|Year : 2018 | Volume
| Issue : 3 | Page : 369-377
Protective effect of obestatin on indomethacin-induced acute gastric ulcer in rats: role of VEGF and TNF-α
Reham 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
|Date of Submission||29-Apr-2018|
|Date of Acceptance||26-Jul-2018|
|Date of Web Publication||07-Jan-2019|
Dr. Reham M Ibrahim
Department of Physiology, Faculty of Medicine, Benha University, Benha, 13511
Source of Support: None, Conflict of Interest: None
Background Gastric ulcer (GU) is one of the most common disorders that affect the gastrointestinal tract. Obestatin, a ghrelin-related peptide, has been shown to exhibit some protective and therapeutic effects in the gut.
Aim This study aimed at investigating the protective effect of obestatin on acute indomethacin (IND)-induced GU, clarifying the role of vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α).
Materials and methods A total of 32 adult Wistar albino male rats were divided into four main groups: control group, obestatin group, GU group, and obestatin+GU group. Obestatin was given by a single intraperotineal injection (30 µg/kg) 1 h before induction of GU by a single oral dose of IND (40 mg/kg). Pyloric ligation was carried out in all animals before IND or distilled water administration. Four hours later after IND treatment, gastric ulcer index, preventive index, gastric juice volume, and free and total acidity were assessed. Nitric oxide, VEGF mRNA, and TNF-α mRNA were measured in gastric tissue, as well as histopathological study of gastric injury.
Results IND caused a significant increase in ulcer index, gastric juice volume, free and total acidity, and TNF-α mRNA, with a significant decrease in nitric oxide and VEGF mRNA. Pretreatment with obestatin reversed these effects.
Conclusion Obestatin pretreatment showed a gastroprotective effect against the IND-induced GU that can be explained by the anti-inflammatory and angiogenic effects of obestatin treatment and reduction of gastric juice volume, free acidity, and total acidity.
Keywords: anti-inflammatory, gastric ulcer, indomethacin, obestatin, vascular endothelial growth factor
|How to cite this article:|
Ibrahim RM, Allam MM, El-Gohary OA, El-Talees AE, El-Hamady MS. Protective effect of obestatin on indomethacin-induced acute gastric ulcer in rats: role of VEGF and TNF-α. Benha Med J 2018;35:369-77
|How to cite this URL:|
Ibrahim RM, Allam MM, El-Gohary OA, El-Talees AE, El-Hamady MS. Protective effect of obestatin on indomethacin-induced acute gastric ulcer in rats: role of VEGF and TNF-α. Benha Med J [serial online] 2018 [cited 2020 Sep 20];35:369-77. Available from: http://www.bmfj.eg.net/text.asp?2018/35/3/369/249432
| Introduction|| |
Gastric ulcer (GU) is a major health hazard in terms of both morbidity and mortality ; ∼14.5 million people worldwide are affected by GU, with a mortality rate of 4.08 million per year .
The pathogenesis of GU has been considered mainly owing to imbalance between defensive factors [mucus secretion, gastroprotective prostaglandins (PGs) synthesis, bicarbonate production, and normal tissue microcirculation] and aggressive factors (excessive secretion of gastric acid, alcohol intake, bile salts, abnormal motility, infection with Helicobacter pylori, and NSAIDs) .
NSAIDs-induced GUs are the second most common etiology of GUs . According to previous reports, the oral administration of indomethacin (IND), a well-known NSAID, in rats causes ulcerative lesions in the gastric mucosa . IND induces its gastrointestinal tract (GIT) toxicity via several mechanisms such as an increase in gastric acid secretion, interfere with mucosal cell regeneration via inhibition of PGE2 synthesis, production of free radicals, reduction of gastric nitric oxide (NO) level, and invasion of activated neutrophils as well as induction of gastric cells apoptosis . In addition, it causes oxidative damage of the stomach and the generation of proinflammatory mediators . Furthermore, NSAIDs were reported to inhibit angiogenesis through downregulation of the pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and upregulation of anti-angiogenic proteins such as endostatin leading to delayed ulcer healing .
Obestatin is a ghrelin-related peptide. It is composed of 23 amino acids . It is expressed in many tissues along the GIT, most notably the stomach, pancreas, duodenum, jejunum, and colon . However, most cells that produce obestatin appear to be concentrated in the oxyntic mucosa of the stomach, and the stomach is considered the major source of circulating obestatin .
Obestatin has been reported to be an anorexigenic hormone, decreasing food intake and body weight. Interestingly, obestatin has been reported to have certain effects on GIT, including decrease in gastric emptying time and jejunal motility, and it exhibits some protective and therapeutic effects in the gut . Moreover, it was reported that obestatin has anti-inflammatory ,,, anti-oxidative stress , and antiapoptotic effects .
| Materials and methods|| |
This study was conducted on 32 adult Wistar albino male rats, 6–8 weeks old, weighing between 180 and 220 g. They were obtained from the Experimental Animal Unit of Moshtohor Faculty of Agriculture, Benha University. The study was approved by our IRB. The animals were acclimatized to the laboratory conditions for 10 days before the initiation of the experiment. They had free access to water and diet. Experimental rats were under complete healthy conditions all over the experiment and under care of a professional technician. No rats died throughout the experiment. At the end of the study, the rats were incinerated at Benha university hospital incinerator.
The rats were deprived of food for 24 h before the experiment in mesh-bottomed cages to minimize coprophagia but allowed free access to water except the last hour before the experiments . All experiments were performed during the same time of the day between 8 a.m. and 12 p.m. to avoid variations owing to diurnal rhythms of putative regulators of gastric functions .
The animals of the experiment were divided into four groups, and each group consisted of eight rats. In group I ‘control group’, animals received single intraperitoneal (i.p.) injection of saline and then after 1 h, a single oral dose of distilled water (the solvent of IND). In group II ‘obestatin group’, animals received a single i.p. injection of obestatin in a dose of 30 µg/kg , and then after 1 h a single oral dose of distilled water. In group III ‘GU group’, animals received single i.p. injection of saline and then after 1 h gastric ulceration was induced by a single oral dose of IND (40 mg/kg) by orogastric gavage . In group IV ‘obestatin+GU group’, animals were pretreated with a single i.p. injection of obestatin (30 µg/kg) 1 h before induction of GU by a single oral dose of IND (40 mg/kg) by orogastric gavage.
Pyloric ligation was carried out in each animal before oral administration of distilled water or IND to collect gastric juice under light di-ethyl ether anesthesia. A mid-line abdominal incision was performed; the pyloric portion of the stomach was gently mobilized and carefully ligated with a silk ligature around the pyloric sphincter taking care not to interfere with gastric blood supply. The abdominal incision was sutured, and the animals were allowed to recover from anesthesia .
Gastric juice was allowed to accumulate for a period of next 4 h . The animals were anesthetized with di-ethyl ether before cervical dislocation. Their stomachs were rapidly removed after clamping the esophagus, opened by an incision along the greater curvature, and the gastric juice was collected and then assessed for volume, free and total acidity. Gastric tissues were washed with ice-cold saline to remove gastric content remnants and blood clots and then assessed macroscopically. Finally, a part of each stomach was immediately kept in formaldehyde to be prepared for histopathological examination with hematoxylin and eosin for detection of the histopathological changes, and the other part was immediately frozen in liquid nitrogen and stored at −80°C for biochemical estimations of tissue NO, tumor necrosis factor-α (TNF-α) mRNA, and VEGF mRNA.
Assessment of gastric mucosal lesions
Gastric tissues were pinned out flat on a cork board and photographed for lesion assessment. The stomachs were examined for macroscopical mucosal lesions with the aid of a magnifier by a pathologist unaware of the treatment protocol. The gastric mucosal lesions were expressed in terms of ulcer index (UI) according to Peskar et al.  which depends on the calculation of the severity of each lesion by using a 0–3 scoring system. The severity factor was defined according to the length of the lesions, where severity factor 0 = no lesions; severity factor 1 = lesions less than 1 mm length, severity factor 2 = lesions 2–4 mm in length, and severity factor 3 = lesions greater than 4 mm in length. The lesion score for each rat was calculated as the number of lesions in the rat multiplied by their respective severity factor. The UI for each group was taken as the mean lesion score of all the rats in that group. The preventive index (PI) of a given drug (obestatin) was calculated by the equation of Hano et al. :
Analysis of gastric juice
Determination of volume
Gastric juice from each animal was centrifuged at 1000g for 10 min to remove any solid debris, and the volume of the supernatant was measured and expressed in ml.
Determination of free and total acidity
A volume 1 ml gastric juice diluted with 10 ml of distilled water was taken into a conical flask, and 2–3 drops of Topfer’s reagent as an indicator was added to it and titrated with 0.01 N NaOH until a canary yellow color was observed. The volume of NaOH consumed corresponding to free acidity was noted. Then, 2–3 drops of phenolphthalein solution were added, and the titration was continued until a permanent pink color was observed. Again, the total volume of NaOH corresponding to total acidity was noted. The acidity was calculated by using the following formula :
Measurement of tissue NO
It was done by Nitric oxide assay kit (catalog no.: no 25 3; Biodiagnostic, Giza, Egypt) according to the manufacturers’ instructions. The NO content in the stomach tissue was determined by measuring its nitrite (an indicator of original NO present). This method depends on reduction of nitrate to nitrite by vanadium trichloride (VCl3), which was followed by addition of Griess reagent .
Assessment of mRNA expression of VEGF and TNFα by real-time RT-PCR
Total RNA extraction: total RNA was extracted using RNeasy mini kit (Qiagen, GmbH, Hilden, Germany) according to the manufacturer’s protocol, and the product of extraction was stored at −80°C. The concentration and purity of RNA were determined spectrophotometrically by the 260/280 nm ratio, which ranged between 1.8 and 2.1.
Reverse transcription reaction: the isolated total RNA was reverse-transcribed into complementary DNA (cDNA) using the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s instructions, and all products were stored at −20°C.
Real-time quantitative qPCR: the expression levels of VEGF and TNF-α genes were analyzed by qPCR using the SYBR Green PCR Master MIX (Applied Biosystems), and the expressions of VEGF and TNF-α were performed in Applied Biosystems 7500 Real-Time PCR. The sequences of the primers used are listed in [Table 1]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. As a relative quantitation, fold changes were calculated following the 2−ΔΔCt method. For each sample, the Ct value of target gene mRNA was normalized against the GAPDH endogenous control as ΔCt (ΔCt=Cttarget gene–CtGAPDH). The fold change of the target gene mRNA in the experimental sample relative to control sample was determined by 2−ΔΔCt, where ΔΔCt=ΔCtExperimental−ΔCtControl.
For histological evaluation, stomach samples were fixed in 10% formalin solution where they remained for 24 h. After fixation, the samples were transferred to a solution of 70% alcohol and processed for paraffin wax embedding. Sections (4 µm thick) were deparaffinized, stained with hematoxylin and eosin, and then examined under a light microscope by an experienced pathologist who was blinded to the treatment.
Indomethacin (Liometacen) (The NILE Co. for Pharm. and Chemical Ind., Cairo, Egypt), obestatin (mouse, rat) (O0266-.5MG) (Lot No.: 020M4807; Sigma Aldrich, St. Louis, Missouri, USA), Topfer Reagent (Lot No.: 0000245747; HiMedia Laboratories Pvt. Ltd, Mumbai, India), and Di-ethyl ether (38132 L05; Sd Fine-Chem Limited, India) were used for the experiment.
The collected data were summarized in terms of mean±SD. Comparisons between the different study groups were carried out using the one-way analysis of variance (F value) followed by post-hoc tests using the LSD method using the statistical package for social science (SPSS; SPSS Inc., Chicago, Illinois, USA) program, version 19. P value less than 0.05 was considered statistically significant.
| Results|| |
Effect of obestatin on ulcer index and preventive index in different experimental groups
There was a nonsignificant change (P>0.05) in UI in group II (0.00±0.00) when compared with that of control group (group I) (0.00±0.00). IND administration in group III at a dose of 40 mg/kg p.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 IV at a dose of 30 µg/kg i.p. significantly decreased UI (P<0.05) when compared with group III from 27±2.98 to 3.25±1.67, with PI 87.96%; however, UI was significantly 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 [Figure 1]).
|Table 2 Effect of obestatin pretreatment on ulcer index and preventive index in different experimental groups (n=8)|
Click here to view
|Figure 1 Photomicrograph showing the macroscopic appearance of the gastric mucosa in different experimental groups. (a) Macroscopic appearance of the gastric mucosa in control group (group I) revealed no lesions in gastric mucosa. (b) Macroscopic appearance of the gastric mucosa in obestatin group (group II) revealed no lesions in gastric mucosa. (c) Macroscopic appearance of the gastric mucosa in indomethacin group (group III) revealed multiple hemorrhagic lesions in gastric mucosa (arrows). (d) Macroscopic appearance of the gastric mucosa in obestatin pretreated group (group IV) revealed minimal lesions in gastric mucosa (arrows).|
Click here to view
Effect of obestatin on gastric juice volume and gastric juice free and total acidity in different experimental groups
Gastric juice volume in obestatin group (group II) showed a nonsignificant (P>0.05) decrease when compared with that of control group (group I). IND administration in group III at a dose of 40 mg/kg p.o. resulted in a significant increase (P<0.05) in gastric juice 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 of 30 µg/kg i.p. resulted in a significant decrease (P<0.05) in gastric juice volume when compared with that of group 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]).
|Table 3 Effect of obestatin pretreatment on gastric juice volume and gastric juice free and total acidity in different experimental groups (n=8)|
Click here to view
There was a nonsignificant decrease (P>0.05) in gastric juice free and total acidity in group II when compared with gastric juice free and total acidity of control group (group I). Group III treated with IND at a dose of 40 mg/kg p.o. showed a significant increase (P<0.05) in gastric juice free and total acidity when compared with gastric juice free and total acidity of control group (group I) and obestatin group (group II). Interestingly, pretreatment with obestatin in group IV in a dose of 30 µg/kg i.p. showed a significant decrease (P<0.05) in gastric juice free and total acidity when compared with group III, but there was a significant increase (P<0.05) in the gastric juice free and total acidity when compared with gastric juice free and total acidity of control group (group I) and obestatin group (group II).
Effect of obestatin on tissue NO level and RQ (fold changes) of VEGF mRNA expression and TNF-α mRNA expression in gastric tissue in different experimental groups
There was a significant (P<0.05) increase in gastric tissue NO in group II when compared with that of control group (group I). Group III treated with IND at a dose of 40 mg/kg p.o. showed a significant decrease (P<0.05) in gastric tissue NO when compared with that of control group (group I) and group II. Pretreatment with obestatin at a dose of 30 µg/kg i.p. in group IV showed a significant increase (P<0.05) in gastric tissue NO when compared with group III and control group (group I), but showed a nonsignificant decrease (P>0.05) when compared with group II ([Table 4]).
|Table 4 Effect of obestatin pretreatment on tissue nitric oxide level and RQ (fold changes) of vascular endothelial growth factor mRNA expression and tumor necrosis factor-α mRNA expression in gastric tissue in different experimental groups (n=8)|
Click here to view
The VEGF mRNA expression in group II showed a significant increase (P<0.05) when compared with that of control group (group I). IND administration in group III at a dose of 40 mg/kg p.o. resulted in a significant decrease (P<0.05) in VEGF mRNA expression when compared with that of control group (group I) and group II. Group IV pretreated with obestatin at a dose of 30 µg/kg i.p. showed a significant increase (P<0.05) in VEGF mRNA expression when compared with that of control group (group I) and group III, respectively, and a nonsignificant increase (P>0.05) when compared with 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 with that of control group (group I). In group III treated with IND at a dose of 40 mg/kg; p.o., there was a significant increase (P<0.05) in the TNF-α mRNA expression when compared with that of control group (group I) and group II correspondingly. Pretreatment with obestatin at a dose of 30 µg/kg i.p. in group IV showed a significant decrease (P<0.05) in TNF-α mRNA expression when compared with that of group III, although it showed a significant increase (P<0.05) when compared with control group (group I) and obestatin group (group II).
The microscopic examination of the stomach of group I (control group) and group II (obestatin treated group) ([Figure 2]a and b, respectively) revealed normal intact gastric mucosa and normal gastric glands. On the other hand, stomach of group III (IND treated group) showed necrosis, loss of epithelial cells, sub mucosal edema, marked infiltration with inflammatory cells and congested blood vessels ([Figure 2]c). Pretreatment with obestatin before IND in group IV (obestatin+IND group) showed improvement of histopathologic changes produced by IND as it showed intact mucosa, improvement of edema and mild inflammatory cells infiltration ([Figure 2]d).
|Figure 2 Histological evaluation (H&E) of gastric mucosa in different experimental 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 normal histopathologic structure of stomach (H&E, ×100). (c) Light microscopy 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 the stomach, and intact mucosa with mild inflammatory cellular infiltration (arrow) (H&E, ×100). H&E, hematoxylin and eosin.|
Click here to view
| Discussion|| |
NSAIDs such as IND have been widely used clinically as anti-inflammatory and analgesic agents. However, ulcerative lesions of the GIT are one of the major adverse effects of NSAIDs and are the major limitation to their use as anti-inflammatory drugs ,.
There are a variety of pathogenic mechanisms that may contribute to the formation of a GU via imbalance between aggressive factors and decreased gastric resistance. IND induces its gastrointestinal toxicity via several mechanisms such as an increase in gastric acid secretion, interference with mucosal cell regeneration via inhibition of PGE2 synthesis, production of free radicals, reduction of gastric NO level, and invasion of activated neutrophils as well as induction of gastric cells apoptosis .
Obestatin is a novel 23-amino acid peptide hormone first identified in the rat stomach as a ghrelin-accompanying peptide . It was reported that obestatin has anti-inflammatory ,,, antioxidative stress , and antiapoptotic effects .
In spite of the accumulating data about the effects of obestatin on GIT, the role of obestatin in the management of the GIT diseases needs to be established. Hence, this study was designed to investigate the protective effect of obestatin on acute GU induced by IND administration with exploration of the possible underlying mechanisms.
Parameters chosen to assess the GU and the protective effect of obestatin included gastric juice volume and acidity, NO, VEGF mRNA, inflammatory markers such as TNF-α mRNA, with measurement of UI and obestatin PI, and finally histopathological evaluation of the gastric mucosa.
The results of the present study showed that administration of IND at a single dose (40 mg/kg p.o.) resulted in multiple hemorrhagic lesions by gross examination along with a significant increase (P<0.05) in gastric UI in group III when compared with control group (group I). This result was in agreement with previous studies ,,, which reported that IND administration caused a remarkably high UI. This may be explained by that, IND comprises polar lipids that have a high affinity for the lipophilic areas of cell membranes, where their polar groups trigger membrane disruption, with loss of structural phospholipids and membrane proteins. In addition, this leads to reduced hydrophobicity of the mucosal coat adherent to the mucosal cell surface. Such loss 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 key role in the development of the gastric mucosal lesions induced by IND ,,.
Moreover, these results were confirmed by histopathological examination of gastric mucosa, as IND-administered rats showed necrosis and loss of epithelial cells, submucosal edema, marked infiltration with inflammatory cells and congested blood vessels. Our results regarding mucosal histological derangement were in accordance with the previous reports ,, which found that the gastric mucosa was focally necrotic, ulcerated, and infiltrated with intense leukocytes beside extensive hemorrhages.
Our results showed that IND induced aggressive factors as it resulted in a significant increase (P<0.05) in gastric juice volume as well as free and total acidity in group III when compared with control group (group I). It was reported that increased gastric acid secretion plays an important role in GU induction and is involved in its etiology . These findings were in line with previous work  which demonstrated that IND induces GU through increasing gastric acidity.
According to the previous study , the abnormal elevation of gastric juice acidity by IND may participate in the augmentation of the severity of GU. This effect may be attributed to either free radicals formation or inhibition of PGs synthesis. Decreased PGs level has been attributed to impaired gastro-protection and increased gastric acid secretion which are important events in the etiology of mucosal ulceration. Moreover, excess acid secretion decreases the process of restitution and ulcer healing via altering angiogenesis .
The results of our study also indicated that there was a significant decrease (P<0.05) in gastric NO level in group III in comparison with control group (group I). These results were in agreement with previous reports  which demonstrated that the decrease in NO could participate in the induction of GU induced by NSAIDs like IND. Moreover, this finding was in accordance with previous reports  which reported that administration of IND was associated with a decrease in NO biosynthesis, as a result of decreased nitric oxide synthase (NOS) activity that was associated with an increase in the extent damage.
The reduction of gastric NO level induced by IND also may be attributed to the ability to increase asymmetric dimethy l-arginine (ADMA), which has been identified as the major endogenous inhibitor of NOS. ADMA has been shown to mediate gastric injury induced by ethanol, stress, H. pylori, and IND . Beside this, IND effect on NO could be explained by the ability to upregulate endothelin-1, a factor that leads to decreased release of endothelial NO, leading to an eventual loss of mucosal integrity .
Moreover, our results showed that IND significantly decreased (P<0.05) VEGF mRNA expression in group III when compared with that of control group (group I). This result was in agreement with previous work  that found that IND administration significantly decreased the mucosal VEGF level. Another study , also agreed with us which founded that IND reduced VEGF expression in gastric mucosa. This result might be explained by the inhibitory effect of IND on gastric mucosal PGE2 level as a result of cyclo-oxygenase inhibition , as previous studies found that VEGF expression decreased during ulceration, which could be because of the unavailability of PGE2, resulting in decreased VEGF mRNA .
In the present study, IND showed a significant increase (P<0.05) in the TNF-α mRNA expression in group III when compared with that of control group (group I). This result was in agreement with previous reports , which reported that inflammatory mediators, such as TNF-α, are involved in the pathogenesis of IND-induced GU and is one of the aggressive factors in ulcerogenesis. Moreover, another study  proved that IND increased pro-inflammatory cytokines such TNF-α, interlukin (IL)-1β, and IL-6 as well as decreased gastric level of the anti-inflammatory cytokine IL-10. This could be explained by the ability of IND to activate nuclear factor-κB (NF-κB); subsequently, NF-κB translocated into the nucleus to upregulate the expression of proinflammatory cytokines genes such as TNF-α and cytokine-induced neutrophil chemoattractant ,. The increase in TNF-α after IND also could be explained by IND-induced inhibition of PGE2, which is known to be a potent inhibitor of TNF-α release from macrophages and mast cells .
On studying the prophylactic effect of obestatin on IND-induced GU, as it was administrated in a dose of 30 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 IV when compared with IND group (group III). These observations were confirmed by histopathological examination that showed nearly normal mucosa with mild inflammatory cells infiltration that illustrate the improvement of histopathological changes produced by IND.
Previous studies , agreed with us as these studies stated that pretreatment with obestatin dose-dependently attenuated stress and ethanol-induced gastric lesions, and this effect was accompanied by an increase in gastric mucosal blood flow. The reduction of GU area may be contributed to the ability of obestatin to enhance the PGE2 generation at ulcer margin .
Interestingly, the results of our study also indicated that pretreatment with obestatin in group IV resulted in a significant decrease (P<0.05) in gastric juice volume as well as free and total acidity when compared with that of group III. To the best of our knowledge, this is the first study describing the effect of obestatin pretreatment on gastric juice volume and acidity in IND-induced GU in rats. The mechanism by which obestatin decreases gastric juice volume and free and total acidity may be explained by the enhancement of the PGE2 generation , and increased NO release ,,, as PGE2 and NO are known to have an inhibitory effect on gastric acid secretion .
Our results also demonstrated a significant increase (P<0.05) in gastric tissue NO in group IV when compared with group III. This result was in agreement with other studies  which reported that obestatin stimulates NOS and NO release from endothelial cells that exerts a vasodilator action on coronary vessels.
The results of our study also indicated that pretreatment with obestatin resulted in a significant increase (P<0.05) in VEGF mRNA expression in group IV when compared with group III. VEGF activates migration and proliferation of cells at the edge of the ulcer and promotes the formation of granulation tissue, angiogenesis , and remodeling of connective tissues during the process of ulcer repair . Moreover, VEGF activates eNOS, leading to downstream release of potent vasodilators, including NO and PGI2 ,. These results were in accordance with previous reports , which found that obestatin protected gastric mucosa against stress-induced gastric lesion via pro-angiogenic actions owing to upregulation of VEGF mRNA. Moreover, another study  found that obestatin helps in skeletal muscle repair by increasing the expression of VEGF and VEGFR2 and the consequent microvascularization.
Our results also showed a significant decrease (P<0.05) in TNF-α mRNA expression in group IV when compared with that of group III. Previous reports , agreed with us as it was documented that obestatin caused a downregulation of gene and protein expression of TNF-α in ethanol-induced gastric injury. This result also was in agreement with the study done in Dembiński et al. , which stated that treatment with obestatin reverses the ulcer-induced increase in mucosal expression of mRNA for IL-1β and TNF-α, indicating that the therapeutic effect of obestatin involves inhibition of mucosal inflammation.
This result may be explained by previous reports  which demonstrated that obestatin reduced the expression on NF-κB during mesenteric ischemia reperfusion. NF-κB is a crucial nuclear transcription factor for the regulation of TNF-α, IL-1β, and IL-6 gene expression .
Moreover, we observed that there was a significant increase (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 previous study , which found that NO production is markedly increased in response to obestatin via a signaling cascade involving NOS activation; however, the significant increase (P<0.05) in VEGF mRNA in obestatin group (group II) may contribute to such a result.
| Conclusion|| |
We concluded that obestatin pretreatment could improve the outcome of IND-induced gastric injury in rats. This protective effect could be explained on the basis of anti-inflammatory, vasodilator, and proangiogenic effects. We also demonstrate for the first time that obestatin has an inhibitory effect on gastric juice volume and free and total acidity, which could contribute to the gastro-protective effect of obestatin.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Chaturvedi A, Kumar MM, Bhawani G, Chaturvedi H, Kumar M, Goel RK. Effect of ethanolic extract of Eugenia jambolana
seeds on gastric ulceration and secretion in rats. Indian J Physiol Pharmacol 2007; 51:131.
Mentis A, Lehours P, Mégraud F. Epidemiology and diagnosis of Helicobacter pylori
infection. Helicobacter 2015; 20(S1):1–7.
Boligon AA, de Freitas RB, de Brum TF, Waczuk EP, Klimaczewski CV, de Ávila DS et al.
Antiulcerogenic activity of Scutia buxifolia
on gastric ulcers induced by ethanol in rats. Acta Pharm Sin B 2014; 4:358–367.
Adinortey MB, Ansah C, Galyuon I, Nyarko A. In vivo models used for evaluation of potential antigastroduodenal ulcer agents. Ulcers 2013; 2013:796405.
Kim JH, Kim BW, Kwon HJ, Nam SW. Curative effect of selenium against indomethacin-induced gastric ulcers in rats. J Microbiol Biotechnol 2011; 21:400–404.
Matsui H, Shimokawa O, Kaneko T, Nagano Y, Rai K, Hyodo I. The pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced mucosal injuries in stomach and small intestine. J Clin Biochem Nutr 2011; 48:107–111.
Kolgazi M, Cantali-Ozturk C, Deniz R, Ozdemir-Kumral ZN, Yuksel M, Sirvanci S et al.
Nesfatin-1 alleviates gastric damage via direct antioxidant mechanisms. J Surg Res 2015; 193:111–118.
Tarnawski AS. Cellular and molecular mechanisms of gastrointestinal ulcer healing. Dig Dis Sci 2005; 50:S24–S33.
Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C et al.
Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 2005; 310:996–999.
Zhao CM, Furnes MW, Stenström B, Kulseng B, Chen D. Characterization of obestatin-and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study. Cell Tissue Res 2008; 331:575–587.
Furnes MW, Stenström B, Tømmerås K, Skoglund T, Dickson SL, Kulseng B et al.
Feeding behavior in rats subjected to gastrectomy or gastric bypass surgery. Eur Surg Res 2008; 40:279–288.
Ceranowicz P, Warzecha Z, Dembinski A. Peptidyl hormones of endocrine cells origin in the gut − their discovery and physiological relevance. J Physiol Pharmacol 2015; 66:11–27.
Aragno M, Mastrocola R, Ghé C, Arnoletti E, Bassino E, Alloatti G et al.
Obestatin induced recovery of myocardial dysfunction in type 1 diabetic rats: underlying mechanisms. Cardiovasc Diabetol 2012; 11:129.
Erşahin M, Özsavcı D, Şener A, Özakpınar ÖB, Toklu HZ, Akakin D et al.
Obestatin alleviates subarachnoid haemorrhage-induced oxidative injury in rats via its anti-apoptotic and antioxidant effects. Brain Inj 2013; 27:1181–1189.
Matuszyk A, Ceranowicz P, Warzecha Z, Cieszkowski J, Bonior J, Jaworek J et al.
Obestatin accelerates the healing of acetic acid-induced colitis in rats. Oxid Med Cell Longev 2016; 2016 (XX):2834386.
Şen LS, Karakoyun B, Yeğen C, Akkiprik M, Yüksel M, Ercan F et al.
Treatment with either obestatin or ghrelin attenuates mesenteric ischemia-reperfusion-induced oxidative injury of the ileum and the remote organ lung. Peptides 2015; 71:8–19.
Granata R, Settanni F, Gallo D, Trovato L, Biancone L, Cantaluppi V et al.
Obestatin promotes survival of pancreatic β-cells and human islets and induces expression of genes involved in the regulation of β-cell mass and function. Diabetes 2008; 57:967–979.
Yamasaki K, Ishiyama H, Imaizumi T, Kanbe T, Yabuuchi Y. Effect of OPC-12759, a novel antiulcer agent, on chronic and acute experimental gastric ulcer, and gastric secretion in rats. Jpn J Pharmacol 1989; 49:441–448.
Noriyoshi S, Masaru Y, Takemi G, Yoshimitsu A. Stimulation of prostaglandin E2 and interleukin-1ß production from periodontal ligament cells of old rat subjected to mechanical stress. J Gerontol 2000; 55:B489–B495.
Konturek PC, Konturek S, Koziel J, Brzozowski T. W1717 Effect of Obestatin on Stress-Induced Gastric Lesions in Rat. Gastroenterology 2010; 138:S–725.
Rainsford KD, Whitehouse MW. Biochemical gastroprotection from acute ulceration induced by aspirin and related drugs. Biochem Pharmacol 1980; 29:1281–1289.
Alumets J, Ekelund M, Håkanson R, Hedenbro J, Rehfeld JF, Sundler F et al.
Gastric acid response to pylorus ligation in rats: is gastrin or histamine involved?. J Physiol 1982; 323:145–156.
Tarique M, Siddiqui HH, Khushtar M, Rahman MA. Protective effect of hydro-alcoholic extract of Ruta graveolens Linn. leaves on indomethacin and pylorus ligation-induced gastric ulcer in rats. J Ayurveda Integr Med 2016; 7:38–43.
Peskar BM, Ehrlich K, Peskar BA. Role of ATP-sensitive potassium channels in prostaglandin-mediated gastroprotection in the rat. J Pharmacol Exp Ther 2002; 301:969–974.
Hano J, Bugajski J, Danek L. Effect of adrenergic blockade on gastric secretion altered by catecholamines in rats. Arch Immunol Ther Exp (Warsz) 1976; 24:507–524.
Hawk PB, Oser BL, Summerson WH. Folin-Wu modified method. Practical physiological chemistry. 12th ed. Philadelphia: The Blakiston Co; 1947. 506.
Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001; 5:62–71.
Suleyman B, Halici Z, Odabasoglu F, Gocer F. The effect of lacidipine on indomethacin induced ulcers in rats. Int J Pharmacol 2012; 8:115–121.
Abbas AM, Sakr HF. Effect of selenium and grape seed extract on indomethacin-induced gastric ulcers in rats. J Physiol Biochem 2013; 69:527–537.
Abdallah IZA, Khattab HAH, Heeba GH. Gastroprotective effect of Cordia myxa
L. fruit extract against indomethacin-induced gastric ulceration in rats. Life Sci J 2011; 8:433–445.
Sabina EP, Rasool M. Therapeutic efficacy of Indian Ayurvedic Herbal Formulation Triphala on Lipid Peroxidation, Antioxidant Status and Inflammatory Mediator TNF-Ot in Adjuvant-induced Arthritic Mice. Int J Biol Chem 2007; 1:149–155.
El-Moselhy MA, Abdel-Hamid NM, Abdel-Raheim SR. Gastroprotective effect of nicorandil in indomethacin and alcohol-induced acute ulcers. Appl Biochem Biotechnol 2009; 152: 449–459.
Valcheva‐Kuzmanova S, Krasnaliev I, Galunska B, Belcheva A. Influence of dl‐alpha‐tocopherol acetate on indomethacin‐induced gastric mucosal injury in rats. Auton Autacoid Pharmacol 2007; 27:131–136.
Musumba C, Pritchard DM, Pirmohamed M. cellular and molecular mechanisms of NSAID‐induced peptic ulcers. Aliment Pharmacol Ther 2009; 30:517–531.
Oluwabunmi IJ, Abiola T. Gastroprotective effect of methanolic extract of Gomphrena celosioides on indomethacin induced gastric ulcer in Wistar albino rats. Int J Appl Basic Med Res 2015; 5:41.
Sabiu S, Garuba T, Sunmonu TO, Sulyman AO, Ismail NO. Indomethacin-induced gastric ulceration in rats: ameliorative roles of Spondias mombin
and Ficus exasperata
. Pharm Biol 2016; 54:180–186.
El-Ashmawy NE, Khedr EG, El-Bahrawy HA, Selim HM. Gastroprotective effect of garlic in indomethacin induced gastric ulcer in rats. Nutrition 2016; 32:849–854.
Adhikary B, Yadav SK, Chand S, Bandyopadhyay SK, Chattopadhyay S. Black tea and theaflavins suppress various inflammatory modulators and i-NOS mediated nitric oxide synthesis during gastric ulcer healing. Free Radic Res 2011; 45:767–778.
Zhang Z, Zou YY, Li FJ, Hu CP. Asymmetric dimethylarginine: a novel biomarker of gastric mucosal injury? World J Gastroenterol 2011; 17:2178.
Abdel‐Raheem IT. Gastroprotective Effect of Rutin against Indomethacin‐Induced Ulcers in Rats. Basic Clin Pharmacol Toxicol 2010; 107:742–750.
Antonisamy P, Kannan P, Aravinthan A, Duraipandiyan V, Valan Arasu M, Ignacimuthu S et al.
Gastroprotective activity of violacein isolated from Chromobacterium violaceum on indomethacin-induced gastric lesions in rats: investigation of potential mechanisms of action. ScientificWorldJournal 2014; 2014:616432.
George MY, Esmat A, Tadros MG, El-Demerdash E. In vivo cellular and molecular gastroprotective mechanisms of chrysin; Emphasis on oxidative stress, inflammation and angiogenesis. Eur J Pharmacol 2018; 818:486–498.
Ghosh AK. Regulation by prostaglandin E2 and histamine of angiogenesis in inflammatory granulation tissue. Yakugaku Zasshi 2003; 123:295–303.
Choi YJ, Kim N, Lee JY, Nam RH, Chang H, Seo JH et al.
Protective effects of garlic extract, PMK-S005, against nonsteroidal anti-inflammatory drugs-induced acute gastric damage in rats. Dig Dis Sci 2014; 59:2927–2934.
Takahashi S, Fujita T, Yamamoto A. Role of nuclear factor-κB in gastric ulcer healing in rats. Am J Physiol Gastrointest Liver Physiol 2001; 280:G1296–G1304.
Yadav SK, Adhikary B, Chand S, Maity B, Bandyopadhyay SK, Chattopadhyay S. Molecular mechanism of indomethacin-induced gastropathy. Free Radic Biol Med 2012; 52:1175–1187.
Abood WN, Abdulla MA, Ismail S. Involvement of inflammatory mediators in the gastroprotective action of Phaleria macrocarpa against ethanol-induced gastric ulcer. World Appl Sci J 2014; 30:344–350.
Konturek PC, Koziel J, Konturek S, Brzozowski T. Sa1735 Obestatin Protects Gastric Mucosa Against Ethanol-Induced Injury. Gastroenterology 2011; 140:S–316.
Brzozowski T, Szlachcic A, Pajdo R, Sliwowski Z, Drozdowicz D, Majka J et al.
(2011). Role of New Appetite Hormones Ghrelin, Orexin-A and Obestatin in the Mechanism of Healing of Chronic Gastric Ulcers. In Peptic Ulcer Disease. InTech
Agnew AJ, Robinson E, McVicar CM, Harvey AP, Ali IH, Lindsay JE et al.
The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium‐dependent NO signalling. Br J Pharmacol 2012; 166:327–338.
Ku JM, Andrews ZB, Barsby T, Reichenbach A, Lemus MB, Drummond GR et al.
Ghrelin-related peptides exert protective effects in the cerebral circulation of male mice through a nonclassical ghrelin receptor (s). Endocrinology 2015; 156:280–290.
Schinzari F, Iantorno M, Campia U, Mores N, Rovella V, Tesauro M et al.
Vasodilator responses and endothelin-dependent vasoconstriction in metabolically healthy obesity and the metabolic syndrome. Am J Physiol Endocrinol Metab 2015; 309:E787–E792.
Rahim NA, Hassandarvish P, Golbabapour S, Ismail S, Tayyab S, Abdulla MA. Gastroprotective effect of ethanolic extract of Curcuma xanthorrhiza leaf against ethanol-induced gastric mucosal lesions in Sprague-Dawley rats. Biomed Res Int 2014; 2014:416409.
Penna C, Tullio F, Femminò S, Rocca C, Angelone T, Cerra MC et al.
Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway. J Cell Mol Med 2017; 21:3670–3678.
Azlina MFN, Qodriyah HMS, Chua KH, Kamisah Y. Comparison between tocotrienol and omeprazole on gastric growth factors in stress-exposed rats. World J Enterol 2017; 23:5887.
Milani S, Calabrò A. Role of growth factors and their receptors in gastric ulcer healing. Microsc Res Tech 2001; 53:360–371.
Luizon MR, Sandrim VC. Hypertension and vascular endothelial growth factors. In: Pathophysiology and pharmacotherapy of cardiovascular disease
. Adis, Cham. 2015; pp. 695–707.
Neagoe PE, Lemieux C, Sirois MG. Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and-2 heterodimer. J Biol Chem 2005; 280:9904–9912.
Gurriarán-Rodríguez U, Santos-Zas I, González-Sánchez J, Beiroa D, Moresi V, Mosteiro CS et al.
Action of obestatin in skeletal muscle repair: stem cell expansion, muscle growth, and microenvironment remodeling. Mol Ther 2015; 23:1003–1021.
Dembiński A, Warzecha Z, Ceranowicz P, Cieszkowski J, Dembiński M, Ptak-Belowska A et al.
Administration of obestatin accelerates the healing of chronic gastric ulcers in rats. Med Sci Monit 2011; 17:BR196.
Tornatore L, Thotakura AK, Bennett J, Moretti M, Franzoso G. The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation. Trends Cell Biol 2012; 22:557–566.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]