Saudi Journal of Gastroenterology
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ORIGINAL ARTICLE Table of Contents   
Year : 2007  |  Volume : 13  |  Issue : 1  |  Page : 17-20
Protective effect of oleic acid against acute gastric mucosal lesions induced by ischemia-reperfusion in rat

Department of Physiology, College of Medicine, King Saud University, Saudi Arabia

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Date of Submission30-Oct-2006
Date of Acceptance13-Nov-2006


Aim: To assess the effect of Linoleic acid and oleic acid on gastric lesions induced by ischemia reperfusion. Materials and Methods: Twenty-six fasted rats were subjected to 30 minutes of gastric ischemia in the presence of 100 mmol/L hydrochloric acid and reperfusion periods of 60 minutes. The vascular permeability was quantified by measuring the extravasated Evans blue in the stomach. Results: There were greater amount of leakage of Evans blue in gastric mucosa of rats treated with linoleic acid as compared to the control. Evans blue levels were significantly higher in gastric fluid as compared to the control. In contrast, Oleic acid significantly reduced the leakage of Evans blue in the gastric mucosa. Conclusion: Oleic acid played a significant role in protecting the gastric mucosa from ischemia/reperfusion injury. Replacement of LA by OA in the diet could be beneficial in protecting the gastric mucosa.

Keywords: Gastric mucosa, oleic acid

How to cite this article:
Alzoghaibi M. Protective effect of oleic acid against acute gastric mucosal lesions induced by ischemia-reperfusion in rat. Saudi J Gastroenterol 2007;13:17-20

How to cite this URL:
Alzoghaibi M. Protective effect of oleic acid against acute gastric mucosal lesions induced by ischemia-reperfusion in rat. Saudi J Gastroenterol [serial online] 2007 [cited 2021 Oct 28];13:17-20. Available from:

A variety of factors produce damage of gastric mucosa, including: systemic events such as thermal stress or local mucosal application of various irritants that are commonly named breakers of gastric mucosal barrier.[1],[2] It has been reported that reactive oxygen species (ROS) play important roles in the pathogenesis of inflammation, atherosclerosis, neurodegenerative disease, diabetes mellitus, apoptosis, and ischemia-reperfusion (I/R).[3],[4] The ischemia itself causes tissue damage and eventual death, but further injuries can occur while oxygen is reintroduced to the tissue. The possible factors that cause ischemia-reperfusion injury are: (1) the over influx of calcium ions into the cells, (2) the increase of ROS production.[5],[6] During ischemia, ATP is catabolized to hypoxanthine and is accumulated within the tissue. With reperfusion xanthine oxidase can utilize hypoxanthine and oxygen to form superoxide ( O 2-) and hydrogen peroxide (H 2 O 2 ).[5],[7] O 2- and H 2 O 2 may then interact to produce the highly reactive hydroxyl radical ( OH). Lipid peroxidation mediated by free radicals is believed to be one of the important causes of cell membrane destruction and cell damage, because the cell membrane contains much lipid, especially unsaturated fatty acids.[5],[8],[9],[10]

The long chain polyunsaturated fatty acid (PUFA), linoleic acid (LA, 18:2 n -6), is the precursor for arachidonic acid and its metabolites,[11] which are important resources of ROS and regulators of inflammatory responses.[12] Recent study showed a significant role of LA in the production of inflammatory marker (interleukin-8, IL-8) from Crohn's intestinal smooth muscle cells. In contrast, oleic acid, OA, a monounsaturated fatty acid, (MUFA, 18:1 n -9) had protective effects.[13] Recent studies reported that an olive oil diet significantly reduced arachidonic acid concentrations in rat macrophages and colonic carcinogenesis.[14],[15]

Therefore the aim of the present study is to test the role of LA and OA in gastric mucosal damage by ischemia reperfusion.

   Materials and methods Top


Fatty acids were purchased from Cayman Chemical Company, Ann Arbor, MI, USA. Linoleic acid (LA) and oleic acid (OA) were initially diluted in 100% ethanol and then diluted to final concentration in phosphate buffered saline (PBS).


Male Wister rats, approximately the same age and weighing between 220 and 240 gram, were obtained from the Animal House, College of Medicine, King Saud University, Riyadh, Saudi Arabia. The animals were maintained on a standard rat chow diet with free access to water and housed at 221oC on a 12 hour day-night cycle. They kept in individual cages with raised mesh floors to prevent coprophagia. The animals were starved for 24 hours before the experiments, but water was allowed. After 24 hours the rats were randomly coded into five groups and water was withdrawn. The groups were then subjected to one of the previously planned studies outlined below. The Ethics Committee at our institute approved the study.

Induction of gastric lesions

Gastric lesions were induced using the method described by Takaishi et al .[16] About 26 rats were fasted for 24 hours. The fasted rats were divided into five groups as following: (1) six control animals, (2) four rats treated with Oleic acid (OA, 4M), (3) six rats treated with Oleic acid (OA, 6M), (4) four rats treated with Linoleic acid (OA, 4M), (5) six rats treated with Linoleic acid (OA, 6M). Each rat was kept separate in an individual cage. Animals were anesthetized with Urethane (125 mg/kg). Following a midline laparotomy incision was made to express the stomach and corresponding vascular supply. A feeding tube was advanced into the stomach via the mouth and use for administration of Hcl (100 m/L in a volume of 1 mL/100 grams body weight). The celiac artery was carefully dissected and a bulldog clamp was then placed on the pyloric end of the stomach. Feeding tube was withdrawn into the lower esophagus. A second bulldog clamp was then positioned around the lower esophagus. HCl was used to maintain the pH of the stomach during various experimental protocols. After the clamps were placed, the celiac artery was occluded with a fine vascular clamp for 25 minutes. Then, the acid was removed and replaced with PBS and ischemia continued for an additional 5 minutes for a total of 30 minutes ischemia. At 30 minutes, the bulldog and vascular clamps were removed and reperfusion of the stomach continued for 60 minutes. OA and LA were injected intravenously through peneal artery during the last 5 minutes of reperfusion.

Determination of vascular permeability ( Evans blue More Details dye)

The Evans blue dye was used as an indicator of increased capillary permeability.[17],[18],[19] When injected into the circulation, the Evans blue bind within seconds to serum albumins, forming a dissociable complex. The extravasated Evans blue due to damaged micro-vascular tissues can be easily quatified by the spectrophotometer. The Evans blue was extracted as previously described.[19] Immediately after the reperfusion, 1 mL of 0.5% Evans blue was injected intravenously and the amount of Evans blue that accumulated in the stomach within the reperfusion period was measured as follows. After the rats were euthanized, the stomach was removed together with the pyloric and esophageal clamps. Gastric contents were collected carefully by lavaging with 5 mL of cold distilled water, the stomach was opened along the greater curvature, and the corpus mucosa scraped off using two glass slides and put into a tube containing 5 mL of distilled water. The extraction of the Evans blue present in gastric contents and mucosa was performed by adding 5 mL of formamide to each tube, which was kept in a shaking water bath at a temperature of 50C for 24 hours incubation. The samples were centrifuged at 3000 rpm for 10 minutes and the absorbance of supernatant was measured at 612 nm using spectrophotometer. The amount of Evans blue present in both gastric juice and mucosa was calculated from previously constructed standard curves and data expressed as micrograms per stomach.[20]

Statistical analysis

Data are expressed as mean standard error of the mean (SEM) values. Comparisons between control rats and treated rats (6M and 4M) were made using anova and results were considered statistically significant when p<0.05. I used post hoc test (Bonferroni test) to see where the significant is. Standard statistical software (SPSS, version 12; Chicago, IL) was used for the analyses.

   Results Top

Amount of Evans blue in gastric mucosa

About 30 minutes of ischemia of gastric mucosa resulted in leakage of 86.83.16 mg/stomach Evans blue, which was considered as the control group for this study ( n =6). However, there was a greater amount of leakage of Evans blue in stomach that had been pre-treated with Linoleic acid, 6M (93.34.1 mg/stomach, n =6, P >0.05) [Figure - 1]. There was no significant difference in leakage of Evans blue between stomachs treated with LA (4M) and control group (81.85.4 vs. 86.83.16, respectively). Neither LA (6M) nor LA (4M) was statistically significantly different from the control group.

Pre-treatment of rats with oleic acid (OA, 6M) significantly reduced the extravasation of Evans blue in the stomach as compared to the control stomach (51.22.1 and 86.83.16 respectively, P <0.0001) [Figure - 2]. However, pre-treatment with OA (4M) did not change the levels of leakage of Evans blue as compared to the control (87.34.6 vs. 86.83.16, respectively, n = 4, P >0.05).

Amount of Evans blue in gastric fluid

LA (6M) significantly increased the levels of Evans blue in gastric fluid as compared to control (41.73.9 vs. 262.4, respectively, P =0.009) [Figure - 3]. However, LA (4M) did not show any significant difference (27.42.5 vs. 262.4). Neither OA (6M) nor OA (4M) was significantly different from the control mucosal fluid (6M: 26.82.4, 4M: 31.73.8 vs. 262.4, P >0.05) [Figure - 4].

   Discussion Top

The results of this study showed that gastric injury was formed during 60 minutes reperfusion following 30 minutes of ischemia but pre-treatment with oleic acid protected the gastric mucosa of rats against the noxious effects of ischemia reperfusion. On the other hand, linoleic acid increased the levels of Evans blue in gastric fluid. It increased the leakage of the dye in gastric mucosa but it was not significant.

Previous studies indicated that ischemia/reperfusion leads to marked injury in the rate of gastric mucosa similar with our findings.[3],[7],[21],[22],[23] Oxygen radical have been reported to be involved in the pathogenesis of acute gastric lesions induced by ischemia reperfusion.[22],[24],[25],[26] Ishii et al . have demonstrated that ROS and NO seem to be implicated in the ischemia/reperfusion injury and reduction of gastric ischemia/reperfusion injury may be via reduction of ROS and NO toxicity.[27]

It is demonstrated that antioxidant agents such as melatonin, vitamin C and vitamin E decreased the total area of lesions induced by ischemia reperfusion in gastric mucosa.[21],[24],[28] Vitamin C and E as well as oleic acid showed a significant reduction in the levels of oxidative stress which were quantitated by measurement of thiobarbituric acid reactive substances in crohn's smooth muscle cells.[13] The fatty acid profile in plasma phospholipids and tissue in many different diseases has attracted widespread attention among researchers. The long chain PUFA, linoleic acid (LA, 18:2 n -6), is the precursor for arachidonic acid (AA) and its metabolites,[11] which are important regulators of inflammatory responses.[12] Recent studies reported that olive oil diet significantly reduced AA concentrations in rat macrophages and colonic carcinogenesis.[14],[15] Olive oil is a (MUFA, 18:1 n -9) that may reduce the inflammatory response. The role of oleic acid, as previously described, as antioxidant and anti-inflammatory agent is consistent with its role as a protective agent against gastric lesion, which was induced by ischemia/reperfusion.

In summary, LA increased the levels of Evans blue die in gastric fluid and increased the leakage of the die in gastric mucosa but not significant after ischemia reperfusion injury of the rat stomach. Oleic acid played a significant role in protecting the gastric mucosa from ischemia/reperfusion injury. The fact that gastric lesion was induced by LA, but not OA suggests that AA may be mediators of the response because LA is a dietary fatty acid precursor for AA. These results suggest that replacement of LA by OA in the diet and increased intake of a diet reach in antioxidants could be beneficial in protecting the gastric mucosa. [29]

   Acknowledgements Top

This work was supported in part by College of Medicine Research Centre (CMRC). I would like to thank Mr. Sabirin for his excellent technical assistance.

   References Top

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2.Brzozowski T, Konturek PC, Sliwowski Z, Drozdowicz D, Hahn EG, Konturek SJ. Importance of nitric oxide and capsaicin-sensitive afferent nerves in healing of stress lesions induced by epidermal growth factor. J Clin Gastroenterol 1997;25:S28-38.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
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4.Nishida K, Ohta Y, Kobayashi T, Ishiguro I. Involvement of the xanthine-xanthine oxidase system and neutrophils in the development of acute gastric mucosal lesions in rats with water immersion restraint stress. Digestion 1997;58:340-51.  Back to cited text no. 4  [PUBMED]  
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13.Alzoghaibi MA, Walsh SW, Willey A, Fowler AA 3rd, Graham MF. Linoleic acid, but not oleic acid, upregulates the production of interleukin-8 by human intestinal smooth muscle cells isolated from patients with Crohn's disease. Clin Nutr 2003;22:529-35.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Bartoli R, Fernandez-Banares F, Navarro E, Castella E, Mane J, Alvarez M, et al . Effect of olive oil on early and late events of colon carcinogenesis in rats: Modulation of arachidonic acid metabolism and local prostaglandin E(2) synthesis. Gut 2000;46:191-9.  Back to cited text no. 14    
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17.Szabo S, Trier JS, Brown A, Schnoor J. Early vascular injury and increased vascular permeability in gastric mucosal injury caused by ethanol in the rat. Gastroenterology 1985;88:228-36.  Back to cited text no. 17    
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20.Takeuchi K, Okada M, Niida H, Okabe S. Role of sulfhydryls in mucosal injury caused by ethanol: Relation to microvascular permeability, gastric motility and cytoprotection. J Pharmacol Exp Ther 1989;248:836-41.  Back to cited text no. 20    
21.De La Lastra CA, Cabeza J, Motilva V, Martin MJ. Melatonin protects against gastric ischemia-reperfusion injury in rats. J Pineal Res 1997;23:47-52.  Back to cited text no. 21    
22.Kwiecien S, Brzozowski T, Konturek SJ. Effects of reactive oxygen species action on gastric mucosa in various models of mucosal injury. J Physiol Pharmacol 2002;53:39-50.  Back to cited text no. 22    
23.Hassan M, Kashimura H, Matsumaru K, Nakahara A, Iwata R, Hayashi T, et al . Gastric mucosal injury induced by local ischemia-reperfusion in rats. Role of endogenous endothelin-1 and free radical. Dig Dis Sci 1997;42:1375-80.  Back to cited text no. 23    
24.Kitano M, Wada K, Kamisaki Y, Nakamoto K, Kishimoto Y, Kawasaki H, et al . Effects of cimetidine on acute gastric mucosal injury induced by ischemia-reperfusion in rats. Pharmacology 1997;55:154-64.   Back to cited text no. 24    
25.Tanaka J, Yuda Y. Role of lipid peroxidation in gastric mucosal lesions induced by ischemia-reperfusion in the pylorus-ligated rat. Biol Pharm Bull 1993;16:29-32.  Back to cited text no. 25    
26.Perry MA, Wadhwa S, Parks DA, Pickard W, Granger DN. Role of oxygen radicals in ischemia-induced lesions in the cat stomach. Gastroenterology 1986;90:362-7.  Back to cited text no. 26    
27.Ishii M, Shimizu S, Nawata S, Kiuchi Y, Yamamoto T. Involvement of reactive oxygen species and nitric oxide in gastric ischemia-reperfusion injury in rats: Protective effect of tetrahydrobiopterin. Dig Dis Sci 2000;45:93-8.  Back to cited text no. 27    
28.Nakamoto K, Kamisaki Y, Wada K, Kawasaki H, Itoh T. Protective effect of acetaminophen against acute gastric mucosal lesions induced by ischemia-reperfusion in the rat. Pharmacology 1997;54:203-10.  Back to cited text no. 28    
29.Wada K, Kamisaki Y, Ohkura T, Kanda G, Nakamoto K, Kishimoto Y, et al . Direct measurement of nitric oxide release in gastric mucosa during ischemia-reperfusion in rats. Am J Physiol 1998;274:G465-71.  Back to cited text no. 29    

Correspondence Address:
Mohammed Alzoghaibi
College of Medicine, Physiology Department, King Saud University, P.O. Box 2925, Riyadh 11461
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1319-3767.30460

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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]


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