Gastroprotective effect of orexin-A and heme oxygenase system

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Gastroprotective effect of orexin-A and heme oxygenase system _ Burcu Gemici, PhD,a Ruken Tan, PhD,a Ilknur Birsen, MSc,b _ ¨ t Uysal, PhDb,* and V. Nimet Izgu a b

Department of Physiology, Faculty of Medicine, Near East University, Lefkosa, Cyprus Department of Physiology, Faculty of Medicine, Akdeniz University, Antalya, Turkey

article info

abstract

Article history:

Background: Orexin-A, besides playing an important role in the mechanism of food intake,

Received 15 April 2014

exhibits a potent gastroprotective action against the formation of acute gastric mucosal

Received in revised form

injury. The aim of the present study was to determine the effect of administered orexin-A

25 July 2014

against ischemiaereperfusion (I/R)-induced gastric injury on the expression of heme

Accepted 27 August 2014

oxygenase (HO)-1 and HO-2 in gastric tissue.

Available online 2 September 2014

Materials and methods: Wistar rats were subjected to 30 min of ischemia followed by 3 h reperfusion. Orexin-A was infused at a dose of 500 pmol/kg/min during the I/R period. The

Keywords:

lesion area was measured by stereomicroscope. The myeloperoxidase activity and

Orexin-A

4-hydroxinonenol-malondialdehyde content of gastric mucosa were evaluated spectro-

Ischemiaereperfusion

photometrically, and the gastric tumor necrosis factor-a was measured by enzyme linked

Gastric

immune sorbent assay. The expression of HO-1 and HO-2 was determined by Western

Lesion

blotting analysis.

Heme oxygenase-1

Results: Orexin-A significantly decreased the I/R-induced gastric lesions, myeloperoxidase

Heme oxygenase-2

activity, and 4-hydroxinonenol-malondialdehyde concentration in gastric tissue exposed to I/R. The gastroprotective effect of orexin-A in gastric I/R model was accompanied by the increase in HO-2 expression and the decrease in HO-1 expression. Conclusions: Orexin-A exerts a protective action on gastric mucosa subjected to I/R, and this effect is associated with the reduction of neutrophil infiltration and lipid peroxidation in gastric tissue in addition to the increase in HO-2 expression due to the administration of orexin-A. ª 2015 Elsevier Inc. All rights reserved.

1.

Introduction

Orexins or hypocretins are novel neuropeptides originally discovered in neurons in the lateral hypothalamus. Orexin peptides are involved in a wide variety of physiological functions such as food intake, sleep and/or awake, energy balance, and the neuroendocrinological response [1]. The orexins act through two G protein-coupled receptors, the orexin-1 (OX1R) and orexin-2 receptors. According to in vitro studies, the OX1R

has a greater affinity for orexin-A over orexin-B, whereas orexin-2 exhibits similar affinity for both ligands [1e3]. Besides the presence in the brain, orexin peptides and receptors are also found in the myenteric plexus of the enteric nervous system, enteric endocrine cells, pancreatic islets [4,5], testis, ovary, kidney, lung, thyroid gland, adrenal gland, spleen, and liver [3,6] in different species including guinea pig, rat, mouse, and humans. Previous studies have revealed that ischemic conditions lead to marked alterations in the orexigenic

* Corresponding author. Department of Physiology, Faculty of Medicine, Akdeniz University, Antalya 07070, Turkey. Tel.: þ90 242 2496962; fax: þ90 242 2274483. _ ¨ t Uysal). E-mail address: [email protected] (V.N. Izgu 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.08.048

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system. Dohi et al. [7] reported that orexin-A concentrations were lower in the cerebrospinal fluid of subarachnoid hemorrhage patients. It was also shown that global or focal cerebral ischemia induced increased expression of OX1R in the brain, which correlated with decreases of orexin-A in the cerebrospinal fluid [8]. The authors concluded that the reduction in orexin-A production and OX1R expression may be associated with alterations in brain orexin-A signaling in response to ischemiaereperfusion (I/R). Ischemia followed by reperfusion has been reported to induce tissue injury in the targeted organs such as the brain, heart, kidney, and stomach [9,10]. Gastric (I/R) injury is an important clinical problem during the pathological events such as hemorrhagic shock [11]. The complex mechanism of I/ R injury is attributed to neutrophil accumulation at the site of tissue injury and the release of proinflammatory mediators such as reactive oxygen species and cytokines, which lead to cellular injury [11e13]. Previous studies have revealed that the administration of orexin-A led to the apparent increase in the messenger RNA expression of an antioxidizing and radical scavenging enzyme superoxide dismutase in rats [14]. Furthermore, Butterick et al. [15] have demonstrated that orexin-A protected immortalized hypothalamic neurons against H2O2-induced toxicity by decreasing lipid peroxidative stress and apoptosis. It has been demonstrated that I/R causes heme oxygenase (HO) overexpression [16e18]. HO is the rate-limiting enzyme in the degradation of heme, catalyzing the cleavage of heme to carbon monoxide, biliverdin, and ferrous iron [16,19]. The HO family consists of three isozymes; the inducible HO-1, the constitutive HO-2, and the third isoform HO-3 is constitutively expressed and the functional role of this protein is unclear [16,20]. HO-1 is an inducible protein whose expression is increased several fold in response to a variety of cellular stresses and stimuli, including hypoxia, oxidative stress, and inflammatory cytokines such as tumor necrosis factor (TNF)-a and IL-1, suggesting an important role for this enzyme in tissue protection [21e23] and is widely distributed throughout the body with high levels of expression in the liver, spleen, and bone morrow [24]. HO-2 is localized primarily in the neural tissues and is highly expressed in the brain and gastrointestinal tract [25]. HO-2 has been demonstrated to be frequently expressed in myenteric and submucosal enteric neurons, Cajal cells, endothelium and mucosal epithelial cells in gastric fundus and gastric antrum [26]. A constitutive form, HO-2 is important in the protection of vascular endothelium against apoptotic changes induced by oxidative stress and cytokinemediated inflammation [27]. Oxidative stress is a common cause of I/R-induced gastric injury, and potent antioxidant defense is absolutely essential for gastric protection. It has been demonstrated in in vivo and in vitro experimental models that HO-2 is a component of antioxidant defense mechanism [28]. HO-2 has been demonstrated to be a cytoprotective enzyme in a variety of in vivo experimental models. However, the role of specific HO-1 or HO-2 in gastric defense mechanism was not completely elucidated. Central, as well as peripheral administration of orexin-A, affects gastrointestinal functions. Orexin-A stimulates intestinal motility, inhibits migrating motor complex, stimulates gastric emptying and gastric secretion, and also exhibits

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protection against the development of gastric lesions [12,29e32]. HO enzymes that are known to have antioxidant and cytoprotective effects were thought to play a role in the mechanism of gastroprotective action of orexin-A. The aim of the present study was to determine the effect of administered orexin-A against gastric I/R injury on HO-1 and HO-2 expression in the gastric tissue.

2.

Methods

2.1.

Animals

A total of 30 adult male Wistar rats, weighing 200e250 g were provided by the Akdeniz University, Faculty of Medicine, Experimental Animals Care and Production Unit and used in this study. The rats were maintained in a temperaturecontrolled environment (22  1 C) and on a 12-h lightedark cycle, with free access to rat chow and tap water ad libitum. Animals were adapted for at least 7 d before the experiments. All experimental procedures were performed in accordance with mandated standards of humane care and were approved by Animal Care and Use Committee of the Akdeniz University.

2.2.

Experimental protocol

Animals were randomly divided into three groups; sham operated (control), I/R, and I/R þ orexin-A. After an 18-h fasting period, the animals were anesthetized with urethane (1 g/ kg intraperitoneally) and catheterized through the jugular vein for drug infusion. The abdomen was opened by a midline incision and the celiac artery was isolated from its’ adjacent tissues. The celiac artery was clamped with a small nontraumatic vascular clamp for 30 min to induce ischemia and then released to allow reperfusion for 3 h. Infusion of orexin-A was started at the same time with the ischemia induction and finished at the end of the reperfusion period. Orexin-A (SigmaeAldrich, St. Louis, MO) was dissolved in saline for a stock solution, and infusion solutions were freshly prepared with saline on every experiment day. Orexin-A 500 pmol/kg/ min was infused at the rate of 0.1 mL/kg/min. Control group animals were infused with saline. After reperfusion, the rats were killed and the stomach removed immediately.

2.3.

Measurement of mucosal injury

To determine the lesion index, the stomachs was removed, incised along the greater curvature, and pinned onto a platform. Lesion areas were measured with a ruler under a stereoscopic microscope (Zeiss StemiSV11, 20; Oberkochen, Germany), and the total area of hemorrhagic erosions was calculated as the lesion index (in squared millimeter). After measuring the lesion index, the stomach was divided into four pieces along the greater curvature for measurement of other parameters.

2.4.

Myeloperoxidase activity assay

Leukocyte infiltration into the gastric mucosa was assessed by determining gastric myeloperoxidase (MPO) activity by using a

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Fig. 1 e Lesion index. The results are the mean ± standard deviation of six experiments per group. ***P < 0.001 versus control group, &&&P < 0.001 versus I/R group.

method of Gemici et al. [33]. Briefly, the stomach specimens were weighted and 250 mg of the tissue was suspended in 2.5 mL of 50 mM phosphate buffer (pH 6.0) containing 1% hexadecyltrimethylammonium bromide. After the homogenization, the homogenate was sonicated three times for 10 s and stored in a deep freezer at 80 C until assayed. The samples were centrifuged at 15,000 rpm for 5 min at 4 C. Aliquots (0.05 mL) of supernatant mixed with 0.79 mL of 80 mM NaH2PO4, 0.1 mL of 1 mM H2O2, and 0.06 mL of 20 mM tetramethylbenzidine. The change in the absorbance at 655 nm during 1 min was measured in a spectrophotometer (Shimadzu Europea Gmbh, Shimadzu UV 1600; Kyoto, Japan). One unit of MPO was defined as the amount of enzyme causing a change in absorbance of 0.001 per min. Results are expressed as U per gram of protein. The total protein concentration of each tissue homogenate was determined by using a Thermo Scientific Pierce Coomasie Plus (Bradford) protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL).

2.4.1.

4- Hydroxinonenol-malondialdehyde assay

To evaluate lipid peroxidation in the gastric mucosa, the concentration of 4- hydroxinonenol-malondialdehyde (4-HNE-MDA) was determined according to the protocol supplied with the colorimetric assay for lipid peroxidation (Bioxytech LPO 586; OxisResearch, Burlingame, CA). Briefly,

tissues (1 g of tissue per 10 mL of buffer) were homogenized in ice-cold phosphate buffered saline (20 mM, pH 7.4). Before homogenization, 10 mL 0.5 M BHT in acetonitrile was added per 1 mL of tissue homogenate to prevent sample oxidation during homogenization. Homogenate was centrifuged at 3000g at 4 C for 10 min. Clean supernatant was used for the assay. Samples were transferred to a colorimetric assay kit according to the manufacturer’s instructions. 4-HNE plus MDA concentration was calculated using a standard curve and the results expressed as nanomolar per milligram of tissue.

2.5.

TNF-a assay

The concentrations of TNF-a in the stomach tissue were measured by commercial enzyme linked immune sorbent assay kit (KRC3011-invitrogen Co, Camarillo, CA). The samples (100 mg of tissue per 1 mL of buffer) were homogenized in icecold phosphate buffered saline (20 mM, pH 7.4). The tissue homogenates were centrifuged at 10,000g at 4 C for 10 min. The clean supernatants were used for the assay. Samples were diluted 1:2 with standard diluent buffer and mixed thoroughly before loading in the microtiter wells. Enzyme linked immune sorbent assay kit was assayed according to the

Fig. 2 e The representative macroscopical appearance of gastric lesions. (A) Control group, (B) I/R group (30 min ischemia, 3 h reperfusion), and (C) I/R D orexin-A (500 pmol/kg/min) group. (Color version of the figure is available online.)

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Fig. 3 e MPO activity. The results are the mean ± standard deviation of six experiments per group. ***P < 0.001 versus control group, &&&P < 0.001 versus I/R group.

manufacturer’s instructions. The results are expressed as picogram per gram of tissue.

2.6.

Western blotting analysis

Samples were prepared by homogenizing the tissues in 300 mL lysis buffer (10 mM Tris at pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N0 ,N0 -tetraacetic acid, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton-X, 10% glycerol, and 0.1% Sodium dodecyl sulfate 0.5% deoxycholate) supplemented with 5 mL protease inhibitor cocktail (P2714; SigmaeAldrich) per 100 mg of gastric tissue. The mixture was incubated on ice for 30 min and sonicated for 30 s (UW2070; Bandelin Electronic, Berlin, Germany). The specimen was centrifuged at 15,000 rpm for 10 min at 4 C. The supernatant was solubilized in Laemmli sample buffer (S3401; SigmaeAldrich). Before electrophoresis, the sample was boiled for 5 min at 95 C. The protein concentration was determined with a commercial kit (R3236; Pierce) according to Bradford [34], and 50 mg of the total protein was loaded onto a Sodium dodecyl sulfate-polyacrylamide gel with accompaniment of a molecular weight marker (SM1811; Fermentas) and run at 80 V, on 12.5% gel and transferred onto a nitrocellulose membrane (1620112; Bio-Rad Laboratories, Hercules, CA) at 4 C overnight blotting and subsequently hybridized with the primary antibody HO-1 (Stressgen, Enzo Life Sciences Inc, Farmingdale, NY, ADI-OSA-110, with dilution 1:1000), HO-2 (Stressgen, ADI-OSA200, with dilution 1:1000), b-actin (Cell Signal, 4967, with dilution 1:500). After incubation with the primary antibody at 4 C overnight, membranes were washed with Tris buffered salineTween-20 buffer for 1 h. The membranes were incubated with the horse radish peroxidaseeconjugated secondary antibodies appropriate for HO-1 and HO-2 primary antibodies (Sigma, A0168, with dilution 1:80,000 and Chemicon, Temecula, CA, AP132P, with dilution 1:3000, respectively) dissolved in Tris buffered saline-Tween-20 buffer containing 5% nonfat milk (170-6404; Bio-Rad Laboratories) at room temperature for 1 h. The blots were visualized using a chemiluminecent detection system kit according to the manufacturer’s instructions (2600; Chemicon). The membranes were exposed to hyperfilm (RPN3103 K; Amersham Biosciences, Buckinghamshire, United

Kingdom), which was subsequently analyzed using image J, 1.37v software.

2.7.

Statistics

The results are presented as the mean  standard deviation. Statistical analyses were performed by using KruskaleWallis and ManneWhitney U test. The level of significance was accepted as P < 0.05.

3.

Results

3.1. Effect of exogenous orexin-A on I/R-induced gastric lesions The gastroprotective effect of orexin-A was observed on I/Rinduced gastric lesions in rats. The gastroprotective effect of orexin-A was determined by measuring the remarkable changes on gastric lesion index. The infusion of orexin-A at the dose of 500 pmol/kg/min caused significant reduction in the I/R-induced gastric lesions (P < 0.001; Fig. 1). Figure 2 shows the representative macroscopic appearance of gastric lesions. There was no generation of gastric lesions in control rats treated with vehicle (saline) (Fig. 2A). However, 30 min ischemia followed by 3 h reperfusion led to gastric ulceration (Fig. 2B). Orexin-A infusion prevented the increase of gastric lesions. We determined the gastric lesions with the smaller size in the I/R þ orexin-A group compared with those in the I/R group.

3.2.

Effect of exogenous orexin-A on MPO activity

As presented in Figure 3, MPO activity in the gastric mucosa, an index of tissue neutrophil infiltration, was significantly higher in rats exposed to I/R compared with the control group (P < 0.001). Orexin-A prevented the rise in the MPO activity due to I/R (P < 0.001).

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Fig. 4 e 4-HNE-MDA concentration. The results are the mean ± standard deviation of six experiments per group. *P < 0.05 versus control group, &P < 0.05 versus I/R group.

3.3. Effect of exogenous orexin-A on gastric 4-HNE-MDA concentration

3.5. Effect of exogenous orexin-A on the expression of HO-1 and HO-2

The exposure of gastric mucosa to I/R resulted in a significant increase in the mucosal 4-HNE-MDA concentration compared with the intact mucosa (P < 0.05; Fig. 4). Orexin-A infusion (500 pmol/kg/min) significantly attenuated in the concentrations of lipid peroxidation products (4-HNE-MDA) compared with respective values recorded in vehicle-treated animals exposed to I/R (P < 0.05).

As shown in Figure 6, the expression of HO-1 was increased two fold in rats exposed to I/R compared with the control group, (P < 0.05). However, there was no difference in expression of HO-2 in the control or the I/R groups (Fig. 7). Pretreatment with orexin-A attenuated the expression of HO1 protein (P < 0.05), whereas it significantly augmented the expression of HO-2 protein (P < 0.05).

3.4. Effect of exogenous orexin-A on gastric TNF-a concentration

4.

The effects of orexin-A on I/R-induced gastric damage were evaluated by the measurement of inflammatory markers. As seen in Figure 5, exposure of gastric mucosa to I/R resulted in a significant increase in the gastric TNF-a concentration (P < 0.05). The administration of orexin-A led to the decrease in TNF-a concentration, but not statistically significant.

Discussion

In the present study, the gastroprotective effect of orexin-A was investigated in the I/R injury model. The rat model of gastric I/R injury was established by clamping the celiac artery for 30 min followed by 3 h of reperfusion. We demonstrated that the exposure of the gastric mucosa to I/R led to the development of widespread acute gastric lesions. I/R-induced lesions in the stomach were associated with an increase in

Fig. 5 e TNF-a concentration. The results are the mean ± standard deviation of six experiments per group. *P < 0.05 versus control group.

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Fig. 6 e The expression of HO-1. The results are the mean ± standard deviation of four experiments per group. *P < 0.05 versus control group, &P < 0.05 versus I/R group.

MPO activity and an increase in the concentration of TNF-a and 4-HNE-MDA. We determined an increase in HO-1 expression in gastric tissue exposed to I/R. Treatment with orexin-A significantly attenuated the lesions in gastric mucosa exposed to I/R. However, a significant decrease in gastric MPO activity and gastric 4-HNE-MDA concentration was observed in treated rats with orexin-A. Orexin-A infusion prevented the increase in HO-1 expression due to I/R and augmented HO-2 expression in gastric tissue. It has been known that ischemia followed by reperfusion induces the gastric injury [11,12,35,36]. Acute gastric mucosal

lesion is accompanied with infiltration of inflammatory cells in the gastric mucosa exposed to I/R [12,37,38]. Neutrophils play an important role in the pathogenesis of gastric mucosal injury induced by I/R [16]. The main source of reactive oxygen species maybe polymorphonuclear leucocytes. Neutrophils produce the superoxide radical anion, one of the reactive oxygen species. The superoxide radical anion reacts with cellular lipids and leads to production of MDA and 4-HNE, which are reactive short-chain aldehydes. As in our previous study, MDA concentration in gastric tissue significantly was increased after ischemia for 30 min and reperfusion for 3 h;

Fig. 7 e The expression HO-2. The results are the mean ± standard deviation of four experiments per group &P < 0.05 versus I/R group.

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orexin-A administration decreased lipid peroxidation in gastric tissue [12]. It is suggested that the decline in lipid peroxidation could possibly be attributed to the antioxidant features of orexin-A. Orexin-A has an antioxidant effect by decreasing the generation of reactive oxygen radicals and by increasing the expression of antioxidizing and radical scavenging enzyme, superoxide dismutase [14,15]. MPO activity in tissue is considered an index of neutrophil infiltration. It has been demonstrated previously that there is a significant increase in MPO activity accompanying gastric mucosal damage by I/R, and treatment with antioxidant drugs significantly decreases the rise in MPO activity [13]. We found that similar to 4-HNE-MDA result, I/R caused the increase in the MPO activity and orexin-A reduced the MPO activity in gastric tissue exposed to I/R. The present finding provides that orexin-A has a suppressor effect on leukocyte infiltration into the gastric mucosa because of I/R. It is known that the HO system has potent cytoprotective and anti-inflammatory properties in many organs [39]. HO-1 seems to have an important role in acute and chronic inflammation of gastrointestinal system [19]. It is known that I/R is an inflammatory gastric injury model in the rat [40]. Thus, gastric HO-1 expression increased in rat gastric mucosa exposed to I/R in the present study, as we expected. It has been known that the expression of HO-1 is usually increased in gastrointestinal inflammation and injury [19]. I/R-induced increase in HO-1 expression in gastric tissue was not determined with orexin-A infusion. HO-1 expression is increased by oxidative stress. In our study, the reducing effect of orexinA on the expression of HO-1 in gastric tissue may largely be explained by its antioxidant effect. Orexin-A has previously been reported to decrease I/R-induced peroxidative damage in neuronal and gastric tissues in vitro and in vivo [15]. However, we also demonstrated gastric HO-2 induction in rats treated with orexin-A and exposed to I/R. HO-2 is thought to play a role as the constitutive HO activity maintaining cell homeostasis [39]. Furthermore, HO-2 has been known to be a cytoprotective enzyme in a variety of in vivo experimental models. Previous studies have shown that the deletion of HO-2 causes the disruption of the acute inflammatory and reparative response after epithelial injury and leads to an exaggerated inflammatory response in antigen-induced peritonitis [41]. HO-2 is widely expressed in the gastrointestinal tract and more specifically in myenteric and submucosal enteric neurons, endothelial cell lining blood vessels, interstitial cells of Cajal, and mucosal epithelial cells [42]. HO-2 in these cell types, through the action of bilirubin, represents a major contribution to the gastric antioxidant defense mechanisms in normal and pathologic conditions [24]. In a rat model of lipopolysaccharide-induced sepsis, HO has been shown to be an active participant in the general response to oxidative stress against the increased oxidative metabolites in the gastrointestinal mucosa [43]. Parfenova et al. [44] demonstrated that HO-2 is cytoprotective against TNF-a-induced oxidative stress-mediated endothelial injury in cerebral circulation. HO-2 activity provides cerebroprotection against inflammation-induced injury. Our findings suggest that the HO-2 mediated mechanism of cytoprotection against injury caused by inflammation maybe related to induction of orexinA in gastric tissue exposed to I/R.

5.

Conclusions

These results demonstrate that the administration of exogenous orexin-A exhibits gastroprotection against the I/R-induced lesions accompanied by an increase in gastric 4-HNE-MDA concentration and MPO activity. HO-2, a cytoprotective and antioxidant protein, is expressed in the gastric tissue, and orexin-A induces HO-2 expression in the rat gastric tissue exposed to I/R. Our findings suggest that HO-2 may play a role in the mechanism of gastroprotective effect of orexin-A by inhibiting lipid peroxidation in the gastric tissue. Additional studies are needed to investigate further mechanism of gastroprotective effect of orexin-A.

Acknowledgment This study is supported by Akdeniz University Scientific Research Projects Coordination Unit (Project number: 2008.03.0122.002). _ Authors’ contributions: B.G., R.T., and I.B. collected animals’ samples, analyzed all biochemical parameters, and _ were involved in editing the article. V.N.I.U. designed the study and wrote the article. All authors read and approved the final article.

Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.

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