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Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury
Life Sciences 73 (2003) 81 – 91 www.elsevier.com/locate/lifescie
Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury ¨ zer Sßehirli a, Ayhan Kacßmaz b Go¨ksel Sßener a,*, Handan Sßatırog˘lu a, A. O a
Marmara University, School of Pharmacy, Department of Pharmacology, Istanbul, Turkey b Haydarpasßa Numune Hospital, Department of Surgery, Istanbul, Turkey Received 29 October 2002; accepted 6 December 2002
Abstract Oxygen free radicals have been implicated in mediating various pathological processes including burninduced organ damage. This study was designed to determine the possible protective effect of aqueous garlic extract against oxidative organ damage distant from the original burn wound. Under ether anaesthesia, rats were subjected to severe skin scald injury covering 30% of total body surface area. Rats were decapitated either 2 h or 24 h after burn injury. Aqueous garlic extract (1 ml/kg) was administered i.p. immediately after burn injury. In the 24-h burn group injection was repeated once more (at 12 hour) following the burn injury. Liver, intestine and lung tissues were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and protein oxidation (PO). Burn injury caused a significant decrease in GSH level, and significant increases in MDA and PO levels, and MPO activity at post-burn 2 and 24 hours. Since garlic extract reversed these oxidant responses it seems likely that garlic extract protects tissues against oxidative damage. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Burn; Garlic; Lipid peroxidation; Glutathione; Myeloperoxidase; Protein oxidation
Introduction . Oxygen free radicals such as hydrogen peroxide (H2O2), superoxide anion (O2 ) and hydroxyl radical . (OH ) have been implicated in mediating various pathological process such as ischemia, inflammatory ¨ . Eczacılık Faku¨ltesi, Farmakoloji Ab. D., Tibbiye Cad. I˙stanbul 34668, Turkey. Tel.: +90-216* Corresponding author. M.U 418-65-92; fax: +90-216-330-10-93. E-mail address: [email protected] (G. Sßener). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00236-4
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diseases, diabetes, and atherosclerosis [1,2]. Thermal trauma, one of the most common problems faced in the emergency room, may cause damage to multiple organs distant from the original burn wound and may lead to multiorgan failure. Following burn injury, all tissues are subjected to ischemia and consequently, during burn shock resuscitation, reperfusion occurs. The role of free oxygen radicals, neutrophils and endothelial cells in ischemic insult or organ failure is well documented [3,4]. Several studies have demonstrated that burn injury is associated with lipid peroxidation, which is an autocatalytic mechanism leading to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic, reactive metabolites and cell death [5,6]. Youn et al. demonstrated that burn injury results in increased lipid peroxidation in the plasma, lung, kidney and liver [7]. Since the major source of the oxidants could be neutrophils sequestered in systemic organs as a systemic inflammatory reaction to a local burn insult [8], it can be expected that agents which inhibit the activation and adherence of neutrophils might also exert protective effects against thermal injury [9]. It has been shown that antioxidants, when given postburn, restored antioxidant defense mechanisms and attenuated tissue injury [10–12]. We recently shown that antioxidant melatonin can protect against burninduced oxidative organ damage [13,14]. Garlic has been used as a folk remedy for a variety of ailments since ancient times. In the past few years, it has been found in certain models that garlic preperations including aged garlic (AGE) prevented tumour promotion [15], cardiovascular diseases [16], liver damage [17], and aging [18] which are considered to be associated with oxygen radical and lipid peroxidation. The intrinsic antioxidant activity of garlic [19], garlic extracts [20,21] and some garlic constituents [22,23], have been widely documented in vivo [24,25] and in vitro [21,23]. Garlic extracts increases SOD [26], GPx [27], and CAT activities [27] in vascular cells in culture, and S- allyl-cysteine sulfoxide (alliin), a garlic compound prevents the decrease in hepatic SOD and CAT activities observed in diabetic rats [24]. Therefore, in this work we studied the effect of aqueous garlic extract against oxidative organ damage induced by thermal trauma.
Materials and methods Animals The Marmara University School of Medicine Animal Care and Use Committee approved all experimental protocols. Wistar albino rats of both sexes weighing 200 to 250 g were fasted for 12 h, but were allowed free access to water, before burn injury. Rats were kept in a room at a constant temperature 22 F 2 jC with 12-h light and dark cycles, in individual wire-bottomed cages and fed standard rat chow. Thermal Injury Under brief ether anesthesia, dorsum of the rats was shaved and exposed to 90 jC water bath for 10 s which resulted in partial-thickness second-degree skin burn involving 30% of the total body surface area [13,14]. The animals were then resuscitated with physiological saline solution (10 mg/kg, s.c.). Rats were decapitated at 2 or 24 h after burn injury in both vehicle and garlic-treated groups: Burn-2 h, Burn24 h and Burn + Gar-2 h, Burn + Gar-24 h respectively. Each group consists of 8 rats. In order to rule out
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the effects of anesthesia, the same protocol was applied in the sham group, except that the dorsums were dipped in a 25 jC water bath for 10 s. Garlic-treatment Peeled garlic (30 g) was crushed with distilled water in a mortar. The crushed material was carefully decanted by pressing and 60 ml of aqueous extract was extracted. 1 ml of aqueous extract contained material from 500 mg of garlic [28]. In the 2-h burn group, either garlic extract (1 ml/kg) or saline (0.1 ml/ kg) was administered i.p. immediately after the burn. In the 24-h burn groups, the injections of garlic extract or vehicle were repeated one more occasion (at 12 h) following burn injury. Biochemical analysis After decapitation, the liver, small intestine and lung tissues were immediately taken and stored at 70 jC. Afterwards, malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and protein carbonyl concentration, a specific marker of oxidative damage of proteins were measured in these samples. Tissue-associated myeloperoxidase (MPO) activity, as an indirect evidence of neutrophil infiltration, was measured in all tissue samples. Malondialdehyde and glutathione assays Tissue samples were homogenized with ice-cold 150 mM KCl for determination of malondialdehyde (MDA) and glutathione levels. The MDA levels were assayed for products of lipid peroxidation [29]. Results were expressed as Amol MDA/g tissue. Glutathione was determined by the spectrophotometric method which was based on the use of Ellman’s reagent [30]. Results were expressed Amol GSH/g tissue. Protein oxidation The protein content of tissue samples was determined by the Lowry assay [31]. Oxidized protein was quantified using the interaction between dinitrophenylhydrazine (DNP) and the carbonyls to yield a chromophore that absorbs strongly at 360 nm. All samples were diluted to 2–4 mg/ml of protein with wash buffer and treated with 1% streptomycin. The carbonyl content was calculated assuming a molar extinction coefficient of 22,000 [32]. Myeloperoxidase activity MPO activity was measured in tissues in a procedure similar to that documented by Hillegas et al. [32]. Liver, lung or intestine samples were homogenized in 50 mM potassium phosphate buffer (PB, pH 6.0), and centrifuged at 41,400 g (10 min); pellets were suspended in 50 mM PB containing 0.5% hexadecyltrimethylammonium bromide (HETAB). After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41.400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of the MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue.
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Statistics Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego; CA; USA). All data were expressed as means F SEM. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Values of p < 0.05 were regarded as significant.
Fig. 1. Glutathione (GSH) levels in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. **p < 0.01,*** p < 0.001 : Compared with Sham group. + p < 0.05, + + p < 0.01, + + + p < 0.001: Garlic-treated groups compared with respective vehicle-treated groups.
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Results Glutathione (GSH) levels The levels of GSH in the liver, intestine and lung tissue showed a tendency to decrease at the 2rd and 24th hours following burn injury, when compared to sham group (p < 0.01–p < 0.001). Administration of garlic extract to burned animals significantly (p < 0.01–p < 0.001) elevated the GSH levels at 2 h and at 24 h (Fig. 1a, b and c).
Fig. 2. Malondialdehyde (MDA) levels in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. ** p < 0.01, *** p < 0.001 : Compared with Sham group. + + p < 0.01, + + + p < 0.001: Garlic -treated groups compared with respective vehicle-treated groups.
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Malondialdehyde (MDA) levels The liver and intestine MDA levels were found to be significantly higher in both 2-h and 24-h burn groups than those of the sham group (p < 0.001). In the lung tissue, burn injury led to a significant rise
Fig. 3. Protein oxidation levels in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. *** p < 0.001: Compared with Sham group. + + + p < 0.001: Garlic -treated groups compared with vehicle-treated groups.
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in MDA at only 24 h (p < 0.001). Treatment with garlic extract significantly reversed the elevations in MDA levels of liver and intestine tissues at 2 h and 24 h after burn injury (p < 0.001); and depressed the lung MDA level in 24-h burn group (p < 0.001), (Fig. 2a, b and c). Protein oxidation levels In both 2-h and 24-h post-burn injury groups, there were significant (p < 0.001) increases in protein oxidation levels when compared with sham group. Garlic treatment significantly abolished the elevations in protein oxidation at both time points after burn injury (p < 0.001), (Fig. 3a, b and c).
Fig. 4. Myeloperoxidase (MPO) activity in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. *** p < 0.001 : Compared with Sham group. + p < 0.05, + + + p < 0.001: Garlic -treated groups compared with respective vehicle-treated groups.
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Myeloperoxidase (MPO) activity In both 2-h and 24-h burn groups, MPO activity was found to be significantly increased, when compared to sham group, while garlic treatment reversed the elevations (p < 0.05–0.001; Fig. 4a, b and c). Discussion Since garlic contains various biochemically active substances including thioallyl compounds, and since its extracts have been known to protect organs from various injuries [17,34,35], we investigated the protective effect of agueous garlic extract against thermal injury which is not studied so far. The results of the present study demonstrate that the burn-induced injury in the liver, intestine and lung tissue was ameliorated by aqueous garlic extract treatment, as evidenced by changes in malondialdehyde and glutathione levels, protein oxidation and myeloperoxidase activity. These findings suggest that garlic extract has a protective role in the burn-induced oxidative injury, which may be attributed to its antioxidant effects. A skin burn is a common traumatic injury the results in both local tissue damage and a systemic mediator-induced response [36]. The local and systemic inflammatory response to thermal injury is extremely complex, resulting in both local burn tissue damage and subsequent edema, as well as marked systemic effects involving organs distant from the burn area itself. The local tissue trauma activates a number of systemic mediator cascades, e.g. a complement activation (interleukin 1, IL-1 and tumor necrosis factor-a; TNF-a) resulting in a generalized neutrophil sequestration and a ‘priming’ of local and systemic neutrophils and macrophages [4]. Activated neutrophils, lead to the formation of toxic oxygen products which further cause tissue damage. Reactive oxygen products can generate hypocholorus acid (HOCl) in the presence of neutrophil-derived myeloperoxidase (MPO) and initiate the deactivation of antiproteases and activation of latent proteases, which lead to tissue damage [37]. MPO activity is used as an indirect evidence of neutrophil infiltration in oxidant induced tissue injury. Our observation that MPO levels were increased in liver, intestine and lung tissues indicate that neutrophil accumulation in this tissues contribute to organ injury distant from the original wound. Furthermore, the results also suggest that garlic extract has a preventive effect in thermal trauma through inhibition of the infiltration of neutrophils, since MPO levels found to be significantly decreased after garlic treatment. Research on animals and clinical studies have shown that, a local burn insult produces oxidantinduced organ changes as evidenced by increased lipid peroxidation in the lung, liver and gut [5,38]. Lipid peroxidation in cell membranes is devastating to the functional integrity of these structures and if the damage is severe, death of the cell is inevitable [39]. Aged garlic extract and diallyl polysulfides inhibit the formation of thiobarbituric acid-reactive substances and fluorescent substances induced by iron-ascorbic acid in isolated liver microsomal . membranes, indicating the protection of lipid peroxidation [34]. Alliin scavenges the OH radical and . garlic powder scavenges both OH and 1,1-diphenyl-2-picrylhydrazyl radicals [33]. Yamasaki et al., shown that an aged garlic extract protects vascular endothelial cells from H2O2-induced oxidative damage by inhibiting lipid peroxidation [40]. Numagami et al., showed the attenuation of rat ischemic brain damage by aged garlic extracts and suggested that garlic compounds, S-allyl cysteine, Allyl sulfide and allyl disulfide may be responsible for this effect [20].
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In the present study, the levels of MDA, an end product of lipid peroxidation, are significantly increased in liver, lung and intestine tissues at both 2 h and 24 h after burn injury. This observation is in agreement with previous studies, in which elevated levels of lipid products were increased from 40 to 80% above basal values [38,41]. It may be suggested that disintegration of intestinal membrane further adds to systemic inflammation. Our results demonstrate that aqueous garlic extract inhibits MDA elevations significantly and reverses back to control levels. Thus, garlic extract could be protective against distant organ damage by preserving the cellular integrity. On the other hand, cellular proteins are also believed to be a target of oxidative injury. That is, the accumulation of oxidized proteins can impair cell function and eventually lead to cell damage. In the present study, protein oxidation, which is an early sign of cellular injury, is found to be reduced by garlic treatment. When taken together, aqueous garlic extract appears to be involved in both the early and late phases of oxidative damage to improve the tissue response. It may be possible to limit oxidative stress-induced tissue damage and, hence, prevent or ameliorate disease progression by favoring the balance towards lower oxidative stress. It has been reported that chronic garlic intake significantly decreased lipid peroxidation and increased endogenous antioxidants such as superoxide dismutase, catalase, glutathione and glutathione peroxidase [42]. Glutathione is an important constituent of intracellular protective mechanisms against various noxious stimuli including oxidative stress. However reduced glutathione as the main component of endogenous non-protein sulfhydryl pool, is known to be a major low molecular weight scavenger of free radicals in the cytoplasm [43,44]. Because of their exposed sulfhydryl groups, non-protein sulphydryls bind a variety of electrophilic radicals and metabolites that may be damaging to cells [45]. The results of the present study further support the notion that depletion of tissue GSH is one of the major factors that permit lipid peroxidation and subsequent tissue damage. Glutathione levels in all tissues both 2 and 24 hours after thermal trauma decresed significantly. Furthermore administration of 1 ml/kg garlic extract maintained the glutathione levels, thus protected tissue against oxidative stress. The following compounds of garlic; S-allyl-cyteine, S-allyl-mercaptocysteine, S-allyl-cysteinesulfoxide and allicin, involved in its radical scavenging ability and it has been shown that these compounds are responsible for the prevention against oxidative injury [22]. Diallyldisulfide (DADS), a metabolit of allicin, includes redox-active sulfhydryl (SH)- or disulfide (-S-S)- groups which have already been proven to act as radical scavengers [46].
Conclusion In conclusion, burn-induced increases in lipid peroxidation products, protein oxidation with a concomitant decrease in glutathione levels suggest that the organ damage distant from original wound induced by burn injury is dependent upon the formation of oxygen radicals. Moreover, garlic-extract, with its ability to directly neutralize a number of reactive oxygen species demonstrated a protective effect in burn-induced tissue injury.
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Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury ¨ zer Sßehirli a, Ayhan Kacßmaz b Go¨ksel Sßener a,*, Handan Sßatırog˘lu a, A. O a
Marmara University, School of Pharmacy, Department of Pharmacology, Istanbul, Turkey b Haydarpasßa Numune Hospital, Department of Surgery, Istanbul, Turkey Received 29 October 2002; accepted 6 December 2002
Abstract Oxygen free radicals have been implicated in mediating various pathological processes including burninduced organ damage. This study was designed to determine the possible protective effect of aqueous garlic extract against oxidative organ damage distant from the original burn wound. Under ether anaesthesia, rats were subjected to severe skin scald injury covering 30% of total body surface area. Rats were decapitated either 2 h or 24 h after burn injury. Aqueous garlic extract (1 ml/kg) was administered i.p. immediately after burn injury. In the 24-h burn group injection was repeated once more (at 12 hour) following the burn injury. Liver, intestine and lung tissues were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and protein oxidation (PO). Burn injury caused a significant decrease in GSH level, and significant increases in MDA and PO levels, and MPO activity at post-burn 2 and 24 hours. Since garlic extract reversed these oxidant responses it seems likely that garlic extract protects tissues against oxidative damage. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Burn; Garlic; Lipid peroxidation; Glutathione; Myeloperoxidase; Protein oxidation
Introduction . Oxygen free radicals such as hydrogen peroxide (H2O2), superoxide anion (O2 ) and hydroxyl radical . (OH ) have been implicated in mediating various pathological process such as ischemia, inflammatory ¨ . Eczacılık Faku¨ltesi, Farmakoloji Ab. D., Tibbiye Cad. I˙stanbul 34668, Turkey. Tel.: +90-216* Corresponding author. M.U 418-65-92; fax: +90-216-330-10-93. E-mail address: [email protected] (G. Sßener). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00236-4
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diseases, diabetes, and atherosclerosis [1,2]. Thermal trauma, one of the most common problems faced in the emergency room, may cause damage to multiple organs distant from the original burn wound and may lead to multiorgan failure. Following burn injury, all tissues are subjected to ischemia and consequently, during burn shock resuscitation, reperfusion occurs. The role of free oxygen radicals, neutrophils and endothelial cells in ischemic insult or organ failure is well documented [3,4]. Several studies have demonstrated that burn injury is associated with lipid peroxidation, which is an autocatalytic mechanism leading to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic, reactive metabolites and cell death [5,6]. Youn et al. demonstrated that burn injury results in increased lipid peroxidation in the plasma, lung, kidney and liver [7]. Since the major source of the oxidants could be neutrophils sequestered in systemic organs as a systemic inflammatory reaction to a local burn insult [8], it can be expected that agents which inhibit the activation and adherence of neutrophils might also exert protective effects against thermal injury [9]. It has been shown that antioxidants, when given postburn, restored antioxidant defense mechanisms and attenuated tissue injury [10–12]. We recently shown that antioxidant melatonin can protect against burninduced oxidative organ damage [13,14]. Garlic has been used as a folk remedy for a variety of ailments since ancient times. In the past few years, it has been found in certain models that garlic preperations including aged garlic (AGE) prevented tumour promotion [15], cardiovascular diseases [16], liver damage [17], and aging [18] which are considered to be associated with oxygen radical and lipid peroxidation. The intrinsic antioxidant activity of garlic [19], garlic extracts [20,21] and some garlic constituents [22,23], have been widely documented in vivo [24,25] and in vitro [21,23]. Garlic extracts increases SOD [26], GPx [27], and CAT activities [27] in vascular cells in culture, and S- allyl-cysteine sulfoxide (alliin), a garlic compound prevents the decrease in hepatic SOD and CAT activities observed in diabetic rats [24]. Therefore, in this work we studied the effect of aqueous garlic extract against oxidative organ damage induced by thermal trauma.
Materials and methods Animals The Marmara University School of Medicine Animal Care and Use Committee approved all experimental protocols. Wistar albino rats of both sexes weighing 200 to 250 g were fasted for 12 h, but were allowed free access to water, before burn injury. Rats were kept in a room at a constant temperature 22 F 2 jC with 12-h light and dark cycles, in individual wire-bottomed cages and fed standard rat chow. Thermal Injury Under brief ether anesthesia, dorsum of the rats was shaved and exposed to 90 jC water bath for 10 s which resulted in partial-thickness second-degree skin burn involving 30% of the total body surface area [13,14]. The animals were then resuscitated with physiological saline solution (10 mg/kg, s.c.). Rats were decapitated at 2 or 24 h after burn injury in both vehicle and garlic-treated groups: Burn-2 h, Burn24 h and Burn + Gar-2 h, Burn + Gar-24 h respectively. Each group consists of 8 rats. In order to rule out
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the effects of anesthesia, the same protocol was applied in the sham group, except that the dorsums were dipped in a 25 jC water bath for 10 s. Garlic-treatment Peeled garlic (30 g) was crushed with distilled water in a mortar. The crushed material was carefully decanted by pressing and 60 ml of aqueous extract was extracted. 1 ml of aqueous extract contained material from 500 mg of garlic [28]. In the 2-h burn group, either garlic extract (1 ml/kg) or saline (0.1 ml/ kg) was administered i.p. immediately after the burn. In the 24-h burn groups, the injections of garlic extract or vehicle were repeated one more occasion (at 12 h) following burn injury. Biochemical analysis After decapitation, the liver, small intestine and lung tissues were immediately taken and stored at 70 jC. Afterwards, malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and protein carbonyl concentration, a specific marker of oxidative damage of proteins were measured in these samples. Tissue-associated myeloperoxidase (MPO) activity, as an indirect evidence of neutrophil infiltration, was measured in all tissue samples. Malondialdehyde and glutathione assays Tissue samples were homogenized with ice-cold 150 mM KCl for determination of malondialdehyde (MDA) and glutathione levels. The MDA levels were assayed for products of lipid peroxidation [29]. Results were expressed as Amol MDA/g tissue. Glutathione was determined by the spectrophotometric method which was based on the use of Ellman’s reagent [30]. Results were expressed Amol GSH/g tissue. Protein oxidation The protein content of tissue samples was determined by the Lowry assay [31]. Oxidized protein was quantified using the interaction between dinitrophenylhydrazine (DNP) and the carbonyls to yield a chromophore that absorbs strongly at 360 nm. All samples were diluted to 2–4 mg/ml of protein with wash buffer and treated with 1% streptomycin. The carbonyl content was calculated assuming a molar extinction coefficient of 22,000 [32]. Myeloperoxidase activity MPO activity was measured in tissues in a procedure similar to that documented by Hillegas et al. [32]. Liver, lung or intestine samples were homogenized in 50 mM potassium phosphate buffer (PB, pH 6.0), and centrifuged at 41,400 g (10 min); pellets were suspended in 50 mM PB containing 0.5% hexadecyltrimethylammonium bromide (HETAB). After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41.400 g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of the MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue.
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Statistics Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego; CA; USA). All data were expressed as means F SEM. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Values of p < 0.05 were regarded as significant.
Fig. 1. Glutathione (GSH) levels in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. **p < 0.01,*** p < 0.001 : Compared with Sham group. + p < 0.05, + + p < 0.01, + + + p < 0.001: Garlic-treated groups compared with respective vehicle-treated groups.
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Results Glutathione (GSH) levels The levels of GSH in the liver, intestine and lung tissue showed a tendency to decrease at the 2rd and 24th hours following burn injury, when compared to sham group (p < 0.01–p < 0.001). Administration of garlic extract to burned animals significantly (p < 0.01–p < 0.001) elevated the GSH levels at 2 h and at 24 h (Fig. 1a, b and c).
Fig. 2. Malondialdehyde (MDA) levels in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. ** p < 0.01, *** p < 0.001 : Compared with Sham group. + + p < 0.01, + + + p < 0.001: Garlic -treated groups compared with respective vehicle-treated groups.
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Malondialdehyde (MDA) levels The liver and intestine MDA levels were found to be significantly higher in both 2-h and 24-h burn groups than those of the sham group (p < 0.001). In the lung tissue, burn injury led to a significant rise
Fig. 3. Protein oxidation levels in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. *** p < 0.001: Compared with Sham group. + + + p < 0.001: Garlic -treated groups compared with vehicle-treated groups.
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in MDA at only 24 h (p < 0.001). Treatment with garlic extract significantly reversed the elevations in MDA levels of liver and intestine tissues at 2 h and 24 h after burn injury (p < 0.001); and depressed the lung MDA level in 24-h burn group (p < 0.001), (Fig. 2a, b and c). Protein oxidation levels In both 2-h and 24-h post-burn injury groups, there were significant (p < 0.001) increases in protein oxidation levels when compared with sham group. Garlic treatment significantly abolished the elevations in protein oxidation at both time points after burn injury (p < 0.001), (Fig. 3a, b and c).
Fig. 4. Myeloperoxidase (MPO) activity in garlic extract or vehicle-treated groups at 2 h and 24 h following burn injury. *** p < 0.001 : Compared with Sham group. + p < 0.05, + + + p < 0.001: Garlic -treated groups compared with respective vehicle-treated groups.
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Myeloperoxidase (MPO) activity In both 2-h and 24-h burn groups, MPO activity was found to be significantly increased, when compared to sham group, while garlic treatment reversed the elevations (p < 0.05–0.001; Fig. 4a, b and c). Discussion Since garlic contains various biochemically active substances including thioallyl compounds, and since its extracts have been known to protect organs from various injuries [17,34,35], we investigated the protective effect of agueous garlic extract against thermal injury which is not studied so far. The results of the present study demonstrate that the burn-induced injury in the liver, intestine and lung tissue was ameliorated by aqueous garlic extract treatment, as evidenced by changes in malondialdehyde and glutathione levels, protein oxidation and myeloperoxidase activity. These findings suggest that garlic extract has a protective role in the burn-induced oxidative injury, which may be attributed to its antioxidant effects. A skin burn is a common traumatic injury the results in both local tissue damage and a systemic mediator-induced response [36]. The local and systemic inflammatory response to thermal injury is extremely complex, resulting in both local burn tissue damage and subsequent edema, as well as marked systemic effects involving organs distant from the burn area itself. The local tissue trauma activates a number of systemic mediator cascades, e.g. a complement activation (interleukin 1, IL-1 and tumor necrosis factor-a; TNF-a) resulting in a generalized neutrophil sequestration and a ‘priming’ of local and systemic neutrophils and macrophages [4]. Activated neutrophils, lead to the formation of toxic oxygen products which further cause tissue damage. Reactive oxygen products can generate hypocholorus acid (HOCl) in the presence of neutrophil-derived myeloperoxidase (MPO) and initiate the deactivation of antiproteases and activation of latent proteases, which lead to tissue damage [37]. MPO activity is used as an indirect evidence of neutrophil infiltration in oxidant induced tissue injury. Our observation that MPO levels were increased in liver, intestine and lung tissues indicate that neutrophil accumulation in this tissues contribute to organ injury distant from the original wound. Furthermore, the results also suggest that garlic extract has a preventive effect in thermal trauma through inhibition of the infiltration of neutrophils, since MPO levels found to be significantly decreased after garlic treatment. Research on animals and clinical studies have shown that, a local burn insult produces oxidantinduced organ changes as evidenced by increased lipid peroxidation in the lung, liver and gut [5,38]. Lipid peroxidation in cell membranes is devastating to the functional integrity of these structures and if the damage is severe, death of the cell is inevitable [39]. Aged garlic extract and diallyl polysulfides inhibit the formation of thiobarbituric acid-reactive substances and fluorescent substances induced by iron-ascorbic acid in isolated liver microsomal . membranes, indicating the protection of lipid peroxidation [34]. Alliin scavenges the OH radical and . garlic powder scavenges both OH and 1,1-diphenyl-2-picrylhydrazyl radicals [33]. Yamasaki et al., shown that an aged garlic extract protects vascular endothelial cells from H2O2-induced oxidative damage by inhibiting lipid peroxidation [40]. Numagami et al., showed the attenuation of rat ischemic brain damage by aged garlic extracts and suggested that garlic compounds, S-allyl cysteine, Allyl sulfide and allyl disulfide may be responsible for this effect [20].
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In the present study, the levels of MDA, an end product of lipid peroxidation, are significantly increased in liver, lung and intestine tissues at both 2 h and 24 h after burn injury. This observation is in agreement with previous studies, in which elevated levels of lipid products were increased from 40 to 80% above basal values [38,41]. It may be suggested that disintegration of intestinal membrane further adds to systemic inflammation. Our results demonstrate that aqueous garlic extract inhibits MDA elevations significantly and reverses back to control levels. Thus, garlic extract could be protective against distant organ damage by preserving the cellular integrity. On the other hand, cellular proteins are also believed to be a target of oxidative injury. That is, the accumulation of oxidized proteins can impair cell function and eventually lead to cell damage. In the present study, protein oxidation, which is an early sign of cellular injury, is found to be reduced by garlic treatment. When taken together, aqueous garlic extract appears to be involved in both the early and late phases of oxidative damage to improve the tissue response. It may be possible to limit oxidative stress-induced tissue damage and, hence, prevent or ameliorate disease progression by favoring the balance towards lower oxidative stress. It has been reported that chronic garlic intake significantly decreased lipid peroxidation and increased endogenous antioxidants such as superoxide dismutase, catalase, glutathione and glutathione peroxidase [42]. Glutathione is an important constituent of intracellular protective mechanisms against various noxious stimuli including oxidative stress. However reduced glutathione as the main component of endogenous non-protein sulfhydryl pool, is known to be a major low molecular weight scavenger of free radicals in the cytoplasm [43,44]. Because of their exposed sulfhydryl groups, non-protein sulphydryls bind a variety of electrophilic radicals and metabolites that may be damaging to cells [45]. The results of the present study further support the notion that depletion of tissue GSH is one of the major factors that permit lipid peroxidation and subsequent tissue damage. Glutathione levels in all tissues both 2 and 24 hours after thermal trauma decresed significantly. Furthermore administration of 1 ml/kg garlic extract maintained the glutathione levels, thus protected tissue against oxidative stress. The following compounds of garlic; S-allyl-cyteine, S-allyl-mercaptocysteine, S-allyl-cysteinesulfoxide and allicin, involved in its radical scavenging ability and it has been shown that these compounds are responsible for the prevention against oxidative injury [22]. Diallyldisulfide (DADS), a metabolit of allicin, includes redox-active sulfhydryl (SH)- or disulfide (-S-S)- groups which have already been proven to act as radical scavengers [46].
Conclusion In conclusion, burn-induced increases in lipid peroxidation products, protein oxidation with a concomitant decrease in glutathione levels suggest that the organ damage distant from original wound induced by burn injury is dependent upon the formation of oxygen radicals. Moreover, garlic-extract, with its ability to directly neutralize a number of reactive oxygen species demonstrated a protective effect in burn-induced tissue injury.
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