Original article 369

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Protective effect of obestatin on indomethacin-induced acutegastric ulcer in rats: role of VEGF and TNF-αReham M. Ibrahim, Mona M. Allam, Ola A. El-Gohary, Alaa E.A. El-Talees,Mohamed S. El-Hamady

Department of Physiology, Benha Faculty of

Medicine, Benha University, Benha, Egypt

Correspondence to Reham M. Ibrahim, MSc,

Department of Physiology, Faculty of Medicine,

Benha University, Benha, 13511, Egypt.

Tel: 0122349445;

e-mail: dr_reham_13@yahoo.com

Received 29 April 2018

Accepted 26 July 2018

Benha Medical Journal 2018, 35:369–377

© 2019 Benha Medical Journal | Published by Wolters K

BackgroundGastric ulcer (GU) is one of the most common disorders that affect thegastrointestinal tract. Obestatin, a ghrelin-related peptide, has been shown toexhibit some protective and therapeutic effects in the gut.AimThis study aimed at investigating the protective effect of obestatin on acuteindomethacin (IND)-induced GU, clarifying the role of vascular endothelialgrowth factor (VEGF) and tumor necrosis factor-α (TNF-α).Materials and methodsA total of 32 adultWistar albinomale rats were divided into four main groups: controlgroup, obestatin group, GU group, and obestatin+GU group. Obestatin was givenby a single intraperotineal injection (30 μg/kg) 1 h before induction of GU by a singleoral dose of IND (40mg/kg). Pyloric ligation was carried out in all animals beforeIND or distilled water administration. Four hours later after IND treatment, gastriculcer index, preventive index, gastric juice volume, and free and total acidity wereassessed. Nitric oxide, VEGF mRNA, and TNF-α mRNA were measured in gastrictissue, as well as histopathological study of gastric injury.ResultsIND caused a significant increase in ulcer index, gastric juice volume, free and totalacidity, and TNF-α mRNA, with a significant decrease in nitric oxide and VEGFmRNA. Pretreatment with obestatin reversed these effects.ConclusionObestatin pretreatment showed a gastroprotective effect against the IND-inducedGU that can be explained by the anti-inflammatory and angiogenic effects ofobestatin treatment and reduction of gastric juice volume, free acidity, and totalacidity.

Keywords:anti-inflammatory, gastric ulcer, indomethacin, obestatin, vascular endothelial growth factor

Benha Med J 35:369–377

© 2019 Benha Medical Journal

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IntroductionGastric ulcer (GU) is a major health hazard in terms ofboth morbidity and mortality [1];∼14.5 million peopleworldwide are affected by GU, with a mortality rate of4.08 million per year [2].

The pathogenesis of GU has been considered mainlyowing to imbalance between defensive factors [mucussecretion, gastroprotective prostaglandins (PGs)synthesis, bicarbonate production, and normal tissuemicrocirculation] and aggressive factors (excessivesecretion of gastric acid, alcohol intake, bile salts,abnormal motility, infection with Helicobacter pylori,and NSAIDs) [3].

NSAIDs-induced GUs are the second most commonetiology of GUs [4]. According to previous reports, theoral administration of indomethacin (IND), a well-known NSAID, in rats causes ulcerative lesions in thegastric mucosa [5]. IND induces its gastrointestinal

luwer - Medknow

tract (GIT) toxicity via several mechanisms such as anincrease in gastric acid secretion, interfere withmucosalcell regeneration via inhibition of PGE2 synthesis,production of free radicals, reduction of gastric nitricoxide (NO) level, and invasion of activated neutrophilsas well as induction of gastric cells apoptosis [6]. Inaddition, it causes oxidative damage of the stomach andthe generation of proinflammatory mediators [7].Furthermore, NSAIDs were reported to inhibitangiogenesis through downregulation of the pro-angiogenic factors such as vascular endothelialgrowth factor (VEGF) and upregulation of anti-angiogenic proteins such as endostatin leading todelayed ulcer healing [8].

DOI: 10.4103/bmfj.bmfj_86_18

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Obestatin is a ghrelin-related peptide. It is composedof 23 amino acids [9]. It is expressed in many tissuesalong the GIT, most notably the stomach, pancreas,duodenum, jejunum, and colon [10]. However, mostcells that produce obestatin appear to be concentratedin the oxyntic mucosa of the stomach, and the stomachis considered the major source of circulating obestatin[11].

Obestatin has been reported to be an anorexigenichormone, decreasing food intake and body weight.Interestingly, obestatin has been reported to havecertain effects on GIT, including decrease in gastricemptying time and jejunal motility, and it exhibitssome protective and therapeutic effects in the gut[12]. Moreover, it was reported that obestatin hasanti-inflammatory [13–15], anti-oxidative stress[16], and antiapoptotic effects [17].

Materials and methodsAnimalsThis study was conducted on 32 adult Wistar albinomale rats, 6–8 weeks old, weighing between 180 and220 g. They were obtained from the ExperimentalAnimal Unit of Moshtohor Faculty of Agriculture,Benha University. The study was approved by ourIRB. The animals were acclimatized to thelaboratory conditions for 10 days before theinitiation of the experiment. They had free access towater and diet. Experimental rats were under completehealthy conditions all over the experiment and undercare of a professional technician. No rats diedthroughout the experiment. At the end of the study,the rats were incinerated at Benha university hospitalincinerator.

Experimental designThe rats were deprived of food for 24 h before theexperiment in mesh-bottomed cages to minimizecoprophagia but allowed free access to water exceptthe last hour before the experiments [18]. Allexperiments were performed during the same timeof the day between 8 a.m. and 12 p.m. to avoidvariations owing to diurnal rhythms of putativeregulators of gastric functions [19].

The animals of the experiment were divided into fourgroups, and each group consisted of eight rats. In groupI ‘control group’, animals received singleintraperitoneal (i.p.) injection of saline and thenafter 1 h, a single oral dose of distilled water (thesolvent of IND). In group II ‘obestatin group’,animals received a single i.p. injection of obestatin in

a dose of 30 μg/kg [20], and then after 1 h a single oraldose of distilled water. In group III ‘GU group’,animals received single i.p. injection of saline andthen after 1 h gastric ulceration was induced by asingle oral dose of IND (40mg/kg) by orogastricgavage [21]. In group IV ‘obestatin+GU group’,animals were pretreated with a single i.p. injectionof obestatin (30 μg/kg) 1 h before induction of GUby a single oral dose of IND (40mg/kg) by orogastricgavage.

Pyloric ligationPyloric ligation was carried out in each animal beforeoral administration of distilled water or IND to collectgastric juice under light di-ethyl ether anesthesia. Amid-line abdominal incision was performed; thepyloric portion of the stomach was gently mobilizedand carefully ligated with a silk ligature around thepyloric sphincter taking care not to interfere withgastric blood supply. The abdominal incision wassutured, and the animals were allowed to recoverfrom anesthesia [22].

Gastric juice was allowed to accumulate for a period ofnext 4 h [23]. The animals were anesthetized with di-ethyl ether before cervical dislocation. Their stomachswere rapidly removed after clamping the esophagus,opened by an incision along the greater curvature, andthe gastric juice was collected and then assessed forvolume, free and total acidity. Gastric tissues werewashed with ice-cold saline to remove gastriccontent remnants and blood clots and then assessedmacroscopically. Finally, a part of each stomach wasimmediately kept in formaldehyde to be prepared forhistopathological examination with hematoxylin andeosin for detection of the histopathological changes,and the other part was immediately frozen in liquidnitrogen and stored at −80°C for biochemicalestimations of tissue NO, tumor necrosis factor-α(TNF-α) mRNA, and VEGF mRNA.

Assessment of gastric mucosal lesionsGastric tissues were pinned out flat on a cork board andphotographed for lesion assessment. The stomachswere examined for macroscopical mucosal lesionswith the aid of a magnifier by a pathologist unawareof the treatment protocol. The gastric mucosal lesionswere expressed in terms of ulcer index (UI) according toPeskar et al. [24] which depends on the calculation ofthe severity of each lesion by using a 0–3 scoringsystem. The severity factor was defined according tothe length of the lesions, where severity factor 0= nolesions; severity factor 1= lesions less than 1mmlength, severity factor 2= lesions 2–4mm in length,

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and severity factor 3= lesions greater than 4mm inlength. The lesion score for each rat was calculated asthe number of lesions in the rat multiplied by theirrespective severity factor. The UI for each group wastaken as the mean lesion score of all the rats in thatgroup. The preventive index (PI) of a given drug(obestatin) was calculated by the equation of Hanoet al. [25]:

PI ¼ UIofINDgroup�UIofpretreatedgroup

UIofINDgroup× 100:

Analysis of gastric juiceDetermination of volume

Gastric juice from each animal was centrifuged at1000g for 10min to remove any solid debris, andthe volume of the supernatant was measured andexpressed in ml.

Determination of free and total acidityA volume 1ml gastric juice diluted with 10ml ofdistilled water was taken into a conical flask, and2–3 drops of Topfer’s reagent as an indicator wasadded to it and titrated with 0.01N NaOH until acanary yellow color was observed. The volume ofNaOH consumed corresponding to free acidity wasnoted. Then, 2–3 drops of phenolphthalein solutionwere added, and the titration was continued until apermanent pink color was observed. Again, the totalvolume of NaOH corresponding to total acidity wasnoted. The acidity was calculated by using thefollowing formula [26]:

Acidity ¼ Volume ofNaOH×N× 100

0:1mEq=l:

Biochemical analysisMeasurement of tissue NO

It was done by Nitric oxide assay kit (catalog no.: no 253; Biodiagnostic, Giza, Egypt) according to themanufacturers’ instructions. The NO content in thestomach tissue was determined by measuring its nitrite(an indicator of original NO present). This methoddepends on reduction of nitrate to nitrite by vanadiumtrichloride (VCl3), which was followed by addition ofGriess reagent [27].

Table 1 Primer sequences used for real-time PCR

Forward

VEGF ATCATGCGGATCAAACCTCACC

TNFα TGCACCACCACCTGCTTAGC

GAPDH AAATGGGCTCCCTCTGATCAGT

TNF-α, tumor necrosis factor-α.

Assessment of mRNA expression of VEGF and TNFα byreal-time RT-PCR

Total RNA extraction: total RNA was extracted usingRNeasy mini kit (Qiagen, GmbH, Hilden, Germany)according to the manufacturer’s protocol, and theproduct of extraction was stored at −80°C. Theconcentration and purity of RNA were determinedspectrophotometrically by the 260/280 nm ratio,which ranged between 1.8 and 2.1.

Reverse transcription reaction: the isolated total RNAwas reverse-transcribed into complementary DNA(cDNA) using the high-capacity cDNA ReverseTranscription Kit (Applied Biosystems, Foster City,California, USA) according to the manufacturer’sinstructions, and all products were stored at −20°C.

Real-time quantitative qPCR: the expression levels ofVEGF and TNF-α genes were analyzed by qPCRusing the SYBR Green PCR Master MIX (AppliedBiosystems), and the expressions of VEGF and TNF-αwere performed in Applied Biosystems 7500 Real-Time PCR. The sequences of the primers used arelisted in Table 1. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as thehousekeeping gene. As a relative quantitation, foldchanges were calculated following the 2−ΔΔCt

method. For each sample, the Ct value of targetgene mRNA was normalized against the GAPDHendogenous control as ΔCt (ΔCt=Cttarget

gene–CtGAPDH). The fold change of the target genemRNA in the experimental sample relative to controlsample was determined by 2−ΔΔCt, whereΔΔCt=ΔCtExperimental−ΔCtControl.

Histopathological examinationFor histological evaluation, stomach samples were fixedin 10% formalin solution where they remained for 24 h.After fixation, the samples were transferred to asolution of 70% alcohol and processed for paraffinwax embedding. Sections (4 μm thick) weredeparaffinized, stained with hematoxylin and eosin,and then examined under a light microscope by anexperienced pathologist who was blinded to thetreatment.

5′–3′ sequences

Reverse

GGTCTGCATTCACATCTGCTATGC

GGCATGGACTGTGGTCATGAC

T TCTGCTTGGTGGTTTGCTACCAC

Table 2 Effect of obestatin pretreatment on ulcer index andpreventive index in different experimental groups (n=8)

UI PI

Group I 0.00±0.00 –

Group II 0.00±0.00 –

Group III 27±2.98*,+ –

Group IV 3.25±1.67*,+,# 87.96%

Data are represented as mean±SD. Group I: control group; groupII: obestatin group; group III: gastric ulcer group; group IV:obestatin (30 μg/kg)+gastric ulcer; PI, preventive index; UI, ulcer

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Chemicals usedIndomethacin (Liometacen) (The NILE Co. forPharm. and Chemical Ind., Cairo, Egypt), obestatin(mouse, rat) (O0266-.5MG) (Lot No.: 020M4807;Sigma Aldrich, St. Louis, Missouri, USA), TopferReagent (Lot No.: 0000245747; HiMediaLaboratories Pvt. Ltd, Mumbai, India), and Di-ethyl ether (38132 L05; Sd Fine-Chem Limited,India) were used for the experiment.

index. P<0.05 is significant tested by one-way analysis ofvariance and post-hoc multiple comparison least significantdifference method. *P<0.05 versus group I. +P<0.05 versus groupII. #P<0.05 versus group III.

Figure 1

Statistical analysisThe collected data were summarized in terms of mean±SD. Comparisons between the different study groupswere carried out using the one-way analysis of variance(F value) followed by post-hoc tests using the LSDmethod using the statistical package for social science(SPSS; SPSS Inc., Chicago, Illinois, USA) program,version 19. P value less than 0.05 was consideredstatistically significant.

Photomicrograph showing the macroscopic appearance of the gas-tric mucosa in different experimental groups. (a) Macroscopic ap-pearance of the gastric mucosa in control group (group I) revealed nolesions in gastric mucosa. (b) Macroscopic appearance of the gastricmucosa in obestatin group (group II) revealed no lesions in gastricmucosa. (c) Macroscopic appearance of the gastric mucosa in

ResultsEffect of obestatin on ulcer index and preventive indexin different experimental groupsThere was a nonsignificant change (P>0.05) in UI ingroup II (0.00±0.00) when compared with that ofcontrol group (group I) (0.00±0.00). INDadministration in group III at a dose of 40mg/kgp.o. resulted in a significant increase (P<0.05) in UI(27±2.98) when compared with that of control group(group I) (0.00±0.00) and obestatin group (group II)(0.00±0.00). Pretreatment with obestatin in group IVat a dose of 30 μg/kg i.p. significantly decreased UI(P<0.05) when compared with group III from 27±2.98to 3.25±1.67, with PI 87.96%; however, UI wassignificantly increased (P<0.05) in group IV (3.25±1.67) compared with both control group (group I)and obestatin group (group II) (0.00±0.00 and 0.00±0.00, respectively) (Table 2 and Fig. 1).

indomethacin group (group III) revealed multiple hemorrhagic lesionsin gastric mucosa (arrows). (d) Macroscopic appearance of thegastric mucosa in obestatin pretreated group (group IV) revealedminimal lesions in gastric mucosa (arrows).

Effect of obestatin on gastric juice volume and gastricjuice free and total acidity in different experimentalgroupsGastric juice volume in obestatin group (group II)showed a nonsignificant (P>0.05) decrease whencompared with that of control group (group I). INDadministration in group III at a dose of 40mg/kg p.o.resulted in a significant increase (P<0.05) in gastricjuice volume when compared with that of control group(group I) and obestatin group (group II). Interestingly,pretreatment with obestatin in group IV at a dose of30 μg/kg i.p. resulted in a significant decrease (P<0.05)in gastric juice volume when compared with that ofgroup III, although it showed a significant increase(P<0.05) in gastric juice volume when compared with

control group (group I) and obestatin group (group II)(Table 3).

There was a nonsignificant decrease (P>0.05) ingastric juice free and total acidity in group II whencompared with gastric juice free and total acidity ofcontrol group (group I). Group III treated with IND ata dose of 40mg/kg p.o. showed a significant increase(P<0.05) in gastric juice free and total acidity whencompared with gastric juice free and total acidity ofcontrol group (group I) and obestatin group (group II).

Table 3 Effect of obestatin pretreatment on gastric juicevolume and gastric juice free and total acidity in differentexperimental groups (n=8)

Gastric juice volume(ml)

Free acidity(mEq/l)

Total acidity(mEq/l)

Group I 2.04±0.35 11.50±2.45 23.88±2.75

GroupII

1.76±0.27 8.44±1.72 23.00±2.27

GroupIII

5.85±0.48*,+ 61.75±5.28*,+ 87.63±5.95*,+

GroupIV

3.01±0.62*,+,# 19.75±5.78*,+,# 37±8.14*,+,#

Data are represented as mean±SD. Group I: control group; groupII: obestatin group; group III: gastric ulcer group; group IV:obestatin (30 μg/kg)+gastric ulcer. P<0.05 is significant tested byone-way analysis of variance and post-hoc multiple comparisonleast significant difference method. *P<0.05 versus group I.+P<0.05 versus group II. #P<0.05 versus group III.

Table 4 Effect of obestatin pretreatment on tissue nitric oxidelevel and RQ (fold changes) of vascular endothelial growthfactor mRNA expression and tumor necrosis factor-α mRNAexpression in gastric tissue in different experimental groups(n=8)

NO (μmol/l) RQ of VEGF RQ of TNF-α

Group I 12.73±1.77 1±0.0 1±0.0

Group II 19.12±2.30* 1.77±0.56* 0.42±0.07

Group III 6.22±1.77*,+ 0.51±0.13*,+ 36.88±4.17*,+

Group IV 18.40±1.58*,# 1.93±0.66*,# 3.89±0.95*,+,#

Data are represented as mean±SD. Group I: control group; groupII: obestatin group; group III: gastric ulcer group; group IV:obestatin (30 μg/kg)+gastric ulcer; NO, nitric oxide; TNF-α, tumornecrosis factor-α; VEGF, vascular endothelial growth factor.P<0.05 is significant tested by one-way analysis of variance andpost-hoc multiple comparison least significant difference method.*P<0.05 versus group I. +P<0.05 versus group II. #P<0.05 versusgroup III.

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Interestingly, pretreatment with obestatin in group IVin a dose of 30 μg/kg i.p. showed a significant decrease(P<0.05) in gastric juice free and total acidity whencompared with group III, but there was a significantincrease (P<0.05) in the gastric juice free and totalacidity when compared with gastric juice free and totalacidity of control group (group I) and obestatin group(group II).

Effect of obestatin on tissue NO level and RQ (foldchanges) of VEGF mRNA expression and TNF-α mRNAexpression in gastric tissue in different experimentalgroupsThere was a significant (P<0.05) increase in gastrictissue NO in group II when compared with that ofcontrol group (group I). Group III treated with IND ata dose of 40mg/kg p.o. showed a significant decrease(P<0.05) in gastric tissue NO when compared withthat of control group (group I) and group II.Pretreatment with obestatin at a dose of 30 μg/kgi.p. in group IV showed a significant increase(P<0.05) in gastric tissue NO when compared withgroup III and control group (group I), but showed anonsignificant decrease (P>0.05) when compared withgroup II (Table 4).

The VEGF mRNA expression in group II showed asignificant increase (P<0.05) when compared with thatof control group (group I). IND administration ingroup III at a dose of 40mg/kg p.o. resulted in asignificant decrease (P<0.05) in VEGF mRNAexpression when compared with that of controlgroup (group I) and group II. Group IV pretreatedwith obestatin at a dose of 30 μg/kg i.p. showed asignificant increase (P<0.05) in VEGF mRNAexpression when compared with that of controlgroup (group I) and group III, respectively, and anonsignificant increase (P>0.05) when comparedwith that of group II.

Moreover, there was a nonsignificant decrease(P>0.05) in the pro-inflammatory cytokine (TNF-α)mRNA expression in group II when compared withthat of control group (group I). In group III treatedwith IND at a dose of 40mg/kg; p.o., there was asignificant increase (P<0.05) in the TNF-α mRNAexpression when compared with that of control group(group I) and group II correspondingly. Pretreatmentwith obestatin at a dose of 30 μg/kg i.p. in group IVshowed a significant decrease (P<0.05) in TNF-αmRNA expression when compared with that ofgroup III, although it showed a significant increase(P<0.05) when compared with control group (group I)and obestatin group (group II).

Histopathological resultsThe microscopic examination of the stomach of groupI (control group) and group II (obestatin treated group)(Fig. 2a and b, respectively) revealed normal intactgastric mucosa and normal gastric glands. On theother hand, stomach of group III (IND treatedgroup) showed necrosis, loss of epithelial cells, submucosal edema, marked infiltration with inflammatorycells and congested blood vessels (Fig. 2c).Pretreatment with obestatin before IND in group IV(obestatin+IND group) showed improvement ofhistopathologic changes produced by IND as itshowed intact mucosa, improvement of edema andmild inflammatory cells infiltration (Fig. 2d).

DiscussionNSAIDs such as IND have been widely used clinicallyas anti-inflammatory and analgesic agents. However,ulcerative lesions of the GIT are one of the majoradverse effects of NSAIDs and are the major limitationto their use as anti-inflammatory drugs [5,28].

There are a variety of pathogenic mechanisms that maycontribute to the formation of a GU via imbalance

Figure 2

Histological evaluation (H&E) of gastric mucosa in different experi-mental groups. (a) Light microscopy of control group (group I)revealed normal histopathologic structure of stomach (H&E, ×100).(b) Light microscopy of obestatin group (group II) revealed normalhistopathologic structure of stomach (H&E, ×100). (c) Light micros-copy of indomethacin group (group III) revealed epithelial cells loss(long arrow), marked inflammatory cells infiltration (arrow head),congested blood vessels (*), and submucosal edema (short arrow)(H&E, ×100). (d) Light microscopy of obestatin pretreated group(group IV) showed nearly normal histopathologic structure of thestomach, and intact mucosa with mild inflammatory cellular infiltration(arrow) (H&E, ×100). H&E, hematoxylin and eosin.

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between aggressive factors and decreased gastricresistance. IND induces its gastrointestinal toxicityvia several mechanisms such as an increase in gastricacid secretion, interference with mucosal cellregeneration via inhibition of PGE2 synthesis,production of free radicals, reduction of gastric NOlevel, and invasion of activated neutrophils as well asinduction of gastric cells apoptosis [6].

Obestatin is a novel 23-amino acid peptide hormonefirst identified in the rat stomach as a ghrelin-accompanying peptide [9]. It was reported thatobestatin has anti-inflammatory [13–15],antioxidative stress [16], and antiapoptotic effects [17].

In spite of the accumulating data about the effects ofobestatin on GIT, the role of obestatin in themanagement of the GIT diseases needs to beestablished. Hence, this study was designed toinvestigate the protective effect of obestatin on acuteGU induced by IND administration with explorationof the possible underlying mechanisms.

Parameters chosen to assess the GU and the protectiveeffect of obestatin included gastric juice volume andacidity, NO, VEGF mRNA, inflammatory markerssuch as TNF-α mRNA, with measurement of UI and

obestatin PI, and finally histopathological evaluation ofthe gastric mucosa.

The results of the present study showed thatadministration of IND at a single dose (40mg/kgp.o.) resulted in multiple hemorrhagic lesions bygross examination along with a significant increase(P<0.05) in gastric UI in group III when comparedwith control group (group I). This result was inagreement with previous studies [5,29,30], whichreported that IND administration caused aremarkably high UI. This may be explained by that,IND comprises polar lipids that have a high affinity forthe lipophilic areas of cell membranes, where theirpolar groups trigger membrane disruption, with lossof structural phospholipids and membrane proteins. Inaddition, this leads to reduced hydrophobicity of themucosal coat adherent to the mucosal cell surface. Suchloss of hydrophobicity facilitates the entry of water-soluble agents of injury (e.g. acid, pepsin, bile salts,etc.), and also alter membrane fluidity that play a keyrole in the development of the gastric mucosal lesionsinduced by IND [5,28,31].

Moreover, these results were confirmed byhistopathological examination of gastric mucosa, asIND-administered rats showed necrosis and loss ofepithelial cells, submucosal edema, marked infiltrationwith inflammatory cells and congested blood vessels.Our results regarding mucosal histologicalderangement were in accordance with the previousreports [32,33], which found that the gastric mucosawas focally necrotic, ulcerated, and infiltrated withintense leukocytes beside extensive hemorrhages.

Our results showed that IND induced aggressivefactors as it resulted in a significant increase(P<0.05) in gastric juice volume as well as free andtotal acidity in group III when compared with controlgroup (group I). It was reported that increased gastricacid secretion plays an important role in GU inductionand is involved in its etiology [34]. These findings werein line with previous work [35] which demonstratedthat IND induces GU through increasing gastricacidity.

According to the previous study [36], the abnormalelevation of gastric juice acidity by INDmay participatein the augmentation of the severity of GU. This effectmay be attributed to either free radicals formation orinhibition of PGs synthesis. Decreased PGs level hasbeen attributed to impaired gastro-protection andincreased gastric acid secretion which are importantevents in the etiology of mucosal ulceration. Moreover,

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excess acid secretion decreases the process of restitutionand ulcer healing via altering angiogenesis [34].

The results of our study also indicated that there was asignificant decrease (P<0.05) in gastric NO level ingroup III in comparison with control group (group I).These results were in agreement with previous reports[37] which demonstrated that the decrease in NOcould participate in the induction of GU induced byNSAIDs like IND. Moreover, this finding was inaccordance with previous reports [38] whichreported that administration of IND was associatedwith a decrease in NO biosynthesis, as a result ofdecreased nitric oxide synthase (NOS) activity thatwas associated with an increase in the extent damage.

The reduction of gastric NO level induced by IND alsomay be attributed to the ability to increase asymmetricdimethy l-arginine (ADMA), which has beenidentified as the major endogenous inhibitor ofNOS. ADMA has been shown to mediate gastricinjury induced by ethanol, stress, H. pylori, and IND[39]. Beside this, IND effect onNO could be explainedby the ability to upregulate endothelin-1, a factor thatleads to decreased release of endothelial NO, leading toan eventual loss of mucosal integrity [40].

Moreover, our results showed that IND significantlydecreased (P<0.05) VEGF mRNA expression ingroup III when compared with that of control group(group I). This result was in agreement with previouswork [41] that found that IND administrationsignificantly decreased the mucosal VEGF level.Another study [42], also agreed with us whichfounded that IND reduced VEGF expression ingastric mucosa. This result might be explained bythe inhibitory effect of IND on gastric mucosalPGE2 level as a result of cyclo-oxygenase inhibition[6], as previous studies found that VEGF expressiondecreased during ulceration, which could be because ofthe unavailability of PGE2, resulting in decreasedVEGF mRNA [43].

In the present study, IND showed a significant increase(P<0.05) in the TNF-α mRNA expression in groupIII when compared with that of control group (groupI). This result was in agreement with previous reports[44], which reported that inflammatory mediators,such as TNF-α, are involved in the pathogenesis ofIND-induced GU and is one of the aggressive factorsin ulcerogenesis. Moreover, another study [41] provedthat IND increased pro-inflammatory cytokines suchTNF-α, interlukin (IL)-1β, and IL-6 as well asdecreased gastric level of the anti-inflammatory

cytokine IL-10. This could be explained by theability of IND to activate nuclear factor-κB (NF-κB); subsequently, NF-κB translocated into thenucleus to upregulate the expression ofproinflammatory cytokines genes such as TNF-αand cytokine-induced neutrophil chemoattractant[45,46]. The increase in TNF-α after IND alsocould be explained by IND-induced inhibition ofPGE2, which is known to be a potent inhibitor ofTNF-α release from macrophages and mast cells [47].

On studying the prophylactic effect of obestatin onIND-induced GU, as it was administrated in a dose of30 μg/kg i.p. before induction of GU by IND,obestatin markedly reduced gastric mucosal lesions,and this was accompanied by a significant decrease(P<0.05) in the gastric UI with PI 87.96% in group IVwhen compared with IND group (group III). Theseobservations were confirmed by histopathologicalexamination that showed nearly normal mucosa withmild inflammatory cells infiltration that illustrate theimprovement of histopathological changes producedby IND.

Previous studies [20,48] agreed with us as these studiesstated that pretreatment with obestatin dose-dependently attenuated stress and ethanol-inducedgastric lesions, and this effect was accompanied byan increase in gastric mucosal blood flow. Thereduction of GU area may be contributed to theability of obestatin to enhance the PGE2 generationat ulcer margin [49].

Interestingly, the results of our study also indicated thatpretreatment with obestatin in group IV resulted in asignificant decrease (P<0.05) in gastric juice volume aswell as free and total acidity when compared with thatof group III. To the best of our knowledge, this is thefirst study describing the effect of obestatinpretreatment on gastric juice volume and acidity inIND-induced GU in rats. The mechanism by whichobestatin decreases gastric juice volume and free andtotal acidity may be explained by the enhancement ofthe PGE2 generation [49], and increased NO release[50–52], as PGE2 and NO are known to have aninhibitory effect on gastric acid secretion [53].

Our results also demonstrated a significant increase(P<0.05) in gastric tissue NO in group IV whencompared with group III. This result was inagreement with other studies [54] which reportedthat obestatin stimulates NOS and NO release fromendothelial cells that exerts a vasodilator action oncoronary vessels.

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The results of our study also indicated thatpretreatment with obestatin resulted in a significantincrease (P<0.05) in VEGF mRNA expression ingroup IV when compared with group III. VEGFactivates migration and proliferation of cells at theedge of the ulcer and promotes the formation ofgranulation tissue, angiogenesis [55], and remodelingof connective tissues during the process of ulcer repair[56]. Moreover, VEGF activates eNOS, leading todownstream release of potent vasodilators, includingNO and PGI2 [57,58]. These results were inaccordance with previous reports [20], which foundthat obestatin protected gastric mucosa against stress-induced gastric lesion via pro-angiogenic actions owingto upregulation of VEGF mRNA. Moreover, anotherstudy [59] found that obestatin helps in skeletal musclerepair by increasing the expression of VEGF andVEGFR2 and the consequent microvascularization.

Our results also showed a significant decrease (P<0.05)in TNF-α mRNA expression in group IV whencompared with that of group III. Previous reports[48], agreed with us as it was documented thatobestatin caused a downregulation of gene andprotein expression of TNF-α in ethanol-inducedgastric injury. This result also was in agreement withthe study done in Dembinski et al. [60], which statedthat treatment with obestatin reverses the ulcer-induced increase in mucosal expression of mRNAfor IL-1β and TNF-α, indicating that thetherapeutic effect of obestatin involves inhibition ofmucosal inflammation.

This result may be explained by previous reports [16]whichdemonstratedthatobestatinreducedtheexpressionon NF-κB during mesenteric ischemia reperfusion. NF-κBisacrucialnucleartranscriptionfactorfortheregulationof TNF-α, IL-1β, and IL-6 gene expression [61].

Moreover, we observed that there was a significantincrease (P<0.05) in NO level in obestatin group(group II) compared with control group (group I),and this increase also was reported in a previousstudy [50], which found that NO production ismarkedly increased in response to obestatin via asignaling cascade involving NOS activation;however, the significant increase (P<0.05) in VEGFmRNA in obestatin group (group II) may contribute tosuch a result.

ConclusionWe concluded that obestatin pretreatment couldimprove the outcome of IND-induced gastric injury

in rats. This protective effect could be explained on thebasis of anti-inflammatory, vasodilator, andproangiogenic effects. We also demonstrate for thefirst time that obestatin has an inhibitory effect ongastric juice volume and free and total acidity, whichcould contribute to the gastro-protective effect ofobestatin.

Financial support and sponsorshipNil.

Conflicts of interestThere are no conflicts of interest.

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