Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease. Despite IBD's unknown etiology, research in animal models, human genetics, basic science, and clinical trials has provided important new insights into the pathogenesis of chronic, immune-mediated intestinal inflammation (1). Many treatment modalities are available for IBD, but these are only effective for ameliorating the signs and symptoms of the disease; there is, as yet, no cure for this condition.
To build knowledge on the mechanisms involved in the development of colitis, several animal models mimicking human colitis have been developed (2). Oral administration of dextran sulfate sodium (DSS) to rodents is an experimental model sharing clinical (diarrhea, bloody stools, and weight loss) and morphological (crypt erosion, goblet cell depletion) features with human ulcerative colitis that can be used to test potential therapeutic agents (3,4). The mechanism of action of DSS is still unknown, but various explanations include direct cytotoxicity, disrupted interaction of intestinal epithelial cells, change of the normal intestinal microflora, and regional inflammation within the colon through the upregulation of TH1 and TH2 type of cytokines (5).
Ghrelin is a growth hormone–releasing peptide secreted predominantly by the endocrine cells of the stomach, and acts on the appetite-regulating center in the hypothalamus to stimulate food intake (6). Ghrelin receptor mRNA expression is present in many peripheral organs, such as heart, lung, liver, kidney, pancreas, stomach, small and large intestines, adipose tissues, and immune cells, which indicate that ghrelin has multiple functions in these tissues (7). Recently, studies have shown anti-inflammatory activity of ghrelin in ischemic reperfusion injuries, endotoxic shock, and sepsis (8,9). The anti-inflammatory action of ghrelin in an experimental model of colitis was also demonstrated (10). It was also demonstrated in patients with IBD that ghrelin level is increased during active disease and decreased during remission (7).
Obestatin is a new peptide derived from preproghrelin, which exhibits opposite effects of ghrelin on energy metabolism and gastrointestinal motility (10–13). Although the studies regarding the effect of obestatin are limited in number, it has been shown that peripherally or intracerebroventricularly administered obestatin has a profound effect in reducing food intake and body weight, whereas it slows gastric emptying and decreases jejunal contractility (12). To our knowledge, the anti-inflammatory effects of obestatin have not been studied. In this study, we aimed to study the anti-inflammatory effects of exogenous obestatin and ghrelin in a murine model of DSS-induced colitis.
Sprague-Dawley rats of both sexes, weighing 230 to 300 g, were obtained from the Marmara University Animal Center. The rats were kept at a constant temperature (22°C ± 1°C) for 12 hour:12 hour light and dark cycles, fed with standard rat chow, and fasted for 12 hours before the experiments, but were allowed free access to water. All experimental protocols were approved by the Marmara University Animal Care and Use Committee.
Induction of Colitis and Experimental Design
Experimental colitis was induced by oral administration of DSS (molecular weight 5000 Da; Sigma Chemicals, St Louis, MO) ad libitum. Acute colitis was induced by adding 3% (wt/vol) DSS to the drinking water of the rats for 5 days, whereas chronic colitis was induced within 10 days. Injections were started 12 hours before drinking DDS-added water and continued daily for 5 days in the acute groups and 10 days in the chronic groups.
In the acute colitis groups, 24 rats were divided into 3 subgroups, each group comprising 8 rats. The first group was injected saline (1 mL/kg) intraperitoneally (IP) (saline group), the second group obestatin IP (50 μg/kg) (obestatin group), and the third group ghrelin IP (20 μg/kg) (ghrelin group) 12 hours before adding 3% DSS into their drinking water, and the injections were continued throughout 5 days of drinking DSS-added water. Similarly, 24 rats were divided into 3 groups and injected with either saline, obestatin, or ghrelin at the same doses, but the rats were given normal tap water for 5 days (control groups).
Rats in the chronic colitis groups were randomized into 3 subgroups, which included 8 rats in each. As in the acute model, saline (1 mL/kg), obestatin (50 μg/kg), or ghrelin (20 μg/kg) was injected IP in the rats 12 hours before and throughout 10 days of drinking DSS-added water. Three control groups that received normal tap water without DSS addition also received saline, obestatin, or ghrelin for 10 days.
Disease Activity Index
Induction and severity of colitis were determined daily by observing the presence of blood in stool, stool consistency, and weight loss. Disease severity was scored using the clinical activity index ranging from 0 to 4 that was calculated by using following parameters: stool consistency, presence or absence of fecal blood, and percentage of weight loss (14).
Macroscopic and Microscopic Evaluation
After decapitation, the distal colon was removed, opened by a longitudinal incision, and was immediately examined, and visible damage was scored on a 0 to 10 scale by using modified Wallace criteria (15). Macroscopic evaluation was done by the same physiologist (B.Y.). Tissue samples taken from ileum and colon were placed in 10% formaldehyde and routinely processed by embedding in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined under a photomicroscope (Olympus BX51, Tokyo, Japan). The modified microscopic scoring was done by the same histologist (F.E.), who was unaware of which treatment the animal was subjected to. The histological score of the organ was calculated using the semiquantitative scale outlined in Table 1 (16,17).
Tissue Myeloperoxidase Activity
Myeloperoxidase (MPO) is an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes (PMNL). Tissue MPO activity is frequently used to estimate tissue PMNL accumulation in inflamed tissues and correlates significantly with the number of PMNL determined histochemically in tissues. MPO activity was measured in tissues with a procedure similar to that documented by Hileglass et al (18). Tissue samples were homogenized in 50 mmol/L potassium phosphate buffer (PB, pH 6.0), and centrifuged at 41,000g (10 minutes); pellets were suspended in 50 mmol/L PB containing 0.5% hexadecyltrimethylammonium bromide. After 3 freeze-and-thaw cycles, with sonication between cycles, the samples were centrifuged at 41,000g for 10 minutes. Aliquots (0.3 mL) were added to 2.3 mL of reaction mixture containing 50-mmol/L PB, o-dianisidine, and 20-mmol/L H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 minutes. MPO activity was expressed as unit per gram of tissue.
Tissue Malondialdehyde and Glutathione Assays
Tissue samples were homogenized with ice-cold 150-mmol/L KCl for the determination of malondialdehyde (MDA) and glutathione levels. The MDA levels were assayed for products of lipid peroxidation as described previously (19). Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1:1.56 × 105 mol−1 cm−1 and results are expressed as nanomoles MDA per gram tissue. Glutathione (GSH) is an antioxidant tripeptide and almost exclusively found in the reduced form in healthy tissues. Its major function is preventing damage to important cellular components caused by reactive oxygen substances such as free radicals and peroxides for the maintenance of cellular proteins and lipids in their functional state (20). GSH measurements were performed using a modification of the Ellman procedure (21). Briefly, after centrifugation at 2000g for 10 minutes, 0.5 mL of supernatant was added to 2 mL of 0.3 mol/L Na2HPO4·2H2O solution. A 0.2-mL solution of dithiobisnitrobenzoate (0.4 mg/mL 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH levels were calculated using an extinction coefficient of 13,600 mol−1 · L · cm−1. Results are expressed in millimoles GSH per gram of tissue.
Tissue Cytokine Assays
For cytokine determination in the colonic mucosa, protein extracts were isolated by homogenization of colonic segments (50-mg tissue per milliliter) in specific buffer including protease inhibitors (Procarta lysis buffer, Affymetrix, Santa Clara, CA). A 150-μL Procarta lysis buffer was used for 10 mg of colonic tissue sample. The tissue was then homogenized in a dounce homogenizer until there were no tissue clumps (approximately 30–40 strokes) and centrifuged at 14,000 rpm (bench top centrifuge) for 10 minutes at 4°C. The supernatant was transferred to a new tube and samples were stored at −80°C until cytokine analysis. Tissue levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, interferon-γ (IFN-γ), transforming growth factor-β (TGF-β), and IL-10 were quantified according to the manufacturer's instructions and guidelines using Procarta Cytokine Assay Kit specific for the rat cytokines. These particular assay kits were selected because of their high degree of sensitivity, specificity, inter- and intraassay precision, and small amount of tissue sample required to conduct the assay.
Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). All data were expressed as means ± standard error of the mean. Groups of data were compared with an analysis of variance followed by Tukey multiple comparison tests. Values of P < 0.05 were regarded as significant.
Effects of Obestatin and Ghrelin on Clinical and Histopathological Evidence of DSS-Induced Colitis
Rats subjected to oral DSS administration developed bloody diarrhea and significant weight loss. Both ghrelin and obestatin treatments ameloriated bloody diarrhea and recovered the body weight loss (Fig. 1A and B). Consequently, in both acute and chronic colitis groups, obestatin and ghrelin injections decreased disease activity index scores, but these reductions were statistically significant only in the acute colitis group (Fig. 1C and D).
In both acute and chronic colitis groups, severe edema, hyperemia, colonic dilatation, hemorrhage, and perforation were observed macroscopically (Fig. 2: I A–C and II A–C, acute and colitis, respectively). Macroscopic findings, hence scores, were significantly decreased in both obestatin- and ghrelin-injected groups; however, there was not statistically significant difference regarding the improvement in the macroscopic changes when the effects of either ghrelin or obestatin were compared.
Microscopic examination of the colonic tissue revealed epithelial cell loss, inflammatory cell infiltration, submucosal edema, and vasculitis in both acute and chronic colitis groups (Fig. 3B and G). In the ghrelin-treated acute colitis group, partial ameloriation of microscopic findings, such as decreases in epithelial cell loss, inflammatory cell infiltration, and submucosal edema were observed; however, histological improvement was much more prominent in the obestatin-treated group (Fig. 3D and F). In the chronic colitis group, only obestatin-treated rats, but not ghrelin-injected rats, showed an improvement in histopathological findings induced by DSS colitis (Fig. 3I).
Myeloperoxidase Activity and Serum MDA and GSH Levels
Oral DSS administration in the saline-treated acute colitis group caused a significant increase in neutrophil infiltration, which was assessed by elevated MPO activity in the colon, compared with the control group (Fig. 4A). Obestatin and ghrelin treatments, however, prevented the DSS-induced increase in MPO activity in the colonic tissue. A major indicator of oxidative injury is the formation of MDA, an end product of lipid peroxidation. Colonic MDA levels were significantly elevated in acute and chronic colitis groups and they were decreased significantly in obestatin- and ghrelin-injected groups (Fig. 4C).
GSH plays a critical role in limiting the propagation of free radical reactions, which would otherwise result in extensive lipid peroxidation. In the saline-treated acute colitis group, there was no significant difference in colonic GSH levels, whereas colonic GSH levels were significantly low in the saline-treated chronic colitis group; however, obestatin-treated rats, but not ghrelin-treated rats, demonstrated a statistically significant increase in colonic GSH level (Fig. 4B).
Tissue Cytokine Levels
Inflammatory cytokines (IL-1β, IFN-γ, TNF-α) in colonic tissue were increased in both acute and chronic groups of DSS-induced colitis (Fig. 5). In the acute colitis group, ghrelin decreased colonic IL-1β levels, but had no significant impact on the colonic levels of the other studied cytokines. Furthermore, in the acute colitis group, obestatin decreased colonic IL-1β and TNF-α levels, but had no effects on the levels of the other cytokines. In the colonic tissues of the chronic colitis groups, ghrelin decreased inflammatory cytokines (IL-1β, IFN-γ, TNF-α) and increased anti-inflammatory cytokine TGF-β without affecting IL-10. Obestatin in the chronic colitis group resulted in decreased colonic inflammatory cytokine response (IL-1β, IFN-γ, TNF-α) with increased colonic levels of anti-inflammatory cytokines (IL-10 and TGF-β).
The results of the present study revealed that both ghrelin and obestatin prevented colitis-induced body weight loss, improved disease activity, and decreased macroscopic and microscopic damage scores. In the acute colitis groups, both ghrelin and obestatin treatments significantly depressed colonic lipid peroxidation and colonic IL-1β, and obestatin further inhibited TNF-α in the acute colitis groups. In the chronic colitis groups, however, elevated MPO activity, increased lipid peroxidation, and increased levels of proinflammatory cytokines were reduced by both ghrelin and obestatin. Accordingly, anti-inflammatory cytokine TGF-β was increased by both hormones, whereas obestatin increased tissue GSH content and anti-inflammatory IL-10 level, showing a more potent anti-inflammatory activity than ghrelin.
Although derived from the same precursor preproghrelin, obestatin exhibits opposite effects of ghrelin in energy metabolism and gastrointestinal motility. In recent years, research has focused on the anti-inflammatory effect of ghrelin in sepsis, endotoxemia, pancreatitis, myocardial ischemia reperfusion injury, and colitis (1–6). According to our knowledge, however, there is no published study demonstrating the role of obestatin in inflammatory colitis. This study is the first showing the role of obestatin in a model of inflammatory colitis and also the first to compare the effects of obestatin and ghrelin in both acute and chronic colitis. In this experimental model of colitis induced by DSS, ghrelin and obestatin were given as therapeutic agents, and the novel anti-inflammatory effects of obestatin and ghrelin were demonstrated. Although obestatin exerted anti-inflammatory effect in both acute and chronic colitis groups, ghrelin was effective only in the acute phase.
Anti-inflammatory action of ghrelin was studied by Gonzalez-Rey et al (10) in experimental models of DSS and trinitrobenzene sulfonic acid–induced colitis. In this study, dose and duration of ghrelin administration was different from our study. They demonstrated anti-inflammatory action of ghrelin by deactivation of intestinal inflammatory response and restoration of mucosal immune tolerance in the gastrointestinal tract. Later, De Smet et al (11) investigated the role of endogenous and exogenous ghrelin in the pathogenesis of colitis, in which DSS was used to induce colitis in ghrelin knockout mice. They suggested that excitatory and inhibitory neural responses were decreased with ghrelin treatment, but ghrelin did not have an anti-inflammatory effect. Instead, the anti-inflammatory effect was caused by the growth hormone, which was increased after single administration, but blunted during continuous treatment.
The inhibition of lipid peroxidation is one of the mechanisms of the anti-inflammatory action of both ghrelin and obestatin (13,22). Imbalance between excess production of free oxygen radicals and inadequate amount of antioxidants causes tissue damage by peroxidation of membrane lipids, which increases cellular permeability and causes ion disequilibrium and leads to intracellular damage (23). MDA is a major indicator of oxidative injury. Its level increases in inflammation and it has been shown that ghrelin treatment decreases tissue MDA level (13,22). In our study, both ghrelin and obestatin decreased tissue MDA levels significantly in acute and chronic colitis. This is the first study showing the anti-inflammatory effect of obestatin in experimental colitis through inhibition of lipid peroxidation. Another important aspect of this study was the use of exogenous ghrelin and obestatin together in the same model, which enabled us to investigate the role and mode of action of these peptides in experimental colitis.
In acute inflammation, recruitment of PMNL into the inflamed tissues is followed by the secretion of several cytotoxic enzymes such as MPO, elastase, protease, and lactoferrin; thus, the tissue MPO levels are directly proportional to the number of PMNL infiltrations (24). In the present study, both ghrelin and obestatin decreased tissue MPO levels significantly in chronic, but not in acute, colitis, suggesting that peptides inhibit PMNL recruitment during the progression of chronic colitis. Previously, it has been shown that ghrelin, when used in higher doses (250 μg/kg), suppressed tissue MPO level significantly (10). It appears that a higher dose in the chronic colitis could be effective in suppressing the tissue neutrophil infiltration.
GSH plays a critical role in limiting the propagation of free radical reactions, which would otherwise result in extensive lipid peroxidation and cellular damage. In the present study, colonic GSH levels were reduced in the chronic colitis group, but not in the acute form. This could be because of the shortness of time for depletion of GSH stores, and this could explain significant GSH depletion in the chronic model. In the obestatin-treated group, colonic GSH levels were increased, which implies the anti-inflammatory effect of obestatin was accompanied by preservation of the cellular antioxidant stores; however, in the ghrelin-treated chronic colitis group, the increase in colonic GSH level did not reach statistical significance. In the previous studies, it has been shown that GSH levels had increased after ghrelin treatment in pancreaticobiliary inflammation (2), gastric ischemia–reperfusion injury (3), biphosphonate-induced gastric damage (22); however, colonic GSH level in the chronic colitis group was replenished by obestatin treatment, suggesting that obestatin supports the maintenance of GSH stores as it protects the colonic tissue against DSS-induced injury. This is the first study investigating the anti-inflammatory activity of both ghrelin and obestatin in a model of experimental colitis and the mode of this effect via particularly GSH.
Both humoral and cellular immune systems play role in the pathogenesis of inflammation in IBD (25–28). Inflammation is caused by imbalance between pro- and anti-inflammatory cytokines. It has been shown that proinflammatory cytokines such as IL-1β, IL-6, and TNF-α were increased in the mucosa of ulcerative colitis, suggesting that they play a role in the pathogenesis of the disease (25–28). Cytokines cause inflammation by several mechanisms such as increasing membrane permeability, inhibiting collagen synthesis, creating local ischemia, and damaging endothelium (25–28); however, as it was demonstrated in tissue cultures obtained from patients with IBD, anti-inflammatory cytokines, for example, IL-10, suppress mononuclear phagocytes (29,30). This was further supported by healing of enterocolitis by injection of IL-10 to transgenic IL-10 knockout mice with chronic colitis (29,30). In the present study, proinflammatory cytokines such as IL-1β, TNF-α, and IFN-γ were increased in both acute and chronic colitis, and the anti-inflammatory IL-10 and TGF-β were decreased in the chronic colitis groups. In our chronic colitis group, ghrelin administration decreased IL-1β, TNF-α, and IFN-γ levels, and increased TGF-β significantly, but in the acute colitis group, ghrelin treatment decreased only IL-1β levels significantly. Ghrelin has a specific and selective inhibitory effect in cytokine production and expression (31). It has been shown that ghrelin infusion inhibited proinflammatory cytokines such as IL-1α, IL-1β, and TNF-α in lipopolysaccharide-administrated rats (31). Gonzalez-Rey et al (10) demonstrated that the TH1 cytokine (TNF-α, IFN-γ, IL-6) response was suppressed and Treg cytokines (IL-10 and TGF-β) were stimulated by administration of ghrelin. In contrast to previous studies, De Smet et al (11) showed that duration of exogenous ghrelin administration may determine whether it reacts as pro- or anti-inflammatory agent. When they used ghrelin for a prolonged period, it exerted an anti-inflammatory effect, whereas shorter periods of application produced no effect at all; however, obestatin administration decreased proinflammatory cytokines in the acute and chronic colitis groups, whereas colonic IL-10 and TGF-β levels were increased in the chronic but not changed in the acute colitis groups. This is the first study investigating the anti-inflammatory effect of obestation, and it has been demonstrated that obestatin treatment suppressed proinflammatory TH1 cytokines in both acute and chronic colitis with a concomitant stimulation of anti-inflammatory cytokines in chronic colitis.
The etiology of IBD is still unknown and still being investigated. Existing treatment modalities are palliative but not curative. In the present study, we demonstrated that ghrelin suppressed neutrophil infiltration, damage caused by lipid peroxidation, and relieved histological damage; in brief, ghrelin prevented tissue inflammation. In chronic colitis, despite its anti-inflammatory effects, ghrelin remained incapable of preventing histopathological damage. This could be because of insufficient dosage and the duration of ghrelin administration. Obestatin alleviated histological damage by suppressing PMNL infiltration, inhibiting reactive oxygen radical generation and proinflammatory cytokine production, and provoking the synthesis of anti-inflammatory cytokines during the progression of both acute and chronic colitis. In contrast to the large number of studies concerning the anti-inflammatory action of ghrelin, the studies regarding the anti-inflammatory effect of obestatin are limited. Because there is still a great medical need for novel pharmacological agents for the treatment of IBD, the present results suggest that ghrelin and obestatin could be promising peptides with anti-inflammatory and merit further experimental and clinical studies to confirm their beneficial and promising effects for future IBD treatment modalities.
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