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Peptides 71 (2015) 8–19

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa g e: www.elsev ier .com/ locate /pept ides

reatment with either obestatin or ghrelin attenuates mesentericschemia–reperfusion-induced oxidative injury of the ileum and theemote organ lung

eyla Semiha S ena,1, Berna Karakoyunb,1, Cumhur Yegena, Mustafa Akkiprikc,eral Yükseld, Feriha Ercane, Ays e Özerc, Berrak C . Yegenf,∗

Marmara University School of Medicine, Department of General Surgery, Istanbul, TurkeyMarmara University Faculty of Health Sciences, Department of Basic Health Sciences, Istanbul, TurkeyMarmara University School of Medicine, Department of Medical Biology, Istanbul, TurkeyMarmara University Vocational School of Health Related Professions, Department of Medical Laboratory, Istanbul, TurkeyMarmara University School of Medicine, Department of Histology and Embryology, Istanbul, TurkeyMarmara University School of Medicine, Department of Physiology, Istanbul, Turkey

r t i c l e i n f o

rticle history:eceived 26 January 2015eceived in revised form 6 April 2015ccepted 14 April 2015vailable online 30 May 2015

eywords:hrelinbestatin

ntestinal ischemia–reperfusion injuryung injuryxidant damage

a b s t r a c t

To evaluate the effects of exogenous ghrelin or obestatin on intestinal injury and accompanying pul-monary injury, intestinal ischemia–reperfusion (I/R) was induced in rats by obstructing the superiormesenteric artery for 60 min, whereas laparotomy was performed in the sham group. At the beginningof the 90-min reperfusion period, the rats were injected with obestatin (100 �g/kg), ghrelin (10 ng/kg),or saline intravenously (iv). At the end of reperfusion, the blood, ileum, and lung samples were takenfor the histological and biochemical assays. In the saline-treated I/R group, the increased serum inter-leukin (IL)-1� level, high damage scores, and elevated tissue malondialdehyde level and collagen contentin both tissues were significantly reduced by obestatin or ghrelin. Increased ileal myeloperoxidaseactivity of the saline-treated I/R group was reduced by treatment with obestatin or ghrelin, whereasincreased pulmonary myeloperoxidase activity was reduced with administration of obestatin. IncreasedDNA fragmentation in the ileum of the saline-treated I/R group was reduced by both peptides. Elevatedluminol–lucigenin chemiluminescence levels and nuclear factor kappa B (NF-�B) messenger RNA (mRNA)

expression in the ileum of the saline-treated-I/R group were significantly decreased by obestatin orghrelin treatment. I/R-induced depletion of the antioxidant glutathione in both ileal and pulmonary tis-sues was prevented with either obestatin or ghrelin treatment. Administration of either obestatin orghrelin exerts similar protective effects against I/R-induced ileal and pulmonary injury, thus warrantingfurther investigation for their possible use against ischemic intestinal injury.

© 2015 Elsevier Inc. All rights reserved.

ntroduction

It is well known that intestinal tissue is extremely susceptible toschemia/reperfusion (I/R) injury [49]. Acute mesenteric ischemia

AMI) for even short periods is a serious vascular emergency with

high mortality rate, because AMI initiates the activation of leuko-ytes and various inflammatory mediators, which in turn cause

∗ Corresponding author at: Department of Physiology, Marmara University, Schoolf Medicine, Temel Tip Bilimleri Binasi, Basibuyuk Yerleskesi, Maltepe, 34854 Istan-ul, Turkey.

E-mail address: byegen@marmara.edu.tr (B.C . Yegen).1 These authors contributed equally to this study.

ttp://dx.doi.org/10.1016/j.peptides.2015.04.014196-9781/© 2015 Elsevier Inc. All rights reserved.

remote organ injury and subsequent death [8]. Although the clinicalawareness of AMI and aggressive diagnostic, pharmacologic, andsurgical approaches to AMI have decreased the mortality and mor-bidity to some extent, novel adjuvant pharmacotherapies are stillrequired to improve the outcome before bowel infarction occurs[26,32].

Ghrelin, an endogenous ligand for the GH secretagogue receptor(GHS-R), is a novel 28-amino acid peptide released principally fromX/A-like cells in the oxyntic mucosa of the stomach [14]. Ghrelinhas several biological actions, including the regulation of cell pro-

liferation and survival [5], cardiovascular activity [59], food intake[45], and immune functions [39] along with a recently definedinhibitory effect on inflammation [21,30,57]. In rat models of sep-sis and intestinal I/R, Wu et al. [64,65] showed that plasma levels

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f ghrelin were significantly decreased, whereas administrationf ghrelin ameliorated intestinal barrier dysfunction, attenuatedrgan injury, and improved survival via the inhibition of pro-nflammatory cytokine release and neutrophil infiltration.

Obestatin, derived from the posttranslational processing of thereproghrelin gene, is secreted by gastric and intestinal cells inumans and several mammals [71]. Although it has been initiallyhown that obestatin decreases gastric and jejunal contractilitynd opposes the effect of ghrelin on food intake by reducing foodntake and body weight [71], the role of obestatin as an inhibitory

odulator of upper GI motility and a satiety signal is largely uncon-rmed [24]. We have previously shown that obestatin exerts potentntioxidant and antiapoptotic effects in brain damage [19], colitis51], or renal I/R injury [36].

Although ghrelin, des-acyl ghrelin, and obestatin are derivedrom preproghrelin and are colocalized in the same endocrineells in the stomach [14,71], they exert contradictory effects onut motility and food intake. However, recent research indicateshat the anti-inflammatory effects of obestatin and ghrelin areroven to be common in some inflammatory models. Based onhese findings, the present study was conducted to test whetherxogenous administration of ghrelin or obestatin has common anti-nflammatory and protective effects on acute intestinal injury andhe accompanying ischemic pulmonary damage.

aterials and methods

nimals and experimental design

All experimental protocols were approved by the Marmara Uni-ersity (MU) Animal Care and Use Committee. Sprague-Dawleyats of both sexes (230–300 g), supplied by the MU Animal Cen-er (DEHAMER), were housed in a temperature-controlled room22 ± 2 ◦C) with standardized light/dark (12/12 h) cycles, where theelative humidity (65–70%) was kept constant. The rats were fedtandard rat pellets and tap water ad libitum.

The rats were randomized into the following groups, each con-aining eight rats: (a) sham-operated control, (b) saline-treated/R (60 min of ischemia followed by 90 min of reperfusion), (c)hrelin-treated I/R, (d) obestatin-treated I/R groups. Obestatin100 �g/kg; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA),hrelin (10 ng/kg; Phoenix Pharmaceuticals, Inc., Burlingame, CA,SA), or saline (1 mL/kg) was administered intravenously (iv) into

he inferior vena cava immediately following ischemia at the begin-ing of reperfusion for 90 min. The rationale for the selected dosesf obestatin [36] and ghrelin [30,31,34,57] was based on our previ-us studies demonstrating their protective action in other oxidativenjury models.

urgery for intestinal I/R

After being fasted overnight (16 h), the rats were anesthetizedith an intraperitoneal (ip) injection of ketamine (100 mg/kg)

nd chlorpromazine (0.75 mg/kg), and a tracheotomy was per-ormed to facilitate breathing. A thermometer was inserted intohe rectum, and body temperature was maintained at 37 ◦C using

heating pad. A midline laparotomy was performed, and theuperior mesenteric artery (SMA) was isolated gently at its ori-in and occluded with an atraumatic microvascular micro-bulldoglamp for 60 min, followed by 90 min of reperfusion [22,48]. Thecclusion was confirmed when pulse stopped and the intestines

ecame pale, whereas the return of pulsatile flow to the mesentericrtery and its branches confirmed the occurrence of reperfusion. Inrder to prevent fluid loss via evaporation, the abdomen was cov-red with a stretch film throughout the experiment. Similarly, the

71 (2015) 8–19 9

experimental protocol was performed in the sham-operated con-trol animals (n = 8), but the SMA blood flow was not occluded.

Collection of samples

At the end of 150 min, blood was collected by cardiac puncturefor the determination of serum interleukin (IL)-1� levels. Smallintestinal segments (from the duodenum to the ileocecal valve) andthe superior lobes of the right lung were removed. After measuringthe wet weights, samples from the ileum and lungs were storedat −80 ◦C for the subsequent measurement of DNA fragmentationratio (%), nuclear factor kappa B (NF-�B) messenger RNA (mRNA)expression, malondialdehyde (MDA) and glutathione (GSH) lev-els, and myeloperoxidase (MPO) activity, whereas the formationof reactive oxygen metabolites (ROMs) in the tissue samples wasmonitored using luminol and lucigenin probes. Additional tissuesamples were obtained for histological evaluation and for the deter-mination of the tissue collagen level.

Blood assay

The serum IL-1� levels were quantified using an enzyme-linked immunosorbent assay (ELISA) kit specific for the ratcytokine according to the manufacturers’ instructions and guide-lines (Biosource Europe S.A., Nivelles, Belgium). These particularassay kits were selected because of their high degree of sensitiv-ity, specificity, and inter- and intra-assay precision, and the smallamount of serum sample required for conducting the assay.

DNA fragmentation assay

The mucosal scraping from the ileum was processed immedi-ately after collection to minimize nonspecific DNA fragmentation.The amount of fragmented DNA was determined according to apreviously described method with some modifications [46]. Inbrief, intestinal mucosal scrapings were homogenized in a lysisbuffer consisting of 5 mM Tris–HCl (pH 8.0), 20 mM ethylenedi-aminetetraacetic acid (EDTA) (Sigma), and 0.5% (wt/vol) TritonX-100 (Sigma). Aliquots of the homogenate (1 mL) from each sam-ple were centrifuged at 27,000 g for 20 min at 4 ◦C to separate theintact chromatin (pellet) from the fragmented DNA (supernatant)[67]. The supernatant was decanted and preserved, and the pelletwas resuspended in 1 mL of buffer consisting of 10 mM Tris–HCl(pH 8.0) and 1 mM EDTA. The DNA contents of the pellet andsupernatant fractions were assayed by a diphenylamine reactionas previously described [10]. Each experiment was performed intriplicate, and the results are expressed as the percentage of frag-mented DNA divided by total DNA.

Purification of mucosal DNA and agarose gel electrophoresis

DNA was extracted from the 27,000-g fraction [67]. The frag-mented DNA from the various fractions was extracted with aphenol–chloroformisoamyl alcohol mixture (25:24:1, vol/vol/vol)sequentially to remove protein. The protein-free DNA extracts weretreated with 100% ethanol in 0.1 mol/L sodium acetate at −20 ◦Covernight to purify the DNA. The precipitated DNA was washedwith 70% ethanol and resuspended in Tris buffer (pH 8.0) with10 mmol/L of Tris–HCl and 10 mmol/L of EDTA. The DNA sampleswere incubated with 100 �g/mL of ribonuclease for 15 min at 37 ◦Cto remove RNA. Resolving agarose gel electrophoresis was per-

formed with 1.5% gel strength containing 1.0 �g/mL of ethidiumbromide. Depending on the experiment, 10 �g of DNA was loadedper well. DNA standards were included to identify the size of theDNA fragments. Electrophoresis was performed for 2 h at 70 V, and

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NA was visualized by ultraviolet (UV) fluorescence. A distinct DNAadder was considered to be characteristic of apoptosis [46,67].

easurement of MDA and GSH levels

The ileal and pulmonary tissue samples were homogenized in0 volumes of ice-cold 10% trichloracetic acid and centrifuged at000 rpm for 15 min at 4 ◦C. The supernatant was removed andecentrifuged at 15,000 rpm at 4 ◦C for 8 min. The GSH level wasetermined using a spectrophotometric method, which is a mod-

fication of the Ellman procedure [3]. The lipid peroxide levels arexpressed in terms of MDA equivalents as nanomoles of MDA perram of tissue [11].

easurement of MPO activity

The tissue MPO activity is frequently used to estimate tissueolymorphonuclear (PMN) leukocyte accumulation in inflamed tis-ues, which is shown to correlate significantly with the numberf PMN leukocytes determined histochemically [9]. The tissue-ssociated MPO activity was determined in 0.2–0.5-g ileal andulmonary samples that were homogenized in 10 volumes of ice-old potassium phosphate-buffered saline (PBS, 20 mM K2HPO4,H 7.4). The homogenate was centrifuged at 12,000 rpm for 10 mint 4 ◦C, and the supernatant was discarded. The pellet was thene-homogenized with an equivalent volume of K2HPO4 containing.5% (w/v) hexadecyltrimethylammonium bromide (HETAB). ThePO activity was assessed by measuring the H2O2-dependent oxi-

ation of o-dianizidine·2HCl. One unit of enzyme activity is defineds the amount of MPO present that causes a change in absorbancef 1.0 unit/min at 460 nm and 37 ◦C, and it is expressed in units perram of tissue [27].

F-�B mRNA expression by reverse transcription-polymerasehain reaction

Total RNA was isolated from tissue specimens of each groupsing an RNeasy Mini Kit (Qiagen). After confirmation of RNA

ntegrity by gel electrophoresis, the total RNA concentration ofample was calculated by spectrophotometric measurements.ccording to instructions on the Omniscript RT kit (Qiagen), 1 �g of

he total RNA was added to a master mix consisting of 10× BufferT, 1 �M OligodT15, 0.5 mM deoxynucleotide triphosphate (dNTP),0 units of Rnase inhibitor, and 4 units of Omniscript reverse trans-riptase. The sequences of specific primers for NF-�B p65 (398 bp)ere as follows: forward primer, 5′-GAAGAAGCGAGACCTGGAG-3′;

nd reverse primer, 5′-TCCGGAACACAATGGCCAC-3′. The house-eeping gene �-actin (190 bp) was used as a control, with a forwardrimer of 5′-TCCTAGCACCATGAAGATC-3′ and a reverse primer of′-AAACGCAGCTCAGTAACAG-3′ [68]. A negative control withoutemplate RNA was also included in all reactions. For the polymerasehain reaction (PCR), 4 �L of complementary DNA (cDNA) was useds a template. The conditions for amplification were as follows: pre-eating at 94 ◦C for 4 min, denaturing at 94 ◦C for 45 s, annealing at5 ◦C for 30 s, extending at 72 ◦C for 1 min, and extending again fornother 5 min at 72 ◦C after 25 cycles. The PCR products were sep-rated by gel electrophoresis on 1.2% agarose containing ethidiumromide and analyzed under UV light. The bands were quantitated

y densitometric scanning.

hemiluminescence assay

To assess the contribution of ROMs in I/R-induced ileal andulmonary injury, the luminol and lucigenin chemiluminescence

71 (2015) 8–19

(CL) levels were measured as indicators of radical formation. Theluminescence of the tissue samples was recorded at room tem-perature using a Junior LB 9509 luminometer (EG&G Berthold, BadWildbad, Germany). The specimens were placed in vials containingPBS–HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)buffer (0.5 mol/L of PBS containing 20 mmol/L of HEPES, pH 7.2).ROMs were quantitated after addition of the enhancers, lucigeninor luminol, for a final concentration of 0.2 mmol/L. Luminol detectsa group of reactive species, that is, OH−, H2O2, and HOCl− radicals,whereas lucigenin is selective for O2

− [15]. Counts were obtainedat 1-min intervals, and the results are given as the area under thecurve for a counting period of 5 min. The counts were corrected forwet tissue weights and expressed as relative light units (rlu) permilligram of tissue.

Microscopic preparation and analysis

For light microscopic investigations, ileal and pulmonary tis-sue specimens were fixed with 10% formaldehyde and processedroutinely for embedding in paraffin. Tissue sections of 5-�m thick-ness were stained with hematoxylin and eosin (H&E) and examinedunder an Olympus BX51 (Tokyo, Japan) photomicroscope. Micro-scopic scoring was done by an experienced histologist (F.E.), whowas blinded to the treatments. Each criterion was scored 0, none; 1,mild; 2, moderate; or 3, severe; the semiquantitative scale included(a) degeneration of surface and crypt epithelium, (b) degenerationof villus structure, and (c) inflammatory cell infiltration in the ilealtissue, and (a) vascular congestion and interstitial edema, (b) alve-olar structural disturbance, and (c) inflammatory cell infiltrationin the lung tissue. The microscopic score of each tissue was cal-culated as the sum of the scores given to each criterion, and atleast five microscopic areas were examined to score each spec-imen. The maximum score that could be given was ‘9’ for eachtissue [12,58].

To demonstrate the topographical alterations of ileal tissues,scanning electron microscopy (SEM) was used. The ileal tissuesamples were fixed for 2 h in a 2.5% phosphate-buffered glutaralde-hyde solution (0.1 M, pH 7.4), postfixed in a 1% phosphate-bufferedosmium tetroxide solution, and passed through an increasing alco-hol and amyl acetate series. After drying the tissue samples witha Bio-Rad critical point dryer and gold-coating with a Bio-Rad SC502, tissue samples were examined under a JEOL 5200 JSM (Tokyo,Japan) scanning electron microscope.

To demonstrate the ultrastructural alterations in lungparenchyma, transmission electron microscopy (TEM) wasused. The pulmonary tissue samples were fixed for 2 h in a 2.5%phosphate-buffered glutaraldehyde solution (0.1 M, pH 7.4),postfixed in a 1% phosphate-buffered osmium tetroxide solution,passed through an increasing alcohol series, and polymerizedin Epon 812 resin. Ultrathin sections were stained with uranylacetate and lead citrate and investigated using a JEOL 1200 SXtransmission electron microscope.

Tissue collagen content measurement

The ileal and pulmonary tissue samples were cut with a razorblade and immediately fixed in 10% formalin in 0.1 M phosphatebuffer (pH 7.2) in paraffin, to obtain approximately 15-�m-thicksections. The collagen content was evaluated according to themethod published by Lopez de Leon and Rojkind [40], based on

selective binding of the dyes Sirius Red and Fast Green to collagenand non-collagenous components, respectively. Both dyes wereeluted readily and simultaneously, using 0.1 M NaOH–methanol(1:1, v/v). Finally, the absorbances at 540 and 605 nm were usedto determine the amount of collagen and protein, respectively.

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tatistical analysis

Calculations were made using GraphPad Prism 6.0 (Graph-ad Software, San Diego, CA, USA). All data are expressed asean ± standard error of measurement (SEM). Groups of dataere compared with an analysis of variance (ANOVA) followed

y Tukey’s multiple comparison tests. The results were consideredignificant when p was less than 0.05.

esults

et weights of the small intestine and lung

Tissue wet weights (�g) were divided by the 100-g body weightsf the rats, which were not statistically different among the exper-mental groups (p > 0.05). The wet weights of the small intestinefrom the duodenum to the ileocecal valve) and the upper lobesf the right lung in the saline-treated I/R group were signifi-antly higher than those of the sham-operated group (p < 0.001 and

< 0.01; Table 1), implying the presence of tissue edema. On thether hand, the wet weights of the small intestine were signifi-antly reduced by obestatin or ghrelin (p < 0.05–0.001), whereas/R-induced lung edema was reduced by ghrelin (p < 0.01), but notbestatin.

icroscopic evaluation of histopathological changes in the ileumnd lung

Compared with the regular ileal mucosa observed in the con-rol group, the ileal mucosa of the saline-treated I/R group showedevere degeneration of the surface epithelium with flattened villi,enuded lamina propria, and inflammatory cell infiltration (Fig. 1).he ileal mucosa of ghrelin-injected I/R groups demonstrated a con-iderably normal epithelium with villi and mild inflammatory cellnfiltration. Similarly, the ileal mucosae of the obestatin-injected/R groups showed a mild degeneration of the surface epithe-ium with flattened villi structures and mild inflammatory cellnfiltration.

A regular lung parenchyma was observed in the sham-operatedontrol group (Fig. 2). In the saline-treated I/R group, massivelveolar damage was evident with degenerated type I and type IIneumocytes and infiltrated inflammatory cells along with severe

nterstitial edema and vascular congestion. The lungs of the ghrelin-njected I/R groups demonstrated mild alveolar degeneration withighly regular ultrastructure of type I and type II pneumocytes,oderate vascular congestion, and inflammatory cell infiltration.

imilarly, the lungs of the obestatin-treated rats with I/R presented

ild alveolar degeneration with a better ultrastructure of type I and

ype II pneumocytes, moderate vascular congestion, and moderatenflammatory cell infiltration (Fig. 2).

able 1issue wet weights (�g/100-g body weight) of the sham-operated control rats andats intravenously treated with saline, obestatin, or ghrelin that had superior mesen-eric artery occlusion and reperfusion (I/R).

Groups (�g/100-g body weight) Small intestine Lung

Sham-operated control 1580 ± 55 30 ± 5Saline-treated I/R 2070 ± 80*** 50 ± 2**

Obestatin-treated I/R 1800 ± 80+ 50 ± 6Ghrelin-treated I/R 1650 ± 30+++ 40 ± 3++

** p < 0.01 and*** p < 0.001, compared with the sham-operated group;

+ p < 0.05,++ p < 0.01 and

+++ p < 0.001, compared with the saline-treated I/R group.

71 (2015) 8–19 11

Microscopic damage scores in the ileum and lung of the saline-treated I/R group were found to be significantly higher than those ofthe sham-operated group (p < 0.001; Figs. 3A and 4A). The damagescores of the ileum were significantly reduced by the injection ofobestatin or ghrelin (p < 0.001; Fig. 3A). Similarly, high microscopicdamage scores recorded in the lungs of the saline-treated I/R groupwere significantly reduced by treatment with obestatin or ghrelin(p < 0.05; Fig. 4A).

MDA levels in the ileum and lung

The ileal and pulmonary MDA levels were significantly elevatedin the saline-treated I/R group compared with the sham-operatedgroup (p < 0.01 and p < 0.001, respectively; Figs. 3B and 4B), indi-cating increased lipid peroxidation. On the other hand, the MDAlevels were significantly reduced in both tissues of the obestatin-or ghrelin-treated I/R groups (p < 0.05–0.01).

DNA fragmentation in the ileum and lung

DNA fragmentation (%) in the ileal and pulmonary tissues wasanalyzed as an indicator of cell death, including apoptosis. DNAfragmentation was elevated in the ileum of the saline-treated I/Rgroup with respect to that of the sham-operated group (p < 0.05),whereas treatment with both obestatin and ghrelin reduced thedegree of apoptosis (p < 0.01–0.001; Fig. 3C). On the other hand,DNA fragmentation in the lungs of the saline-treated I/R group wasnot different from that in the sham-operated group, and treatmentwith neither obestatin nor ghrelin changed the degree of apoptosis(Fig. 4C).

Collagen contents in the ileum and lung

The tissue collagen content was measured as a free radical-induced fibrosis marker. The collagen content was elevated in boththe ileal and pulmonary tissues of the saline-treated I/R group ascompared with the sham-operated group (p < 0.01 and p < 0.001).Treatment with either obestatin or ghrelin in the I/R-inducedrats significantly reduced the collagen contents of both the ileum(p < 0.01) and lung (p < 0.001; Figs. 3D and 4D).

CL levels in the ileum and lung

As an indicator of the involvement of ROMs in I/R-induced ilealdamage, CL levels detected in the ileal samples by both luminol andlucigenin probes showed significant increases in the saline-treatedgroup compared with the sham-operated group (p < 0.05; Fig. 5Aand B). Ghrelin-induced reduction in ileal luminol CL did not reachstatistical significance. However, the elevation in ileal lucigenin CLdue to I/R injury was canceled out with ghrelin treatment (p < 0.01).On the other hand, obestatin reduced I/R-induced elevation in theileal luminol CL level (p < 0.05), but obestatin did not significantlychange lucigenin CL levels in the ileum.

The pulmonary CL levels as detected by both luminol and luci-genin probes were not different from those of the sham-operatedgroup, and neither obestatin nor ghrelin changed the pulmonaryluminol or lucigenin CL levels (Fig. 6A and B).

MPO activities in the ileum and lung

MPO activity, which is accepted as an indicator of neutrophilinfiltration to the inflamed tissue, was significantly higher in the

ileum and lung of the saline-treated I/R group as compared withthe sham-operated group (p < 0.001; Figs. 5C and 6C). In rats treatedwith obestatin (p < 0.001) or ghrelin (p < 0.01), the ileal MPO activi-ties were significantly reduced. Similarly, the elevation in lung MPO

12 L.S. S en et al. / Peptides 71 (2015) 8–19

Fig. 1. Photomicrographs of ileum showing H&E staining on the left panel and SEM on the right panel. Control group (A): Normal villi and lamina propria morphology;Saline-treated I/R group (B): Severely degenerated surface epithelium (arrows) with flattened villi, severe inflammatory cell infiltration (*) (left), and denuded lamina propria( arrowg and mv

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*) (right). Ghrelin-treated I/R group (C): Regular epithelium with villi structures (roup (D): Mild degenerations of epithelium with flattened villi structures (arrows)illi (*) (right).

ctivity due to I/R was decreased in the obestatin-treated (p < 0.01)roups. However, the reductions observed in the pulmonary MPOctivities of ghrelin-treated rats did not reach statistical signifi-ance.

SH levels in the ileum and lung

The level of GSH, the endogenous antioxidant, in the smallntestine of saline-treated rats was significantly decreased as

s) and mild inflammatory cell infiltration (*) (left and right); Obestatin-treated I/Rild inflammatory cell infiltration (*) (left and right). Degeneration of the tips of the

compared with the sham-operated group (p < 0.001). Ghrelin treat-ment replenished the tissue GSH contents as compared with thesaline-treated I/R group (p < 0.05) (Fig. 5D), but the GSH level wasnot changed when treated with obestatin.

The pulmonary GSH level was reduced significantly in the

saline-treated I/R group as compared with the sham-operatedgroup (p < 0.001) (Fig. 6D). This marked reduction in the pulmonaryGSH level was reversed when the rats were treated with obestatin(p < 0.05), but not ghrelin.

L.S. S en et al. / Peptides 71 (2015) 8–19 13

Fig. 2. Photomicrographs of lung showing H&E staining on the left panel and TEM on the right panel. Control group (A): Normal appearance of the lung parenchyma. Saline-treated I/R group (B): Severe alveolar degeneration (arrowheads), inflammatory cell infiltration (*), vascular congestion (arrows) and inflammatory cell infiltration (left), anddecreased number of apical microvilli (arrow) and empty large lamellar bodies (*) in type II pneumocytes (right). Ghrelin-treated I/R group (C): Inflammatory cell infiltration( rrowsm ion (a( r of ap

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*) and mild alveolar degeneration (arrowheads), moderate vascular congestion (aicrovilli (arrow) (right). Obestatin-treated I/R group (D): Mild alveolar degenerat

*) (left), better type II pneumocytes with lamellar bodies (*), and increased numbe

evels of serum IL-1 and tissue NF-�B mRNA

In the saline-treated I/R group, the serum IL-1� level was signif-cantly increased when compared with that of the sham-operatedroup (p < 0.05), whereas the I/R-induced elevation in the serum IL-

� level was canceled out with administration of either obestatinr ghrelin (p < 0.05; Fig. 7A).

The ileal NF-�B mRNA expression level (band density) ofhe saline-treated I/R group was significantly higher than that

) (left), and highly regular type II pneumocytes with lamellar bodies (*) and apicalrrowheads), moderate vascular congestion (arrows), inflammatory cell infiltrationical microvilli (arrow) (right).

measured in the sham-operated group (p < 0.05; Fig. 7B). Thiselevated mRNA level in the ileum was significantly reduced byobestatin (p < 0.05), but the reduction observed with ghrelin didnot reach statistical significance.

Induction of I/R in the saline-treated group increased pulmonary

NF-�B mRNA expression without reaching statistical significance(Fig. 7C), and the pulmonary NF-�B mRNA levels in all the treatmentgroups were similar and they did not differ from the saline-treatedgroup.

14 L.S. S en et al. / Peptides 71 (2015) 8–19

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iscussion

The severity of ileal and pulmonary I/R-injury, as evidencedy increased levels of plasma IL-1�, tissue MDA, and collagen,as alleviated by obestatin or ghrelin administration. Moreover,

leal neutrophil accumulation due to I/R was reduced by treatmentith both obestatin and ghrelin, whereas increased pulmonaryPO activity was reduced with obestatin. In accordance with these

iochemical changes, the morphologic evaluation of the tissuesevealed that obestatin and ghrelin were also effective in protec-ing the ileum and lung against I/R-induced degenerative changes.he elevated luminol and lucigenin CL levels in the ileal samples,ndicative of increased generation of ROMs, showed significantncreases in the I/R group, whereas obestatin or ghrelin treatmentecreased the elevations in CL levels, indicating the scavenging rolef both peptides. Furthermore, ROM-mediated activation of NF-B verifying ileal injury was suppressed by obestatin. Accordingly,

/R-induced depletion of the antioxidant GSH in both the ileal andulmonary tissues was inhibited by treatment with either obestatinr ghrelin. Thus, the results of the current study demonstrated thatbestatin and ghrelin, which are derived from the same precur-or proghrelin, have similar protective effects against mesenteric/R-induced oxidative injury of the ileum and the remote organung.

Ghrelin plays a crucial role in the regulation of food intake,nergy homeostasis, and gastric emptying [38,56], but the effect of

bestatin on food intake and gastric emptying is not clear yet [63].reatments with both ghrelin and obestatin were suggested to beardioprotective in rats [43,44] and in isolated rat hearts with I/Rnjury [1,13]. We have reported that ghrelin [20,21,30,31,34,51,57]

ghrelin (n = 8) that had superior mesenteric artery occlusion and reperfusion (I/R). < 0.01 and +++p < 0.001 compared with the saline-treated I/R group.

and obestatin [19,36,51] attenuate inflammation in several inflam-matory models by exerting protective effects against the oxidativeinjury of the brain, kidney, and gut. The serum ghrelin levels andcolonic ghrelin expression were found to be higher in patientswith chronic inflammation, such as ulcerative colitis and ilealCrohn’s disease [2,29,33], whereas a positive correlation was shownbetween the ghrelin levels and the severity of the intestinalmucosal lesions of patients with celiac disease [54]. On the otherhand, the plasma ghrelin levels were reduced significantly in acuteinflammatory processes (e.g., sepsis or intestinal I/R) [65,66], sug-gesting that exogenous ghrelin treatment could exert protectiveeffects against intestinal I/R-induced injury. Accordingly, ghrelinadministration following gut I/R was shown to attenuate organinjury and improve survival by inhibiting neutrophil infiltrationand pro-inflammatory cytokine release [65]. Likewise, intestinalinflammation and dysfunction induced by traumatic brain injurywere prevented by ghrelin treatment [7]. In parallel to the involve-ment of ghrelin in inflammation, obestatin was also correlatedpositively with the levels of inflammatory markers, IL-6 and TNF-�,in some chronic inflammatory diseases [35]. In the current study,both ghrelin and obestatin were almost equally effective in exertinganti-inflammatory effects on the ileal and pulmonary tissues.

Following intestinal I/R, acute lung injury occurs as a major com-plication, contributing to a high mortality rate [70]. Neutrophils,ROMs, and pro-inflammatory substances released by the injuredintestines enter the systemic circulation and initiate a systemic

inflammatory reaction mainly affecting the lungs, liver, and kidneys[42]. Accordingly, anti-inflammatory agents that prevent mucosalinjury and improve gut function are expected to facilitate lungrecovery after I/R injury. Thus, by initially alleviating oxidative

L.S. S en et al. / Peptides 71 (2015) 8–19 15

A

0

2

4

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10

12 obestatin/ghreli n

Sham

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Obestatin Ghreli n

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salin e

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Sham Obestatin Ghrelin

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)

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mol

/g)

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0.05

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Sham Obestati n Ghrelin

+++ ++ +

I/R

Col

lage

n co

nten

t(µ

g co

llage

n/m

g pr

otei

n)

Fig. 4. Microscopic lesion score (A), tissue malondialdehyde (MDA) level (B), percentage of DNA fragmentation (C), and collagen content (D) in the lung tissues of sham-operated control rats (n = 4) and rats treated with saline (n = 4), obestatin (n = 8), or ghrelin (n = 8) that had superior mesenteric artery occlusion and reperfusion (I/R).***p < 0.001 compared with the sham-operated group; +p < 0.05 and +++p < 0.001 compared with the saline-treated I/R group.

A

0

10

20

30

40 obestatin /gh reli n

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Sham Obestatin Ghrelin

saline

I/R

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inol

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(rlu

/mg)

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150

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Sha m Obestati n Ghreli n

I/R

Luci

geni

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L (r

lu/m

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0.0

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1.0

1.5

2.0

2.5

3.0

***

Sham Obestatin Ghrelin

+

I/R

GSH

( μm

ol/g

)

Fig. 5. Luminol (A) and lucigenin (B) chemiluminescence (CL) levels, myeloperoxidase (MPO) activity (C), and glutathione (GSH) levels (D) in the intestinal tissues of sham-operated control rats (n = 4) and rats treated with saline (n = 4), obestatin (n = 8), or ghrelin (n = 8) that had superior mesenteric artery occlusion and reperfusion (I/R).rlu = relative light units. *p < 0.05 and ***p < 0.001 compared with the sham-operated group; +p < 0.05, ++p < 0.01, and +++p < 0.001 compared with the saline-treated I/R group.

16 L.S. S en et al. / Peptides 71 (2015) 8–19

A

0

10

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30

Sham Obestati n Ghrelin

obe statin /ghreli nsalin e

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inol

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/mg)

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25

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75

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125

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Sham

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g)

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I/R

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geni

n C

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0.0

0.5

1.0

1.5

2.0

***

+

Sha m Obestatin Ghrelin

I/R

GSH

( μm

ol/g

)

Fig. 6. Luminol (A) and lucigenin (B) chemiluminescence (CL) levels, myeloperoxidase (MPO) activity (C), and glutathione (GSH) levels (D) in the lung tissues of sham-operatedc 8) thu comp

ivo

a[wtilsibtmieeatitatat

fIa

ontrol rats (n = 4) and rats treated with saline (n = 4), obestatin (n = 8), or ghrelin (n =nits. ***p < 0.001 compared with the sham-operated group; +p < 0.05 and ++p < 0.01

njury of the ileum, ghrelin and obestatin protect against lung injuryia the inhibition of neutrophil infiltration and associated releasef inflammatory mediators.

Ghrelin has been previously shown to reduce oxidative stressnd inhibit the production of reactive oxygen species in the gut18,72]. Using a luminol- and lucigenin-enhanced CL method,hich is a simple but reproducible technique, allowed us to quan-

itate the free radical generation induced by I/R injury. Luminols known to detect H2O2, OH−, hypochlorite, peroxynitrite, andipid peroxyl radicals, whereas lucigenin is specifically sensitive touperoxide radical [25,47]. The CL data revealed that I/R-inducedleal damage involves the generation of toxic metabolites detectedy both probes, which can severely disrupt cell membrane func-ion and lead to DNA damage and cell death in the gastrointestinal

ucosa [4,55]. The elevations in all toxic oxygen metabolites in theleum were decreased by ghrelin, but obestatin appears to have noffect on I/R-induced superoxide generation. Thus, the protectiveffects of ghrelin and obestatin in the ileum involve their inhibitoryction on free radical formation. On the other hand, both pep-ides suppressed the accumulation of neutrophils, indicating annhibitory effect on the possible source of free radicals. Althoughhe remote organ lung demonstrated significant microscopic injurynd increased lipid peroxidation with a concomitant reduction inhe antioxidant GSH levels, these inflammatory responses were notssociated with the local generation of free radicals and neitherreatment altered the CL level.

The inhibition of NF-�B, which is a crucial nuclear transcriptionactor for the regulation of tumor necrosis factor alpha (TNF-�),L-1�, and IL-6 gene expressions [62], was shown to attenu-te I/R-induced intestinal [61] and hepatic [23] injury. Ghrelin

at had superior mesenteric artery occlusion and reperfusion (I/R). rlu = relative lightared with the saline-treated I/R group.

provides significant protection against I/R injury via the inhibi-tion of NF-�B activation, and it shows potent anti-inflammatoryeffects by suppressing cytokine production in the rat myocardiumand human endothelial cells [37,39]. Ghrelin promotes an antiox-idative defense mechanism in the rat liver, not only by reducingthe NF-�B protein levels but also by increasing the proteinexpression of antioxidative enzymes (e.g., superoxide dismutase,catalase, and GSH peroxidase) [17]. Accordingly, in the presentstudy, suppression of NF-�B activation by ghrelin or obestatinduring mesenteric I/R also enhanced the antioxidant content ofboth the ileal and pulmonary tissues and protected them againstinjury.

Ghrelin downregulates pro-inflammatory cytokines in humanT lymphocytes and monocytes [16] and in the plasma of septicrats [64], whereas the antioxidant superoxide dismutase activityin the plasma increases [69]. In the current study, I/R-induced ele-vation in plasma IL-1� and ileal MDA levels as well as increasedmRNA expression of ileal NF-�B were reduced by both ghrelinand obestatin, while the depleted ileal GSH content was replen-ished. We have previously shown that treatments with bothghrelin and obestatin significantly ameliorated the severity ofcolitis, and this therapeutic effect was associated with reducedcolonic IL-1� and TNF-� levels, along with increased tissue GSHcontent and anti-inflammatory IL-10 level [51]. Similarly, our stud-ies on subarachnoid hemorrhage-induced brain injury revealedthat increased levels of plasma IL-1�, TNF-�, and IL-6 with con-

comitant decreases in antioxidant enzymes were reversed byeither obestatin [19] or ghrelin [21]. In renal I/R, obestatin treat-ment reduced the severity of injury, whereas renal GSH levelswere significantly increased [36]. When taken together with the

L.S. S en et al. / Peptides 71 (2015) 8–19 17

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obestatin /gh rel in

Obe statin Ghrel in

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IL-1β

(pg/

ml)

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Sham Obestatin Ghreli n

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NF-Κ

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and d

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Sham Obe statin Ghrel in

I/R

NF-Κ

B (B

and d

ensit

y)

F lung

o reperw

aa

tmcibrl

micAttttbiu

C

F

ig. 7. Serum IL-1� level (A) and NF-�B mRNA expressions in the intestinal (B) andbestatin (n = 8), or ghrelin (n = 8) that had superior mesenteric artery occlusion andith the saline-treated I/R group.

forementioned studies, the present data confirm the antioxidative,nti-inflammatory, and antiapoptotic properties of both peptides.

Pawlik et al. [53] suggested that ghrelin-induced intestinal pro-ection involves the vasodilatatory effect of the peptide on the

esenteric circulation, which could have played a role in theurrent results. Although the protection afforded by obestatin inndomethacin-induced gastric mucosal injury was not found toe associated with mucosal blood flow [41], further studies areequired to elucidate the effect of obestatin on mesenteric circu-ation.

Ghrelin receptors are distributed in the pituitary, hypothala-us, brain stem, lungs, blood vessels, heart, stomach, pancreas,

ntestines, kidneys, and adipose tissue [52,60], but neither the spe-ific receptor for obestatin nor its distribution is clear yet [28].s both ghrelin and obestatin were previously shown to cross

he blood–brain barrier [6,50], it can be suggested that both cen-ral and peripheral receptors possibly mediate the effects of thewo peptides encoded by the common ghrelin gene. In conclusion,he results of the present study implicate ghrelin and obestatin ineing almost equally effective at attenuating I/R-induced oxidative

njury, therefore meriting further investigation for their possiblese against ischemic intestinal injury.

ompeting financial interests

This work was supported by the Marmara University Researchund, Istanbul, Turkey.

(C) tissues of sham-operated control rats (n = 4) and rats treated with saline (n = 4),fusion (I/R). *p < 0.05 compared with the sham-operated group; +p < 0.05 compared

Acknowledgments

This work was partially presented at Digestive Disease Week2008 and published in abstract form in Gastroenterology 2008;134(4): A58. The authors would like to thank Ilker Parmaksız andProf. Önder S irikc i for running the serum samples for the determi-nation of IL-1�.

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