMS" Project (to B. C. Kone).
Address for reprint requests and other correspondence: B. C. Kone, Departments of Medicine and of Integrative Biology and Pharmacology, University of Texas Medical School at Houston, 6431 Fannin, Suite 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 March 13, 2002;10.1152/ajpgi.00073.2001
Received 20 February 2001; accepted in final form 24 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aw, TY.
Molecular and cellular responses to oxidative stress and changes in oxidation-reduction imbalance in the intestine.
Am J Clin Nutr
70:
557-565,
1999
2.
Bernard, MA,
Hogue DA,
Cole WG,
Sanford T,
Snuggs MB,
Montufar-Solis D,
Duke PJ,
Carson DD,
Scott A,
Van Winkle WB,
and
Hecht JT.
Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.
J Bone Miner Res
15:
442-450,
2000[ISI][Medline].
3.
Chandrasekar, B,
Colston JT,
Geimer J,
Cortez D,
and
Freeman GL.
Induction of nuclear factor-B but not
B-responsive cytokine expression during myocardial reperfusion injury after neutropenia.
Free Radic Biol Med
28:
1579-1588,
2000[ISI][Medline].
4.
Chiao, H,
Foster S,
Thomas R,
Lipton J,
and
Star RA.
-Melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation.
J Clin Invest
97:
2038-2044,
1996
5.
Chiao, H,
Kohda Y,
McLeroy P,
Craig L,
Housini I,
and
Star RA.
-Melanocyte-stimulating hormone protects against renal injury after ischemia in mice and rats.
J Clin Invest
99:
1165-1172,
1997
6.
Chiao, H,
Kohda Y,
McLeroy P,
Craig L,
Linas S,
and
Star RA.
-Melanocyte-stimulating hormone inhibits renal injury in the absence of neutrophils.
Kidney Int
54:
765-774,
1998[ISI][Medline].
7.
Chiu, CJ,
McArdle AH,
Brown R,
Scott HJ,
and
Gurd FN.
Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal.
Arch Surg
101:
478-483,
1970[ISI][Medline].
8.
Cuzzocrea, S,
De Sarro G,
Costantino G,
Ciliberto G,
Mazzon E,
De Sarro A,
and
Caputi AP.
IL-6 knock-out mice exhibit resistance to splanchnic artery occlusion shock.
J Leukoc Biol
66:
471-480,
1999[Abstract].
9.
De Plaen, IG,
Tan XD,
Chang H,
Qu XW,
Liu QP,
and
Hsueh W.
Intestinal NF-B is activated, mainly as p50 homodimers, by platelet-activating factor.
Biochim Biophys Acta
1392:
185-192,
1998[ISI][Medline].
10.
De Plaen, IG,
Tan XD,
Chang H,
Wang L,
Remick DG,
and
Hsueh W.
Lipopolysaccharide activates nuclear factor-B in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor.
Br J Pharmacol
129:
307-314,
2000
11.
Deryckere, F,
and
Gannon F.
A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues.
Biotechniques
16:
405,
1994[ISI][Medline].
12.
Gantz, I,
Konda Y,
Tashiro T,
Shimoto Y,
Miwa H,
Munzert G,
Watson SJ,
DelValle J,
and
Yamada T.
Molecular cloning of a novel melanocortin receptor.
J Biol Chem
268:
8246-8250,
1993
13.
Granger, DN,
Hollwarth ME,
and
Parks DA.
Ischemia-reperfusion injury: role of oxygen-derived free radicals.
Acta Physiol Scand Suppl
548:
47-63,
1986.
14.
Greenwood, B,
and
Palmer JM.
Neural integration of jejunal motility and ion transport in nematode-infected ferrets.
Am J Physiol Gastrointest Liver Physiol
271:
G48-G55,
1996
15.
Gupta, AK,
Diaz RA,
Higham S,
and
Kone BC.
-MSH inhibits induction of C/EBP
-DNA binding activity and NOS2 gene transcription in macrophages.
Kidney Int
57:
2239-2248,
2000[ISI][Medline].
16.
Hassoun, HT,
Kone BC,
Mercer DW,
Moody FG,
Weisbrodt NW,
and
Moore FA.
Post-injury multiple organ failure: the role of the gut.
Shock
15:
1-10,
2001[ISI].
17.
Haycock, JW,
Rowe SJ,
Cartledge S,
Wyatt A,
Ghanem G,
Morandini R,
Rennie IG,
and
MacNeil S.
-Melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines.
J Biol Chem
275:
15629-15636,
2000
18.
Hebra, A,
Brown MF,
McGeehin K,
Broussard D,
and
Ross AJ, 3rd.
The effects of ischemia and reperfusion on intestinal motility.
J Pediatr Surg
28:
362-365,
1993[ISI][Medline].
19.
Hierholzer, C,
Kalff JC,
Audolfsson G,
Billiar TR,
Tweardy DJ,
and
Bauer AJ.
Molecular and functional contractile sequelae of rat intestinal ischemia/reperfusion injury.
Transplantation
68:
1244-1254,
1999[ISI][Medline].
20.
Ichiyama, T,
Sakai T,
Catania A,
Barsh GS,
Furukawa S,
and
Lipton JM.
Systemically administered -melanocyte-stimulating peptides inhibit NF-
B activation in experimental brain inflammation.
Brain Res
836:
31-37,
1999[ISI][Medline].
21.
Ichiyama, T,
Zhao H,
Catania A,
Furukawa S,
and
Lipton JM.
-Melanocyte-stimulating hormone inhibits NF-
B activation and I
B
degradation in human glioma cells and in experimental brain inflammation.
Exp Neurol
157:
359-365,
1999[ISI][Medline].
22.
Josephs, MD,
Cheng G,
Ksontini R,
Moldawer LL,
and
Hocking MP.
Products of cyclooxygenase-2 catalysis regulate postoperative bowel motility.
J Surg Res
86:
50-54,
1999[ISI][Medline].
23.
Kalff, JC,
Carlos TM,
Schraut WH,
Billiar TR,
Simmons RL,
and
Bauer AJ.
Surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus.
Gastroenterology
117:
378-387,
1999[ISI][Medline].
24.
Kalff, JC,
Schraut WH,
Billiar TR,
Simmons RL,
and
Bauer AJ.
Role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents.
Gastroenterology
118:
316-327,
2000[ISI][Medline].
25.
Kalff, JC,
Schraut WH,
Simmons RL,
and
Bauer AJ.
Surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus.
Ann Surg
228:
652-663,
1998[ISI][Medline].
26.
Kemler, I,
and
Fontana A.
Role of IB
and I
B
in the biphasic nuclear translocation of NF-
B in TNF
-stimulated astrocytes and in neuroblastoma cells.
Glia
26:
212-220,
1999[ISI][Medline].
27.
Khalil, S,
Weisbrodt N,
Russell D,
and
Moody F.
Time course of intestinal transit and inducible nitric oxide synthase (iNOS) after lipopolysaccharide (LPS) administration in the rat (Abstract).
FASEB J
11:
A34,
1997.
28.
Kim, FJ,
Moore EE,
Moore FA,
Biffl WL,
Fontes B,
and
Banerjee A.
Reperfused gut elaborates PAF that chemoattracts and primes neutrophils.
J Surg Res
58:
636-640,
1995[ISI][Medline].
29.
Lawrence, T,
Gilroy DW,
Colville-Nash PR,
and
Willoughby DA.
Possible new role for NF-B in the resolution of inflammation.
Nat Med
7:
1291-1297,
2001[ISI][Medline].
30.
Lille, ST,
Lefler SR,
Mowlavi A,
Suchy H,
Boyle EM, Jr,
Farr AL,
Su CY,
Frank N,
and
Mulligan DC.
Inhibition of the initial wave of NF-B activity in rat muscle reduces ischemia/reperfusion injury.
Muscle Nerve
24:
534-541,
2001[ISI][Medline].
31.
Lipton, JM,
and
Catania A.
Mechanisms of antiinflammatory action of the neuroimmunomodulatory peptide -MSH.
Ann NY Acad Sci
840:
373-380,
1998
32.
Mangino, MJ,
Anderson CB,
Murphy MK,
Brunt E,
and
Turk J.
Mucosal arachidonate metabolism and intestinal ischemia-reperfusion injury.
Am J Physiol Gastrointest Liver Physiol
257:
G299-G307,
1989
33.
McNally, JG,
Karpova T,
Cooper J,
and
Conchello JA.
Three-dimensional imaging by deconvolution microscopy.
Methods
19:
373-385,
1999[ISI][Medline].
34.
Miller, MS,
Galligan JJ,
and
Burks TF.
Accurate measurement of intestinal transit in the rat.
J Pharmacol Methods
6:
211-217,
1981[ISI][Medline].
35.
Morooka, H,
Bonventre JV,
Pombo CM,
Kyriakis JM,
and
Force T.
Ischemia and reperfusion enhance ATF-2 and c-Jun binding to cAMP response elements and to an AP-1 binding site from the c-jun promoter.
J Biol Chem
270:
30084-30092,
1995
36.
Pan, J,
and
McEver RP.
Regulation of the human P-selectin promoter by Bcl-3 and specific homodimeric members of the NF-B/Rel family.
J Biol Chem
270:
23077-23083,
1995
37.
Panes, J,
and
Granger DN.
Leukocyte-endothelial cell interactions: molecular mechanisms and implications in gastrointestinal disease.
Gastroenterology
114:
1066-1090,
1998[ISI][Medline].
38.
Pritts, TA,
Moon MR,
Wang Q,
Hungness ES,
Salzman AL,
Fischer JE,
and
Hasselgren PO.
Activation of NF-B varies in different regions of the gastrointestinal tract during endotoxemia.
Shock
14:
118-122,
2000[ISI][Medline].
39.
Schwarz, NT,
Kalff JC,
Turler A,
Engel BM,
Watkins SC,
Billiar TR,
and
Bauer AJ.
Prostanoid production via COX-2 as a causative mechanism of rodent postoperative ileus.
Gastroenterology
121:
1354-1371,
2001[ISI][Medline].
40.
Sheppard, KA,
Rose DW,
Haque ZK,
Kurokawa R,
McInerney E,
Westin S,
Thanos D,
Rosenfeld MG,
Glass CK,
and
Collins T.
Transcriptional activation by NF-B requires multiple coactivators.
Mol Cell Biol
19:
6367-78,
1999
41.
Siebenlist, U,
Franzoso G,
and
Brown K.
Structure, regulation and function of NF-B.
Annu Rev Cell Biol
10:
405-455,
1994[ISI].
42.
Stark, ME,
and
Szurszewski JH.
Role of nitric oxide in gastrointestinal and hepatic function and disease.
Gastroenterology
103:
1928-1949,
1992[ISI][Medline].
43.
Udassin, R,
Eimerl D,
Schiffman J,
and
Haskel Y.
Postischemic intestinal motility in rat is inversely correlated to length of ischemia. An in vivo animal model.
Dig Dis Sci
40:
1035-1038,
1995[ISI][Medline].
44.
Yeh, KY,
Yeh M,
Glass J,
and
Granger DN.
Rapid activation of NF-B and AP-1 and target gene expression in postischemic rat intestine.
Gastroenterology
118:
525-534,
2000[ISI][Medline].
45.
Zwacka, RM,
Zhang Y,
Zhou W,
Halldorson J,
and
Engelhardt JF.
Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor-B independently of I
B degradation.
Hepatology
28:
1022-1030,
1998[ISI][Medline].