alpha -Melanocyte-stimulating hormone protects against mesenteric ischemia-reperfusion injury

Heitham T. Hassoun1,4, Lei Zou2,4, Frederick A. Moore1,4, Rosemary A. Kozar1,4, Norman W. Weisbrodt3,4, and Bruce C. Kone2,3,4

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mesenteric ischemia-reperfusion (I/R) injury to the intestine is a common and often devastating clinical occurrence for which there are few therapeutic options. alpha -Melanocyte-stimulating hormone (alpha -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 alpha -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-kappa B (NF-kappa 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-kappa B in ileal nuclear extracts. In contrast, rats treated with alpha -MSH before I/R exhibited intestinal transit and histological injury scores comparable to those of sham-operated controls. In addition, the alpha -MSH-treated rats demonstrated less I/R-induced activation of intestinal NF-kappa B and myeloperoxidase activity after prolonged (6 h) reperfusion. We conclude that alpha -MSH significantly limits postischemic injury to the rat small intestine.

transcription factor; nuclear factor-kappa B; ileus; small intestine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa B) in most cell types.

NF-kappa B is a well-known redox-sensitive and cytokine-inducible cytoplasmic transcriptional factor that belongs to the Rel family of inducible transcriptional factors. NF-kappa 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-kappa B in postischemic rat intestine (44). Under basal conditions, NF-kappa B is sequestered in the cytoplasm as a ternary complex tethered with a family of inhibitory proteins known as Ikappa Bs, whose expression varies in different cell types. On activation, a large multiprotein signalsome, which contains NF-kappa B-inducing kinase (homologous Ikappa B kinases), phosphorylates Ikappa Balpha , targeting it for ubiquitination and subsequent proteosome-dependent degradation. This rapidly frees NF-kappa B to translocate to the nucleus, where it binds to specific DNA sequences located in the promoter regions of a number of proinflammatory genes.

alpha -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 alpha -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). alpha -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 alpha -MSH was shown to inhibit NF-kappa B activation in a model of lipopolysaccharide (LPS)-induced brain inflammation (20). The beneficial effects of alpha -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 alpha -MSH on intestinal injury after mesenteric I/R. We hypothesized that alpha -MSH would limit NF-kappa B activation and protect the gut from I/R-mediated injury.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 either alpha -MSH (Phoenix Pharmaceuticals, Mountain View, CA) as a 50-µg iv bolus followed by 15 µg/h infused over 5 h (or shorter periods as indicated in the text and figure legends) via the internal jugular vein (n = 6) or an equal volume of sterile saline (n = 5) beginning 30 min before superior mesenteric artery (SMA) clamping. The bolus dose of alpha -MSH was selected on the basis of the experience of previous in vivo studies in rat (4-6, 20). The constant infusion dose was arbitrarily estimated with the goal of maximizing the protective effects of the peptide. Each animal underwent midline laparotomy and placement of Silastic catheters via needle puncture into the duodenum for subsequent measurement of intestinal transit. The catheter was then passed through the musculature of the left abdominal wall and subcutaneous tissue toward the back of the neck where it was exteriorized through an interscapular incision and fixed to the skin with 4-0 silk suture. The SMA was then isolated, and a bulldog clamp was applied at the aortic origin. The intestines were carefully returned to the abdomen, and the incision was covered with moist gauze and a sterile plastic sheet. The rats were placed on a heating blanket for the duration of the procedure. After 45 min, the clamp was removed, the incision was closed, and the rats were allowed to awaken. The control (sham-operated, n = 5) animals underwent the same procedure but without arterial clamping. Thus four groups of animals were studied, all of whom had laparotomies and duodenal catherization: sham-operated, sham + alpha -MSH, I/R (with various lengths of reperfusion), and I/R (with various lengths of reperfusion) alpha -MSH. NF-kappa B DNA binding activity and histology were performed after 30 min to 6 h of reperfusion as indicated in the text and figures. Intestinal transit studies, immunolocalization, and myeloperoxidase (MPO) activity studies were performed after 6-h reperfusion during which the animals were fasted but allowed free access to water.

Intestinal 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 + alpha -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-kappa B in ileal nuclear extracts was determined by electrophoretic mobility shift assay (EMSA). The NF-kappa B consensus oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (Promega, Madison, WI) was end labeled with [gamma -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-kappa B oligonucleotide was added to the binding reaction. For supershift assays, 2 µl of antibody to NF-kappa 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) + alpha -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -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 alpha -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 alpha -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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Fig. 1.   Protective effects of alpha -melanocyte-stimulating hormone (alpha -MSH) on small intestinal transit after ischemia-reperfusion (I/R) injury. The sham group of rats had no arterial clamp. The second group was subjected to 45-min superior mesenteric artery (SMA) clamping followed by 6-h I/R or sham surgery. A third group was pretreated with intravenous alpha -MSH and subjected to sham surgery. A fourth group was treated with intravenous alpha -MSH before SMA clamping. Intestinal transit of tracer along intestinal segments was then measured as detailed in MATERIALS AND METHODS. Data are means ± SE, n = 5 rats per group.

alpha -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) + alpha -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) + alpha -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) alpha -MSH-treated (Grade 0.8 ± 0.3) animals.


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Fig. 2.   Protective effects of alpha -MSH on ileal injury after I/R. Rats were subjected to sham surgery or 45-min SMA occlusion followed by 6-h I/R. A third group of rats were pretreated with intravenous alpha -MSH before SMA occlusion. Ileal samples were then obtained. Formalin-fixed, paraffin-embedded sections of ileum were stained with hematoxylin and eosin. Magnification ×20; n = 4 rats per group.

alpha -MSH limits late NF-kappa B p50/p50 activation in ileum after SMA I/R. Time-course experiments after various durations of reperfusion for NF-kappa B DNA binding activity in nuclear extracts harvested from sham, mesenteric I/R, and mesenteric I/R + alpha -MSH treated animals were performed. NF-kappa B DNA binding activities on EMSAs were quantified by scanning densitometry. As seen in Fig. 3A, NF-kappa 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-kappa B DNA binding activity levels 40% greater than sham-operated controls. In contrast, NF-kappa 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-kappa 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 + alpha -MSH treated rats exhibited a pattern of NF-kappa 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 + alpha -MSH treated animals exhibited ~30% lower levels of NF-kappa B DNA binding activity (Fig. 3A). A 2-h reperfusion time point was not obtained for I/R + alpha -MSH treated animals, because downregulation of NF-kappa B was not a focus of the study.


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Fig. 3.   Effect of alpha -MSH on nuclear factor-kappa B (NF-kappa B) activation after I/R of the ileum. A: densitometric analysis of bands representing NF-kappa B-specific protein DNA complexes from the ileum of sham, I/R (0-, 0.5-, 1-, 2-, and 6-h reperfusion), and I/R (0-, 0.5-, 1-, and 6-h reperfusion) + alpha -MSH treated rats. Data are means ± SE (n = 4 to 6 rats). * P < 0.05 vs. sham control. ** P < 0.05 vs. sham + alpha -MSH. # P < 0.05 vs. I/R (6-h reperfusion) control. B: representative electrophoretic mobility shift assay (EMSA) demonstrating NF-kappa B DNA binding activity in nuclear extracts from the ileum of sham, I/R (6-h reperfusion), and I/R (6-h reperfusion) + alpha -MSH treated rats. Lanes 1-2, control; lanes 3-4, I/R; lanes 5-6, I/R + alpha -MSH. C: representative autoradiogram displaying results of EMSA specificity experiments and supershift assays in samples from I/R animals. To demonstrate binding specificity, reactions were conducted in the absence (-) and presence (+) of a 100-fold molar excess of unlabeled NF-kappa B oligomers. Polyclonal IgGs specific for NF-kappa B p50, p65, p52, c-Rel, or RelB were used in supershift experiments (n >=  3 rats).

A representative EMSA of nuclear extracts from full-thickness ileal samples of sham, mesenteric I/R (6-h reperfusion), and mesenteric I/R (6-h reperfusion) + alpha -MSH treated animals is depicted in Fig. 3B. A small amount of constitutive NF-kappa B DNA binding activity (complex I) was present in ileal extracts of all animal groups. This constitutive activity was also present in control animals that did not undergo laparotomy (data not shown). Mesenteric I/R animals exhibited induction of a higher molecular weight kappa B-specific complex, complex II (lanes 3 and 4). Systemic administration of alpha -MSH during I/R (6-h reperfusion) partially abrogated the induction of NF-kappa B complex II (lanes 5 and 6) compared with I/R alone (lanes 3 and 4). The radioactive signal for NF-kappa B-DNA complexes I and II was not evident when a 100-fold molar excess of unlabeled kappa B oligomer was added to the reaction, indicating specificity of the binding reactions (Fig. 3C, lane 2). Supershift assays revealed that anti-p50 completely supershifted NF-kappa B-DNA complex II, whereas anti-p65 partially supershifted complex I (Fig. 3C). Antibodies against NF-kappa B p52, c-Rel, and RelB failed to supershift either complex. The same pattern and response of p50 and p65 NF-kappa B complexes were apparent at the early and late time points, so that "switching" of NF-kappa B complexes of different composition did not appear to occur. In the aggregate, these results indicate that in ileum, NF-kappa B complex II, which is induced during I/R, contains NF-kappa B p50/p50 homodimers, and complex I, which is constitutively expressed, contains NF-kappa B p65. The fact that complex I was only partially supershifted by NF-kappa B p65 antibodies and not supershifted by antibodies to the other NF-kappa B family members, suggests that the complex may include a p65 binding partner from another transcription factor family (40).

Because NF-kappa B p50 DNA binding activity was specifically induced in the postischemic ileum, deconvolution immunofluorescence microscopy of ileal sections with NF-kappa B p50 antibodies was performed to localize this transcription factor. Little NF-kappa B p50 immunofluorescence was evident in the enterocytes (Fig. 4A), lamina propria (Fig. 4A), or muscularis (Fig. 4B) of the sham animals. In contrast, the mesenteric I/R (6-h reperfusion) animals demonstrated strong NF-kappa B p50 immunofluorescence in the nuclei, as judged by colocalization with the nuclear stain DAPI, of the majority of enterocytes as well as in cells of the lamina propria (Fig. 4A). Induction of nuclear p50 immunofluorescence after I/R is in agreement with the EMSA data. No significant NF-kappa B p50 immunofluorescence was observed in the muscularis, except for an occasional cell, presumably a muscularis macrophage (Fig. 4B). The mesenteric I/R (6-h reperfusion) alpha -MSH animals exhibited no significant NF-kappa B p50 immunofluorescence in the enterocytes (Fig. 4A) or muscularis (Fig. 4B) but prominent immunofluorescence in the nuclei of the lamina propria cells, which was comparable to that of the I/R animals (Fig. 4A).


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Fig. 4.   Immunolocalization of NF-kappa B p50 subunit in rat ileum. Sections of ileum harvested from sham, I/R (6-h reperfusion), and I/R (6-h reperfusion) + alpha -MSH treated animals were fixed as described in MATERIALS AND METHODS. Images of NF-kappa B p50 immunofluorescence were obtained by automated deconvolution microscopy and image analysis. p50 Was labeled with a Texas red-conjugated secondary antibody. F-actin was labeled with FITC-conjugated phalloidin (fluoresces as green). Nuclei were labeled with 4',6-diamidine-2-phenylindole, which yields blue fluorescence. p50 Is localized in the cytoplasm in some epithelial cells in the villi and in some lamina propria cells (A), but not in the smooth muscle cells (B) in the sham-operated group. After I/R, there is massive increase in nuclear expression of p50 in the enterocytes and lamina propria cells (A), but p50 is absent in the smooth muscle cells (B). In the ileum of rats treated with alpha -MSH before I/R, p50 expression was evident in the nuclei of lamina propria cells (A), but not in enterocytes (A) or smooth muscle cells (B). Arrows point to representative enterocytes. Numbers with red bars = µm.

alpha -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) alpha -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 + alpha -MSH-treated rats exhibited MPO activity that was not different from sham or alpha -MSH-treated sham controls.


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Fig. 5.   Effect of alpha -MSH on myeloperoxidase activity after reperfusion of the ischemic ileum. Myeloperoxidase activity in ileal homogenates prepared from sham, I/R (6-h reperfusion), sham + alpha -MSH, and I/R (6-h reperfusion) + alpha -MSH treated animals was measured as detailed in MATERIALS AND METHODS (n = 4 to 8 rats; * P < 0.05 vs. sham control; ** P < 0.05 vs. I/R control). Two error bars were too small to be seen.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B p50/p50 homodimers in ileal extracts, nuclear localization of NF-kappa 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 alpha -MSH during mesenteric I/R protected the ileum from morphologic and functional injury, as judged by histology and intestinal transit. In addition, alpha -MSH limited the late activation of NF-kappa 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 alpha -MSH. alpha -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 alpha -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-kappa B in postinjury ileus. The ability of alpha -MSH to suppress late activation of NF-kappa B in intestinal mucosa and to ameliorate villous injury in response to I/R suggests local protective mechanisms. However, alpha -MSH partially ameliorated I/R-induced inhibition of intestinal transit although there was no suggestion of an I/R-induced activation of NF-kappa 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-kappa B p50 activation, perhaps enough to affect transit. It remains possible that NF-kappa 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 alpha -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 alpha -MSH preserves intestinal transit after mesenteric I/R needs further investigation.

Relatively little is known about the role of NF-kappa B in the postischemic intestine. In a similar model of mesenteric I/R injury, Yeh et al. (44) recently demonstrated activation of NF-kappa B in the jejunum. They found that NF-kappa 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-kappa 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-kappa B activation in our model are unclear. Decline in NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa B activity was due to rapid degradation of Ikappa Balpha , whereas after 1 h, Ikappa Balpha was resynthesized to levels exceeding the amounts present in unstimulated cells leading to low levels of nuclear NF-kappa B binding activity. Degradation of both Ikappa Balpha and Ikappa Bbeta contributed to the late phase of induction. Further studies will be needed to elucidate the complex regulation of NF-kappa B in the postischemic intestine.

In addition to I/R, both platelet activating factor and bacterial LPS have been shown to activate intestinal NF-kappa B p50/p50 homodimers (9, 10). In the case of LPS administration, both NF-kappa 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-kappa 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-kappa B p50/p50 homodimers in response to I/R in this tissue. Whereas DNA binding activity of NF-kappa 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 kappa 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-kappa 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, alpha -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-kappa 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 alpha -MSH in this setting.

Chiao et al. (5) showed that alpha -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 alpha -MSH was effective even when administered <= 6 h after the ischemic insult. In a follow-up study (6), these authors demonstrated that alpha -MSH reduced renal I/R injury in ICAM-1 knockout mice that had 75% less neutrophil infiltration than background mice after ischemia, suggesting that alpha -MSH inhibits neutrophil-independent pathways of injury as well. In a recent study, Ichiyama et al. (20) demonstrated that systemically administered alpha -MSH reduced the activation of NF-kappa B in brain tissue after an intracerebroventricular injection of LPS. Activation of NF-kappa B requires phosphorylation, ubiquitination, and proteasomal degradation of the Ikappa B subunit. Ikappa B release allows the NF-kappa B complex to migrate to the nucleus and induce transcription. In vitro studies suggested that alpha -MSH prevents the degradation of Ikappa Balpha and thereby blocks activation of NF-kappa B (21). Our study lends further support to the concept that alpha -MSH exerts its broad anti-inflammatory properties upstream in inflammatory cascades by limiting the activation of inducible transcriptional factors such as NF-kappa B and CCAAT/enhancer binding protein-beta (15) that are known to transactivate a variety of proinflammatory genes. Clearly, however, the rather small effects (30% inhibition) of alpha -MSH on NF-kappa 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-kappa B p50 after I/R in both the epithelial cells and some of the cells of the lamina propria of the ileum. NF-kappa B p65 has been shown to immunolocalize in a similar distribution in the rat ileum after LPS treatment (10). Interestingly, whereas the alpha -MSH + I/R rats demonstrated no appreciable immunoreactivity for NF-kappa 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 alpha -MSH-responsive signaling machinery leading to activation of NF-kappa B remains open to investigation.

In summary, I/R results in activation of NF-kappa B, mucosal injury, MPO activity, and functional compromise of the rat ileum. alpha -MSH partially prevents these events. In addition, alpha -MSH selectively blocks I/R-induced activation of NF-kappa B p50/p50 in the enterocytes of the ileum. These data suggest therapeutic potential for alpha -MSH, an endogenously produced and safe molecule, in clinical settings associated with mesenteric I/R injury.


    ACKNOWLEDGEMENTS

The expert technical assistance of Tri Phan and Mark Snuggs is gratefully acknowledged.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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