Abstract
Alendronate sodium, a primary amino bisphosphonate, is widely used in the treatment of various diseases that are associated with bone resorption, such as postmenopausal osteoporosis and Paget’s disease of bone. Although the adverse effects of biphosphonates on the gastrointestinal system have been demonstrated in experimental and clinical studies, the exact mechanisms underlying this damage are not clear yet. Ghrelin, a 28 amino acid peptide produced predominantly by the stomach, was shown to exert a potent protective action on the stomach of rats exposed to ethanol or stress.
Our objective was to evaluate the possible anti-oxidant and anti-inflammatory effects of ghrelin against alendronate-induced gastric damage. Wistar albino rats were administered alendronate (20 mg/kg) by gavage for 4 days, along with either ghrelin (10 ng/kg per day) or saline given i.p. After decapitation, stomach tissues were removed for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and tissue collagen content, while the extent of tissue damage was analyzed microscopically. Formation of reactive oxygen species was determined by chemiluminesence using a luminol probe in fresh gastric tissues. Serum tumor necrosis factor (TNF-α) and lactate dehydrogenase levels were assessed in trunk blood.
Oral administration of alendronate-induced significant gastric damage, accompanied by increased MPO activity, collagen content, MDA and luminol levels (P< 0.01–P< 0.001), while tissue GSH was decreased (P< 0.01). On the other hand, ghrelin treatment reversed these alterations (P< 0.05–P<0.001) as well as elevating serum TNF-α levels significantly (P< 0.001).
The findings of the present study suggest that alendronate induces oxidative gastric damage by a local irritant effect, and ghrelin ameliorates this damage by its possible antioxidant and anti-inflammatory properties.
Introduction
The bisphosphonates are a class of compounds known for their ability to prevent osteoclast-mediated bone resorption (Geddes et al. 1994) and recommended for the treatment of various disease conditions with enhanced bone resorption, including postmenopausal osteoporosis, Paget’s disease, hypercalcemia of malignancy and osteolytic bone metastases (Fleisch 1997, Meunier et al. 1999). These agents are analogs of pyrophosphate, an endogenous inhibitor of bone mineralization (Rosen & Kessenich 1996). Alendronate sodium (ALD) is a primary amino-bisphosphonate and one of the most commonly used members of this group. However, oral administration of bisphosphonates have been associated with gastrointestinally adverse effects including gastritis, gastric ulcer and erosive esophagitis, and it has been demonstrated that bisphosphonates differ in their potential to damage the gastroesophageal mucosa, ALD having a higher potential for such effects (Blank et al. 1997, Lanza et al. 1998, Graham & Malaty 1999). It was reported that the rate of acute mucosal injury due to ALD was comparable with that observed with non-steroidal anti-inflammatory drugs (Lowe et al. 2000). Generally, these gastrointestinal sideeffects were attributed to high local concentrations of the drug in patients who did not follow dosing instructions properly, or to their having an underlying esophageal or gastric disease (Lanza et al. 1998, Lowe et al. 2000, Graham 2002). Moreover, two large clinical studies revealed that when standard doses of bisphosphonates were administered in the proper manner in female volunteers without gastrointestinal symptoms or initial endoscopic pathology, erosions and ulceration in both the stomach and duodenum developed (Lanza et al. 2000, Thomson et al. 2002).
The exact mechanism underlying ALD-induced gastric damage is not clear yet. Marshall et al.(2000) conducted a randomized controlled trial to investigate the effect of ALD on gastric mucosal prostaglandin (PG) synthesis, but no association was found between the ALD-induced gastric mucosal damage and gastric mucosal PGE2 synthesis. Elliott et al.(1998) studied the effect of ALD in rats and rabbits and observed that the damaging effects of the drug were not attributable to changes in gastric acid secretion, blood flow or PG synthesis; they concluded that the damaging effects of ALD on the stomach were due to a topical irritant effect.
Ghrelin is a novel 28 amino acid peptide discovered in rat and human gastrointestinal tract, particularly gastric mucosa, as an endogenous ligand for growth hormone (GH) secretagogue receptor (GHS-R) (Kojima et al. 1999, Ariyasu et al. 2001). This peptide was also shown to affect gastric motility and gastric secretion (Masuda et al. 2000, Date et al. 2001). It has been demonstrated that feeding, food intake and obesity decrease circulating ghrelin levels in humans and rodents (Toshinai et al. 2001, Tschop et al. 2001). Ghrelin secretion is pulsatile and directly related to feeding behavior (Tolle et al. 2002) and it is upregulated during negative nutritional status, fasting and insulin-induced hypoglycemia (Toshiani et al. 2001), suggesting that ghrelin plays an important role in the regulation of feeding behavior, energy balance and obesity. Previous reports have indicated that ghrelin protects against ethanol-induced gastric ulcer (Sibilia et al. 2003) and exerts both specific and selective inhibitory effects on the expression and production of the inflammatory cytokines (Dixit et al. 2004). On the other hand, ghrelin infusions in lipopolysaccharide-challenged mice resulted in a significant attenuation of anorexia and led to inhibition of the proinflammatory cytokines, interleukin (IL)-6, IL-1β and TNF-α (Dixit et al. 2004), which are known to play an important critical role in the development of anorexia–cachexia syndrome (Inui 1999).
On the basis of this background, the present study was designed to elucidate the putative protective effect of ghrelin treatment on ALD-induced damage in the rat gastric mucosa.
Materials and Methods
Animals
Adult Wistar albino rats (250–300 g) of both sexes were kept in a light- and temperature-controlled room (12 h light:12 h darkness, 22 ± 0.5 °C) with relative humidity (65–70%) kept constant. The animals were fed a standard pellet. Experiments were approved by the Marmara University School of Medicine, Animal Care and Use Committee.
Based on our previous studies (Sener et al. 2004, 2005), ALD was given by gavage (20 mg/kg) once a day for 4 days following overnight fasting periods. Either ghrelin (10 ng/kg per day) or saline was given i.p. immediately after and 6 h after ALD administration. This anti-inflammatory dose of ghrelin was chosen based on our earlier experiments in rats with acute pancreatitis (Kasimay et al. unpublished observations). A control group had no orogastric or i.p. treatments. Each group consisted of six animals. On the 4th day, rats were decapitated 1 h after the last treatment, and the trunk blood was collected and centrifuged (4000 g, 20 min) for the measurement of serum TNF-α and lactate dehydrogenase (LDH) levels. Serum LDH activity was measured as a marker of systemic tissue injury. The gastric tissue samples were immediately taken and stored at −70 °C. Formation of reactive oxygen species in tissue samples was monitored by using a chemiluminescence method. Malondialdehyde (MDA, an end product of lipid peroxidation) and glutathione (GSH, a key antioxidant) levels were measured in another set of tissue samples. Tissue-associated myeloperoxidase (MPO) activity, as indirect evidence of neutrophil infiltration, was measured in both tissue samples, while oxidant-induced tissue fibrosis was determined by tissue collagen content.
Biochemical analysis
Measurement of serum TNF-α and LDH levels
TNF-α was evaluated by an RIA-IRMA method. All samples were assayed in duplicate using a commercial kit (Biosource Europe SA, Nivelles, Belgium). The activity of radioactive assays was measured by a gamma counter (LKB Wallac 1270 Rack Gamma Counter; Turku, Finland). TNF-α in the serum samples was expressed as ng/ml. LDH was determined spectrophotometrically using an automated analyzer (OpeRA, Bayer, USA).
Tissue MPO activity
MPO is an enzyme that is found predominantly in the azurophilic granules of polymor-phonuclear leukocytes (PMNs) and tissue MPO activity correlates significantly with the number of PMNs determined histochemically in inflamed tissues (Bradley et al. 1982). Thus, tissue-associated MPO activity was utilized as an indication of accumulation of neutrophils. All reagents for MPO assay were obtained from Sigma (St Louis, MO, USA). The tissue samples (0.2–0.3 g) were homogenized in 10 volumes of ice-cold potassium phosphate buffer (50 mM K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5% w/v). The homogenate was centrifuged at 12 000 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was then re-homogenized with an equivalent volume of 50 mM K2HPO4 containing 0.5% (w/v) HETAB and 10 mM EDTA (Sigma). MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine·2 HCl. One unit (U) of enzyme activity was defined as the amount of the MPO present/g tissue weight that caused a change in absorbance of 1.0/min at 460 nm and 37 °C (Bradley et al. 1982).
Tissue GSH and MDA assays
Stomach samples were homogenized in ice-cold 150 mM KCl for determination of MDA and GSH levels. The MDA levels were assayed for products of lipid peroxidation (Beuge & Aust 1978). Results are expressed as nmol MDA/g tissue. GSH was determined by a spectrophotometric method using Ellman’s reagent (Beutler 1975). Results are expressed as μmol GSH/g tissue.
Tissue collagen measurement
Gastric collagen contents were determined as a marker of oxidant-induced fibrosis. Tissue samples were cut with a razor blade and immediately fixed in 10% formalin in 0.1 M phosphate buffer (pH 7.2) in paraffin and sections of 15 μm thick were obtained. Collagen content was measured according to a method described by Lopez de Leon & Rojkind (1985). The method is based on selective binding of the dyes Sirius Red and Fast Green FCF to collagen and non-collagenous components respectively. Both dyes were eluted readily and simultaneously by using 0.1 M NaOH–methanol (1:1). Finally, the absorbances at 540 and 605 nm were used to determine the amount of collagen and protein respectively.
Chemiluminescence assay
To assess the role of reactive oxygen species in ALD-induced gastric damage, luminol chemiluminescence, which measures the formation of hydroxyl radical, H2O2, hypochloride and peroxynitrite (ONOO−), was monitored at room temperature using a Mini Lumat LB 9506 luminometer (EG & G Berthold, Bad Wildbad, Germany). Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) was obtained from Sigma. Tissue samples were put into a vial and chemiluminescence counts were obtained after the addition of 0.2 mM luminol (Haklar et al. 2002). Counts were obtained at 15 s intervals for 10 min and corrected for mg of tissue sample. Data were expressed as area under the curve of relative light units (rlu).
Histopathological analysis
Samples were fixed in 10% formalin solution and following dehydration in a descending series of ethyl alcohol, were cleared in xylene and embedded in paraffin. Paraffin sections of tissues were cut at 6 μm on a rotary microtome, mounted on slides, and stained with hematoxylin-eosin (H&E) and examined under an Olympus BH-2 photomicroscope.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). All data are expressed as means ± s.e.m. Groups of data were compared by ANOVA followed by Tukey’s multiple comparison tests. Values of P< 0.05 were regarded as significant.
Results
Serum TNF-α and LDH levels
In the ALD-administered rats treated with saline, serum TNF-α levels were significantly increased (23.0 ± 2.5 pg/ml; P< 0.001) when compared with the control group (7.6 ± 0.9 pg/ml), while ghrelin treatment abolished this elevation (6.4 ± 0.6 pg/ml; P< 0.001) (Fig. 1A). On the other hand, significantly increased serum LDH levels (2558 ± 196.2 U/l; P< 0.001) in the ALD plus saline administered rats as compared with the control group (1543 ± 126.1 U/l) were not reduced by the ghrelin treatment (2518 ± 153.1 U/l; P< 0.01) (Fig. 1B).
Tissue MDA and GSH levels
Tissue MDA levels were increased in the ALD-treated group (14.3 ± 1.1 nmol/g; P< 0.05) when compared with the control group (10.3 ± 1.0 nmol/g). Ghrelin treatment reduced the ALD-induced increase in MDA levels (9.7 ± 0.9 nmol/g; P< 0.05) back to control levels (Fig. 2A). Oral ALD administration significantly reduced gastric GSH levels (0.9 ± 0.05 μmol/g; P< 0.05) (Fig. 2B), while ghrelin treatment significantly increased the GSH levels (1.4 ± 0.1 μmol/g; P< 0.05) back to control levels (1.4 ± 0.06 μmol/g).
Tissue MPO activity
Oral ALD administration caused a significant increase in gastric neutrophil infiltration, as assessed by elevated MPO activity in the stomach (26.39 ± 4.2 U/g; P< 0.01) when compared with the control group (9.9 ± 1.3 U/g; Fig. 2C). On the other hand, ghrelin treatment prevented the ALD-induced increase in the gastric MPO activity (15.1 ± 2.4 U/g; P< 0.05).
Tissue collagen content
Gastric collagen content was elevated in the ALD-administered group (8.7 ± 0.1 μg/mg protein; P< 0.001) when compared with the control group (6.1 ± 0.1 μg/mg protein; Fig. 2D). Ghrelin treatment, on the other hand, reduced the ALD-induced elevation in collagen level (7.2 ± 0.3 μg/mg protein; P< 0.001).
Tissue luminol levels
ALD administration in the saline-treated group significantly increased the gastric luminol level (40.4 ± 2.7 rlu/mg; P< 0.01) compared with the control group (22.4 ± 3.4 rlu/mg), while ghrelin treatment reduced luminol (28.6 ± 2.1 rlu/mg; P< 0.05) back to the control level (Fig. 2E).
Microscopic evaluation
The control group had a regular contour of both surface epithelium and fundic glands (Fig. 3A) with regular interglandular spaces. In the saline-treated ALD group, severe congestion of vessels in the connective tissue and prominent dilations among the fundic glands were observed (Fig. 3B). Besides, there was a moderate density of inflammatory cells in the lamina propria. In the ghrelin-treated ALD group, congestion of vessels in the connective tissue was reduced and fewer inflammatory cells were present, while the dilations in the surface and neck epithelia were still present (Fig. 3C).
Discussion
The results of the present study revealed that ALD-induced gastric injury, as evidenced by the changes in gastric GSH and MDA levels, MPO activity and luminol levels, appears to be an oxidative damage. Ghrelin, which demonstrated anti-inflammatory and antioxidant effects, protected the stomach against ALD-induced oxidative injury. Furthermore, the increase in serum TNF-α, which plays a pivotal role in the inflammatory processes, was also reversed by ghrelin treatment.
Bisphosphonates are a choice of effective treatment for osteoporosis but have been associated with gut mucosal injury. The anatomical distribution of both the gastric ulcers and esophageal damage is consistent with the topical irritant effect of bisphosphonates, but its clinical significance is still uncertain (Graham 2002). We have previously investigated the mechanism(s) underlying the gastrointestinal damaging property of chronic treatment with ALD, and suggested that ALD causes gastric damage by a direct effect on the mucosa, resulting in a direct oxidative damage, and also by an indirect oxidative damage through induction of neutrophil infiltration (Sener et al. 2005). It has been previously shown that at pH< 2, ALD sodium is converted to its acid form (Peter et al. 1998), which is known to be more irritating than the sodium salt. Since in the present study, ALD was administered orally in fasted rats, it seems likely that the gastric damage is, at least in part, due to the direct irritating effect of the drug in its acid form.
Despite the difficulty in quantifying reactive oxygen metabolites because of their reactive nature and short lives, chemiluminescence as used in the present study, is a simple but a reproducible technique. Luminol-enhanced chemiluminescence data revealed that the toxic metabolites that participate in the ALD-induced gastric damage, are mainly H2O2, OH• hypochlorite, peroxynitrite and lipid peroxyl radicals, which can severely disrupt cell membrane function and lead to DNA damage and cell death in the gastrointestinal mucosa (Bagchi et al. 1998, Salvemini et al. 1999). Lipid peroxidation mediated by reactive oxygen metabolites is believed to be an important cause of destruction and damage to gastric mucosa (Bandyopadhyay et al. 2002). In the present study, gastric MDA levels that indicate lipid peroxidation of the membranes were significantly increased after ALD, demonstrating tissue damage. The present results also demonstrated that ghrelin ameliorates the oxidative injury of the gastric tissue, which appears to involve the inhibition of toxic oxygen metabolite generation, suggesting the presence of an antioxidative function of ghrelin. In accordance with the lipid peroxidation results, the histological findings also support the belief that ghrelin can prevent the gastric damage induced by oral ALD treatment. Moreover, ALD-induced increase in fibrotic activity, as assessed by the collagen contents in the stomach, is reduced by ghrelin treatment, suggesting an additional protective effect on inflammation-induced production and deposition of extracellular matrix components. Similarly, ghrelin was shown to protect against ethanol- (Sibilia et al. 2003) and stress-induced (Brzozowski et al. 2004) gastric ulcers in rats. Since the damaging effects of both ethanol and stress are known to be closely associated with the impairment of mucosal barrier mechanisms, which involves the generation of reactive oxygen species, it is possible that ghrelin, via its antioxidant effects, reinforces the mucosal barrier function, as evidenced in the present study. Furthermore, ghrelin was implicated as having an anti-inflammatory role in Helicobacter pylori infection, where persisting gastric inflammation was associated with lower ghrelin levels (Murray et al. 2003), while the correction of infection has upregulated ghrelin secretion.
There are many studies focusing on the role of oxygen metabolite-mediated lipid peroxidation and neutrophil infiltration in the development of gastric mucosal injury (Ichikawa et al. 2002, Jimenez et al. 2004, Sener et al. 2004). Besides their direct damaging effects on tissues, it is well established that oxygen metabolites play a role in the recruitment of neutrophils, preferentially PMNs, into injured tissues (Zimmerman et al. 1990, Kettle & Winterbourn 1997). Activated PMNs are also a potential source of oxygen metabolites (Sullivan et al. 2000), initiating the deactivation of antiproteases (Swantek et al. 2000) and activating cytotoxic enzymes including elastase, proteases, lactoferrin and MPO (Kettle & Winterbourn 1997), thereby generating hypochlorous acid (HOCl). In the present study, elevated serum TNF-α levels along with increased MPO activity indicate the contribution of PMN infiltration and the impact of pro-inflammatory TNF-α in the oxidative gastric damage. Since ghrelin inhibited the ALD-induced increase in MPO activity and abolished the TNF-α response concomitantly, our results suggest that neutrophil accumulation contributes to ALD-induced gastric injury and the protective effect of ghrelin may be, in part, dependent on its inhibitory effect on tissue neutrophil infiltration and the neutrophil-associated TNF-α response. Previous studies have shown that ghrelin exerts potent anti-inflammatory effects by inhibiting the expression of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α (Dixit et al. 2004).
GSH, the main intracellular non-protein sulfhydryl, plays an important role in the maintenance of cellular proteins and lipids in their functional state, and provides major protection by participating in the cellular defense systems against oxidative damage (Ross 1988). GSH and other antioxidants play a critical role in limiting the propagation of oxygen metabolite-mediated lipid peroxidation. It is known that mitochondrial damage may lead to severe GSH depletion, because due to lack of γ-glutamylcysteine synthetase or GSH synthetase activities mitochondria cannot synthesize GSH, and GSH is transported from the cytosol (Reiter et al. 2000). Thus, gastric GSH depletion in the ALD-treated group of the present study may be attributable to enhanced lipid peroxidation. On the other hand, restoration of tissue GSH levels by ghrelin administration reduced the severity of gastric injury. Thus, it is likely that ghrelin has an important role in the maintenance of gastric GSH, either by increasing the synthesis or by inhibiting the depletion of this crucial antioxidant against the irritant drug-induced lipid peroxidation.
The present data suggest that ghrelin may be of therapeutic use in preventing ALD-induced oxidative gastric injury, by depressing neutrophil infiltration and inhibiting the release of inflammatory cytokines. In conclusion, the findings of the current study illustrate that exogenously administered ghrelin is capable of ameliorating the gastric side-effect of the anti-osteoporotic drug, ALD. Furthermore, it was suggested that treatment with the endogenous ligand for the GHS-R affects biochemical markers of bone turnover and increases growth velocity in selected short children with or without GH deficiency, while in rodents, GHS treatment increased bone mineral content (Svensson et al. 2001). Although appropriate clinical studies are necessary to test the direct beneficial effect of ghrelin in the treatment of osteoporosis, its protective role in the pharmacotherapy of bone resorption by ALD also merits consideration.
(A) Serum TNF-α and (B) LDH levels of control (‘C’), and alendronate-administered (‘ALD’, 20 mg/kg per day) rats, treated with either ghrelin (10 ng/kg per day) or saline for 4 days. In each group n=6; mean ± s.e.m.**P< 0.01 and ***P< 0.001 compared with control group; +++P< 0.001 compared with saline-treated ALD group.
Citation: Journal of Endocrinology 187, 3; 10.1677/joe.1.06432
(A) Malondialdehyde (MDA) levels, (B) glutathione (GSH) levels, (C) myeloperoxidase (MPO) activity, (D) collagen levels and (E) luminol levels in the gastric tissues of control (‘C’), and alendronate-administered (‘ALD’, 20 mg/kg per day) rats, treated with either ghrelin (10 ng/kg per day) or saline for 4 days. In each group n=6; mean ± s.e.m. *P< 0.05, **P< 0.01 and ***P< 0.001 compared with control group; +P< 0.05 and +++P< 0.001 compared with saline-treated ALD group. rlu: relative light units.
Citation: Journal of Endocrinology 187, 3; 10.1677/joe.1.06432
(A) Control group. The epithelium shows regular integrity (*) with interepithelial dilations (arrow); (B) Alendronate-treated group. Severe congestion of vessels in the connective tissue of both the subepithelium and lamina propria (arrow); prominent dilations in the surface and neck epithelia (*) and moderate density of inflammatory cells were observed. (C) Ghrelin-treated group. Congestion of vessels in the connective tissues was reduced (arrow) and fewer inflammatory cells were present; dilations in the surface and neck epithelia were still ongoing (*). H&E × 200.
Citation: Journal of Endocrinology 187, 3; 10.1677/joe.1.06432
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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