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Effect of Aronia melanocarpa fruit juice on indomethacin-induced gastric mucosal damage and oxidative stress in rats
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 56 (2005) 385–392
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Effect of Aronia melanocarpa fruit juice on indomethacin-induced gastric mucosal damage and oxidative stress in rats S. Valcheva-Kuzmanovaa,, K. Marazovaa,1, I. Krasnalievb, B. Galunskaa, P. Borisovaa, A. Belchevaa a
Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University, 55 Marin Drinov Str., 9002 Varna, Bulgaria b Department of General and Clinical Pathology, Medical University, Varna, Bulgaria Received 26 October 2004; accepted 12 January 2005
Abstract Aronia melanocarpa fruits are rich in phenolic substances—mainly flavonoids from the anthocyanin subclass. The anthocyanins are water-soluble plant pigments with antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, gastroprotective and other activities. We studied the effect of A. melanocarpa fruit juice (AMFJ) on indomethacininduced gastric mucosal damage in rats and its possible relation to the oxidative status. AMFJ (5, 10 and 20 ml kg1) was applied orally as a pretreatment 1 h before the subcutaneous administration of indomethacin (30 mg kg1). Gastric ulcer formation was estimated morphometrically and histopathologically 4 h after the indomethacin administration. Malondialdehyde (MDA) in rat plasma and gastric mucosa and also reduced glutathione (GSH) and oxidized glutathione (GSSG) in gastric mucosa were determined and used as biochemical markers of the oxidative status. AMFJ-pretreatment diminished the number and area of indomethacin-induced gastric lesions. Histopathological examination of rat stomachs demonstrated that AMFJ induced an increase in gastric mucus production and a reduction of the depth and severity of indomethacin-induced mucosal lesions. AMFJ dose-dependently reduced the elevated indomethacin plasma and gastric MDA levels and at the doses of 10 and 20 ml kg1 they were not significantly different from the control values. Neither indomethacin-treatment, nor AMFJ-pretreatment had a significant influence on GSH and GSSG gastric mucosal levels. These results demonstrated that indomethacin-induced gastric mucosal damage was accompanied by the development of oxidative stress, evidenced by the accumulation of MDA. AMFJ-pretreatment decreased the gastric lesions caused by indomethacin. It could be suggested that this effect of AMFJ was probably due to the increased mucus production and interference with oxidative stress development as evidenced by the decreased plasma and gastric mucosal MDA. r 2005 Elsevier GmbH. All rights reserved. Keywords: Aronia melanocarpa; Indomethacin; Gastric mucosal damage; Oxidative stress; Lipid peroxidation; Antioxidant activity
Corresponding author. Fax: +359 52 65 0019. 1
E-mail address: [email protected] (S. Valcheva-Kuzmanova). Present address: INSERM SC11, Hopital Broussais, 75014 Paris, France.
0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.01.001
ARTICLE IN PRESS 386
S. Valcheva-Kuzmanova et al. / Experimental and Toxicologic Pathology 56 (2005) 385–392
Introduction The genesis of peptic ulcer is complex and involves infection of the gastric mucosa with Helicobacter pylori plus other factors such as imbalance between the mucosal damaging mechanisms (acid, pepsin), and the mucosal protecting mechanisms (mucus, bicarbonate, local prostaglandin synthesis, gastric mucosal blood flow). It has been established that reactive oxygen species (ROS), especially the superoxide anion (Od 2 ) and the hydroxyl radical (OHd), play an important role in the pathogenesis of acute experimental gastric lesions induced by stress (Das and Banerjee, 1993), ethanol (Hernandez-Munoz et al., 2000), pyloric ligation (Tanaka and Yuda, 1993), H. pylori (Davis et al., 1994), non-steroidal anti-inflammatory drugs (NSAIDs) (Pihan et al., 1987; Das and Banerjee, 1993; Yoshikawa et al., 1993; Naito et al., 1998). Indomethacin like other NSAIDs is known to induce gastric mucosal damage in animals and humans. Although the inhibition of cyclooxygenase (COX) and deficiency of endogenous prostaglandins (PGs) is accepted as a main mechanism implicated in indomethacininduced gastropathy, there is increasing body of evidence suggesting the involvement of oxidative stress in this pathology (Yoshikawa et al., 1993; Naito et al., 1998). Aronia melanocarpa Elliot is a bush brought to Eastern Europe from North America. The fruits are very rich in flavonoids from the anthocyanin subclass. The anthocyanins in A. melanocarpa fruits (560 mg/100 g of fruits) are cyanidin-3-O-galactoside, cyanidin-3-Oarabinoside, cyanidin-3-O-xyloside and cyanidin-3-Oglucoside (Oszmian´ski and Sepia, 1988). These are water-soluble plant pigments, responsible for the purple and even black colour of the fruits. Some recent reports have indicated that many flavonoids are gastroprotective due to their antioxidant properties (Martin et al., 1998; La Casa et al., 2000). The antioxidant activity of anthocyanins has been demonstrated in many studies (Tsuda et al., 1996; Wang et al., 1999; Noda et al., 2002). The aim of the present study was to investigate the effect of A. melanocarpa fruit juice (AMFJ) in a rat model of indomethacin-induced gastric mucosal damage and its possible interference with the gastric oxidative status. Gastric mucosal concentrations of malondialdehyde (MDA) and of reduced glutathione (GSH) and oxidized glutathione (GSSG) and blood plasma concentrations of MDA were used as biochemical markers of the oxidative stress.
Materials and methods Animals Male Wistar rats (200–250 g) were used in the experiment. The animals were kept under the standard
conditions of the animal house with 12-h light-dark cycle (light 7700–19700) at a temperature 23–25 1C. They were fasted 36 h before the experiment but had free access to water. All procedures concerning animal treatment and experimentation were in accordance with the Guiding Principles in the Care and Use of Animals, approved by the Council of the American Physiological Society, with European Communities Council Directives 86/609/EEC and the National regulations, adopted by the local Ethical Commission in the Medical University of Varna.
Chemicals Indomethacin (Indo) was from Fluka Chemie AG. It was prepared as a suspension in a vehicle (2 drops of Tween 80 per 5 ml distilled water). All chemicals used for the biochemical analyses and histopathological examination were of analytical grade and were obtained from Merck (Germany).
AMFJ preparation Fruits from A. melanocarpa Elliot were handpicked, crushed and squeezed. The juice was filtered, pasteurized and stored at 0 1C.
Experimental procedure The experimental animals were randomly divided in eight experimental groups each of six rats according to the experimental setting in Table 1. The rats were orally pretreated by direct stomach intubation with water (10 ml kg1) or AMFJ (5, 10 or 20 ml kg1). One hour later the rats were treated subcutaneously either with indomethacin (30 mg kg1) (Tanaka and Yuda, 1996) or the vehicle for indomethacin in a total volume of 2 ml kg1 body wt.
Tissue preparation and fractionation The animals were anaesthetized with diethylether 4 h after the indomethacin treatment. Blood was collected from the sublingual veins in heparinized tubes. It was centrifuged at 2000g rpm for 10 min and plasma was obtained for the measurement of MDA content and stored at 20 1C until analysis. After the animals decapitation the stomachs were removed immediately, opened along the great curvature, gently washed in physiological salt solution, spread over the pad and observed macroscopically for appearance of mucosal lesions. The length of each lesion was measured. In the case of petechia, five of them were considered as a 1mm lesion. Mean ulcer number and area were calculated. Gastric mucosa was gently separated from the underlying tissue, homogenized in 1:5 w/v 50 mM
ARTICLE IN PRESS S. Valcheva-Kuzmanova et al. / Experimental and Toxicologic Pathology 56 (2005) 385–392
Table 1.
387
Experimental setting of indomethacin-induced gastric mucosal damage in rats
Animal group
Pretreatment
Dose (ml kg1)
Treatment
Dose
1. 2. 3. 4. 5. 6. 7. 8.
Water Water AMFJ AMFJ AMFJ AMFJ AMFJ AMFJ
10 10 5 10 20 5 10 20
Vehicle Indomethacin Vehicle Vehicle Vehicle Indomethacin Indomethacin Indomethacin
2 ml kg1 30 mg kg1 2 ml kg1 2 ml kg1 2 ml kg1 30 mg kg1 30 mg kg1 30 mg kg1
Control Indo AMFJ 5 AMFJ 10 AMFJ 20 AMFJ 5+Indo AMFJ 10+Indo AMFJ 20+Indo
phosphate buffer (pH 7.4) containing 0.1 mM EDTA, at 4000 rpm for 10 min. The homogenate was centrifuged at 800g rpm for 15 min to discard the sediment and the supernatant was frozen until analysis. All manipulations were performed at 4–8 1C. MDA, GSH and GSSG were determined immediately after thawing the samples.
Histopathological study Pieces of the stomachs were fixed in 10% neutralbuffered formaldehyde solution. Fixed tissues were embedded in paraffin, cut into sections and placed on microscope slides. Staining of the slides with hematoxylin-eosin (H & E) and periodic acid-Schiff (PAS) was used for the histomorphological examination which was performed under light microscopy and documented by an Olympus microphotocamera.
Biochemical analysis Membrane lipid peroxidation was monitored by MDA measured by its thiobarbituric acid (TBA) reactivity in rat plasma and gastric mucosal homogenates using the method of Porter et al. (1976). MDA values in nM ml1 plasma and nM g1 gastric mucosal tissue were determined using the extinction coefficient of the MDA-TBA complex at 532 nm ¼ 1.56 105 cm1 M1 solution. GSH and GSSG contents in the gastric mucosal homogenates were analysed according to the method of Hissin and Hilf (1986), using o-phthaldialdehyde as the fluorescent agent. Standard solutions of GSH and GSSG were applied to calculate the glutathione content which was measured in mM g1 gastric mucosal tissue.
Statistical analysis Data were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett,s multiple comparison post test. Two independent groups were compared by Student’s t-test. A value of po0:05 was considered as statistically significant. Data are expressed
as mean7SEM. GraphPad Prism statistical software was used.
Results Macroscopic evaluation of gastric mucosal damage No mucosal lesions were detected in rats which were not treated with indomethacin. Indomethacin induced multiple gastric mucosal lesions in the glandular part of the stomach, most often 1–4 mm2 in size or petechial, bleeding at the moment of observation. The mean ulcer number in the Indo-group was 14.5572.75 (Fig. 1A), and the mean ulcer area was 50.86714.4 mm2 (Fig. 1B). Pretreatment with AMFJ (5, 10 and 20 ml kg1) dosedependently and significantly reduced the ulcer number by 58.8%, 57.0% and 70.0% (Fig. 1A), and the ulcer area by 76.6%, 77.7% and 85.8% (Fig. 1B), respectively.
Histopathological study The microscopic appearance of the gastric mucosa of the control rats was quite normal (Fig. 2A and B). AMFJ induced an increase in mucus production which was most demonstrative in rats that were not treated with indomethacin. In these rats the mucus appeared as a thick continuous layer covering the mucosal surface (Fig. 3A and B). The experimental lesions evoked by indomethacin were manifested by erosive defects comprising about 2/3 of the mucosal layer thickness. The defects were filled with blood coagulates and haematin materials (Fig. 4). In the rats pretreated with AMFJ before the indomethacin administration the gastric erosions were more superficial and in some cases only bleeding and focal desquamation of the epithelium were found (Fig. 5A). The mucus production in the zones of the lesions was reduced (Fig. 5B) while in the zones without lesions it was preserved and even increased.
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A
Biochemical examination
B
In the Indo-group plasma MDA and gastric mucosal MDA increased by 36.3% and by 31.8%, respectively, versus the control group (Fig. 6A and B). The application of AMFJ to the animals (AMFJ 5, AMFJ 10 and AMFJ 20) did not cause statistically significant changes in MDA concentrations in plasma and gastric mucosa (Fig. 6A and B). In the groups pretreated with AMFJ before the indomethacin treatment the plasma and mucosal MDA levels dose-dependently decreased in comparison with those of the Indo-group and at the doses of 10 and 20 ml kg1 they did not differ significantly from the control levels (Fig. 6A and B). Neither indomethacin treatment, nor AMFJ pretreatment caused any significant changes in the gastric mucosal concentrations of GSH and GSSG (Fig. 7A and B).
10
°
75
AMFJ 20 + Indo
AMFJ 10 + Indo
AMFJ 5 + Indo
Indo
0
50
Discussion
°°
°° AMFJ 20 + Indo
0
°°
AMFJ 10 + Indo
25
AMFJ 5 + Indo
Ulcer area [mm2]
° °°
Indo
Ulcer number
20
Fig. 1. Effect of AMFJ pretreatment on the number (A) and area (B) of gastric mucosal lesions in rats with indomethacininduced ulceration. Results are mean7SEM. Statistical significance: 1po0:05; 11po0:01 versus Indo-group.
Indomethacin in the present experiment induced gastric lesions probably due to several mechanisms. It is generally accepted that the ulcerogenic activity of NSAIDs is related to their ability to inhibit endogenous PG synthesis due to the inhibition of COX (Whittle, 1981). PGs form a vital component of gastric mucosal defense. They are produced throughout the gut in high concentrations locally, and are known to have an antisecretory effect on gastric acid production, to stimulate mucus and bicarbonate secretion and to improve the mucosal blood flow (Whittle, 1989). Studies have shown that chronic exposure to indomethacin suppresses the gastric PG synthesis (Shorrock and Rees, 1992). Saad et al. (2002) demonstrated that a
Fig. 2. Microscopic appearance of a control rat stomach following the application of water (10 ml kg1) and vehicle (2 ml kg1). H & E staining (A) and PAS-staining (B); magnification 10 12.5.
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Fig. 3. Microscopic appearance of a rat stomach following the application of AMFJ (20 ml kg1) and vehicle (2 ml kg1). H & Estaining (A) and PAS-staining (B); magnification 10 12.5.
Fig. 4. Microscopic appearance of a rat stomach following the application of water (10 ml kg1) and indomethacin (30 mg kg1). H & E-staining; magnification 10 12.5.
single dose of indomethacin did not inhibit PG synthesis and there was no difference in gastric PGE2 content after a single exposure. They concluded that the complete inhibition of COX leading to decrease in PGE2 content probably consumed a much longer time and did not occur 6 h after a single dose. Having in mind these findings we might suppose that in the present experiment the inhibition of the PG secretion is not the main pathogenic factor for the mucosal damage. There are data that ROS are involved in the development of mucosal damage by NSAIDs (Pihan et al., 1987; Naito et al., 1998). These ROS induce lipid peroxidation which is believed to be an important cause
of destruction and damage of the cellular membranes. In the current study the involvement of extensive lipid peroxidation in indomethacin-induced gastric mucosal damage was evidenced by the accumulation of MDA in gastric mucosa. Similar are the results obtained by Tanaka and Yuda (1996) who studied the peroxidation of lipids and changes in the activities of related enzymes in the gastric mucosa in a rat model of gastric mucosal injury induced by indomethacin (30 mg kg1). In their experiment the amount of thiobarbituric acid reactive substances in the gastric mucosa was significantly increased 4 h after indomethacin administration. Indomethacin activated the polymorphonuclear leukocytes in peripheral blood and enhanced release of oxygen radicals from these cells. In our experiment the administration of indomethacin did not lead to a depletion of GSH, as might be expected having in mind the participation of GSH in the free radical scavenging through its conversion to GSSG by glutathione peroxidase. A possible reason for this effect might be the reduction of glutathione peroxidase activity by the administration of indomethacin (Yoshikawa et al., 1993; Tanaka and Yuda, 1996). The decreased glutathione peroxidase activity would aggravate the injury due to accelerated accumulation of hydrogen peroxide and lipid peroxides in gastric mucosal cells. In the present work we found that AMFJ reduced the morphometrical and histomorphologiocal signs of gastric mucosal damage by indomethacin. The histopathological examination of the stomachs showed that AMFJ caused an increase in mucus production. There are similar data for the anthocyanins from bilberry (cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside and cyanidin-3-O-glucoside) (Magistretti et al., 1988). This effect on the mucus production might be due to an
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Fig. 5. Microscopic appearance of a rat stomach following the application of AMFJ (20 ml kg1) and indomethacin (30 mg kg1). H & E-staining (A) and PAS-staining (B); magnification 10 12.5.
A
5
**
**
MDA [nM ml-1]
4 3 2
50
AMFJ 20 + Indo
AMFJ 10 + Indo
B *
*
40
MDA [nM g-1]
AMFJ 5 + Indo
AMFJ 20
AMFJ 10
AMFJ 5
Contol
0
Indo
1
30 20
AMFJ 20 + Indo
AMFJ 10 + Indo
AMFJ 5 + Indo
AMFJ 20
AMFJ 10
AMFJ 5
Indo
0
Control
10
Fig. 6. Effect of AMFJ on malondialdehyde (MDA) values in rat plasma (A) and gastric mucosa (B) in a model of indomethacin-induced gastric ulceration. Results are mean7SEM. Statistical significance: *po0:05; **po0:01 vs. control.
increase in mucosal PGE2. An anti-ulcerogenic activity of flavonoids associated with an increased mucus secretion and PGE2 release has been established by Beil et al. (1995), Khayyal et al. (2001). Other studies have demonstrated that the increased mucus production by flavonoids is not accompanied by an increase in PGE2 level (Alarcon et al., 1994; Perez-Guerrero et al., 1994). These observations suggest that the gastroprotective effect of AMFJ flavonoids could partly be explained through non-prostanoid-dependent mechanisms. Another possible mechanism of the ulceroprotection by AMFJ is an anti-secretory effect. Flavonoids reportedly (Parmar, 1983) inhibit histidine decarboxylase and reduce gastric mucosal content of histamine, which stimulates the gastric acid secretion. Beil et al. (1995) established an inhibitory action of flavonoids on H+/K+-ATPase activity. Inhibition of H+/K+-ATPase activity is dependent on the ATP concentration. The inhibitory action of flavonoids on H+/K+-ATPase activity is related to their ability to complex ATP. As it has already been pointed out, ROS play a very important role in the pathogenesis of acute experimental gastric lesions. Anthocyanins are exogenous antioxidants acting by scavenging free radicals (Yamasaki et al., 1996; Zheng and Wang, 2003) and by inhibiting lipoprotein oxidation (Tsuda et al., 1996). The results from our experiment showed that AMFJ dose-dependently reduced the lesion area accompanied by decreased MDA levels in gastric mucosa and blood plasma. In previous experiments Martin et al. (1998) and La Casa et al. (2000) established that the gastroprotective effect of quercetin and of rutin was due to the enhancement of the anti-oxidant enzymatic (glutathione peroxidase) activity and anti-lipoperoxidant effect.
ARTICLE IN PRESS S. Valcheva-Kuzmanova et al. / Experimental and Toxicologic Pathology 56 (2005) 385–392 0.5
the increased mucus production and the interference with gastric oxidative status could be suggested as possible ways through which AMFJ was protective against indomethacin-induced gastric ulceration.
A
GSH [µM g-1]
0.4 0.3 0.2
References
Indo
AMFJ 5
AMFJ 10
AMFJ 20
AMFJ 5 + Indo
AMFJ 10 + Indo
AMFJ 20 + Indo
AMFJ 5
AMFJ 10
AMFJ 20
AMFJ 5 + Indo
AMFJ 10 + Indo
AMFJ 20 + Indo
0.75
Indo
0.0
Control
0.1
B
0.50
0.25
0.00
Control
GSSG [µM g-1]
391
Fig. 7. Effect of AMFJ on rat gastric mucosal values of (A) reduced glutathione (GSH) and (B) oxidized glutathione (GSSG) in a model of indomethacin-induced gastric ulceration. Results are mean7SEM.
AMFJ did not significantly influence the GSH and GSSG levels. The influence of AMFJ on glutathione concentrations might be a result of two processes having opposite effects on GSH/GSSG. On the one hand, there are data that anthocyanins in experimental situations of increased lipid peroxidation preserve (Chidambara Murthy et al., 2002) or even increase (Kowalczyk et al., 2002) the glutathione peroxidase activity which would increase the conversion of GSH to GSSG. On the other hand, anthocyanins in AMFJ probably help to maintain the gastric GSH level acting as antioxidants instead of GSH in the process of free radical scavenging. In conclusion, we found that AMFJ reduced indomethacin-induced gastric mucosal injury. The gastroprotective effect of AMFJ was accompanied by a significant decrease in lipid peroxidation (evaluated as gastric and plasma MDA concentrations). Several mechanisms might be implicated in this effect. Our study demonstrated that
Alarcon DLLC, Martin MJ, LaCasa C, et al. Antiulcerogenic activity of flavonoids and gastric protection. J Ethnopharmacol 1994;42:161–70. Beil W, Birkholz C, Sewing KF. Effects of flavonoids on parietal cell acid secretion, gastric mucosal prostaglandin production and Helicobacter pylori growth. Arzneim Forsch 1995;45:697–700. Chidambara Murthy KN, Jayaprakasha GK, Singh RP. Studies on antioxidant activities of pomegranate (Punica granatum) peel extract using in vivo models. J Agric Food Chem 2002;50:4791–5. Das KD, Banerjee RK. Effect of stress on the antioxidant enzymes and gastric ulceration. Mol Cell Biochem 1993;125:115–25. Davis GR, Simmonds NJ, Stevens NJ, et al. Helocobacter pylori stimulates antral mucosal reactive metabolite production in vivo. Gut 1994;35:179–85. Hernandez-Munoz R, Montiel-Ruiz C, Vazquez-Martinez O. Gastric mucosal cell proliferation in ethanol-induced chronic mucosal injury is related to oxidative stress and lipid peroxidation in rats. Lab Invest 2000;80:1161–9. Hissin P, Hilf A. A fluorimetric method for determination of oxidized and reduced glutathione in tissues. Annal Biochem 1986;74:214–26. Khayyal MT, el-Ghalazy MA, Kenawy SA, et al. Antiulcerogenic effect of some gastrointestinally acting plant extracts and their combination. Arzneim Forsch 2001;51:545–53. Kowalczyk E, Kopff A, Niedworok J, et al. Anthocyanins—an adjunct to cardiovascular therapy? Kadiologia Polska 2002;57:332–4. La Casa C, Villegas I, Alarcon de la Lastra C, et al. Evidence of protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions. J Ethnopharamacol 2000;71:45–53. Magistretti MJ, Conti M, Cristoni A. Anti-ulcer activity of an anthocyanidin from Vaccinium myrtillus (bilberry) fruits and cell cultures. Plant Sci 1988;131:95–103. Martin MJ, La-Casa C, Alarcon-de-la-Lastra C, et al. Antioxidant mechanisms involved in gastroprotective effects of quercetin. Z Naturforsch [C] 1998;53:82–8. Naito Y, Yoshikawa T, Yoshida N, et al. Role of oxygen radical and lipid peroxidation in indomethacin-induced gastric mucosal injury. Dig Dis Sci 1998;43:30S–4S. Noda Y, Kaneyuki T, Mori A, et al. Antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin. J Agric Food Chem 2002;50:166–71. Oszmian´ski J, Sepia JC. Anthocyanins in fruits of Aronia Melanocarpa (Chokeberry). J Food Sci 1988;53:1241–2. Parmar NS. The gastric anti-ulcer activity of naringenin, a specific histidine decarbohylase inhibitor. Int J Tissue React 1983;4:415–20.
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Perez-Guerrero C, Martin MJ, Marhuenda E. Prevention by rutin of gastric lesions induced by ethanol in rats: role of endogenous prostaglandins. Gen Pharmacol 1994;25: 575–80. Pihan G, Regillo C, Szabo S. Free radicals and lipid peroxidation in ethanol- and aspirin-induced gastric mucosal injury. Dig Dis Sci 1987;32:1395–401. Porter N, Norton J, Ramdas J. Cyclic peroxidase and thiobarbituric assay. Biochem Biophys Acta 1976;441: 596–9. Saad QHM, Kassim NM, Top GM, et al. Tocotrienol-rich fraction and its effects on parameters affecting gastric mucosal integrity after a single exposure to indomethacin. Pakistan J Nutr 2002;1:89–92. Shorrock CJ, Rees WDW. Mucosal adaptation to indomethacin induced gastric damage in man—studies on morphology, blood flow and prostaglandin E2 metabolism. Gut 1992;33:164–9. 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. Tanaka J, Yuda Y. Lipid peroxidation in gastric mucosal lesions induced by indomethacin in rat. Biol Pharm Bull 1996;19:716–20.
Tsuda T, Shiga K, Oshima K, et al. Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L. Biochem Pharmacol 1996;52:1033–9. Wang H, Nair MG, Strasburg GM, et al. Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, cyanidin, from tart cherries. J Nat Prod 1999;62:294–6. Whittle BJR. Temporal relationship between cyclooxygenase inhibition, as measured by prostacyclin biosynthesis and the gastrointestinal damage induced by indomethacin in the rat. Gastroenterol 1981;80:81–94. Whittle BR. The defensive role played by the gastric microcirculation. Methods Find Exp Clin Pharmacol 1989; 11(Suppl 1):35S–43S. Yamasaki H, Uefuji H, Sakihama Y. Bleaching of the red anthocyanin induced by superoxide radical. Arch Biochem Biophys 1996;332:183–6. Yoshikawa T, Naito Y, Kishi A, et al. Role of active oxygen, lipid peroxidation, and antioxidants in the pathogenesis of gastric mucosal injury induced by indomethacin in rats. Gut 1993;34:732–7. Zheng W, Wang S. Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries. J Agric Food Chem 2003;51:502–9.
Experimental and Toxicologic Pathology 56 (2005) 385–392
EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY www.elsevier.de/etp
Effect of Aronia melanocarpa fruit juice on indomethacin-induced gastric mucosal damage and oxidative stress in rats S. Valcheva-Kuzmanovaa,, K. Marazovaa,1, I. Krasnalievb, B. Galunskaa, P. Borisovaa, A. Belchevaa a
Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University, 55 Marin Drinov Str., 9002 Varna, Bulgaria b Department of General and Clinical Pathology, Medical University, Varna, Bulgaria Received 26 October 2004; accepted 12 January 2005
Abstract Aronia melanocarpa fruits are rich in phenolic substances—mainly flavonoids from the anthocyanin subclass. The anthocyanins are water-soluble plant pigments with antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, gastroprotective and other activities. We studied the effect of A. melanocarpa fruit juice (AMFJ) on indomethacininduced gastric mucosal damage in rats and its possible relation to the oxidative status. AMFJ (5, 10 and 20 ml kg1) was applied orally as a pretreatment 1 h before the subcutaneous administration of indomethacin (30 mg kg1). Gastric ulcer formation was estimated morphometrically and histopathologically 4 h after the indomethacin administration. Malondialdehyde (MDA) in rat plasma and gastric mucosa and also reduced glutathione (GSH) and oxidized glutathione (GSSG) in gastric mucosa were determined and used as biochemical markers of the oxidative status. AMFJ-pretreatment diminished the number and area of indomethacin-induced gastric lesions. Histopathological examination of rat stomachs demonstrated that AMFJ induced an increase in gastric mucus production and a reduction of the depth and severity of indomethacin-induced mucosal lesions. AMFJ dose-dependently reduced the elevated indomethacin plasma and gastric MDA levels and at the doses of 10 and 20 ml kg1 they were not significantly different from the control values. Neither indomethacin-treatment, nor AMFJ-pretreatment had a significant influence on GSH and GSSG gastric mucosal levels. These results demonstrated that indomethacin-induced gastric mucosal damage was accompanied by the development of oxidative stress, evidenced by the accumulation of MDA. AMFJ-pretreatment decreased the gastric lesions caused by indomethacin. It could be suggested that this effect of AMFJ was probably due to the increased mucus production and interference with oxidative stress development as evidenced by the decreased plasma and gastric mucosal MDA. r 2005 Elsevier GmbH. All rights reserved. Keywords: Aronia melanocarpa; Indomethacin; Gastric mucosal damage; Oxidative stress; Lipid peroxidation; Antioxidant activity
Corresponding author. Fax: +359 52 65 0019. 1
E-mail address: [email protected] (S. Valcheva-Kuzmanova). Present address: INSERM SC11, Hopital Broussais, 75014 Paris, France.
0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.01.001
ARTICLE IN PRESS 386
S. Valcheva-Kuzmanova et al. / Experimental and Toxicologic Pathology 56 (2005) 385–392
Introduction The genesis of peptic ulcer is complex and involves infection of the gastric mucosa with Helicobacter pylori plus other factors such as imbalance between the mucosal damaging mechanisms (acid, pepsin), and the mucosal protecting mechanisms (mucus, bicarbonate, local prostaglandin synthesis, gastric mucosal blood flow). It has been established that reactive oxygen species (ROS), especially the superoxide anion (Od 2 ) and the hydroxyl radical (OHd), play an important role in the pathogenesis of acute experimental gastric lesions induced by stress (Das and Banerjee, 1993), ethanol (Hernandez-Munoz et al., 2000), pyloric ligation (Tanaka and Yuda, 1993), H. pylori (Davis et al., 1994), non-steroidal anti-inflammatory drugs (NSAIDs) (Pihan et al., 1987; Das and Banerjee, 1993; Yoshikawa et al., 1993; Naito et al., 1998). Indomethacin like other NSAIDs is known to induce gastric mucosal damage in animals and humans. Although the inhibition of cyclooxygenase (COX) and deficiency of endogenous prostaglandins (PGs) is accepted as a main mechanism implicated in indomethacininduced gastropathy, there is increasing body of evidence suggesting the involvement of oxidative stress in this pathology (Yoshikawa et al., 1993; Naito et al., 1998). Aronia melanocarpa Elliot is a bush brought to Eastern Europe from North America. The fruits are very rich in flavonoids from the anthocyanin subclass. The anthocyanins in A. melanocarpa fruits (560 mg/100 g of fruits) are cyanidin-3-O-galactoside, cyanidin-3-Oarabinoside, cyanidin-3-O-xyloside and cyanidin-3-Oglucoside (Oszmian´ski and Sepia, 1988). These are water-soluble plant pigments, responsible for the purple and even black colour of the fruits. Some recent reports have indicated that many flavonoids are gastroprotective due to their antioxidant properties (Martin et al., 1998; La Casa et al., 2000). The antioxidant activity of anthocyanins has been demonstrated in many studies (Tsuda et al., 1996; Wang et al., 1999; Noda et al., 2002). The aim of the present study was to investigate the effect of A. melanocarpa fruit juice (AMFJ) in a rat model of indomethacin-induced gastric mucosal damage and its possible interference with the gastric oxidative status. Gastric mucosal concentrations of malondialdehyde (MDA) and of reduced glutathione (GSH) and oxidized glutathione (GSSG) and blood plasma concentrations of MDA were used as biochemical markers of the oxidative stress.
Materials and methods Animals Male Wistar rats (200–250 g) were used in the experiment. The animals were kept under the standard
conditions of the animal house with 12-h light-dark cycle (light 7700–19700) at a temperature 23–25 1C. They were fasted 36 h before the experiment but had free access to water. All procedures concerning animal treatment and experimentation were in accordance with the Guiding Principles in the Care and Use of Animals, approved by the Council of the American Physiological Society, with European Communities Council Directives 86/609/EEC and the National regulations, adopted by the local Ethical Commission in the Medical University of Varna.
Chemicals Indomethacin (Indo) was from Fluka Chemie AG. It was prepared as a suspension in a vehicle (2 drops of Tween 80 per 5 ml distilled water). All chemicals used for the biochemical analyses and histopathological examination were of analytical grade and were obtained from Merck (Germany).
AMFJ preparation Fruits from A. melanocarpa Elliot were handpicked, crushed and squeezed. The juice was filtered, pasteurized and stored at 0 1C.
Experimental procedure The experimental animals were randomly divided in eight experimental groups each of six rats according to the experimental setting in Table 1. The rats were orally pretreated by direct stomach intubation with water (10 ml kg1) or AMFJ (5, 10 or 20 ml kg1). One hour later the rats were treated subcutaneously either with indomethacin (30 mg kg1) (Tanaka and Yuda, 1996) or the vehicle for indomethacin in a total volume of 2 ml kg1 body wt.
Tissue preparation and fractionation The animals were anaesthetized with diethylether 4 h after the indomethacin treatment. Blood was collected from the sublingual veins in heparinized tubes. It was centrifuged at 2000g rpm for 10 min and plasma was obtained for the measurement of MDA content and stored at 20 1C until analysis. After the animals decapitation the stomachs were removed immediately, opened along the great curvature, gently washed in physiological salt solution, spread over the pad and observed macroscopically for appearance of mucosal lesions. The length of each lesion was measured. In the case of petechia, five of them were considered as a 1mm lesion. Mean ulcer number and area were calculated. Gastric mucosa was gently separated from the underlying tissue, homogenized in 1:5 w/v 50 mM
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Table 1.
387
Experimental setting of indomethacin-induced gastric mucosal damage in rats
Animal group
Pretreatment
Dose (ml kg1)
Treatment
Dose
1. 2. 3. 4. 5. 6. 7. 8.
Water Water AMFJ AMFJ AMFJ AMFJ AMFJ AMFJ
10 10 5 10 20 5 10 20
Vehicle Indomethacin Vehicle Vehicle Vehicle Indomethacin Indomethacin Indomethacin
2 ml kg1 30 mg kg1 2 ml kg1 2 ml kg1 2 ml kg1 30 mg kg1 30 mg kg1 30 mg kg1
Control Indo AMFJ 5 AMFJ 10 AMFJ 20 AMFJ 5+Indo AMFJ 10+Indo AMFJ 20+Indo
phosphate buffer (pH 7.4) containing 0.1 mM EDTA, at 4000 rpm for 10 min. The homogenate was centrifuged at 800g rpm for 15 min to discard the sediment and the supernatant was frozen until analysis. All manipulations were performed at 4–8 1C. MDA, GSH and GSSG were determined immediately after thawing the samples.
Histopathological study Pieces of the stomachs were fixed in 10% neutralbuffered formaldehyde solution. Fixed tissues were embedded in paraffin, cut into sections and placed on microscope slides. Staining of the slides with hematoxylin-eosin (H & E) and periodic acid-Schiff (PAS) was used for the histomorphological examination which was performed under light microscopy and documented by an Olympus microphotocamera.
Biochemical analysis Membrane lipid peroxidation was monitored by MDA measured by its thiobarbituric acid (TBA) reactivity in rat plasma and gastric mucosal homogenates using the method of Porter et al. (1976). MDA values in nM ml1 plasma and nM g1 gastric mucosal tissue were determined using the extinction coefficient of the MDA-TBA complex at 532 nm ¼ 1.56 105 cm1 M1 solution. GSH and GSSG contents in the gastric mucosal homogenates were analysed according to the method of Hissin and Hilf (1986), using o-phthaldialdehyde as the fluorescent agent. Standard solutions of GSH and GSSG were applied to calculate the glutathione content which was measured in mM g1 gastric mucosal tissue.
Statistical analysis Data were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett,s multiple comparison post test. Two independent groups were compared by Student’s t-test. A value of po0:05 was considered as statistically significant. Data are expressed
as mean7SEM. GraphPad Prism statistical software was used.
Results Macroscopic evaluation of gastric mucosal damage No mucosal lesions were detected in rats which were not treated with indomethacin. Indomethacin induced multiple gastric mucosal lesions in the glandular part of the stomach, most often 1–4 mm2 in size or petechial, bleeding at the moment of observation. The mean ulcer number in the Indo-group was 14.5572.75 (Fig. 1A), and the mean ulcer area was 50.86714.4 mm2 (Fig. 1B). Pretreatment with AMFJ (5, 10 and 20 ml kg1) dosedependently and significantly reduced the ulcer number by 58.8%, 57.0% and 70.0% (Fig. 1A), and the ulcer area by 76.6%, 77.7% and 85.8% (Fig. 1B), respectively.
Histopathological study The microscopic appearance of the gastric mucosa of the control rats was quite normal (Fig. 2A and B). AMFJ induced an increase in mucus production which was most demonstrative in rats that were not treated with indomethacin. In these rats the mucus appeared as a thick continuous layer covering the mucosal surface (Fig. 3A and B). The experimental lesions evoked by indomethacin were manifested by erosive defects comprising about 2/3 of the mucosal layer thickness. The defects were filled with blood coagulates and haematin materials (Fig. 4). In the rats pretreated with AMFJ before the indomethacin administration the gastric erosions were more superficial and in some cases only bleeding and focal desquamation of the epithelium were found (Fig. 5A). The mucus production in the zones of the lesions was reduced (Fig. 5B) while in the zones without lesions it was preserved and even increased.
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A
Biochemical examination
B
In the Indo-group plasma MDA and gastric mucosal MDA increased by 36.3% and by 31.8%, respectively, versus the control group (Fig. 6A and B). The application of AMFJ to the animals (AMFJ 5, AMFJ 10 and AMFJ 20) did not cause statistically significant changes in MDA concentrations in plasma and gastric mucosa (Fig. 6A and B). In the groups pretreated with AMFJ before the indomethacin treatment the plasma and mucosal MDA levels dose-dependently decreased in comparison with those of the Indo-group and at the doses of 10 and 20 ml kg1 they did not differ significantly from the control levels (Fig. 6A and B). Neither indomethacin treatment, nor AMFJ pretreatment caused any significant changes in the gastric mucosal concentrations of GSH and GSSG (Fig. 7A and B).
10
°
75
AMFJ 20 + Indo
AMFJ 10 + Indo
AMFJ 5 + Indo
Indo
0
50
Discussion
°°
°° AMFJ 20 + Indo
0
°°
AMFJ 10 + Indo
25
AMFJ 5 + Indo
Ulcer area [mm2]
° °°
Indo
Ulcer number
20
Fig. 1. Effect of AMFJ pretreatment on the number (A) and area (B) of gastric mucosal lesions in rats with indomethacininduced ulceration. Results are mean7SEM. Statistical significance: 1po0:05; 11po0:01 versus Indo-group.
Indomethacin in the present experiment induced gastric lesions probably due to several mechanisms. It is generally accepted that the ulcerogenic activity of NSAIDs is related to their ability to inhibit endogenous PG synthesis due to the inhibition of COX (Whittle, 1981). PGs form a vital component of gastric mucosal defense. They are produced throughout the gut in high concentrations locally, and are known to have an antisecretory effect on gastric acid production, to stimulate mucus and bicarbonate secretion and to improve the mucosal blood flow (Whittle, 1989). Studies have shown that chronic exposure to indomethacin suppresses the gastric PG synthesis (Shorrock and Rees, 1992). Saad et al. (2002) demonstrated that a
Fig. 2. Microscopic appearance of a control rat stomach following the application of water (10 ml kg1) and vehicle (2 ml kg1). H & E staining (A) and PAS-staining (B); magnification 10 12.5.
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Fig. 3. Microscopic appearance of a rat stomach following the application of AMFJ (20 ml kg1) and vehicle (2 ml kg1). H & Estaining (A) and PAS-staining (B); magnification 10 12.5.
Fig. 4. Microscopic appearance of a rat stomach following the application of water (10 ml kg1) and indomethacin (30 mg kg1). H & E-staining; magnification 10 12.5.
single dose of indomethacin did not inhibit PG synthesis and there was no difference in gastric PGE2 content after a single exposure. They concluded that the complete inhibition of COX leading to decrease in PGE2 content probably consumed a much longer time and did not occur 6 h after a single dose. Having in mind these findings we might suppose that in the present experiment the inhibition of the PG secretion is not the main pathogenic factor for the mucosal damage. There are data that ROS are involved in the development of mucosal damage by NSAIDs (Pihan et al., 1987; Naito et al., 1998). These ROS induce lipid peroxidation which is believed to be an important cause
of destruction and damage of the cellular membranes. In the current study the involvement of extensive lipid peroxidation in indomethacin-induced gastric mucosal damage was evidenced by the accumulation of MDA in gastric mucosa. Similar are the results obtained by Tanaka and Yuda (1996) who studied the peroxidation of lipids and changes in the activities of related enzymes in the gastric mucosa in a rat model of gastric mucosal injury induced by indomethacin (30 mg kg1). In their experiment the amount of thiobarbituric acid reactive substances in the gastric mucosa was significantly increased 4 h after indomethacin administration. Indomethacin activated the polymorphonuclear leukocytes in peripheral blood and enhanced release of oxygen radicals from these cells. In our experiment the administration of indomethacin did not lead to a depletion of GSH, as might be expected having in mind the participation of GSH in the free radical scavenging through its conversion to GSSG by glutathione peroxidase. A possible reason for this effect might be the reduction of glutathione peroxidase activity by the administration of indomethacin (Yoshikawa et al., 1993; Tanaka and Yuda, 1996). The decreased glutathione peroxidase activity would aggravate the injury due to accelerated accumulation of hydrogen peroxide and lipid peroxides in gastric mucosal cells. In the present work we found that AMFJ reduced the morphometrical and histomorphologiocal signs of gastric mucosal damage by indomethacin. The histopathological examination of the stomachs showed that AMFJ caused an increase in mucus production. There are similar data for the anthocyanins from bilberry (cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside and cyanidin-3-O-glucoside) (Magistretti et al., 1988). This effect on the mucus production might be due to an
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Fig. 5. Microscopic appearance of a rat stomach following the application of AMFJ (20 ml kg1) and indomethacin (30 mg kg1). H & E-staining (A) and PAS-staining (B); magnification 10 12.5.
A
5
**
**
MDA [nM ml-1]
4 3 2
50
AMFJ 20 + Indo
AMFJ 10 + Indo
B *
*
40
MDA [nM g-1]
AMFJ 5 + Indo
AMFJ 20
AMFJ 10
AMFJ 5
Contol
0
Indo
1
30 20
AMFJ 20 + Indo
AMFJ 10 + Indo
AMFJ 5 + Indo
AMFJ 20
AMFJ 10
AMFJ 5
Indo
0
Control
10
Fig. 6. Effect of AMFJ on malondialdehyde (MDA) values in rat plasma (A) and gastric mucosa (B) in a model of indomethacin-induced gastric ulceration. Results are mean7SEM. Statistical significance: *po0:05; **po0:01 vs. control.
increase in mucosal PGE2. An anti-ulcerogenic activity of flavonoids associated with an increased mucus secretion and PGE2 release has been established by Beil et al. (1995), Khayyal et al. (2001). Other studies have demonstrated that the increased mucus production by flavonoids is not accompanied by an increase in PGE2 level (Alarcon et al., 1994; Perez-Guerrero et al., 1994). These observations suggest that the gastroprotective effect of AMFJ flavonoids could partly be explained through non-prostanoid-dependent mechanisms. Another possible mechanism of the ulceroprotection by AMFJ is an anti-secretory effect. Flavonoids reportedly (Parmar, 1983) inhibit histidine decarboxylase and reduce gastric mucosal content of histamine, which stimulates the gastric acid secretion. Beil et al. (1995) established an inhibitory action of flavonoids on H+/K+-ATPase activity. Inhibition of H+/K+-ATPase activity is dependent on the ATP concentration. The inhibitory action of flavonoids on H+/K+-ATPase activity is related to their ability to complex ATP. As it has already been pointed out, ROS play a very important role in the pathogenesis of acute experimental gastric lesions. Anthocyanins are exogenous antioxidants acting by scavenging free radicals (Yamasaki et al., 1996; Zheng and Wang, 2003) and by inhibiting lipoprotein oxidation (Tsuda et al., 1996). The results from our experiment showed that AMFJ dose-dependently reduced the lesion area accompanied by decreased MDA levels in gastric mucosa and blood plasma. In previous experiments Martin et al. (1998) and La Casa et al. (2000) established that the gastroprotective effect of quercetin and of rutin was due to the enhancement of the anti-oxidant enzymatic (glutathione peroxidase) activity and anti-lipoperoxidant effect.
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the increased mucus production and the interference with gastric oxidative status could be suggested as possible ways through which AMFJ was protective against indomethacin-induced gastric ulceration.
A
GSH [µM g-1]
0.4 0.3 0.2
References
Indo
AMFJ 5
AMFJ 10
AMFJ 20
AMFJ 5 + Indo
AMFJ 10 + Indo
AMFJ 20 + Indo
AMFJ 5
AMFJ 10
AMFJ 20
AMFJ 5 + Indo
AMFJ 10 + Indo
AMFJ 20 + Indo
0.75
Indo
0.0
Control
0.1
B
0.50
0.25
0.00
Control
GSSG [µM g-1]
391
Fig. 7. Effect of AMFJ on rat gastric mucosal values of (A) reduced glutathione (GSH) and (B) oxidized glutathione (GSSG) in a model of indomethacin-induced gastric ulceration. Results are mean7SEM.
AMFJ did not significantly influence the GSH and GSSG levels. The influence of AMFJ on glutathione concentrations might be a result of two processes having opposite effects on GSH/GSSG. On the one hand, there are data that anthocyanins in experimental situations of increased lipid peroxidation preserve (Chidambara Murthy et al., 2002) or even increase (Kowalczyk et al., 2002) the glutathione peroxidase activity which would increase the conversion of GSH to GSSG. On the other hand, anthocyanins in AMFJ probably help to maintain the gastric GSH level acting as antioxidants instead of GSH in the process of free radical scavenging. In conclusion, we found that AMFJ reduced indomethacin-induced gastric mucosal injury. The gastroprotective effect of AMFJ was accompanied by a significant decrease in lipid peroxidation (evaluated as gastric and plasma MDA concentrations). Several mechanisms might be implicated in this effect. Our study demonstrated that
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