- Email: [email protected]
Effects of Apocynin on Liver Ischemia-Reperfusion Injury in Rats
Effects of Apocynin on Liver Ischemia-Reperfusion Injury in Rats A. Yücela, M.S. Aydogana,*, M. Ucara, K.B. Sarıcıb, and M.G. Karaaslanc a Department of Anesthesiology, Faculty of Medicine, Inonu University, Malatya, Turkey; bDepartment of General Surgery, Faculty of Medicine, Inonu University, Malatya, Turkey; and cDepartment of Chemistry, Inonu University, Malatya, Turkey
ABSTRACT Objective. Ischemia-reperfusion (IR) injury is associated with various clinical conditions, such as myocardial infarction, shock, and surgery under vascular occlusion. We aimed to investigate the protective and therapeutic effects of apocynin (AP) on liver injury induced by IR in an in vivo rat model. Methods. Thirty-two rats were randomly divided into 4 experimental groups with n ¼ 8 in each group: sham, IR, AP, and IR þ AP. AP (20 mg/kg) was intraperitoneally administered to rats in the AP and IR þ AP groups for 30 minutes before 60 minutes of ischemia and followed by 60 minutes of reperfusion. All rats were killed on the same day to evaluate tissue levels of oxidants and antioxidants (catalase, malondialdehyde, myeloperoxidase, superoxidedismutase (SOD), and total glutathione). Results. IR decreased SOD levels in IR group when compared with the sham group. AP supplementation to IR group significantly ameliorated SOD levels (P < .05). Also, IR caused elevation of myeloperoxidase production when compared with the sham group, whereas AP treatment prevented these hazardous effects (P < .05). However, plasma total glutathione, catalase, and malondialdehyde levels did not differ between the AP þ IR and the IR rats. Conclusion. The main finding of the present study was that AP may be protective against liver IR injury. Our results suggested that AP pretreatment suppressed oxidative stress and increased antioxidant levels in an rat model of liver IR.
L
IVER ischemia-reperfusion injury (IRI) occurs during transplantation and major hepatic surgery and may contribute to postoperative liver dysfunction and negatively affect graft function [1,2]. Cessation of blood flow, which occurs during ischemia, causes oxygen and nutrition deficiency, in turn resulting in tissue damage, including inflammatory cell infiltration, production of oxygen-derived reactive oxygen species (ROS) or nitrogen-derived reactive nitrogen species, and microvascular damage, also process the during reperfusion period [3]. Antioxidant enzymes are protective against IRI. For these reasons, treatment with exogenous antioxidants, particularly in the early stages of reperfusion, markedly reduces the severity of liver IRI [4]. Apocynin (4-hydroxy-3-methoxyacetophenone) (AP), which is obtained from the roots of the Apocynum cannabinum plant, has the effect of an NADPH oxidase inhibitor [5]. AP is one of the most promising selective NADPH oxidases and it has a role in the production of superoxide
0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.01.108
1180
inhibitors. Apocynin strengthens antioxidant defensive systems, enhances decreased GSH, and limits the cellular stress triggered by ischemia [6,7]. This experimental study was designed to investigate the protective and therapeutic effects of AP on liver injury induced by ischemia-reperfusion (IR) in an in vivo rat model. To examine this, we evaluated biochemical analyses (including tissue malondialdehyde [MDA], superoxide dismutase [SOD], catalase [CAT], total glutathione [tGSH], myeloperoxidase [MPO]).
*Address correspondence to Mustafa Said Aydogan, MD, Associated Professor of Anesthesiology and Reanimation, Department of Anesthesiology and Reanimation School of Medicine, Inonu University, Fırıncı St, Elazıg Way 8. km. 44280 Malatya, Turkey. Tel: (þ90) 4223410660-3160; Fax: (þ90) 4223410728. E-mail: [email protected] ª 2019 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1180e1183 (2019)
APOCYNIN LIVER ISCHEMIA-REPERFUSION INJURY
MATERIALS AND METHODS Animals A total of 32 Sprague-Dawley female rats (10e12 weeks) were obtained, with weights ranging between 250 to 300 g, from our laboratory animal research center. The rats were maintained at 21 C 2 C with a relative humidity of 60% 5% under 12-hour light and dark cycles. They were housed in plastic cages (50 35 20 cm; 8 animals per cage). The experiments were performed according to the standards of animal research of the National Health Research Institute and with the approval of our university ethical committee.
Experimental Design Rats were randomly divided into 4 groups, each consisting of 8 animals: group 1, sham group: exposure of the hepatic artery, portal vein, and bile duct region but no IR; group 2, IR group: 60 minutes of ischemia followed by 60 minutes of reperfusion; group 3, AP group: 20 mg/kg AP (Sigma-Aldrich, St. Louis, MO, United States) given intraperitoneally; group 4, IR þ AP group: 20 mg/kg AP given intraperitoneally 30 minutes before 60 minutes of ischemia, followed by 60 minutes of reperfusion. The dosage of AP was chosen according to previous dose-response studies [8,9].
Surgical Procedure Following a 12-hour fast, rats were anesthetized with intraperitoneally administered ketamine (40 mg/kg) and xylazine (10 mg/kg). The abdominal region was sterilized with povidone-iodine solution and was explored through midline laparotomy using minimal dissection. Only midline laparotomy was performed for the sham group; the abdomen was closed without any further procedure. In the IR and IR þ AP groups, total hepatic ischemia was induced for 60 minutes by occluding the hepatic artery, portal vein, and bile duct using a nontraumatic vascular clamp. The liver was reperfused for 60 minutes after removing the clamp. After declamping, we confirmed restored hepatic blood flow before closure of the incision. During the surgery, body temperature was maintained at approximately 37.5 C with a heating lamp. Fluid loss was replaced by intraperitoneal injection of 3 mL warm (37 C) saline before abdominal closure. After 60 minutes of reperfusion, the abdomen was reopenedand and 3 mL of blood was drawn from the heart into heparinized microcentrifuge tubes. Subsequently, the animals were sacrificed to perform a hepatectomy. The plasma was stored at 80 C for biochemical analyses. All coded specimens were evaluated by individuals blinded to the group assignments.
Determination of Enzyme Activities CAT, SOD, and MPO activity were determined spectrophotometrically. CAT activity was measured at 37 C by following the rate of disappearance of hydrogen peroxide at 240 nm (ε240 ¼ 40 ¼ 40 M1cm1) [10]. One unit of CAT activity was defined as the amount of enzyme catalyzing the degradation of 1 mmol of hydrogen peroxide/min at 37 C. CAT activity was expressed as U/mg protein in the tissue. SOD (Cu, Zn-SOD) activity in the supernatant fraction was measured using the xanthine oxidase-cytochrome C method [11], in which 1 unit (U) of activity was the amount of enzyme needed to cause half-maximal inhibition of cytochrome C reduction. The amount of SOD in the extract was determined as U of enzyme mg-1 protein, utilizing a commercial SOD as the standard. For MPO activity, samples were weighed 0.1 g and put into 1 mL of phosphate buffer (50 mM, pH 6), and all tissues were homogenized under ice with a IKA-Werke T25 homogenizer (Hettich
1181 Universal 320). Then, homogenates were centrifuged with Hettich Universal 320 microcentrifuge at 15,000 G for 15 minutes at 40 C. The pellets were separated from the supernatant and added 500 mL of HETAB solution (0.5% weight for weight in 50 mM, pH 6 phosphate buffer). The solutions were allowed to freeeze-thaw twice and sonication for a period of 15 seconds (Sonics VCX130). Then, samples were then centrifuged at 15,000 G for 15 minutes. Twenty-five mL of supernatant and 200 mL of reaction mixture (0.167 mg/mL o-dianisidine and 0.0005% volume for volume hydrogen peroxide in 50 mM phosphate buffer, pH: 6) were added into 96-well plates and the measurement was performed at a wavelength of 460 nm with BioTek Eon Eliza microplate reader. MPO activity results were given in unit per gram of wet tissue [12].
tGSH Assay The formation of 5-thio-2-nitrobenzoate (TNB) is followed spectrophotometrically at 412 nm [13]. The amount of tGSH in the extract was determined as nmol/mg protein by utilizing a commercial GSH as the standard.
MDA Assay As a marker of lipid peroxidation production, the MDA concentration was measured as described by Buege and Aust [14], with a minor modification. The reaction mixture was prepared by adding 250 mL of homogenate into 2 mL of reaction solution (15% trichloroacetic acid: 0.375% thiobarbituric acid: 0.25 N hydrochloric acid, 1:1:1, weight to volume) and heated at 100 C for 30 minutes. The mixture was cooled to room temperature, centrifuged (10,000 g for 10 minutes), and the absorbance of the supernatant was recorded at 532 nm. MDA results were expressed as nmol/mg protein in the homogenate.
Determination of Protein Protein levels of the tissue samples were measured by the Bradford method [15]. The absorbance measurement was taken at 595 nm using a UV-VIS spectrophotometer. Bovine serum albumin was used as the protein standard.
Statistical Analysis Data were analyzed using the SPSS software program for Windows, version 17.0 (SPSS Inc., Chicago, Ill, United States). The data were expressed as either median (min-max) values or mean standard deviation, depending on the overall variable distribution. The normality of the distribution was confirmed using the Shapiro-Wilk test. The normally distributed data were analyzed by one-way anaylsis of variance, followed by the Tamhane post hoc test. The non-normally distributed data were compared by the Kruskal-Wallis H test among the groups. When significant differences were determined, multiple comparisons were carried out using the Mann-Whitney U test with Bonferroni correction. The results are expressed as the median (min-max). Values of P < .05 were considered significant.
RESULTS
The biochemical results are presented in Table 1. In brief, IR decreased SOD levels in IR group when compared with the sham group. AP supplementation to IR group significantly ameliorated SOD levels (P < .05). Also, IR caused elevation of MPO production when compared with the
1182
YÜCEL, AYDOGAN, UCAR ET AL Table 1. The Biochemical Results
Group
MPO tGSH MDA SOD CAT
Sham
16.60 9.84 28.31 34.17 172.0
3.20 1.37 3.62 3.43 34.2
AP
16.52 88.13 26.52 35.52 178.9
AP þ IR
IR
3.61 0.74 3.20 3.51 42.3
40.16 9.12 29.65 21.32 188.6
‡
4.53 1.54 2.32 2.74* 35.4
24.32 9.92 28.35 29.42 181.5
3.16‡ 1.23 2.35 2.65† 35.8
Abbreviations: AP, apocynin; CAT, catalase; IR, ischemia-reperfusion; MDA, malondialdehyde; MPO, myeloperoxidase; SOD, superoxidedismutase; tGSH, total glutathione. *Significant decrease (P < .05) vs sham group. † Significant increase (P < .05) vs IR group. ‡ Significant increase (P < .05) vs sham group.
sham group, whereas AP treatment prevented these hazardous effects (P < .05). However, plasma CAT, tGSH, and MDA levels did not differ between the AP þ IR and the IR rats.
DISCUSSION
The main finding of the present study was that AP may be protective against liver IRI. In this study, we found that IR caused a decrease in the SOD level and an increase in MPO activitiy in liver tissue. However, administration of AP to the IR group reduced the SOD level while increasing the MPO activitiy. These findings indicated that a 20 mg/kg dose of AP protected against IR-induced damage in IR rat models. Evidence shows that overproduction of ROS, which is a part of host defense mechanism, gives rise to oxidative stress that has a major role in the formation of IR injury, thus causing membrane lipid peroxidation, oxidation of cell proteins, damage to the DNA helix, and cell death [16,17]. The reperfusion period initiates an inflammatory response cascade that can take a long time and causes irreversible tissue damage [18]. AP, a naturally occurring methoxy-substituted catechol oxidized by peroxides to a more potent dimer in the cell, is a selective nitrogen oxide (NOX) inhibitor, which generates superoxide. AP, which has a low toxicity and specificity, may be of promising potential therapy for variable clinical conditions via antioxidant and anti-inflammatory effects [19,20]. Also, it has been used in many experimental studies related to IR injury. MPO is a major indicator of oxidative stress, and particularly oxidase in polymorphonuclear leukocytes (PMNs) in tissue is used to estimate the PMN chemotaxis and infiltration [21]. PMN infiltration during the reperfusion period may cause generation and releasing of additional large amounts of oxidants that exacerbate this harmful cascade. AP, which is also activated by MPO [22], reduces the generation of inflammatory mediators by inhibiting NOX. In our study, the MPO levels were significantly increased by the IR procedure when compared with the control group. AP usage ameliorated the MPO activity in the treatment group. SOD is antioxidant enzyme that is a component of the defense mechanism against ROS activities. The levels of these enzymes within the host increase to protect the tissues
during IR injury [23]. Any agent used to increase the level of these enzymes can prevent the possible damages that occur during oxidative stress. Also, AP, a significant inhibitor of NOX that uses the NOX pathway to suppress oxidative stress, shows its effect by increasing these enzyme levels in the present study. There were some limitations of the study. The first limitation was that the sample size was small. Secondly, AP dose-related or longer time-dependent responses to IR injury were not evaluated. However, AP attenuated IR injury was established even if it was an incomplete protection. In conclusion, the beneficial effects of AP on liver IR were evaluated for the first time. AP improved the liver damage, which occurred after IR injury, in the treatment groups. According to our results, AP could be used to prevent the liver damage, induced by IR injury occurring in many ways, after further clinical and experimental trials, including trials using different treatment dosages and timing for inductions. REFERENCES [1] Aydogan MS, Erdogan MA, Polat A, Yücel A, Ozgül U, Parlakpinar H, et al. Protective effects of melatonin and b-d-glucan against liver injury in rats—a comparative study. Adv Clin Exp Med 2013;22:621e7. [2] Aydogan MS, Yucel A, Erdogan MA, Polat A, Cetin A, Ucar M, et al. Effects of oral b- glucan on liver ischemia/reperfusion injury in rats. Transplant Proc 2013;45:487e91. [3] Hayes JD, McLellan LI. Glutathione and glutathione dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 1999;31:273e300. [4] Dogan S, Aslan M. Hepatic ischemia-reperfusion injury and therapeutic strategies to alleviate cellular damage. Hepatol Res 2011;41:103e17. [5] Liu PG, He SQ, Zhang YH, Wu J. Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice. World J Gastroenterol 2008;14:2832e7. [6] Connell BJ, Saleh MC, Khan BV, Saleh TM. Apocynin may limit total cell death following cerebral ischemia and reperfusion by enhancing apoptosis. Food Chem Toxicol 2011;49:3063e9. [7] Stolk J, Hiltermann TJ, Dijkman JH, Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol 1994;11:95e102. [8] Altintas R, Polat A, Vardi N, Oguz F, Beytur A, Sagir M, et al. The protective effects of apocynin on kidney damage caused by renal ischþemia/reperfusion. J Endourol 2013;27:617.
APOCYNIN LIVER ISCHEMIA-REPERFUSION INJURY [9] Ozbek O, Altintas R, Polat A, Vardi N, Parlakpinar H, Sagir M, et al. The protective effect of apocynin on testicular ischemia-reperfusion injury. J Urol 2015;193:1417e22. [10] Luck H. Methods of enzymatic analysis. Verlag Chemie Academic Press; 1963. p. 885e8. [11] McCord JM, Fridovich I. Superoxide dismutase: an an enzymatic function for erythrocupreinhemocuprein. J Biol Chem 1969;44:6049e55. [12] Hillegas LM, Griswold DE, Brickson B, AlbrightsonWinslow C. Assessment of myeloperoxidase activity in whole rat kidney. J Pharmacol Methods 1990;24:285e95. [13] Theodorus P, Akerboom M, Sies H. Assay of glutathione, glutathione disulfide and glutathione mixed disulfides in biological samples. Meth Enzymol 1981;77:373e83. [14] Buege AJ, Aust SD. Microsomal lipid peroxidation. Meth Enzymol 1978;52:302e10. [15] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248e54. [16] Chatterjee PK. Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review. Naunyn Schmiedebergs Arch Pharmacol 2007;376:1e43. [17] de Groot H, Rauen U. Ischemia-reperfusion injury: processes in pathogenetic networks: a review. Transplant Proc 2007;39:481e4.
1183 [18] Filho DW, Torres MA, Bordin AL, Crezcynski-Pasa TB, Boveris A. Spermatic cord torsion, reactive oxygen and nitrogen species and ischemia-reperfusion injury. Mol Aspects Med 2004;25: 199e210. [19] Hougee S, Hartog A, Sanders A, Graus YM, Hoijer MA, Grassen J, et al. Oral administration of the NADPH-oxidase inhibitor apocynin partially restores diminished cartilage proteoglycan synthesis and reduces inflammation in mice. Eur J Pharmacol 2006;531:264e9. [20] Hu L, Zhang Y, Lim PS, Miao Y, Tan C, McKenzie KU, et al. Apocynin but not L-arginine prevents and reverses dexamethasone-induced hypertension in the rat. Am J Hypertens 2006;19:413e8. [21] Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206e9. [22] Simons JM, Hart BA, Ip Vai Ching TRAM, Van Dijk H, Labadie RP. Metabolic activation of natural phenols into selective oxidative burst agonists by activated human neutrophils. Free Radic Biol Med 1990;8:251e8. [23] Parlakpinar H, Olmez E, Acet A, Ozturk F, Tasdemir S, Ates B. Beneficial effects of apricot-feeding on myocardial ischemia-reperfusion injury in rats. Food Chem Toxicol 2009;49: 802e8.
ABSTRACT Objective. Ischemia-reperfusion (IR) injury is associated with various clinical conditions, such as myocardial infarction, shock, and surgery under vascular occlusion. We aimed to investigate the protective and therapeutic effects of apocynin (AP) on liver injury induced by IR in an in vivo rat model. Methods. Thirty-two rats were randomly divided into 4 experimental groups with n ¼ 8 in each group: sham, IR, AP, and IR þ AP. AP (20 mg/kg) was intraperitoneally administered to rats in the AP and IR þ AP groups for 30 minutes before 60 minutes of ischemia and followed by 60 minutes of reperfusion. All rats were killed on the same day to evaluate tissue levels of oxidants and antioxidants (catalase, malondialdehyde, myeloperoxidase, superoxidedismutase (SOD), and total glutathione). Results. IR decreased SOD levels in IR group when compared with the sham group. AP supplementation to IR group significantly ameliorated SOD levels (P < .05). Also, IR caused elevation of myeloperoxidase production when compared with the sham group, whereas AP treatment prevented these hazardous effects (P < .05). However, plasma total glutathione, catalase, and malondialdehyde levels did not differ between the AP þ IR and the IR rats. Conclusion. The main finding of the present study was that AP may be protective against liver IR injury. Our results suggested that AP pretreatment suppressed oxidative stress and increased antioxidant levels in an rat model of liver IR.
L
IVER ischemia-reperfusion injury (IRI) occurs during transplantation and major hepatic surgery and may contribute to postoperative liver dysfunction and negatively affect graft function [1,2]. Cessation of blood flow, which occurs during ischemia, causes oxygen and nutrition deficiency, in turn resulting in tissue damage, including inflammatory cell infiltration, production of oxygen-derived reactive oxygen species (ROS) or nitrogen-derived reactive nitrogen species, and microvascular damage, also process the during reperfusion period [3]. Antioxidant enzymes are protective against IRI. For these reasons, treatment with exogenous antioxidants, particularly in the early stages of reperfusion, markedly reduces the severity of liver IRI [4]. Apocynin (4-hydroxy-3-methoxyacetophenone) (AP), which is obtained from the roots of the Apocynum cannabinum plant, has the effect of an NADPH oxidase inhibitor [5]. AP is one of the most promising selective NADPH oxidases and it has a role in the production of superoxide
0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.01.108
1180
inhibitors. Apocynin strengthens antioxidant defensive systems, enhances decreased GSH, and limits the cellular stress triggered by ischemia [6,7]. This experimental study was designed to investigate the protective and therapeutic effects of AP on liver injury induced by ischemia-reperfusion (IR) in an in vivo rat model. To examine this, we evaluated biochemical analyses (including tissue malondialdehyde [MDA], superoxide dismutase [SOD], catalase [CAT], total glutathione [tGSH], myeloperoxidase [MPO]).
*Address correspondence to Mustafa Said Aydogan, MD, Associated Professor of Anesthesiology and Reanimation, Department of Anesthesiology and Reanimation School of Medicine, Inonu University, Fırıncı St, Elazıg Way 8. km. 44280 Malatya, Turkey. Tel: (þ90) 4223410660-3160; Fax: (þ90) 4223410728. E-mail: [email protected] ª 2019 Published by Elsevier Inc. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 1180e1183 (2019)
APOCYNIN LIVER ISCHEMIA-REPERFUSION INJURY
MATERIALS AND METHODS Animals A total of 32 Sprague-Dawley female rats (10e12 weeks) were obtained, with weights ranging between 250 to 300 g, from our laboratory animal research center. The rats were maintained at 21 C 2 C with a relative humidity of 60% 5% under 12-hour light and dark cycles. They were housed in plastic cages (50 35 20 cm; 8 animals per cage). The experiments were performed according to the standards of animal research of the National Health Research Institute and with the approval of our university ethical committee.
Experimental Design Rats were randomly divided into 4 groups, each consisting of 8 animals: group 1, sham group: exposure of the hepatic artery, portal vein, and bile duct region but no IR; group 2, IR group: 60 minutes of ischemia followed by 60 minutes of reperfusion; group 3, AP group: 20 mg/kg AP (Sigma-Aldrich, St. Louis, MO, United States) given intraperitoneally; group 4, IR þ AP group: 20 mg/kg AP given intraperitoneally 30 minutes before 60 minutes of ischemia, followed by 60 minutes of reperfusion. The dosage of AP was chosen according to previous dose-response studies [8,9].
Surgical Procedure Following a 12-hour fast, rats were anesthetized with intraperitoneally administered ketamine (40 mg/kg) and xylazine (10 mg/kg). The abdominal region was sterilized with povidone-iodine solution and was explored through midline laparotomy using minimal dissection. Only midline laparotomy was performed for the sham group; the abdomen was closed without any further procedure. In the IR and IR þ AP groups, total hepatic ischemia was induced for 60 minutes by occluding the hepatic artery, portal vein, and bile duct using a nontraumatic vascular clamp. The liver was reperfused for 60 minutes after removing the clamp. After declamping, we confirmed restored hepatic blood flow before closure of the incision. During the surgery, body temperature was maintained at approximately 37.5 C with a heating lamp. Fluid loss was replaced by intraperitoneal injection of 3 mL warm (37 C) saline before abdominal closure. After 60 minutes of reperfusion, the abdomen was reopenedand and 3 mL of blood was drawn from the heart into heparinized microcentrifuge tubes. Subsequently, the animals were sacrificed to perform a hepatectomy. The plasma was stored at 80 C for biochemical analyses. All coded specimens were evaluated by individuals blinded to the group assignments.
Determination of Enzyme Activities CAT, SOD, and MPO activity were determined spectrophotometrically. CAT activity was measured at 37 C by following the rate of disappearance of hydrogen peroxide at 240 nm (ε240 ¼ 40 ¼ 40 M1cm1) [10]. One unit of CAT activity was defined as the amount of enzyme catalyzing the degradation of 1 mmol of hydrogen peroxide/min at 37 C. CAT activity was expressed as U/mg protein in the tissue. SOD (Cu, Zn-SOD) activity in the supernatant fraction was measured using the xanthine oxidase-cytochrome C method [11], in which 1 unit (U) of activity was the amount of enzyme needed to cause half-maximal inhibition of cytochrome C reduction. The amount of SOD in the extract was determined as U of enzyme mg-1 protein, utilizing a commercial SOD as the standard. For MPO activity, samples were weighed 0.1 g and put into 1 mL of phosphate buffer (50 mM, pH 6), and all tissues were homogenized under ice with a IKA-Werke T25 homogenizer (Hettich
1181 Universal 320). Then, homogenates were centrifuged with Hettich Universal 320 microcentrifuge at 15,000 G for 15 minutes at 40 C. The pellets were separated from the supernatant and added 500 mL of HETAB solution (0.5% weight for weight in 50 mM, pH 6 phosphate buffer). The solutions were allowed to freeeze-thaw twice and sonication for a period of 15 seconds (Sonics VCX130). Then, samples were then centrifuged at 15,000 G for 15 minutes. Twenty-five mL of supernatant and 200 mL of reaction mixture (0.167 mg/mL o-dianisidine and 0.0005% volume for volume hydrogen peroxide in 50 mM phosphate buffer, pH: 6) were added into 96-well plates and the measurement was performed at a wavelength of 460 nm with BioTek Eon Eliza microplate reader. MPO activity results were given in unit per gram of wet tissue [12].
tGSH Assay The formation of 5-thio-2-nitrobenzoate (TNB) is followed spectrophotometrically at 412 nm [13]. The amount of tGSH in the extract was determined as nmol/mg protein by utilizing a commercial GSH as the standard.
MDA Assay As a marker of lipid peroxidation production, the MDA concentration was measured as described by Buege and Aust [14], with a minor modification. The reaction mixture was prepared by adding 250 mL of homogenate into 2 mL of reaction solution (15% trichloroacetic acid: 0.375% thiobarbituric acid: 0.25 N hydrochloric acid, 1:1:1, weight to volume) and heated at 100 C for 30 minutes. The mixture was cooled to room temperature, centrifuged (10,000 g for 10 minutes), and the absorbance of the supernatant was recorded at 532 nm. MDA results were expressed as nmol/mg protein in the homogenate.
Determination of Protein Protein levels of the tissue samples were measured by the Bradford method [15]. The absorbance measurement was taken at 595 nm using a UV-VIS spectrophotometer. Bovine serum albumin was used as the protein standard.
Statistical Analysis Data were analyzed using the SPSS software program for Windows, version 17.0 (SPSS Inc., Chicago, Ill, United States). The data were expressed as either median (min-max) values or mean standard deviation, depending on the overall variable distribution. The normality of the distribution was confirmed using the Shapiro-Wilk test. The normally distributed data were analyzed by one-way anaylsis of variance, followed by the Tamhane post hoc test. The non-normally distributed data were compared by the Kruskal-Wallis H test among the groups. When significant differences were determined, multiple comparisons were carried out using the Mann-Whitney U test with Bonferroni correction. The results are expressed as the median (min-max). Values of P < .05 were considered significant.
RESULTS
The biochemical results are presented in Table 1. In brief, IR decreased SOD levels in IR group when compared with the sham group. AP supplementation to IR group significantly ameliorated SOD levels (P < .05). Also, IR caused elevation of MPO production when compared with the
1182
YÜCEL, AYDOGAN, UCAR ET AL Table 1. The Biochemical Results
Group
MPO tGSH MDA SOD CAT
Sham
16.60 9.84 28.31 34.17 172.0
3.20 1.37 3.62 3.43 34.2
AP
16.52 88.13 26.52 35.52 178.9
AP þ IR
IR
3.61 0.74 3.20 3.51 42.3
40.16 9.12 29.65 21.32 188.6
‡
4.53 1.54 2.32 2.74* 35.4
24.32 9.92 28.35 29.42 181.5
3.16‡ 1.23 2.35 2.65† 35.8
Abbreviations: AP, apocynin; CAT, catalase; IR, ischemia-reperfusion; MDA, malondialdehyde; MPO, myeloperoxidase; SOD, superoxidedismutase; tGSH, total glutathione. *Significant decrease (P < .05) vs sham group. † Significant increase (P < .05) vs IR group. ‡ Significant increase (P < .05) vs sham group.
sham group, whereas AP treatment prevented these hazardous effects (P < .05). However, plasma CAT, tGSH, and MDA levels did not differ between the AP þ IR and the IR rats.
DISCUSSION
The main finding of the present study was that AP may be protective against liver IRI. In this study, we found that IR caused a decrease in the SOD level and an increase in MPO activitiy in liver tissue. However, administration of AP to the IR group reduced the SOD level while increasing the MPO activitiy. These findings indicated that a 20 mg/kg dose of AP protected against IR-induced damage in IR rat models. Evidence shows that overproduction of ROS, which is a part of host defense mechanism, gives rise to oxidative stress that has a major role in the formation of IR injury, thus causing membrane lipid peroxidation, oxidation of cell proteins, damage to the DNA helix, and cell death [16,17]. The reperfusion period initiates an inflammatory response cascade that can take a long time and causes irreversible tissue damage [18]. AP, a naturally occurring methoxy-substituted catechol oxidized by peroxides to a more potent dimer in the cell, is a selective nitrogen oxide (NOX) inhibitor, which generates superoxide. AP, which has a low toxicity and specificity, may be of promising potential therapy for variable clinical conditions via antioxidant and anti-inflammatory effects [19,20]. Also, it has been used in many experimental studies related to IR injury. MPO is a major indicator of oxidative stress, and particularly oxidase in polymorphonuclear leukocytes (PMNs) in tissue is used to estimate the PMN chemotaxis and infiltration [21]. PMN infiltration during the reperfusion period may cause generation and releasing of additional large amounts of oxidants that exacerbate this harmful cascade. AP, which is also activated by MPO [22], reduces the generation of inflammatory mediators by inhibiting NOX. In our study, the MPO levels were significantly increased by the IR procedure when compared with the control group. AP usage ameliorated the MPO activity in the treatment group. SOD is antioxidant enzyme that is a component of the defense mechanism against ROS activities. The levels of these enzymes within the host increase to protect the tissues
during IR injury [23]. Any agent used to increase the level of these enzymes can prevent the possible damages that occur during oxidative stress. Also, AP, a significant inhibitor of NOX that uses the NOX pathway to suppress oxidative stress, shows its effect by increasing these enzyme levels in the present study. There were some limitations of the study. The first limitation was that the sample size was small. Secondly, AP dose-related or longer time-dependent responses to IR injury were not evaluated. However, AP attenuated IR injury was established even if it was an incomplete protection. In conclusion, the beneficial effects of AP on liver IR were evaluated for the first time. AP improved the liver damage, which occurred after IR injury, in the treatment groups. According to our results, AP could be used to prevent the liver damage, induced by IR injury occurring in many ways, after further clinical and experimental trials, including trials using different treatment dosages and timing for inductions. REFERENCES [1] Aydogan MS, Erdogan MA, Polat A, Yücel A, Ozgül U, Parlakpinar H, et al. Protective effects of melatonin and b-d-glucan against liver injury in rats—a comparative study. Adv Clin Exp Med 2013;22:621e7. [2] Aydogan MS, Yucel A, Erdogan MA, Polat A, Cetin A, Ucar M, et al. Effects of oral b- glucan on liver ischemia/reperfusion injury in rats. Transplant Proc 2013;45:487e91. [3] Hayes JD, McLellan LI. Glutathione and glutathione dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 1999;31:273e300. [4] Dogan S, Aslan M. Hepatic ischemia-reperfusion injury and therapeutic strategies to alleviate cellular damage. Hepatol Res 2011;41:103e17. [5] Liu PG, He SQ, Zhang YH, Wu J. Protective effects of apocynin and allopurinol on ischemia/reperfusion-induced liver injury in mice. World J Gastroenterol 2008;14:2832e7. [6] Connell BJ, Saleh MC, Khan BV, Saleh TM. Apocynin may limit total cell death following cerebral ischemia and reperfusion by enhancing apoptosis. Food Chem Toxicol 2011;49:3063e9. [7] Stolk J, Hiltermann TJ, Dijkman JH, Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol 1994;11:95e102. [8] Altintas R, Polat A, Vardi N, Oguz F, Beytur A, Sagir M, et al. The protective effects of apocynin on kidney damage caused by renal ischþemia/reperfusion. J Endourol 2013;27:617.
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