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Departments of Biochemistry and Molecular Biology and * Pharmacology, School of Pharmacy, University of Granada, Spain
2To whom correspondence should be addressed. E-mail: omartine{at}ugr.es.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-casein–derived glycomacropeptide has immunomodulatory and bacterial toxin binding effects. The intestinal anti-inflammatory activity of glycomacropeptide was assessed in trinitrobenzenesulfonic acid–induced colitis in rats. Rats were administered glycomacropeptide daily starting either 2 d before (pretreatment) or 3 h after (post-treatment) colitis induction. Pretreatment with glycomacropeptide had a dose-dependent anti-inflammatory effect, characterized by lower body weight loss, decreased anorexia (57%), colonic damage (65%), and weight to length ratio (32%), as well as a reduction in colonic alkaline phosphatase activity (42%) and interleukin 1, trefoil factor 3, and inducible nitric oxide synthase mRNA levels (P < 0.05). The mechanism of action of glycomacropeptide is unknown but is consistent with an inhibition of the activation of immune cells. The magnitude of the anti-inflammatory effect was generally comparable to that of sulfasalazine, an established drug used in the treatment of inflammatory bowel disease. Bovine glycomacropeptide may play a role in the management of patients with inflammatory bowel disease.
KEY WORDS: • glycomacropeptide • casein macropeptide • trinitrobenzenesulfonic acid • inflammatory bowel disease
Glycomacropeptide (GMP),3 also referred to as casein macropeptide, is a 64-amino acid peptide that contains varying amounts (0 to 5 U) of N-acetylneuraminic (sialic) acid. This peptide results from the enzymatic hydrolysis of milk -casein in the stomach of neonates due to the action of chymosin (1). In addition, GMP is present at 10–15% in milk whey as a result of the action of the same enzyme during the cheese-making process. GMP is included among the biologically active components of milk that have the ability to control the growth of host friendly colonic microflora and to modulate immune functions, thus helping to control infections. GMP may therefore act as an anti-infectious factor, promoting the growth of bifidobacteria while inhibiting the proliferation of pathogens (2,3). In addition, GMP may combat infection by binding to lectins, viruses, and mycoplasma (1). The effects of GMP on immune function are complex, however. GMP has been reported to increase the proliferation and phagocytic activity of the macrophage-like cell line U937 (4), whereas other authors showed that GMP inhibits the proliferation of several immune cells such as spleen cells and Peyer’s patch cells (5). An increase in IgG production by mouse B-lymphocytes (6) and the ability to interfere with interleukin-1ß (IL-1ß) receptor binding (7) have also been described.
In addition to its abilities to modulate immunity, GMP is quite unique in that its amino acid profile is high in BCAA and lacking in the aromatic amino acids, including phenylalanine, tryptophan, and tyrosine (8). Therefore, it is one of the few naturally occurring proteins safe for individuals with phenylketonuria. Because of its high BCAA content, several authors have indicated that this peptide could be useful in the management of some liver diseases (9).
The expression "inflammatory bowel disease" (IBD) refers to 2 distinct but closely related conditions, ulcerative colitis and Crohn’s disease. Both are chronic relapsing diseases of the intestine. They cause a significant deterioration of the quality of life of patients and are highly (and increasingly) prevalent (10,11). Despite intense investigative efforts, IBD etiology remains unknown, but it is believed to involve an interplay of genetic, environmental, microbial, and immunological factors. A current view of IBD is that it may represent an uncontrolled and exacerbated response to luminal antigens that are innocuous for the normal population. Thus, intestinal inflammation would be the culmination of a cascade of events and processes initiated by antigens due to inadequate handling by the host’s immune system. The net result is a self-perpetuating process in which disruption of the epithelial layer and absorption of luminal antigens play a central role. Although IBD can often be successfully managed pharmacologically, the drugs used, such as corticoids, aminosalicylates, or azathioprine, have a plethora of serious adverse effects that limit their application. Hence, the search for new treatments with a low profile of adverse effects is clearly warranted (11).
GMP has an excellent safety record as an integral part of the normal human diet. In addition, its reported immunomodulatory properties may be beneficial in IBD. Thus, we set out to verify this hypothesis by testing the anti-inflammatory effect of GMP in a widespread model of IBD, namely, trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats (12). We present evidence that GMP, preferably administered as a pretreatment, attenuates experimental hapten-induced colonic inflammation. Furthermore, the anti-inflammatory effect of GMP was comparable to that of an established drug used to treat IBD, namely, sulfasalazine.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals. Female Wistar rats (175–225 g) obtained from the Laboratory Animal Service of the University of Granada were used; they were housed in macrolon cages (6/cage) and maintained in our laboratory in air-conditioned animal quarters with a 12-h light:dark cycle. The rats had free access to tap water and food (Panlab A04, Panlab). This study was carried out in accordance with the Directive for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of the European Union (86/609/EEC).
Induction of colitis. Colitis was induced following the method of Morris et al. (13) with minor modifications. Briefly, rats were deprived of food overnight and anesthetized with halothane. Under these conditions, the rats were administered 10 mg of TNBS dissolved in 0.25 mL of 50% ethanol (v:v) by means of a Teflon cannula inserted 8 cm through the anus. Rats were kept in a head-down position for an additional 30 s and returned to their cage.
Experimental design. Rats were randomly assigned to 8 different groups (n = 6). Two groups, C and CG500, were control groups and did not receive the TNBS challenge but 0.25 mL of PBS intrarectally. In addition, group CG500 was administered GMP orally [500 mg/(kg · d) in 1% methylcellulose] from the beginning of the study and for 7 d, whereas group C was administered vehicle only. Colitis was induced in all remaining groups with TNBS as described above. The T group was administered only TNBS to induce colitis and 1% methylcellulose orally, serving as disease control. Groups G500, G50 and G5 were administered different doses of GMP orally [500, 50 or 5 mg/(kg · d) in 1% methylcellulose, respectively] starting 2 d before the TNBS challenge. Group PG500 was administered 500 mg/(kg · d) of GMP orally in 1% methylcellulose, starting 3 h after the TNBS challenge. Two sulfasalazine groups were administered 500 mg/(kg · d) of that drug starting 2 d before colitis (group SZ) or 3 h after colitis induction (group PSZ). Treatments continued for 5 d after the TNBS challenge. An esophageal catheter was used to deliver all GMP and sulfasalazine treatments. A second experiment with groups C, T, and G500 (n = 6) was conducted to obtain additional samples required for the study (because of the limits imposed by the use of longitudinally cut samples). All data were pooled and presented as such. In all cases, body weights as well as group water and food intake were recorded daily.
Assessment of colonic damage. Animals were killed by cervical dislocation and the entire colon was removed, gently flushed with saline, placed on an ice-cold plate, cleaned of fat and mesentery, and blotted on filter paper. Each specimen was weighed and its length measured under a constant load (2 g). The large intestine was opened longitudinally and scored for visible damage by an observer unaware of treatment on a 0–25 scale according to the criteria detailed in Table 1. The colon was subsequently divided longitudinally in several pieces for biochemical determinations. The fragments were immediately frozen in liquid nitrogen except for the sample used to determine total glutathione content, which was immediately weighed and frozen in 1 mL of 5% (wt/v) trichloroacetic acid (see below). All of the samples were kept at –80°C until analysis.
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Western blot. The colonic levels of inducible oxide nitric synthase (iNOS) were determined by immunoblotting. Colonic samples were homogenized in lysis buffer (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100 in PBS) with protease inhibitors (1 mmol/L 1,10-phenanthroline, 1 mmol/L phenylmethylsulfonyl fluoride, 18 mg/L aprotinin). The supernatants obtained after centrifugation (7000 x g, 10 min at 4°C) were boiled for 4 min in Laemmli buffer, separated by SDS-PAGE (10%), electroblotted to nitrocellulose membranes, and probed with the corresponding antibodies (1:1000 overnight at 4°C). The bands were detected by enhanced chemiluminescence (PerkinElmer) and quantitated with NIH software (Scion Image). After the transference of the samples to nitrocellulose membranes, equal loading was checked routinely by Coomassie blue staining. The composition of the Laemmli buffer (5X) was: 312 mmol/L SDS, 50% v:v glycerol, 1% v:v 2-mercaptoethanol, 22.5 mmol/L EDTA trisodium salt, 220 mmol/L Tris, and traces of bromphenol blue (pH = 6.8).
RT-PCR. The expressions of IL-1ß, interleukin 1 receptor antagonist (IL-1ra), transforming growth factor ß (TGF-ß), mucins (MUCs) 2, 3 and 4 as well as trefoil factor 3 (TFF3) were examined by RT-PCR. Total RNA was extracted with Trizol (Life Technologies). RNA (5 µg/sample) was subjected to reverse transcription using the First-strand cDNA synthesis kit (Amersham Biosciences). PCR amplification was performed using 2 µL of cDNA for a final PCR reaction volume of 25 µL. The expression of the ribosomal 18S unit was routinely examined as a loading standard. The primers and size of the amplified fragments were: MUC2 (sense 5'-GCT CAA TCT CAG AAG GCG ACA G-3'; antisense 5'-CCA GAT AAC AAT GAT GCC AGA GC-3' 875 bp); MUC3 (sense 5'-CAC AAA GGC AAG AGT CCA GA-3'; antisense 5'-ACT GTC CTT GGT GCT GAA TG-3' 515 bp); MUC4 (sense 5'-CGT ACT AGA GAA CTT GGA CAT G-3'; antisense 5'-GGT AGG AGA ACT TGT TCA TGG-3' 638 bp); TFF3 (sense 5'-ATG GAG ACC AGA GCC TTC TG-3'; antisense 5'-ACA GCC TTG TGC TGA CTG TA-3' 403 bp); IL-1ß (sense 5'-AAT GAC CTG TTC TTT GAG GCT G-3'; antisense 5'-CGA GAT GCT GCT GTG AGA TTT GAA G-3' 115 bp; IL-1ra (sense 5'-GAG TCA GCT GGC CAC CCT G-3'; antisense 5'-CAG ACT TGA CAC AAG ACA GGC A-3' 230 bp); TGF-ß (sense 5'-GCT AAT GGT GGA CCG CAA CAA C-3'; antisense 5'-CAC TGC TTC CCG AAT GTC TGA C-3' 200 bp); ribosomal 18S unit (sense 5'-CCA TTG GAG GGC AAG TCT GGT G-3'; antisense 5'-CGC CGG TCC AAG AAT TTC ACC-3' 389 bp). To set up the PCR conditions, different amounts of colonic RNA from a pool of samples and different number of cycles were assayed (data not shown). The cycle numbers and hybridization temperatures for each PCR reaction were as follows: 23 cycles and 56°C (for MUC2, MUC3 and TFF3); 27 cycles and 57°C (for MUC4, TGFß, IL-1ß, and IL-1ra); 17 cycles and 60°C (for the ribosomal 18S unit). After the PCR amplification, 5 µL of each reaction was resolved in 2.5% (wt:v) agarose gels using ethidium bromide. Bands were quantitated with NIH software (Scion Image).
Statistical analysis. Results are expressed as means ± SEM. Differences among means were tested for statistical significance by 1-way ANOVA and a posteriori Fisher least significance tests on preselected pairs (treatments vs. C and T groups). All analyses were carried out with the SigmaStat program (Jandel). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Effect of sulfasalazine. Our data show a clear anti-inflammatory effect of this established drug administered as a pretreatment; this was characterized by a decrease in the colonic damage score, necrotic extension, and colonic weight:length ratio (Table 3, P < 0.05). However, sulfasalazine did not reduce anorexia or body weight loss, and colonic length was also unchanged (Tables 2, and 3). Of note, the aforementioned significant effects of sulfasalazine were generally comparable to those of GMP at the same dose, i.e., 500 mg/(kg · d). On the other hand, when administered as a post-treatment, differences in colonic length (P = 0.091) and weight to length ratio (P = 0.075) with sulfasalazine were not significant.
Biochemical characterization of the effects of GMP and sulfasalazine. TNBS colitis was associated with a significant increase in AP activity, a parameter recently proposed as marker of inflammation (16) (Fig. 1A). GMP, but not sulfasalazine pretreatment, reduced AP colonic activity (57%, P < 0.05). However, when administered as a post-treatment, only sulfasalazine had a significant effect (62%, P < 0.05).
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Further examination of the status of the rats after 5 d of colitis revealed that the production of iNOS and proliferating cell nuclear antigen, as well as the mRNA levels of IL-1ß and IL-1ra, were upregulated by the TNBS challenge (Figs. 2and 3 and data not shown). Administration of 500 mg GMP/(kg · d) to rats before the administration of TNBS resulted in a decrease in the expression of iNOS. This was not a uniform effect, i.e., some rats exhibited very low levels, whereas others had iNOS levels comparable to rats from the T group. When GMP was given after TNBS, the response was similar (Fig. 2). Sulfasalazine had a more uniform effect, resulting in a virtual normalization of iNOS as a pretreatment. When administered as a post-treatment, the effect was clearly lower but still significant (P < 0.05).
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We also measured the levels of TGF-ß, a protecting peptide that induces migration, differentiation, and apoptosis and inhibits proliferation. Five days after the induction of colitis, TGF-ß mRNA abundance did not differ among the groups studied (Fig. 3C).
We characterized the mucin expression profile at the mRNA level. Our results indicate that only MUC4 mRNA production was increased 5 d after the induction of colitis, whereas MUC2 or MUC3 mRNA production did not differ (Fig. 4). MUC4 mRNA levels in groups G500 and PG500 were lower than those of the T group (P < 0.05), indicating that the administration of the highest dose of GMP both before and after the TNBS challenge normalized the levels of this mucin. Unfortunately, we could not measure MUC4 mRNA in the sulfasalazine groups. Interestingly, both GMP-treated groups also showed diminished production of MUC3, although this change was not significant in the case of the PG500 group (P = 0.114). On the other hand, TFF3, an important peptide produced by goblet cells and involved in the maintenance and repair of the intestinal mucosal barrier, was also markedly induced in the T group (P < 0.05, Fig. 4A). Interestingly, this effect was completely prevented by GMP administered either as a pretreatment or as a post-treatment. Sulfasalazine, on the other hand, produced a nonsignificant decrease when given before TNBS (P = 0.086), whereas it had no effect when administered after colitis induction.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Our results demonstrate that GMP gavage results in significant protection from inflammatory damage in TNBS-induced colitic rats. Thus GMP reduced the colonic damage score, weight:length ratio, and extent of necrosis, and increased AP colonic activity and iNOS expression. Furthermore, these effects did not differ from those obtained with sulfasalazine, an established drug treatment for IBD (11), suggesting that the therapeutic effect observed with GMP is quantitatively relevant. The effect was clearly dose dependent because the G50 and G5 treatment had a much smaller effect on intestinal inflammation. Consequently, only the G500 group was considered for further analysis. GMP exerted a clearly superior therapeutic effect when given before colitis induction than as a post-treatment; this is a common observation because most therapies offer superior effects as a pretreatment in experimental colitis, including sulfasalazine in the present study. It is interesting to note, however, that even after colitis induction, some of the beneficial effects of GMP were retained, suggesting that patients may benefit from GMP even when taken during inflammatory bouts of their condition.
Unlike sulfasalazine, GMP had a significant effect on anorexia and weight loss. In TNBS-induced colitis as well as in human IBD, anorexia occurs as a consequence of actions on the hypothalamus of systemically elevated IL-1ß and other factors (32,33). Thus a greater food intake as detected in several GMP-treated groups may be interpreted as reflecting a lowering of the levels of these mediators secondary to prevention of colitic damage. Consistent with this hypothesis, peripheral blood mononuclear cells from GMP-treated rats produced lower levels of tumor necrosis factor than those obtained from untreated colitic rats (data not shown).
Interestingly, the expression of IL-1ß, as judged by its mRNA level, was significantly diminished as a consequence of GMP treatment. This is in agreement with the previous observation by Monnai and Otani (7) that GMP stimulates IL-1ra release from monocytes. IL-1ra is a natural cytokine inhibitor that prevents IL-1ß signaling through its receptor by competitive binding with the latter, thus modulating the inflammatory cascade. This mechanism would be expected to reduce the activation of monocytes, themselves a major source of IL-1ß. Hence we measured IL-1ra mRNA levels, which were reduced by GMP pre- and post-treatment. Because both IL-1ß and IL-1ra are subject to post-transcriptional in addition to transcriptional regulation (34,35), our data cannot establish whether the IL-1ß:IL-1ra ratio is modified by GMP treatment. However, it is interesting to note that the IL-1ra mRNA levels were reduced only by GMP post-treatment, whereas those of IL-1ß were unchanged. This suggests that the IL-1ß:IL-1ra ratio may be reduced with GMP pretreatment but not with GMP post-treatment, a circumstance that would be consistent with the reduced anti-inflammatory activity in the latter case. Of note, very similar results were obtained with sulfasalazine, indicating that the same mechanism might account for its diminished effect when given after colitis induction.
We also measured other inflammatory markers in an attempt to define the mechanism of action of GMP. TFF3 is a bioactive peptide involved in the maintenance of gastrointestinal tissue as well as in tissue repair, particularly at the epithelial level (36). Although the mechanisms that mediate TFF3 actions are ill defined, TFF3 appears to be upregulated in experimental colitis and is thought to exert a beneficial role in this context (37–39). Our data are in agreement with these findings because TFF3 expression [which appears to be transcriptionally regulated (40)] was significantly increased in the T group 5 d after colitis induction. However, the GMP-treated groups, G500 and PG500, had TFF3 mRNA levels comparable to those of the control group. This probably reflects the protection of these rats from colonic inflammation brought about by GMP rather than a mechanism of action, although this cannot be ruled out because the timing of TFF3 induction and effects in TNBS colitis are not characterized. On the other hand, it can be assumed that the mechanism of action of GMP is not related to antioxidative activity (because glutathione levels were unchanged) or to actions on TGF-ß, an anti-inflammatory peptide that opposes Th-1 responses but that can produce excessive scarring of tissue (also unaffected by GMP treatment) (41).
iNOS upregulation was greatly reduced by GMP pretreatment and only modestly by GMP post-treatment (P < 0.05). Thus, iNOS inhibition may be a relevant mechanism of action of GMP. Of note, the effect of sulfasalazine on iNOS was very similar, although of a greater magnitude. Sulfasalazine is expected to decrease iNOS induction via its inhibitory effects on nuclear factor-B (42).
The effects of GMP also include a normalization of MUC4 expression. MUC4 is a heterodimeric glycoprotein complex that consists of a peripheral O-glycosylated mucin subunit, ascites sialoglycoprotein-1 (ASGP-1), tightly but noncovalently linked to a N-glycosylated transmembrane subunit, ASGP-2 (43). The complex is expressed in a number of normal, vulnerable epithelial tissues, including the mammary gland, uterus, colon, cornea, and trachea. MUC4 was shown to bind ErbB2, a receptor tyrosine kinase, triggering a specific phosphorylation in the absence of other ErbB ligands and potentiating phosphorylation and signaling by the latter. Hence MUC4 may be involved in epithelial homeostasis in addition to its classically assigned protective function. The fact that neutrophil elastase has the potential to increase MUC4 expression (44) is consistent with its upregulation in TNBS-induced colitis because this model is characterized by intense neutrophil infiltration (13). Thus, the normalization of MUC4 expression observed in the G500 and PG500 groups likely reflects the amelioration of the inflammatory response. These results should be interpreted cautiously because of the existence of post-transcriptional regulation of the MUC4 gene. Furthermore, we do not know whether MUC4 is involved in sulfasalazine action because we were unable to amplify its mRNA. It is obvious, however, that the effects of GMP on TFF3, MUC3, and MUC4 are not as well correlated with the anti-inflammatory activity as the effects on AP and IL-1ß and IL-1ra expression because these are the only variables affected significantly in the pretreatment group (G500) but not in the post-treatment group (PG500). Because the period of colitis was relatively short, it is possible that the effects on mucins and TFF3 may play a more important role in the long term. However, as explained above, the TNBS model is not well suited for this type of mechanism.
In conclusion, GMP exerts a dose-dependent anti-inflammatory effect in TNBS-induced colitis, with a potency and efficacy similar to those of sulfasalazine. Its mechanism of action is unknown but may be related to actions on IL-1ß and iNOS. GMP may be useful for the dietary management of IBD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AP, alkaline phosphatase; ASGP, ascites sialoglycoprotein; C, control (noncolitic) group; CG500, group of noncolitic rats treated with 500 mg/kg of GMP; GMP, glycomacropeptide; GMP500, GMP50, GMP5, groups of colitis rats pretreated with 500, 50, or 5 mg/kg of GMP; IBD, inflammatory bowel disease; IL-1ß, interleukin 1 ß; IL-1ra, interleukin 1 receptor antagonist; iNOS, inducible nitric oxide synthase; MUC, mucin; PG500, group of colitic rats post-treated with 500 mg/kg of GMP; SZ and PSZ, groups of colitic rats pre- or post-treated with 500 mg/kg sulfasalazine, respectively; T, TNBS control group; TFF, trefoil factor; TGF-ß, transforming growth factor ß; TNBS, trinitrobenzenesulfonic acid.
Manuscript received 29 October 2004. Initial review completed 14 November 2004. Revision accepted 28 February 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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