Scholarly article on topic 'Effects of β-glucan pretreatment on acetylsalicylic acid-induced gastric damage: An experimental study in rats'

Effects of β-glucan pretreatment on acetylsalicylic acid-induced gastric damage: An experimental study in rats Academic research paper on "Veterinary science"

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{β-glucan / "lipid peroxidation" / "gastric damage" / "acetylsalicylic acid"}

Abstract of research paper on Veterinary science, author of scientific article — Orhan Veli Ozkan, Oktay Hasan Ozturk, Mehmet Aydin, Nigar Yilmaz, Ibrahim Yetim, et al.

Abstract Background: NSAIDs have been found to induce gastrointestinal tract damage. Recently, it has been suggested that this might be mediated by lipid peroxidation. Objective: The aim of this study was to assess the potential protective effects of β-glucan against acetylsalicylic acid (ASA-induced gastric damage by means of its antioxidant capacity in an experimental rat model. Methods: Thirty-two male Wistar albino rats (200–250 g) were randomized into 4 groups consisting of 8 rats each. The β-glucan group received 50 mg/kg β-glucan once a day for 10 days and 30 minutes before anesthesia. The ASA group received saline once a day for 10 days and 300 mg/kg (20 mg/mL) ASA as a single dose, 4 hours before anesthesia. The ASA+β-glucan group was administered 50 mg/kg β-glucan once a day for 10 days and 30 minutes before anesthesia. Additionally, 300 mg/kg (20 mg/mL) ASA was administered as a single dose, 4 hours before anesthesia. The control group received saline once a day for 10 days and 30 minutes before anesthesia. All medications were administered by intragastric gavage. The stomach from each rat was dissected and divided into 2 parts for histologic and biochemical analysis. Gastric tissue malondialdehyde (MDA), nitric oxide (NO) levels, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were determined for oxidative parameter analysis. Results: The gastroprotective and antioxidant effects of β-glucan appeared to attenuate the ASA-induced gastric tissue damage. Compared with the control group, MDA and NO levels and CAT and GSH-Px activities were significantly increased in the stomachs of ASA-treated rats (MDA, 4.12 [0.44] to 13.41 [1.05] μmol/L; NO, 8.04 [7.25–9.10] vs 30.35 [22.34–37.95] μmol/g protein; CAT, 0.050 [0.004] to 0.083 [0.003] k/g protein; GSH-Px, 0.57 [0.42–0.66] to 1.55 [1.19–1.76] U/L; all, P < 0.001), whereas SOD activity was significantly decreased in the same group (291 [29] to 124 [6] U/mL; P < 0.001). In the ASA+β-glucan group, MDA and NO levels and CAT and GSH-Px activities were found to be significantly lower, while SOD activity was found to be significantly higher, in comparison with the ASA-treated group (all, P < 0.001). Conclusion: β-Glucan appeared to attenuate the gastric damage caused by ASA in these rats.

Academic research paper on topic "Effects of β-glucan pretreatment on acetylsalicylic acid-induced gastric damage: An experimental study in rats"

Volume 71, Number 6, December 2010

Effects of ß-Glucan Pretreatment on Acetylsalicylic Acid-Induced Gastric Damage: An Experimental Study in Rats

Orhan Veli Ozkan, MD1; Oktay Hasan Ozturk, MD2; Mehmet Aydin, MD3; Nigar Yilmaz, MD2; Ibrahim Yetim, MD1; Ahmet Nacar, MD4; Suleyman Oktar, MD5; and Sadik Sogut, MD2

1Department of General Surgery, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey; 2Department of Biochemistry, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey; 3Department of Physiology, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey; 4Department of Histology and Embryology, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey; and 5Department of Pharmacology, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey

ABSTRACT

Background: NSAIDs have been found to induce gastrointestinal tract damage. Recently, it has been suggested that this might be mediated by lipid peroxidation.

Objective: The aim of this study was to assess the potential protective effects of P-glucan against acetylsalicylic acid (ASA)-induced gastric damage by means of its antioxidant capacity in an experimental rat model.

Methods: Thirty-two male Wistar albino rats (200—250 g) were randomized into 4 groups consisting of 8 rats each. The P-glucan group received 50 mg/kg P-glucan once a day for 10 days and 30 minutes before anesthesia. The ASA group received saline once a day for 10 days and 300 mg/kg (20 mg/mL) ASA as a single dose, 4 hours before anesthesia. The ASA+P-glucan group was administered 50 mg/kg P-glucan once a day for 10 days and 30 minutes before anesthesia. Additionally, 300 mg/kg (20 mg/mL) ASA was administered as a single dose, 4 hours before anesthesia. The control group received saline once a day for 10 days and 30 minutes before anesthesia. All medications were administered by intragastric gavage. The stomach from each rat was dissected and divided into 2 parts for histologic and biochemical analysis. Gastric tissue malondialdehyde (MDA), nitric oxide (NO) levels, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were determined for oxidative parameter analysis.

Results: The gastroprotective and antioxidant effects of P-glucan appeared to attenuate the ASA-induced gastric tissue damage. Compared with the control group, MDA and NO levels and CAT and GSH-Px activities were significantly increased

in the stomachs of ASA-treated rats (MDA, 4.12 [0.44] to 13.41 [1.05] pmol/L; NO, 8.04 [7.25-9.10] vs 30.35 [22.34-37.95] pmol/g protein; CAT, 0.050 [0.004] to 0.083 [0.003] k/g protein; GSH-Px, 0.57 [0.42-0.66] to 1.55 [1.19-1.76] U/L; all, P < 0.001), whereas SOD activity was significantly decreased in the same group

Accepted for publication October 25, 2010. © 2010 Elsevier HS Journals, Inc. All rights reserved.

doi:10.1016/j.curtheres.2010.12.007 0011-393X/$ - see front matter

(291 [29] to 124 [6] U/mL; P < 0.001). In the ASA+P-glucan group, MDA and NO levels and CAT and GSH-Px activities were found to be significantly lower, while SOD activity was found to be significantly higher, in comparison with the ASA-treated group (all, P < 0.001).

Conclusion: P-Glucan appeared to attenuate the gastric damage caused by ASA in these rats. (Curr Ther Res Clin Exp. 2010;71:369-383) © 2010 Elsevier HS Journals, Inc.

Key words: P-glucan, lipid peroxidation, gastric damage, acetylsalicylic acid.

INTRODUCTION

An imbalance between the offensive factors such as acid and pepsin secretion and defensive factors namely mucin secretion, cellular shedding and proliferation and prostaglandin (PG) synthesis, has been suggested to play a role in the pathogenesis of gastrointestinal ulcers.1

NSAIDs such as acetylsalicylic acid (ASA) are widely used antiinflammatory and analgesic agents. Unfortunately, they have been found to result in gastrointestinal ulcers through a number of mechanisms which still remain to be elucidated.2 Suppressed PG synthesis due to inhibition of cyclooxygenase, direct irritant action disturbing the mucosal permeability, and neutrophil recruitment and activation have been suggested to be responsible for gastric ulcers. Recently, lipid peroxidation (LPO) mediated by reactive oxygen species (ROS) has been implicated in playing a crucial role in ulcer-related oxidative damage.3-5

Endogenous antioxidant enzymes in tissues, namely, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) act to scavenge ROS in order to prevent LPO-mediated tissue damage.1 Impaired balance between those ROS and endogenous antioxidants may be responsible for some clinical situations such as sepsis, ischemia reperfusion injury, and gastrointestinal ulcers. A variety of compounds may prove beneficial for preventing gastric ulcers through scavenging highly destructive ROS and inhibiting LPO.1,6

P-Glucan is a complex carbohydrate derived from broken cell walls of yeast, fungi, and cereals by a purification procedure that yields P-glucan polymer as a soluble form.7,8 It has been found to be associated with decreasing unhealthy amounts of cholesterol and boosting the immune system.9,10 It acts on macrophages, B lymphocytes, supressor T cells, and natural killer cells. It enhances neutrophil mobilization and migration by binding to the receptors located on monocytes.11 It has also been found to be an efficacious antioxidant and free-radical scavenger.12,13 It has been reported to be a critical potentiator for mucosal immunity in the digestive tract.14 Therefore, P-glucan might be beneficial for a variety of clinical situations through its complex interactions with pro-inflammatory cytokines and endogenous antioxidants.15,16

The present study assessed the potential protective effects of P-glucan against experimentally induced gastric ulcers in rats through its free radical scavenger action based on gastric tissue malondialdehyde (MDA) and nitric oxide (NO) levels, and CAT, GSH-Px, and SOD activities.

MATERIALS AND METHODS

ExpERimeNTAL DesigN

The experiments were carried out on a total of 32 male Wistar albino rats weighing between 200-250 g. All rats were maintained under the same conditions before the surgical procedure. The animals were housed at 22°C ± 2°C and 40% to 45% relative humidity in wire bottom colony cages (4 rats/cage) with a 12-hour light/dark cycle (7:00 am-7:00 pm) and fed standard rat chow. Food was withdrawn for 18 hours before the experiments, but access to water was ad libitum. Care was taken to prevent co-prophagy. The experiments were in compliance with the Principles of Laboratory Animal Care formulated by the National Institutes of Health.17 The study protocol was approved by the committee for the ethical care and use of laboratory animals of Mustafa Kemal University, School of Medicine.

Animal Treatment

The animals were randomized into 4 groups consisting of 8 rats each according to body weight (each group had the same relative mean weight). The P-glucan-treated group was treated with 50 mg/kg P-glucan* in microparticulate form (suspended with saline) once a day for 10 days and 30 minutes before anesthesia. The ASA-treated group received oral saline once a day for 10 days and 30 minutes before anesthesia and was administered a single dose of ASA 300 mg/kg (20 mg/mL; Bayer, Istanbul, Turkey) 4 hours before anesthesia. The ASA+P-glucan-treated group received 50 mg/kg P-glucan once a day for 10 days and 30 minutes before anesthesia and a single dose of ASA 300 mg/kg (20 mg/mL) 4 hours before anesthesia. The control group received oral saline once a day for 10 days and 30 minutes before anesthesia.

All the medications were administered orally by gavage through an intragastric tube. In each of the groups, fluids were administered at 6 mL/kg with medication.

Anesthesia of the animals was achieved through intramuscular injection of a mixture involving ketamine (80 mg kg-1) (Ketalar, Pfizer Inc, Istanbul, Turkey) and xylazine hydrochloride (1.5 mg/kg) (Rompun, Bayer). Laparotomy was conducted and pylorus ligation was performed with no damage to the blood supply, followed by the esophageal clamping. The stomach was dissected and the rats were euthanized by cervical dislocation. The stomachs were cut open along the greater curvature and the mucosae were rinsed with saline solution to remove the blood. Photographs of both forestomach and the glandular parts demonstrating the macroscopic appearance of the stomach were taken. The stomachs were divided into 2 parts for histologic and biochemical studies.

There were 2 primary outcome measures. The first was the demonstration of LPO, oxidative injury induced by ASA treatment by means of measuring MDA, NO levels, CAT, SOD, and GSH-Px activities in gastric tissue. The second was the determination of potential preventative effects of P-glucan against ASA-induced gastric injury through examination of the alterations in oxidative injury parameters following the

*Trademark: Immunex® (Mustafa Nevzat Company, Istanbul, Turkey).

use of P-glucan. Secondary outcome measures were the histologic changes in gastric tissue following ASA exposure and the potential protective role of P-glucan in the case of ASA-induced gastric damage with respect to histologic consequences.

Histologic ANALysis

The freshly excised stomachs were examined macroscopically for hemorrhagic lesions developing in the glandular mucosa. For histologic examination, stomach tissue specimens were fixed in 10% formalin solution. Following embedding in paraffin, sections from blocks (4—5 mm) were stained with hematoxylin and eosin and examined with a light microscope (BH-2, Olympus America Inc., Melville, New York).3,18

ULcer Scoring

Ulcer scoring was made by summing 2 scores. First, a macroscopic score was obtained by adding the total number of ulcers per stomach and the total severity of ulcers per stomach in each group. For macroscopic evaluation, all 8 samples in each group were observed. The sum of the total macroscopy score in each group was then divided by the number of rats in that group and a mean ulcer index (MUI) 1 was calculated (Table I). Secondly, at least 3 sections from each specimen were randomly selected and evaluated microscopically and scored (Table II). Points from all 3 sections in each group were summed and a microscopic score was obtained for each rat. The scores for each rat were then summed and divided by the number of rats to obtain the MUI 2 score. Finally, MUI 1 and MUI 2 were summed to derive a total ulcer score (TUS). Macroscopic and microscopic analysis was performed by an investigator who was not involved in the experimental procedures.

BiocHEmicaL Analysis

Oxidative Parameters

The gastric tissue samples were stored at —30°C until assayed for gastric tissue MDA, NO levels, CAT, SOD, and GSH-Px activities.

Stomach tissues were homogenized (for 2 minutes at 5000 rpm) in 4 volumes of ice-cold Tris-HCl buffer (50 mmol, pH 7.4) using a glass Teflon homogenizer (T10 Basic Ultra Turrax, IKA Werke GmbH & Co. KG, Staufen, Germany). MDA, NO,

Table I. Macroscopic scoring of gastric lesions.

Number of Macroscopic

Lesion Type Lesions Score

Linear hemorrhagic >5 5

Dot-like or patchy >3 2

None - 0

Table II. Microscopic scoring of gastric lesions.

Lesion Microscopic Score

Significant erosion 3 Swollen gastric glands with hemorrhagic

areas and exfoliation of epithelial cells 2

Two or fewer lesions with significant erosion 1

None 0

and protein levels were measured at the homogenate level. Then, the homogenate was centrifuged at 5000g for 60 minutes to remove debris. Supernatant fluid was collected and CAT and GSH-Px activities, as well as protein concentration, were measured. The supernatant solutions were used for the assay. The supernatant solutions were mixed with an equal volume of an ethanol/chloroform mixture (5/3, v/v). After centrifuga-tion at 5000g for 30 minutes, the clear upper layer (the ethanol phase) was collected and used in the analysis of SOD activity and protein assays. All preparation procedures were carried out at 4°C.

Determination of MDA Levels

The thiobarbituric acid reactive substance level was determined by a method based on the reaction with TBA at 90°C to 100°C.19 In the TBA test reaction, MDA or MDA-like substances (ie, the byproduct of lipid peroxidation process of the poly-unsaturated fatty acids) and TBA react together for production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed at pH 2 to 3 at 90°C for 15 minutes. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid to precipitate protein. The precipitate was pelleted by cen-trifugation and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water bath for 10 minutes. After cooling, the absor-bance was read at 532 nm (UV-1800, Shimadzu Corp., Tokyo, Japan). The results were expressed as nmol/g protein.

Determination of NO Levels

Because NO is a very labile molecule, its direct measurement in biological samples is difficult. In aqueous solution, NO reacts with molecular oxygen and accumulates in the plasma as nitrite (NO2~) and nitrate (NO3~) ions. Therefore, the stable oxidation end products of NO, NO2", and NO3~ can be readily measured in biological fluids and have been used in vitro and in vivo as indicators of NO production. Tissue nitrite plus nitrate concentrations as an index of plasma NO levels were determined by the method described previously.20 Quantification of nitrite and nitrate was based on the Griess reaction, in which a chromophore with a strong absorbance at 540 nm is formed by reaction of nitrite with a mixture of naphthylethylenediamine and sulfa-nilamide. The absorbance was measured in a spectrophotometer (UV-1800, Shimadzu Corp.) to assess the nitrite concentration. For nitrate detection, a second sample was

treated with copperized cadmium in glycine buffer at pH 9.7 to reduce nitrate to nitrite, the concentration of which represented the total nitrite plus nitrate. A standard curve was established with a set of serial dilutions (10-8-10-3 mol/L) of sodium nitrite. Results were expressed as pmoL/g protein.

Determination of CAT Activity

CAT (EC 1.11.1.6) activity was measured according to the method of Aebi et al.21 The principle of the assay is based on the determination of the rate constant k (dimension: s-1, k) of hydrogen peroxide (H2O2) decomposition. By measuring the absor-bance changes per minute, the rate constant of the enzyme was determined. Activities were expressed as k/mg protein.

Determination of GSH-Px Activity

GSH-Px (EC 1.6.4.2) activity was measured using the method of Paglia and Valen-tine.22 The enzymatic reaction was initiated by the addition of H2O2 to the reaction mixture containing glomerulus-stimulating hormone, nicotinamide adenine dinucleo-tide phosphate, and glutathione reductase. The change in the absorbance at 340 nm was monitored by a spectrophotometer. Activity was reported in U/g protein.

Determination of SOD Activity

The principle of the total SOD (EC 1.15.1.1) activity method is based, briefly, on the inhibition of nitroblue tetrazolium (NBT) reduction by O2" generated by a xanthine/ xanthine oxidase system.23 Activity was assessed in the ethanol phase of the serum after 1.0 mL ethanol/chloroform mixture (5/3, v/v) was added to the same volume of serum and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. Activity was expressed as U/g protein.

Statistical Analysis

Data were analyzed by using SPSS for Windows version 15.0 (SPSS Inc., Chicago, Illinois). Distribution of the groups was analyzed with the 1-sample Kolmogrov-Smirnov test. Groups showed normal distribution for the level of MDA and the activities of CAT and SOD so that parametric statistical methods were used to analyze the data. A 1-way ANOVA test was performed and post hoc multiple comparisons were made using least squares differences. Results are presented as mean (SEM).

The distribution was not normal for NO and GSH-Px levels in groups. The level of NO and the activity of GSH-Px were assessed between groups using the Kruskal-Wallis test. The Mann-Whitney U test was used for the difference between 2 groups. The results were presented as median and 25th and 75th interquartile range. P < 0.05 was considered to be statistically significant for all data.

RESULTS

Histologic Analysis

In the macroscopic analysis, no damage was noted in the forestomachs of rats in the P-glucan, ASA+P-glucan, and control groups. On the other hand, all ASA group

members showed patchy and dot-like pale lesions. When compared with the glandular regions, lesions were fewer, and there were no linear hemorrhagic lesions.

In the glandular stomach, the most evident finding was linear hemorrhagic lesions in the ASA group (Figure, B). Eight rats in this group had multiple linear lesions and 1 showed dot-like lesions. Patchy pale lesions accompanied hemorrhagic lesions in all samples.

In the ASA+P-glucan group, glandular region showed few dot-like and pale lesions with no linear hemorrhagic lesions compared with the ASA group (Figure, C). Both glandular mucosa and forestomach regions in the ASA+ P-glucan group were no different than those of the control (Figure, A) and P-glucan groups.

In the microscopic examination, the most striking changes occurred in the ASA group. Mucosa was disrupted with erosions. Lesions were extending deep into the mucosa but did not penetrate the muscularis mucosa (Figure, D). Exfoliation of

Figure. Macroscopic and microscopic examination of stomachs: (A) control rat showing normal forestomach and glandular stomach; (B) linear hemorrhagic lesions in glandular stomach of the acetylsalicylic acid (ASA) group; (C) few dot-like and pale lesions with no linear hemorrhagic ulcerations in the ASA+P-glucan group; (D) hematoxylin and eosin staining of stomach tissue in rats administered ASA showing the severity of the erosion (*). Note that muscularis mucosa is intact (magnification x40); (E) hematoxylin and eosin staining of stomach tissue of the ASA group showing hemorrhagic areas and impaired gastric glands (*) (magnification x200); and (F) swollen gastric glands (*) and hemorrhagic areas in the ASA+P-glucan group (magnification x400).

gastric epithelial cells, disruption in the glandular structure, and hemorrhagic lesions were observed (Figure, E).

Pretreatment with P-glucan was associated with fewer gastric lesions when compared with the ASA group. No erosions were noted. However, a few changes such as swollen gastric glands and hemorrhagic areas were observed (Figure, F).

In statistical analysis, histologic findings as formulated as TUS scores were found to be 0.37 (0.26), 7.12 (0.29), 2.87 (0.35), and 0.62 (0.32) in the P-glucan, ASA, ASA+P-glucan, and control groups, respectively. TUS scores significantly differed among control and ASA, ASA and ASA+P-glucan, and control and ASA+P-glucan groups (all, P < 0.001) (Table III).

Biochemical ANALysis

MDA levels and CAT and SOD enzyme activities are shown in Table IV. MDA levels and CAT activity were found to be significantly increased in the stomachs of rats treated with ASA, whereas SOD activity was found to be significantly decreased in the same group in comparison with the control group (MDA, 4.12 [0.44] to 13.41 [1.05]; CAT, 0.050 [0.004] to 0.083 [0.003]; SOD, 291 [29] to 124 [6]; all, P < 0.001). In the group that received P-glucan alone, CAT activity was significantly decreased when compared with the control group (0.050 [0.004] to 0.036 [0.003]; P = 0.02). MDA levels and CAT activity in the ASA+P-glucan group were found to be significantly lower when compared with the ASA-treated group (13.41 [1.05] vs 5.24 [0.47], 0.083 [0.003] vs 0.044 [0.004]; both, P < 0.001). SOD activity in the ASA+P-glucan group was found to be significantly higher in comparison with the ASA-treated group (124 [6] vs 278 [14]; P < 0.001). As shown in Table V, ASA was associated with significantly increased median (interquartile range) levels of NO in comparison with the control group (8.04 [7.25-9.10] vs 30.35 [22.34-37.95]; P < 0.001) (Table V). NO was found to be significantly lower in the ASA+P-glucan group (8.52 [7.60-9.84]; P < 0.001) when compared with the ASA-treated group.

ASA treatment alone was associated with a significant rise in GSH-Px activity in comparison with the control group (0.57 [0.42-0.66] to 1.55 [1.19-1.76]; P < 0.001) (Table V). GSH-Px activity was found to be significantly lower in the ASA+ P-glucan group (0.58 [0.51-0.64]; P < 0.001) when compared with the ASA-treated group.

Table III. Total ulcer score (TUS) comparison between groups. Data are mean (SD).

Variable ß-Glucan (n = 8) ASA (n = 8) ASA+ß-Glucan (n = 8) Control (n = 8) p*

TUS 0.37 (0.26) 7.12 (0.29)f 2.87 (0.35)" 0.62 (0.32) P < 0.001 (F = 101.79)

ASA = acetylsalicylic acid. *One-way ANOVA.

T P < 0.001 versus the control group (least squares difference). f P < 0.001 versus the ASA group (least squares difference).

Table IV. The levels of malondialdehyde (MDA) and the activities of catalase (CAT) and superoxide dismutase (SOD) in groups. Data are mean (SEM).

Parameter ß-Glucan (n = 8) ASA (n = 8) ASA+ ß-Glucan (n = 8) Control (n = 8) p*

MDA, nmoL/g 4.45 (0.57) 13.41 (1.05)f 5.24 (0.47)^ 4.12 (0.44) P < 0.001

protein (F = 41.75)

CAT, k/mg 0.036 0.083 0.044 0.050 P < 0.001

protein (0.003)§ (0.003)f (0.004)? (0.004) (F = 28.26)

SOD, U/g 302 (20) 124 (6)t 278 (14)? 291 (29) P < 0.001

protein (F = 17.66)

ASA = acetylsalicylic acid. *One-way ANOVA.

T P < 0.001 versus the control group (least squares difference). ■ P < 0.001 versus the ASA group (least squares difference). § P < 0.02 versus the control group (least squares difference).

DISCUSSION

ASA-induced gastric mucosal injury has been recently suggested to occur via LPO associated with ROS.3'24 Membrane peroxidation mediated by ROS leads to altered membrane permeability and protein degradation, followed by cell lysis. MDA, the product of LPO, has been adopted as a measure of free radical production and, therefore, an "index of LPO." SOD and GSH-Px activities in tissues are other parameters that may be used to monitor the extent of LPO.9,25

A variety of natural and synthetic antioxidant compounds, namely, melatonin,3 Asparagus racemosus,24 rebamipide,2 and fish oil,1 have been found to inhibit oxidative damage by enhancing the activity of endogenous antioxidant enzymes and scavenging the highly destructive free radical species and, therefore, restoring the balance in favor of antioxidant enzymes counteracting the free radicals.9,24 Antiulcerogenic activity

Table V. Nitric oxide (NO) levels and glutathione peroxidase (GSH-Px) activities in the study groups. Data are median (25th-75th percentile).

Parameter ß-Glucan (n = 8) ASA (n = 8) ASA+ ß-Glucan (n = 8) Control (n = 8)

NO, pmol/g protein 7.55 30.35 8.52 8.04

(5.85-9.24) (22.34-37.95)* (7.60-9.84)? (7.25-9.10)

GSH-Px, U/g protein 0.44 1.55 0.58 0.57

(0.31-0.60) (1.19-1.76)* (0.51-0.64)? (0.42-0.66)

ASA = acetylsalicylic acid.

*P < 0.001 versus the control group.

T P < 0.001 versus the ASA group.

of the aforementioned agents might be attributed to the antioxidant action of those molecules.

P-Glucan has been found to inhibit tumor development,26 enhance defense against infections,8'27 activate macrophages, induce production and release of cytokines, NO, and arachidonic acid metabolites, increase hematopoeisis,28-30 exert radioprotective effects, be effective against thermal trauma, improve wound healing,18,31 and lower serum cholesterol levels.18 It has been reported to be an effective antioxidant and free-radical scavenger, which are among the several mechanisms proposed for the protective effects of P-glucan in all those processes.9,18,25

Based on the free-radical scavenging activity and potential protective effects of P-glucan in different pathologic processes where LPO plays a crucial role, we investigated the impact of P-glucan on acidified gastric mucosa by ASA use and potential antiulcerogenic activity of the drug.

In the present study, we observed that ASA administration was associated with significant changes in gastric tissue both macroscopically and microscopically. The most striking findings were linear hemorrhagic lesions, erosions limited to the mus-cularis mucosa, and impaired gastric glands. Similar histologic findings were also reported by Sener-Muratoglu et al,3 Sairam et al,24 and Choi et al.32 Another important observation was that P-glucan was associated with significantly improved gastric glandular structure. This might be due to the gastroprotective and antioxidant effects of P-glucan in addition to its capacity to modulate the mucosal immunity of the intestinal tract.14 We developed our own ulcer scoring method that combined both macroscopic and microscopic findings. In previous studies, different scoring methods have been used. Millar et al33 performed both microscopic and macroscopic scorings and evaluated them separately. Szabo et al34 used a macroscopic index and Sarkar and Buha35 measured mucosal thickness to obtain a microscopic score. We preferred to combine both findings with an adaptation based on the literature. As supported by the literature, we gave top scores to evident erosions and ulcerations and rated other findings. Our results supported this new scoring system with significant values between the ASA and P-glucan groups.

Regarding the analysis of oxidative-damage parameters indicating LPO in this study, gastric tissue levels of MDA, NO levels, and CAT and GSH-Px activities were found to be significantly increased in ASA-induced gastric damage in rats; however, SOD activity was found to be significantly decreased in the same group. Subsequently, P-glucan pretreatment in ASA-induced gastric ulcers (ASA+P-glucan group) was found to depress LPO as it resulted in a higher SOD activity and lower MDA levels in comparison with the group administered only ASA, suggesting the protective effect of P-glucan against oxidative tissue damage. Previous studies have suggested similar findings. Local or systemic use of P-glucan was found to inhibit MDA elevations in cases of sepsis- or burn-induced oxidative tissue damage.8,18 Toklu et al,18 who investigated the putative protective effect of local or systemic P-glucan treatment on burn-induced remote organ injury in rats, found that severe skin-scald injury resulted in decreased glomerulus-stimulating hormone levels of the liver and intestinal tissues (P < 0.01 and P < 0.001). MDA levels were significantly increased at 6 and

48 hours post burn (P < 0.01 and P < 0.001). Local and systemic P-glucan treatment was associated with reducing MDA and increasing glomerulus stimulating hormone levels back to control levels. The same investigators later reported an inhibitory effect of P-glucan with respect to MDA production that implied a reduction in LPO-mediated cellular injury and protection of the liver against acetaminophen-induced oxidative damage in mice.36 Sener et al37 investigated oxidative organ injury following a single dose of methotrexate injection in rats. Methotrexate was associated with a significant decrease in glomerulus stimulating hormone levels while MDA levels, LPO activity, and collagen content were significantly increased in the ileum, liver, and renal tissues. P-Glucan use following methotrexate-induced oxidative organ injury was also found to eliminate the depletion of GSH-Px and inhibit the increases in MDA levels, LPO activity, and collagen content in addition to attenuation of tissue damage observed in histologic analysis. The findings of Senoglu et al7 are also in accordance with our data that P-glucan treatment was associated with significantly reversing the diminished SOD activity and high MDA levels in hepatic tissue of a rat model where sepsis was induced by cecal ligation and puncture. Another experimental study of ischemia reperfusion injury in rabbits supported the conclusions of the previous studies.9 P-Glucan administration was associated with decreased MDA and NO levels, whereas increased SOD and GSH-Px activities suggested that antioxidant medication might help lower limb ischemia reperfusion injury. However, Kayali et al25 reported no effect of P-glucan on SOD and MDA activities, and GSH-Px levels were decreased to preinjury levels (P = 0.002) in case of spinal cord injury in an experimental rat model.

In the present study, ASA was associated with significantly increased levels of NO in gastric tissue. ASA has been found to induce NO release from vascular endotheli-um.38 It was suggested that ASA directly stimulates the activity of endothelial NO synthase without affecting the expression of endothelial NO synthase (NOS).39 Inducible NOS (iNOS) expression and iNOS-derived NO synthesis were observed in leucocytes of the mesentery of ASA-treated rats.40 However, NO functions as a smooth muscle-relaxing agent and counteracts the reduction in gastric blood flow caused by inhibitors of prostaglandin synthesis. Therefore, stimulation of NO formation through ASA might help to reduce gastric injury or irritation.39 Some researchers have reported that P-glucans stimulate the production of NO,41 whereas other researchers have found that P-glucan has no effect on the production of NO in vivo or in vitro.42 P-Glucan administration alone did not induce any NO production in the present study, but P-glucan was associated with decreased ASA-induced NO production. Most likely, P-glucan does not affect the basal release of NO, but reduces the increased NO production. Indeed, Young et al43 reported that lipopolysaccharide-induced NO production by alveolar macrophage was decreased by in vivo pretreatment with zymosan (1^3—P-glucan).

Comparable to the present study, P-glucan decreased GSH-Px levels to the preinjury levels in spinal cord—injured rats and the administration of P-glucan alone did not change GSH-Px levels.25 Researchers have found that P-glucan inhibits CAT activity and dramatically increases H2O2 accumulation suspension-cultured cells of potato and

tobacco leaf explants.44,45 However, there are some studies that do not support our results. Reverberi et al46 reported that P-glucans stimulate GSH-Px, CAT, and other antioxidant enzyme activities.9 The literature regarding this issue appears to be controversial. According to the findings of Bobek and Galbavy,47 diet containing pleuran (P-glucan) reduced GSH-Px activity and increased CAT activity in erythrocytes. Moreover, CAT levels in the liver tissue were significantly increased by P-glucan administration.7 P-glucan at concentrations of 42.5, 85, 170, and 340 mg x 100 mL-1 markedly increased the activities of antioxidant enzymes such as CAT in human red blood cell hemolysates and in fish erythrocytes.48,49 It may be a partial explanation for the conflicting results: the effect of P-glucan on antioxidant enzyme activity may be dose-dependent. The doses of P-glucan are similar in the supporting studies related to GSH-Px. An explanation is more difficult for different CAT activities. The effect of P-glucan on CAT enzyme activity may be dose-dependent or its cellular effects may vary. Indeed, the effect of P-glucan on CAT activity is variable in different cell types in vitro.44,45,47-49 P-Glucan might directly reduce CAT activity, but indirectly reduce the activity of GSH-Px, because P-glucan itself scavenges free radicals.

Further parameters of oxidative injury such as myeloperoxidase activity and tissue collagen content (a free radical-induced fibrosis marker)3,36,37 and additional assessments such as determination of gastric mucus secretion and gastric acidity1,3 and estimation of cellular mucin as glycoproteins1,24 are lacking in the present study. Those variables may be promising contributory parameters for future investigation. Based on its potential antioxidant capacity, P-glucan may be considered of therapeutic value for the prevention and/or treatment of ASA-induced gastric ulcers. Future investigation with rigorous scientific design is required to clarify the role of P-glucan pretreatment for the prevention of ASA-induced gastric damage.

CONCLUSION

P-Glucan appeared to attenuate the gastric damage caused by ASA in these rats. ACKNOWLEDGMENTS

This study was supported by The Research Fund of Mustafa Kemal University (No: 03 M 0107). The authors have indicated that they have no conflicts of interest regarding the content of this article.

Drs. Sogut, Ozkan, and Aydin designed the study. Drs. Ozkan, Ozturk, Aydin, Yilmaz, Nacar, Oktar, and Yetim performed the research and carried out the acquisition, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript, and statistical analysis.

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Address correspondence to: Orhan Veli Ozkan, MD, Department of General Surgery, Mustafa Kemal University, Hatay 31100, Turkey. E-mail: veliorhan@ hotmail.com