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reoxygenation–induced intestinal injury
Journal of Pediatric Surgery (2011) 46, 685–690
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Protective effects of Ginkgo biloba extract in rats with hypoxia/reoxygenation–induced intestinal injury Özmert M.A. Özdemir a,⁎, Hacer Ergin a , Çigdem Yenisey b , Nilay Şen Türk c a
Departments of Pediatrics, Faculty of Medicine, Pamukkale University, 20100 Denizli, Turkey Department of Biochemistry, Faculty of Medicine, Adnan Menderes University, 09100 Aydın, Turkey c Departments of Pathology, Faculty of Medicine, Pamukkale University, 20100 Denizli, Turkey b
Received 21 June 2010; revised 22 August 2010; accepted 23 September 2010
Key words: EGb 761; H/R-induced intestinal injury; Oxidative stress; Nitric oxide; Lipid peroxidation
Abstract Background: The purpose of this study is to investigate the protective effects of Ginkgo biloba extract (EGb 761) in rat pups with hypoxia/reoxygenation (H/R)–induced bowel injury. Methods: One-day-old Wistar albino rat pups (n = 21) were randomly divided into 3 groups: group 1 (control, untreated and not exposed to H/R, n = 7), group 2 (untreated but exposed to H/R, n = 7), and group 3 (EGb 761 + H/R, n = 7). Ginkgo biloba extract was administered (100 mg/kg per day, subcutaneously) to group 3 for 3 days. On the fourth day, all animals except controls were exposed to H/R and were killed 6 hours after H/R. Histopathologic injury scores (HIS), malondialdehyde, glutathione (GSH), GSH-peroxidase (Px) activities, and nitric oxide (NO) levels were measured on intestinal samples. Results: Although the control group had normal HIS, group 2 had grade 3 HIS. In contrast, group 3 had minimal HIS, and these results were significantly better than those of group 2 (P b .001). Malondialdehyde and NO levels of group 3 were significantly lower than those of group 2 (P b .01). Glutathione and GSH-Px activities of group 1 were higher than those of groups 2 and 3 (P b .05). However, there were no significant differences for GSH and GSH-Px activities between groups 2 and 3. Conclusions: This study showed that hypoxia and NO contributed to the pathogenesis of H/R-induced intestinal injury and that prophylactically administered EGb 761 had a protective effect on bowel injury. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.
Necrotizing enterocolitis (NEC) is the most common surgical gastrointestinal emergency among premature infants in the neonatal intensive care unit [1]. Although NEC is an important cause of neonatal morbidity and mortality, its pathogenesis remains incompletely understood [2]. The
⁎ Corresponding author. Department of Pediatrics, School of Medicine, Pamukkale University, Bayramyeri, 20100 Denizli, Turkey. Tel.: +90 258 4440728, +90 532 3841133 (GSM); fax: +90 258 2410040. E-mail address: [email protected] (Ö.M.A. Özdemir).
common final pathway of mucosal injury is linked to formula feeding, bacterial colonization, hypoxia, and intestinal ischemia [3]. Hypoxia and ischemia appear to play an important role in the disruption of mucosal integrity. Investigators have reported that the metabolites of oxidative stress produced during reperfusion may play a critical role in the pathophysiology of NEC [4-9]. Nitric oxide (NO), through its toxic metabolite, may play a major role in the initiation of intestinal mucosal injury [10]. Glutathione (GSH) metabolism is an essential antioxidative defense system, and GSH-peroxidase (Px) is a dominant
0022-3468/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.09.053
686 enzyme dealing with toxic metabolites of oxidative stress [8,11-13]. Ginkgo biloba extract (EGb 761), which is the leaf extract of G biloba, has many pharmacologic effects. For example, antioxidant properties, reduction of myeloperoxidase activity, inhibition of malondialdehyde (MDA) and NO production, increase in superoxide dismutase and GSH activities, antiinflammatory effects and decreasing NO, tumor necrosis factor α, and prostaglandin E2 levels have been reported [14-18]. This study was designed (1) to investigate the role of NO and lipid peroxidation and, hence, the MDA production, as an indicator of the oxidative stress index and to observe the consequent alterations in GSH (total thiol) and antioxidant enzyme GSH-Px in the pathogenesis of hypoxia/reoxygenation (H/R)–induced bowel injury in rat pups, and (2) to evaluate the potential benefits of prophylactically administered EGb 761.
Ö.M.A. Özdemir et al.
2.3. Histopathologic examination A section of distal ileum from each rat pup was removed, fixed in 10% buffered formalin, placed in paraffin blocks, sectioned at 5 μm, and stained with hematoxylin and eosin for histologic evaluation. Histopathologic changes in intestinal architecture were scored by a pathologist in a blinded fashion and graded as follows: grade 1, normal histology; grade 2 (minimal), hydropic degeneration and/or separation of the surface epithelial cells from lamina propria; grade 3 (mild), epithelial cell necrosis confined to the tips of the villi; grade 4 (moderate), complete villus necrosis; and grade 5 (severe), transmural necrosis [19]. The remaining segments of rat pup distal ileum were stored at –70°C until biochemical analysis and used for determination of MDA, NO, GSH (total thiol), and GSH-Px activities. All measurements for biochemical analysis were done at 20°C.
2.4. Lipid peroxidation analysis
2. Materials and methods 2.1. Animals This study was performed on newborn (1-4 days old) Wistar albino rat pups (approximate weight, 6 g each) whose mothers were maintained under standard conditions. All experiments were approved by the Pamukkale University Animal Research Committee.
2.2. Experimental design Twenty-one 1-day-old Wistar albino rat pups were randomly divided into 3 groups: group 1 (control group, untreated and not exposed to H/R, n = 7), group 2 (untreated but exposed to H/R, n = 7), and group 3 (EGb 761 + H/R, n = 7). All animals were returned to their mothers' cages, kept in a normothermic environment (at 22°-23°C), and breast-fed. Ginkgo biloba extract (G biloba Hevert inject, Dil.D3 2 mL; Hevert-Arzneimittel GmbH & Co, KG Nussbaum, Deutschland) was administered subcutaneously (100 mg/kg body weight, once daily) to group 3 rat pups for 3 days. Fourday-old rat pups in groups 2 and 3 were exposed to H/R procedure described by Okur et al [6]. Hypoxia was accomplished by placing the rat pups in an airtight Plexiglass chamber, which was perfused with 100% CO2 for 5 minutes. At the end of this period, the animals were cyanotic and gasping. After hypoxia was induced, the animals were reoxygenated for 5 minutes with 100% oxygen. This protocol was not performed on the control group. At the sixth hour after H/R, rat pups in all groups were killed on the fourth day of life [8]. Histopathologic injury score (HIS) and MDA, NO, GSH (total thiol), and GSH-Px activities for biochemical examinations were measured on intestinal tissue samples. The weight of the rats was also evaluated throughout the experiment.
The degree of lipid peroxidation based on MDA production in the intestinal tissue homogenates was assessed by the method of Ohkawa et al [20]. The principle of this method is that MDA forms a colored complex in the presence of thiobarbituric acid, which is detectable by measurement of absorbance at 532 nm. Absorbance was measured with Shimadzu UV-160 spectrophotometer. 1,1,3,3-Tetramethoxypropane was used as a standard, and the results were expressed as micromoles per gram of wet tissue.
2.5. NO determination Nitric oxide (nitrite + nitrate) was assayed by a modification of the Cd-reduction method of NavarroGonzalves et al [21]. The nitrite produced was determined by diazotization of sulfanilamide and coupling to naphthlethylene diamine. For the measurement of NO, 400 μL samples were denatured by adding 80 μL 30% ZnSO4 solution, stirring, and then centrifuging at 10,000 × g for 20 minutes at 4°C. First, we activated Cd granules using CuSO4 solution in glycine-NaOH buffer. Then, 100 μL of deproteinized samples and standards was added. This reaction used pretreatment of samples to reduce nitrate to nitrite, which can be accomplished by catalytic reactions using enzyme or Cd. The samples were analyzed spectrophotometrically using a microplate reader and quantified automatically against KNO3 standard curve, and the results were expressed as micromoles per gram of wet tissue.
2.6. GSH (total thiol) measurement Glutathione (total thiol) content in intestinal tissue samples was determined using metaphosphoric acid to precipitate the protein and 5.5′-dithiobis(2-nitrobenzoic
Protective effects of Ginkgo biloba extract
687
acid) for color development. A standard curve was used to calculate GSH (total thiol) content, and results were expressed as milligrams per gram of wet tissue [22].
2.7. GSH-Px activity measurement The assay mixture consisted of 2.0 mL of 75 mmol phosphate buffer (pH 7.0), 50 μL of 60-mmol GSH, 0.1 mL of 30-U/mL GSH reductase, 0.1 mL of 15 mmol disodium salt of EDTA, 0.1 mL of 3 mmol reduced nicotinamide adenine dinucleotide phosphate (NADPH), and appropriate amount of tissue supernatant to a final volume of 3.0 mL. The reaction was started by addition of 0.1 mL of 7.5-mmol H2O2. The rate of change of absorbance was determined by using the conversion of NADPH to NADP+. It was recorded spectrophotometrically at 340 nm for 3 minutes. Glutathione peroxidase activity was expressed as micromoles of NADPH oxidized to NADP+. The results were expressed in milliunits per gram of wet tissue [23].
2.8. Statistical analysis For statistical analysis, the results were subjected to nonparametric tests (Kruskal-Wallis and Mann-Whitney U tests) using Statistical Packages for Social Sciences for Windows (Version 10.0, SPSS Inc, Chicago, IL), as appropriate. All values are expressed as median, minimummaximum. P b .05 was considered significant.
3. Results The experimental model was tolerated very well because no animals died after the procedure. The gross findings of H/ R-induced intestinal injury, such as intestinal discoloration, intestinal hemorrhage, and distension, were observed in group 2 in which the rat pups were stressed experimentally, but no evidence of bowel lesions was found in the control group (group 1). There was no significant difference in weight among the groups (P N .05). Histopathologically, all of the rat pups in the control group had grade 1 HIS, normal histology (Fig. 1A). The group 2 rat pups had epithelial cell necrosis confined to the tips of the villi, grade 3 HIS (Fig. 1B). In contrast, median HIS was 2.0 (range, grade 1-2) in the group 3 pups treated with EGb 761, and this was significantly lower than that in group 2 (P b .001) (Fig. 1C and Table 1). Intestinal tissue MDA levels of group 2 animals were significantly higher than those of group 3 (P b .01). In addition, NO levels of group 2 rat pups were significantly higher than those in groups 1 and 3 (P b .05 and P b .01, respectively). Although GSH (total thiol) and GSH-Px activities of group 1 rats were higher than those of groups 2 and 3 (P b .05), there were no statistically significant differences for GSH (total thiol) and GSH-Px activities
Fig. 1 Microscopic appearances of the distal small bowel from rat pups. A, Grade 1 injury: normal histology. B, Grade 3 injury: epithelial cell necrosis confined to the tips of the villi. C, Grade 2 injury: hydropic degeneration and/or separation of the surface epithelial cells from lamina propria (hematoxylin and eosin ×200).
between groups 2 and 3. The concentrations of MDA and NO and the activities of GSH (total thiol) and GSH-Px in intestinal tissues of the groups are shown in Table 2.
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Ö.M.A. Özdemir et al.
Table 1
HIS of intestinal tissue samples in the groups
Groups
HIS median grade (minimum-maximum)
Group 1 (control, n = 7) Group 2 (H/R, n = 7) Group 3 (EGb 761 + H/R, n = 7)
1.0 (1-1) 3.0 (3-3) a 2.0 (1-2) b
a b
Group 2 N groups 1 and 3 (P b .001). Group 3 N group 1 (P b .01).
4. Discussion Necrotizing enterocolitis involves mostly premature infants in 90% of cases and is much less common in term infants. The incidence increases with decreasing gestational age, and risk of NEC remains high until the postconceptual age of 36 weeks [1,3]. A 1-day-old rat corresponds to human fetus at about 22- to 24-week gestation; and a 3-day-old rat to a human fetus at about 28 to 32 weeks [24,25]. Therefore, we designed this study to investigate the effects of EGb 761 on the biochemical and histopathologic alterations on 1- to 4day-old rat pups exposed to H/R procedures. Although the pathogenesis of NEC remains uncertain, hypoxia and ischemia appear to play an important role. [4-9]. Intestinal ischemia and the effects of hypoxia have been studied in several animal models [5-10]. Okur et al [6] reported that histopathologic lesions in newborn rats with H/ R were similar to those found in early NEC. Thus, we used the method described by Okur et al for H/R in this study. Investigators have suggested that hypoxia is associated with decreased intestinal perfusion and mucosal ischemic changes [5-10]. It has been reported that inflammatory mediators and oxygen-derived free radicals are involved in the pathophysiologic mechanism of H/R-induced intestinal injury [6,9]. Reperfusion of the tissue supplies molecular oxygen, which results in a burst of superoxide radical production that overwhelms the normal balance of free radical production [6]. The classic histologic finding of NEC is coagulation necrosis, present in more than 90% of specimens [26]. Previous studies in mice and rats with an H/R model show Table 2 groups
that the histopathologic lesions ranged from normal histology to transmural necrosis and that the prominent microscopic lesions were located in the distal small intestine [6,8,9,19]. We used the method described by Ozkan et al [19] to evaluate histopathologic results, which demonstrated that ischemic lesions occurred mainly in the ileocolic region after the H/R. Although the control rat pups showed no evidence of any mucosal lesions, there was epithelial cell necrosis of the villi in intestinal samples of H/R-induced groups. In contrast, there was minimal intestinal injury in group 3 rat pups receiving EGb 761. This study confirmed findings in previous studies that hypoxia is an important risk factor for intestinal injury. Furthermore, EGb 761 has a protective effect on intestinal injury on histopathologic studies. Glutathione metabolism is one of the most essential antioxidative defense mechanisms present in both tissues and blood, and its depletion accelerates intestinal injury because of oxidative stress. Glutathione peroxidase is also one of the dominant enzymes dealing with toxic metabolites of oxidative stress. Enzyme distribution may vary in tissues [8,11-13]. In the present study, we showed that GSH (total thiol) and GSH-Px activities significantly decreased in group 2 rat pups exposed to H/R, when compared with the control group. Controversial results concerning these parameters are noted in the literature [8,14,15]. The study of Louajri et al [14] performed on hypoxic rats treated with EGb 761 (25 and 50 mg/kg body weight) showed that this had no effect on antioxidant enzyme activities (Cu-Zn SOD-GSH-Px), whereas the study of Shenoy et al [15] performed on rats with hepatic injury induced by carbon tetrachloride showed that G biloba (50 mg/kg body weight) significantly increased GSH activity. In contrast, we used EGb 761 (100 mg/kg body weight) in this study and demonstrated that EGb 761 had no effect on GSH metabolism. Lipid peroxidation measurement is a more practical and safer method to evaluate the factors causing cellular injury and activation of the common pathway. Tissue MDA content, the last product of lipid breakdown caused by oxidative stress, is a good indicator of free radical–induced lipid peroxidation [6,8,9]. In experimental H/R studies,
Biochemical evaluation of MDA, NO, GSH (total thiol), and GSH-Px levels in intestinal tissue samples of the experimental
Groups
MDA (μmol/g wet tissue) median, (minimum-maximum)
NO (μmol/g wet tissue), median (minimum-maximum)
GSH (total thiol) (mg/g wet tissue), median (minimum-maximum)
GSH-Px (mU/g wet tissue), median (minimum-maximum)
Group 1 (control, n = 7) Group 2 (H/R, n = 7) Group 3 (EGb 761 + H/R, n = 7)
0.23 (0.11-0.63) 0.33 d (0.24-0.72) 0.23 (0.18-0.25)
0.34 a (0.25-1.17) 0.55 e (0.41-0.65) 0.10 (0.06-0.28)
4.1 b (2.5-7.6) 2.8 (2.5-3.4) 2.7 (2.6-2.9)
2165.5 c (1347.0-2732.5) 1385.5 (749.2-1893.5) 1105.3 (676.9-1690.7)
a b c d e
Group 1 N group 3 (P b .01). Group 1 N groups 2 and 3 (P b .05). Group 1 N groups 2 and 3 (P b .05 and P b .01, respectively). Group 2 N group 3 (P b .01). Group 2 N groups 1 and 3 (P b .05 and P b .01, respectively).
Protective effects of Ginkgo biloba extract MDA levels in intestinal tissue were significantly higher than those in the control and treated groups [6,8,9,19]. We determined the TBARS for evaluation of intestinal tissue MDA levels. Although the highest MDA levels were found in group 2, no statistical significance was observed between groups 1 and 2. In contrast, MDA levels of group 3 were significantly decreased when compared with group 2. Recently, several studies showed that EGb 761 decreased MDA and NO levels [17,27,28]. In the study of Liu et al [17] in rats with lung injury induced by intestinal ischemia/ reperfusion, when EGb 761 (100 mg/kg per day) was given before intestinal ischemia/reperfusion injury, it significantly decreased MDA and NO production. Nitric oxide is the product of the conversion of arginine to citruline by NO synthase (NOS) [10]. Nitric oxide is a small, highly reactive gas that acts as a signaling molecule and forms reactive intermediates that alter cell function and is a critical mediator of the inflammatory response in the pathogenesis of NEC. In NEC, NO is released by the inflammatory cells that are recruited to the sites of mucosal disruption and the enterocytes in response to cytokine induction of the enzyme inducible NOS. High levels of exogenous NO exert cytopathic effects on the intestine that worsens the degree of mucosal injury [10,29]. Potoka et al [10] suggested that peroxynitrite could induce enterocyte apoptosis through several mechanisms, including inhibition of mitochondrial function and adenosine triphosphate depletion, activation of caspases via cytochrome c and apoptosis-activating factor-1 release from mitochondria, and activation of poly(ADP-ribose) synthetase, in addition to inhibition of enterocyte proliferation and differentiation within the intestinal crypts by interfering with tyrosine kinase signaling cascades. Nitric oxide, through its toxic metabolite peroxynitrite, plays a major role in the initiation of intestinal mucosal injury in clinical conditions associated with sustained inducible NOS up-regulation in the gut [10,29]. Liu et al [17] showed that EGb 761 significantly reduced the generation of NO accompanied by the downregulation of inducible NOS expression. In the present study, NO levels of group 2 rat pups were significantly higher than those of groups 1 and 3. In addition, group 2 animals had grade 3 intestinal injury score. Moreover, NO levels of group 3 pups were significantly lower than group 1. Thus, we also showed that NO was a critical mediator of the inflammatory response for development of intestinal injury and that EGb 761 significantly decreased the intestinal tissue levels of NO. We demonstrated that EGb 761 reduced HIS and intestinal tissue levels of MDA and NO but had no effect on GSH metabolism (GSH and GSH-Px) in this experimental study. We conclude that the protective effect of EGb 761 observed in this study is primarily because of the decreasing MDA and NO production. These observations suggest that oxidative stress contributes to the pathogenesis of NEC. Nitric oxide, directly or through its toxic metabolite peroxynitrite, likely plays a critical role in the initiation of intestinal mucosal injury and
689 the inflammatory response in the development of NEC. This study is the first to show that prophylactically administered EGb 761 has a protective effect in this model of hypoxiainduced bowel injury.
Acknowledgments The authors thank Barbaros Sahin and Pamukkale University Animal Research Laboratory for their help with experimental techniques.
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Ö.M.A. Özdemir et al. oxidative stress and nitric oxide. World J Gastroenterol 2007;13: 299-305. Ilieva I, Ohgami K, Shiratori K, et al. The effects of ginkgo biloba extract on lipopolysaccharide-induced inflammation in vitro and in vivo. Exp Eye Res 2004;79:181-7. Ozkan KU, Ozokutan BH, Inanç F, et al. Does maternal nicotine exposure during gestation increase the injury severity of small intestine in the newborn rats subjected to experimental necrotizing enterocolitis. J Ped Surg 2005;40:484-8. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxidase in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. Navarro-Gonzalves JA, Garcia-Benayas C, Arenas J. Semiautomated measurement of nitrate in biological fluids. Clin Chem 1998;44: 679-81. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;51:882-8. Kakkar R, Mantha SV, Radhi J, et al. Increased oxidative stress in rat liver and pancreas during progression of streptozotocin-induced diabetes. Clin Sci (Lond) 1998;94:623-32.
[24] Hagberg H, Bona E, Gilland E, et al. Hypoxia-ischemia model in the 7-day-old rat: possibilities and shortcomings. Acta Paediatr Suppl 1997;422:85-8. [25] Yossuck P, Kraszpulski M, Salm AK. Perinatal corticosteroid effect on amygdala and hippocampus volume during brain development in the rat model. Early Hum Dev 2006;82:267-72. [26] Ballance WA, Dahms BB, Shenker N, et al. Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 1990;117: S6-S13. [27] Calapai G, Crupi A, Frenzuoli F, et al. Neuroprotective effects of ginkgo biloba extract in brain ischemia are mediated by inhibition of nitric oxide synthesis. Life Sci 2000;67:2673-83. [28] Gulec M, Iraz M, Yilmaz HR, et al. The effects of ginkgo biloba extract on tissue adenosine deaminase, xanthine oxidase, myeloperoxidase, malondialdehyde, and nitric oxide in cisplatin-induced nephrotoxicity. Toxicol Ind Health 2006;22:125-30. [29] Upperman JS, Potoka D, Grishin A, et al. Mechanisms of nitric oxide– mediated intestinal barrier failure in necrotizing enterocolitis. Semin Pediatr Surg 2005;14:159-66.
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Protective effects of Ginkgo biloba extract in rats with hypoxia/reoxygenation–induced intestinal injury Özmert M.A. Özdemir a,⁎, Hacer Ergin a , Çigdem Yenisey b , Nilay Şen Türk c a
Departments of Pediatrics, Faculty of Medicine, Pamukkale University, 20100 Denizli, Turkey Department of Biochemistry, Faculty of Medicine, Adnan Menderes University, 09100 Aydın, Turkey c Departments of Pathology, Faculty of Medicine, Pamukkale University, 20100 Denizli, Turkey b
Received 21 June 2010; revised 22 August 2010; accepted 23 September 2010
Key words: EGb 761; H/R-induced intestinal injury; Oxidative stress; Nitric oxide; Lipid peroxidation
Abstract Background: The purpose of this study is to investigate the protective effects of Ginkgo biloba extract (EGb 761) in rat pups with hypoxia/reoxygenation (H/R)–induced bowel injury. Methods: One-day-old Wistar albino rat pups (n = 21) were randomly divided into 3 groups: group 1 (control, untreated and not exposed to H/R, n = 7), group 2 (untreated but exposed to H/R, n = 7), and group 3 (EGb 761 + H/R, n = 7). Ginkgo biloba extract was administered (100 mg/kg per day, subcutaneously) to group 3 for 3 days. On the fourth day, all animals except controls were exposed to H/R and were killed 6 hours after H/R. Histopathologic injury scores (HIS), malondialdehyde, glutathione (GSH), GSH-peroxidase (Px) activities, and nitric oxide (NO) levels were measured on intestinal samples. Results: Although the control group had normal HIS, group 2 had grade 3 HIS. In contrast, group 3 had minimal HIS, and these results were significantly better than those of group 2 (P b .001). Malondialdehyde and NO levels of group 3 were significantly lower than those of group 2 (P b .01). Glutathione and GSH-Px activities of group 1 were higher than those of groups 2 and 3 (P b .05). However, there were no significant differences for GSH and GSH-Px activities between groups 2 and 3. Conclusions: This study showed that hypoxia and NO contributed to the pathogenesis of H/R-induced intestinal injury and that prophylactically administered EGb 761 had a protective effect on bowel injury. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.
Necrotizing enterocolitis (NEC) is the most common surgical gastrointestinal emergency among premature infants in the neonatal intensive care unit [1]. Although NEC is an important cause of neonatal morbidity and mortality, its pathogenesis remains incompletely understood [2]. The
⁎ Corresponding author. Department of Pediatrics, School of Medicine, Pamukkale University, Bayramyeri, 20100 Denizli, Turkey. Tel.: +90 258 4440728, +90 532 3841133 (GSM); fax: +90 258 2410040. E-mail address: [email protected] (Ö.M.A. Özdemir).
common final pathway of mucosal injury is linked to formula feeding, bacterial colonization, hypoxia, and intestinal ischemia [3]. Hypoxia and ischemia appear to play an important role in the disruption of mucosal integrity. Investigators have reported that the metabolites of oxidative stress produced during reperfusion may play a critical role in the pathophysiology of NEC [4-9]. Nitric oxide (NO), through its toxic metabolite, may play a major role in the initiation of intestinal mucosal injury [10]. Glutathione (GSH) metabolism is an essential antioxidative defense system, and GSH-peroxidase (Px) is a dominant
0022-3468/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.09.053
686 enzyme dealing with toxic metabolites of oxidative stress [8,11-13]. Ginkgo biloba extract (EGb 761), which is the leaf extract of G biloba, has many pharmacologic effects. For example, antioxidant properties, reduction of myeloperoxidase activity, inhibition of malondialdehyde (MDA) and NO production, increase in superoxide dismutase and GSH activities, antiinflammatory effects and decreasing NO, tumor necrosis factor α, and prostaglandin E2 levels have been reported [14-18]. This study was designed (1) to investigate the role of NO and lipid peroxidation and, hence, the MDA production, as an indicator of the oxidative stress index and to observe the consequent alterations in GSH (total thiol) and antioxidant enzyme GSH-Px in the pathogenesis of hypoxia/reoxygenation (H/R)–induced bowel injury in rat pups, and (2) to evaluate the potential benefits of prophylactically administered EGb 761.
Ö.M.A. Özdemir et al.
2.3. Histopathologic examination A section of distal ileum from each rat pup was removed, fixed in 10% buffered formalin, placed in paraffin blocks, sectioned at 5 μm, and stained with hematoxylin and eosin for histologic evaluation. Histopathologic changes in intestinal architecture were scored by a pathologist in a blinded fashion and graded as follows: grade 1, normal histology; grade 2 (minimal), hydropic degeneration and/or separation of the surface epithelial cells from lamina propria; grade 3 (mild), epithelial cell necrosis confined to the tips of the villi; grade 4 (moderate), complete villus necrosis; and grade 5 (severe), transmural necrosis [19]. The remaining segments of rat pup distal ileum were stored at –70°C until biochemical analysis and used for determination of MDA, NO, GSH (total thiol), and GSH-Px activities. All measurements for biochemical analysis were done at 20°C.
2.4. Lipid peroxidation analysis
2. Materials and methods 2.1. Animals This study was performed on newborn (1-4 days old) Wistar albino rat pups (approximate weight, 6 g each) whose mothers were maintained under standard conditions. All experiments were approved by the Pamukkale University Animal Research Committee.
2.2. Experimental design Twenty-one 1-day-old Wistar albino rat pups were randomly divided into 3 groups: group 1 (control group, untreated and not exposed to H/R, n = 7), group 2 (untreated but exposed to H/R, n = 7), and group 3 (EGb 761 + H/R, n = 7). All animals were returned to their mothers' cages, kept in a normothermic environment (at 22°-23°C), and breast-fed. Ginkgo biloba extract (G biloba Hevert inject, Dil.D3 2 mL; Hevert-Arzneimittel GmbH & Co, KG Nussbaum, Deutschland) was administered subcutaneously (100 mg/kg body weight, once daily) to group 3 rat pups for 3 days. Fourday-old rat pups in groups 2 and 3 were exposed to H/R procedure described by Okur et al [6]. Hypoxia was accomplished by placing the rat pups in an airtight Plexiglass chamber, which was perfused with 100% CO2 for 5 minutes. At the end of this period, the animals were cyanotic and gasping. After hypoxia was induced, the animals were reoxygenated for 5 minutes with 100% oxygen. This protocol was not performed on the control group. At the sixth hour after H/R, rat pups in all groups were killed on the fourth day of life [8]. Histopathologic injury score (HIS) and MDA, NO, GSH (total thiol), and GSH-Px activities for biochemical examinations were measured on intestinal tissue samples. The weight of the rats was also evaluated throughout the experiment.
The degree of lipid peroxidation based on MDA production in the intestinal tissue homogenates was assessed by the method of Ohkawa et al [20]. The principle of this method is that MDA forms a colored complex in the presence of thiobarbituric acid, which is detectable by measurement of absorbance at 532 nm. Absorbance was measured with Shimadzu UV-160 spectrophotometer. 1,1,3,3-Tetramethoxypropane was used as a standard, and the results were expressed as micromoles per gram of wet tissue.
2.5. NO determination Nitric oxide (nitrite + nitrate) was assayed by a modification of the Cd-reduction method of NavarroGonzalves et al [21]. The nitrite produced was determined by diazotization of sulfanilamide and coupling to naphthlethylene diamine. For the measurement of NO, 400 μL samples were denatured by adding 80 μL 30% ZnSO4 solution, stirring, and then centrifuging at 10,000 × g for 20 minutes at 4°C. First, we activated Cd granules using CuSO4 solution in glycine-NaOH buffer. Then, 100 μL of deproteinized samples and standards was added. This reaction used pretreatment of samples to reduce nitrate to nitrite, which can be accomplished by catalytic reactions using enzyme or Cd. The samples were analyzed spectrophotometrically using a microplate reader and quantified automatically against KNO3 standard curve, and the results were expressed as micromoles per gram of wet tissue.
2.6. GSH (total thiol) measurement Glutathione (total thiol) content in intestinal tissue samples was determined using metaphosphoric acid to precipitate the protein and 5.5′-dithiobis(2-nitrobenzoic
Protective effects of Ginkgo biloba extract
687
acid) for color development. A standard curve was used to calculate GSH (total thiol) content, and results were expressed as milligrams per gram of wet tissue [22].
2.7. GSH-Px activity measurement The assay mixture consisted of 2.0 mL of 75 mmol phosphate buffer (pH 7.0), 50 μL of 60-mmol GSH, 0.1 mL of 30-U/mL GSH reductase, 0.1 mL of 15 mmol disodium salt of EDTA, 0.1 mL of 3 mmol reduced nicotinamide adenine dinucleotide phosphate (NADPH), and appropriate amount of tissue supernatant to a final volume of 3.0 mL. The reaction was started by addition of 0.1 mL of 7.5-mmol H2O2. The rate of change of absorbance was determined by using the conversion of NADPH to NADP+. It was recorded spectrophotometrically at 340 nm for 3 minutes. Glutathione peroxidase activity was expressed as micromoles of NADPH oxidized to NADP+. The results were expressed in milliunits per gram of wet tissue [23].
2.8. Statistical analysis For statistical analysis, the results were subjected to nonparametric tests (Kruskal-Wallis and Mann-Whitney U tests) using Statistical Packages for Social Sciences for Windows (Version 10.0, SPSS Inc, Chicago, IL), as appropriate. All values are expressed as median, minimummaximum. P b .05 was considered significant.
3. Results The experimental model was tolerated very well because no animals died after the procedure. The gross findings of H/ R-induced intestinal injury, such as intestinal discoloration, intestinal hemorrhage, and distension, were observed in group 2 in which the rat pups were stressed experimentally, but no evidence of bowel lesions was found in the control group (group 1). There was no significant difference in weight among the groups (P N .05). Histopathologically, all of the rat pups in the control group had grade 1 HIS, normal histology (Fig. 1A). The group 2 rat pups had epithelial cell necrosis confined to the tips of the villi, grade 3 HIS (Fig. 1B). In contrast, median HIS was 2.0 (range, grade 1-2) in the group 3 pups treated with EGb 761, and this was significantly lower than that in group 2 (P b .001) (Fig. 1C and Table 1). Intestinal tissue MDA levels of group 2 animals were significantly higher than those of group 3 (P b .01). In addition, NO levels of group 2 rat pups were significantly higher than those in groups 1 and 3 (P b .05 and P b .01, respectively). Although GSH (total thiol) and GSH-Px activities of group 1 rats were higher than those of groups 2 and 3 (P b .05), there were no statistically significant differences for GSH (total thiol) and GSH-Px activities
Fig. 1 Microscopic appearances of the distal small bowel from rat pups. A, Grade 1 injury: normal histology. B, Grade 3 injury: epithelial cell necrosis confined to the tips of the villi. C, Grade 2 injury: hydropic degeneration and/or separation of the surface epithelial cells from lamina propria (hematoxylin and eosin ×200).
between groups 2 and 3. The concentrations of MDA and NO and the activities of GSH (total thiol) and GSH-Px in intestinal tissues of the groups are shown in Table 2.
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Ö.M.A. Özdemir et al.
Table 1
HIS of intestinal tissue samples in the groups
Groups
HIS median grade (minimum-maximum)
Group 1 (control, n = 7) Group 2 (H/R, n = 7) Group 3 (EGb 761 + H/R, n = 7)
1.0 (1-1) 3.0 (3-3) a 2.0 (1-2) b
a b
Group 2 N groups 1 and 3 (P b .001). Group 3 N group 1 (P b .01).
4. Discussion Necrotizing enterocolitis involves mostly premature infants in 90% of cases and is much less common in term infants. The incidence increases with decreasing gestational age, and risk of NEC remains high until the postconceptual age of 36 weeks [1,3]. A 1-day-old rat corresponds to human fetus at about 22- to 24-week gestation; and a 3-day-old rat to a human fetus at about 28 to 32 weeks [24,25]. Therefore, we designed this study to investigate the effects of EGb 761 on the biochemical and histopathologic alterations on 1- to 4day-old rat pups exposed to H/R procedures. Although the pathogenesis of NEC remains uncertain, hypoxia and ischemia appear to play an important role. [4-9]. Intestinal ischemia and the effects of hypoxia have been studied in several animal models [5-10]. Okur et al [6] reported that histopathologic lesions in newborn rats with H/ R were similar to those found in early NEC. Thus, we used the method described by Okur et al for H/R in this study. Investigators have suggested that hypoxia is associated with decreased intestinal perfusion and mucosal ischemic changes [5-10]. It has been reported that inflammatory mediators and oxygen-derived free radicals are involved in the pathophysiologic mechanism of H/R-induced intestinal injury [6,9]. Reperfusion of the tissue supplies molecular oxygen, which results in a burst of superoxide radical production that overwhelms the normal balance of free radical production [6]. The classic histologic finding of NEC is coagulation necrosis, present in more than 90% of specimens [26]. Previous studies in mice and rats with an H/R model show Table 2 groups
that the histopathologic lesions ranged from normal histology to transmural necrosis and that the prominent microscopic lesions were located in the distal small intestine [6,8,9,19]. We used the method described by Ozkan et al [19] to evaluate histopathologic results, which demonstrated that ischemic lesions occurred mainly in the ileocolic region after the H/R. Although the control rat pups showed no evidence of any mucosal lesions, there was epithelial cell necrosis of the villi in intestinal samples of H/R-induced groups. In contrast, there was minimal intestinal injury in group 3 rat pups receiving EGb 761. This study confirmed findings in previous studies that hypoxia is an important risk factor for intestinal injury. Furthermore, EGb 761 has a protective effect on intestinal injury on histopathologic studies. Glutathione metabolism is one of the most essential antioxidative defense mechanisms present in both tissues and blood, and its depletion accelerates intestinal injury because of oxidative stress. Glutathione peroxidase is also one of the dominant enzymes dealing with toxic metabolites of oxidative stress. Enzyme distribution may vary in tissues [8,11-13]. In the present study, we showed that GSH (total thiol) and GSH-Px activities significantly decreased in group 2 rat pups exposed to H/R, when compared with the control group. Controversial results concerning these parameters are noted in the literature [8,14,15]. The study of Louajri et al [14] performed on hypoxic rats treated with EGb 761 (25 and 50 mg/kg body weight) showed that this had no effect on antioxidant enzyme activities (Cu-Zn SOD-GSH-Px), whereas the study of Shenoy et al [15] performed on rats with hepatic injury induced by carbon tetrachloride showed that G biloba (50 mg/kg body weight) significantly increased GSH activity. In contrast, we used EGb 761 (100 mg/kg body weight) in this study and demonstrated that EGb 761 had no effect on GSH metabolism. Lipid peroxidation measurement is a more practical and safer method to evaluate the factors causing cellular injury and activation of the common pathway. Tissue MDA content, the last product of lipid breakdown caused by oxidative stress, is a good indicator of free radical–induced lipid peroxidation [6,8,9]. In experimental H/R studies,
Biochemical evaluation of MDA, NO, GSH (total thiol), and GSH-Px levels in intestinal tissue samples of the experimental
Groups
MDA (μmol/g wet tissue) median, (minimum-maximum)
NO (μmol/g wet tissue), median (minimum-maximum)
GSH (total thiol) (mg/g wet tissue), median (minimum-maximum)
GSH-Px (mU/g wet tissue), median (minimum-maximum)
Group 1 (control, n = 7) Group 2 (H/R, n = 7) Group 3 (EGb 761 + H/R, n = 7)
0.23 (0.11-0.63) 0.33 d (0.24-0.72) 0.23 (0.18-0.25)
0.34 a (0.25-1.17) 0.55 e (0.41-0.65) 0.10 (0.06-0.28)
4.1 b (2.5-7.6) 2.8 (2.5-3.4) 2.7 (2.6-2.9)
2165.5 c (1347.0-2732.5) 1385.5 (749.2-1893.5) 1105.3 (676.9-1690.7)
a b c d e
Group 1 N group 3 (P b .01). Group 1 N groups 2 and 3 (P b .05). Group 1 N groups 2 and 3 (P b .05 and P b .01, respectively). Group 2 N group 3 (P b .01). Group 2 N groups 1 and 3 (P b .05 and P b .01, respectively).
Protective effects of Ginkgo biloba extract MDA levels in intestinal tissue were significantly higher than those in the control and treated groups [6,8,9,19]. We determined the TBARS for evaluation of intestinal tissue MDA levels. Although the highest MDA levels were found in group 2, no statistical significance was observed between groups 1 and 2. In contrast, MDA levels of group 3 were significantly decreased when compared with group 2. Recently, several studies showed that EGb 761 decreased MDA and NO levels [17,27,28]. In the study of Liu et al [17] in rats with lung injury induced by intestinal ischemia/ reperfusion, when EGb 761 (100 mg/kg per day) was given before intestinal ischemia/reperfusion injury, it significantly decreased MDA and NO production. Nitric oxide is the product of the conversion of arginine to citruline by NO synthase (NOS) [10]. Nitric oxide is a small, highly reactive gas that acts as a signaling molecule and forms reactive intermediates that alter cell function and is a critical mediator of the inflammatory response in the pathogenesis of NEC. In NEC, NO is released by the inflammatory cells that are recruited to the sites of mucosal disruption and the enterocytes in response to cytokine induction of the enzyme inducible NOS. High levels of exogenous NO exert cytopathic effects on the intestine that worsens the degree of mucosal injury [10,29]. Potoka et al [10] suggested that peroxynitrite could induce enterocyte apoptosis through several mechanisms, including inhibition of mitochondrial function and adenosine triphosphate depletion, activation of caspases via cytochrome c and apoptosis-activating factor-1 release from mitochondria, and activation of poly(ADP-ribose) synthetase, in addition to inhibition of enterocyte proliferation and differentiation within the intestinal crypts by interfering with tyrosine kinase signaling cascades. Nitric oxide, through its toxic metabolite peroxynitrite, plays a major role in the initiation of intestinal mucosal injury in clinical conditions associated with sustained inducible NOS up-regulation in the gut [10,29]. Liu et al [17] showed that EGb 761 significantly reduced the generation of NO accompanied by the downregulation of inducible NOS expression. In the present study, NO levels of group 2 rat pups were significantly higher than those of groups 1 and 3. In addition, group 2 animals had grade 3 intestinal injury score. Moreover, NO levels of group 3 pups were significantly lower than group 1. Thus, we also showed that NO was a critical mediator of the inflammatory response for development of intestinal injury and that EGb 761 significantly decreased the intestinal tissue levels of NO. We demonstrated that EGb 761 reduced HIS and intestinal tissue levels of MDA and NO but had no effect on GSH metabolism (GSH and GSH-Px) in this experimental study. We conclude that the protective effect of EGb 761 observed in this study is primarily because of the decreasing MDA and NO production. These observations suggest that oxidative stress contributes to the pathogenesis of NEC. Nitric oxide, directly or through its toxic metabolite peroxynitrite, likely plays a critical role in the initiation of intestinal mucosal injury and
689 the inflammatory response in the development of NEC. This study is the first to show that prophylactically administered EGb 761 has a protective effect in this model of hypoxiainduced bowel injury.
Acknowledgments The authors thank Barbaros Sahin and Pamukkale University Animal Research Laboratory for their help with experimental techniques.
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