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Protective effects of herbimycin A on hepatic reperfusion injury
Protective Effects of Herbimycin A on Hepatic Reperfusion Injury T. Hatakeyama, H. Sakai, M. Yamaguchi, H. Shimura, M. Kuzume, T. Matsumoto, A. Matsumiya, Y. Yoshizawa, T. Midorikawa, K. Kumada, and Y. Sanada
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XYGEN-derived free radicals are key mediators of reperfusion injury in liver transplantation, although the involved cellular mechanisms have not been fully defined. Primary targets for free radicals are thought to be the unsaturated bonds in membrane lipids. Consequent peroxidation causes a loss of membrane fluidity and receptor alignment, and this may lead to cellular lysis.1 Free radicals also induce apoptosis in a variety of cell types.2–7 Herbimycin A, a potent tyrosine kinase inhibitor, inhibits the induction of apoptosis in some types of cells.8,9 Therefore, we investigated the effects of herbimycin A on hepatic ischemia-reperfusion injury in vivo and on the radicalinduced injury of hepatocytes in vitro. MATERIALS AND METHODS Hepatic Ischemia-Reperfusion Injury Eighteen male Wistar rats (7 weeks old, weighing 150 to 200 g) were subjected to hepatic ischemia-reperfusion injury. Rats were anesthetized with intraperitoneal administration of sodium pentobarbital (50 mg/kg). Partial hepatic ischemia was performed for 1 hour by partial clamp of hepatic hilar vessels supplying to the cephalad three lobes with or without pretreatment of herbimycin A (0, 0.1, 10 g/kg) (Sigma Chemical, St. Louis, MO). After reper-
fusion for 4 hours, serum levels of liver transaminases, hepatic tissue levels of malondialdehyde, and reduced form of glutathione (GSH) were measured.
Measurement of Serum Liver Transaminase Serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were determined using an automatic JEOLRX40 analyzer (Japan Electron Optic Laboratory, Tokyo, Japan).
Measurement of Hepatic Tissue Levels of Malondialdehyde Hepatic lipid peroxidation caused by ischemia-reperfusion was analyzed by measuring the tissue levels of malondialdehyde with From the Department of Surgery, Showa University Fujigaoka Hospital, Yokohama, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education; by a Grant-in-Aid for Cancer Research from the Ministry of Health Welfare; and by the High-Technology Research Center Project from the Ministry of Education, Science, Sport and Culture of Japan. Address reprint requests to Masahiko Yamaguchi, MD, Department of Surgery, Showa University Fujigaoka Hospital, 1-30, Fujigaoka, Aoba-ku, Yokohama, Japan.
Fig 1. Protective effects of herbimycin A on increased serum levels of AST (A) and ALT (B) at 4 hours of reperfusion after partial hepatic ischemia for 1 hour (n ⫽ 18). Open column indicates sham operation group and hatched columns indicate hepatic ischemia-reperfusion group. *P ⬍ .05 compared with hepatic ischemia-reperfusion without herbimycin A.
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01674-2
Transplantation Proceedings, 32, 2303–2305 (2000)
2303
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HATAKEYAMA, SAKAI, YAMAGUCHI ET AL
Fig 2. Effects of herbimycin A on hepatic tissue levels of malondialdehyde (A) and GSA (B) at 4 hours of reperfusion after partial hepatic ischemia for 1 hour (n ⫽ 18). Open column indicates sham operation group and hatched columns indicate hepatic ischemia-reperfusion group. the thiobarbituric acid method (Lipid Peroxide-Test Wako®, Wako Pure Chemical, Osaka, Japan). The liver specimen taken from the reperfused lobe was washed with cold saline and homogenized with 9 volumes of 1.15% KCl. The homogenate was mixed with 3 mL of 1% phosphate and 1 mL of thiobarbituric acid reagent, and boiled for 45 minutes. After cooling the mixture, 4 mL of n-butanol was added to the sample, then centrifugation was performed at 3000 rpm for 10 minutes. The optical density of the supernatant was determined at 535 nm using spectrophotometer (Hitachi 100-40 spectrophotometer, Hitachi, Tokyo, Japan).
exposed to superoxide anion (O2⫺) generated by addition of hypoxanthine (250 mol/L, Sigma) and xanthine oxidase (100 mU, Sigma), with or without pretreatment of herbimycin A (doses of 0.1 and 1 mol/L) for 90 minutes. Viable hepatocytes were counted
Measurement of Hepatic Tissue Levels of Reduced Form of Glutathione Hepatic oxidative stress by ischemia-reperfusion was analyzed by measuring GSH in liver tissue. Small cubes of the liver tissue taken from 30-minute reperfused lobe, weighing 150 to 200 mg, were subjected to the measurement of the concentrations of GSH using high-performance liquid chromatography (L-7000, Hitachi, Tokyo, Japan) as described previously.10
Hepatocyte Radical Injury Hepatocytes were isolated from male Wistar rats (7 weeks old, weighing 150 to 200 g) by the collagenase perfusion method.11 The liver was perfused with an oxygen-saturated Ca2⫹/Mg2⫹-free Hank’s balanced salt solution containing 0.5 mmol/L EGTA and 10 mmol/L HEPES (pH 7.5) at 37°C via the portal vein at a rate of 15 mL/min for 10 minutes. The liver was then recirculated with oxygen-saturated Ca2⫹/Mg2⫹-free Hank’s balanced salt solution with 0.05% collagenase IV (Wako), 5 mol/L CaCl2 and 10 mmol/L HEPES (pH 7.5) at the same flow rate for 5 minutes. Isolated hepatocytes were suspended at a concentration of 1 ⫻ 105 cells/mL in William’s E medium (ICN Biochemicals, Costa Mesa, CA). Hepatocytes were incubated in Williams’ E medium containing 10% fetal calf serum, 100 mg/mL penicillin, 100 U/mL streptomycin, 25 mg/mL fungizone, 1 ⫻ 106 mol/L dexamethasone at 37°C for 12 hours in collagen-coated 24-well plates and were
Fig 3. Protective effects of herbimycin A on hepatocyte radical injury generated with hypoxanthine and xanthine oxidase (n ⫽ 4). Open column indicates no-treatment hepatocytes and hatched columns indicate hepatic superoxide-treated hepatocytes. *P ⬍ .01 compared with superoxide-treated hepatocytes without herbimycin A.
HERBIMYCIN A AND HEPATIC REPERFUSION with the trypan blue dye exclusion method. Experiments were performed at least in triplicate and repeated at least three times with similar results. Each data point represents the mean ⫾ SE from a representative experiment.
Statistics Results were expressed as mean ⫾ SE, and statistical analysis was carried out with the Mann-Whitney U test. P values less than .05 were considered to be significant.
RESULTS Effects of Herbimycin A on Serum Levels of AST and ALT
Herbimycin A inhibited an increase in the serum levels of AST and ALT in a dose-dependent fashion after 4 hours of reperfusion, especially 10 g/kg of herbimycin A, which significantly inhibited the increased levels of AST (Fig 1). Effects of Herbimycin A on Tissue Malondialdehyde and GSH Levels
Either 0.1 or 10 g/kg of herbimycin A did not decrease the hepatic tissue malondialdehyde levels, which were increased by hepatic ischemia-reperfusion injury (Fig 2A). Pretreatment of herbimycin A could not increase the hepatic tissue GSH levels after hepatic ischemia-reperfusion (Fig 2B). Effects of Herbimycin A on Hepatocyte Radical Injury
Herbimycin A at doses of 0.1 and 1 mol/L significantly decreased superoxide-induced radical injury of hepatocytes (Fig 3). CONCLUSIONS
We have shown that herbimycin A reduced liver transaminase levels increased by hepatic ischemia-reperfusion and prevented hepatocyte injury induced by superoxide, but did not change the hepatic tissue levels of malondialdehyde and GSH. It is suggested that herbimycin A may reduce hepatic ischemia-reperfusion injury via the protection of hepatocyte from radical injury, but not via the prevention of radicalinduced lipid peroxidation and oxidative stress. Antioxidants such as N-acetylcysteine,10 superoxide dismutase, and allopurinol12 decrease hepatic ischemia-reperfusion injury via the prevention of radical-induced lipid peroxidation and/or oxidative stress, and they are used as
2305
the constituents of the preservation solution in liver transplantation. Herbimycin A did not inhibit lipid peroxidation or oxidative stress in the reperfused liver tissue, but reduced the liver damage and hepatocyte injury. It is reported that oxygen radicals can mediate intracellular signaling via tyrosine phosphorylation.9 Herbimycin A has been reported to inhibit the induction of apoptosis,8 which is mediated by intracellular signaling. If the radical-induced cell injury is also mediated by intracellular signaling, it is suggested that herbimycin A, a potent tyrosine kinase inhibitor, may inhibit radical-mediated intracellular signaling via tyrosine phosphorylation. Because herbimycin A reduced hepatic reperfusion injury through the different mechanism from antioxidants, synergistic protective effects may be achieved by the addition of herbimycin A into preservation solution. Our previous study13 demonstrated that herbimycin A also has protective effects on radical-induced injury in human vascular endothelial cells. Protection of vascular endothelial cells in the graft is also important in liver transplantation. Therefore, it is concluded that herbimycin A has protective effect on hepatic reperfusion injury in liver transplantation via protection of both hepatocytes and vascular endothelial cells. REFERENCES 1. Maeda K, Kimura M, Hayashi S: Br J Pharmacol 108:1077, 1993 2. Rao GN, Berk BC: Circ Res 70:593, 1992 3. Buttke TM, Sandstrom PA: Immunol Today 15:7, 1994 4. Ueda N, Shah SV: J Clin Invest 90:2593, 1992 5. Hansson M, Asea A, Ersson U, et al: J Immunol 156:42, 1996 6. Sandstrom PA, Mannie MD, Buttke TM: J Leukoc Biol 55:221, 1994 7. Watson RWG, Redmond HP, Wang JH, et al: J Immunol 156:3986, 1996 8. Khar A, Pardhasaradhi BV, Varalakshmi C, et al: Immunol Lett 46:165, 1995 9. Watson RWG, Rotstein OD, Nathens AB, et al: Surgery 120:150, 1996 10. Nakano H, Boudjema K, Alexandre E, et al: Hepatology 22:539, 1995 11. Nakano H, Monden M, Umeshita K, et al: Surgery 116:883, 1994 12. Matsumoto F, Sakai H, Yamaguchi M, et al: Eur Surg Res 29:429, 1997 13. Shimura H, Yamaguchi M, Kuzume M, et al: Eur Surg Res (in press)
O
XYGEN-derived free radicals are key mediators of reperfusion injury in liver transplantation, although the involved cellular mechanisms have not been fully defined. Primary targets for free radicals are thought to be the unsaturated bonds in membrane lipids. Consequent peroxidation causes a loss of membrane fluidity and receptor alignment, and this may lead to cellular lysis.1 Free radicals also induce apoptosis in a variety of cell types.2–7 Herbimycin A, a potent tyrosine kinase inhibitor, inhibits the induction of apoptosis in some types of cells.8,9 Therefore, we investigated the effects of herbimycin A on hepatic ischemia-reperfusion injury in vivo and on the radicalinduced injury of hepatocytes in vitro. MATERIALS AND METHODS Hepatic Ischemia-Reperfusion Injury Eighteen male Wistar rats (7 weeks old, weighing 150 to 200 g) were subjected to hepatic ischemia-reperfusion injury. Rats were anesthetized with intraperitoneal administration of sodium pentobarbital (50 mg/kg). Partial hepatic ischemia was performed for 1 hour by partial clamp of hepatic hilar vessels supplying to the cephalad three lobes with or without pretreatment of herbimycin A (0, 0.1, 10 g/kg) (Sigma Chemical, St. Louis, MO). After reper-
fusion for 4 hours, serum levels of liver transaminases, hepatic tissue levels of malondialdehyde, and reduced form of glutathione (GSH) were measured.
Measurement of Serum Liver Transaminase Serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were determined using an automatic JEOLRX40 analyzer (Japan Electron Optic Laboratory, Tokyo, Japan).
Measurement of Hepatic Tissue Levels of Malondialdehyde Hepatic lipid peroxidation caused by ischemia-reperfusion was analyzed by measuring the tissue levels of malondialdehyde with From the Department of Surgery, Showa University Fujigaoka Hospital, Yokohama, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education; by a Grant-in-Aid for Cancer Research from the Ministry of Health Welfare; and by the High-Technology Research Center Project from the Ministry of Education, Science, Sport and Culture of Japan. Address reprint requests to Masahiko Yamaguchi, MD, Department of Surgery, Showa University Fujigaoka Hospital, 1-30, Fujigaoka, Aoba-ku, Yokohama, Japan.
Fig 1. Protective effects of herbimycin A on increased serum levels of AST (A) and ALT (B) at 4 hours of reperfusion after partial hepatic ischemia for 1 hour (n ⫽ 18). Open column indicates sham operation group and hatched columns indicate hepatic ischemia-reperfusion group. *P ⬍ .05 compared with hepatic ischemia-reperfusion without herbimycin A.
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/00/$–see front matter PII S0041-1345(00)01674-2
Transplantation Proceedings, 32, 2303–2305 (2000)
2303
2304
HATAKEYAMA, SAKAI, YAMAGUCHI ET AL
Fig 2. Effects of herbimycin A on hepatic tissue levels of malondialdehyde (A) and GSA (B) at 4 hours of reperfusion after partial hepatic ischemia for 1 hour (n ⫽ 18). Open column indicates sham operation group and hatched columns indicate hepatic ischemia-reperfusion group. the thiobarbituric acid method (Lipid Peroxide-Test Wako®, Wako Pure Chemical, Osaka, Japan). The liver specimen taken from the reperfused lobe was washed with cold saline and homogenized with 9 volumes of 1.15% KCl. The homogenate was mixed with 3 mL of 1% phosphate and 1 mL of thiobarbituric acid reagent, and boiled for 45 minutes. After cooling the mixture, 4 mL of n-butanol was added to the sample, then centrifugation was performed at 3000 rpm for 10 minutes. The optical density of the supernatant was determined at 535 nm using spectrophotometer (Hitachi 100-40 spectrophotometer, Hitachi, Tokyo, Japan).
exposed to superoxide anion (O2⫺) generated by addition of hypoxanthine (250 mol/L, Sigma) and xanthine oxidase (100 mU, Sigma), with or without pretreatment of herbimycin A (doses of 0.1 and 1 mol/L) for 90 minutes. Viable hepatocytes were counted
Measurement of Hepatic Tissue Levels of Reduced Form of Glutathione Hepatic oxidative stress by ischemia-reperfusion was analyzed by measuring GSH in liver tissue. Small cubes of the liver tissue taken from 30-minute reperfused lobe, weighing 150 to 200 mg, were subjected to the measurement of the concentrations of GSH using high-performance liquid chromatography (L-7000, Hitachi, Tokyo, Japan) as described previously.10
Hepatocyte Radical Injury Hepatocytes were isolated from male Wistar rats (7 weeks old, weighing 150 to 200 g) by the collagenase perfusion method.11 The liver was perfused with an oxygen-saturated Ca2⫹/Mg2⫹-free Hank’s balanced salt solution containing 0.5 mmol/L EGTA and 10 mmol/L HEPES (pH 7.5) at 37°C via the portal vein at a rate of 15 mL/min for 10 minutes. The liver was then recirculated with oxygen-saturated Ca2⫹/Mg2⫹-free Hank’s balanced salt solution with 0.05% collagenase IV (Wako), 5 mol/L CaCl2 and 10 mmol/L HEPES (pH 7.5) at the same flow rate for 5 minutes. Isolated hepatocytes were suspended at a concentration of 1 ⫻ 105 cells/mL in William’s E medium (ICN Biochemicals, Costa Mesa, CA). Hepatocytes were incubated in Williams’ E medium containing 10% fetal calf serum, 100 mg/mL penicillin, 100 U/mL streptomycin, 25 mg/mL fungizone, 1 ⫻ 106 mol/L dexamethasone at 37°C for 12 hours in collagen-coated 24-well plates and were
Fig 3. Protective effects of herbimycin A on hepatocyte radical injury generated with hypoxanthine and xanthine oxidase (n ⫽ 4). Open column indicates no-treatment hepatocytes and hatched columns indicate hepatic superoxide-treated hepatocytes. *P ⬍ .01 compared with superoxide-treated hepatocytes without herbimycin A.
HERBIMYCIN A AND HEPATIC REPERFUSION with the trypan blue dye exclusion method. Experiments were performed at least in triplicate and repeated at least three times with similar results. Each data point represents the mean ⫾ SE from a representative experiment.
Statistics Results were expressed as mean ⫾ SE, and statistical analysis was carried out with the Mann-Whitney U test. P values less than .05 were considered to be significant.
RESULTS Effects of Herbimycin A on Serum Levels of AST and ALT
Herbimycin A inhibited an increase in the serum levels of AST and ALT in a dose-dependent fashion after 4 hours of reperfusion, especially 10 g/kg of herbimycin A, which significantly inhibited the increased levels of AST (Fig 1). Effects of Herbimycin A on Tissue Malondialdehyde and GSH Levels
Either 0.1 or 10 g/kg of herbimycin A did not decrease the hepatic tissue malondialdehyde levels, which were increased by hepatic ischemia-reperfusion injury (Fig 2A). Pretreatment of herbimycin A could not increase the hepatic tissue GSH levels after hepatic ischemia-reperfusion (Fig 2B). Effects of Herbimycin A on Hepatocyte Radical Injury
Herbimycin A at doses of 0.1 and 1 mol/L significantly decreased superoxide-induced radical injury of hepatocytes (Fig 3). CONCLUSIONS
We have shown that herbimycin A reduced liver transaminase levels increased by hepatic ischemia-reperfusion and prevented hepatocyte injury induced by superoxide, but did not change the hepatic tissue levels of malondialdehyde and GSH. It is suggested that herbimycin A may reduce hepatic ischemia-reperfusion injury via the protection of hepatocyte from radical injury, but not via the prevention of radicalinduced lipid peroxidation and oxidative stress. Antioxidants such as N-acetylcysteine,10 superoxide dismutase, and allopurinol12 decrease hepatic ischemia-reperfusion injury via the prevention of radical-induced lipid peroxidation and/or oxidative stress, and they are used as
2305
the constituents of the preservation solution in liver transplantation. Herbimycin A did not inhibit lipid peroxidation or oxidative stress in the reperfused liver tissue, but reduced the liver damage and hepatocyte injury. It is reported that oxygen radicals can mediate intracellular signaling via tyrosine phosphorylation.9 Herbimycin A has been reported to inhibit the induction of apoptosis,8 which is mediated by intracellular signaling. If the radical-induced cell injury is also mediated by intracellular signaling, it is suggested that herbimycin A, a potent tyrosine kinase inhibitor, may inhibit radical-mediated intracellular signaling via tyrosine phosphorylation. Because herbimycin A reduced hepatic reperfusion injury through the different mechanism from antioxidants, synergistic protective effects may be achieved by the addition of herbimycin A into preservation solution. Our previous study13 demonstrated that herbimycin A also has protective effects on radical-induced injury in human vascular endothelial cells. Protection of vascular endothelial cells in the graft is also important in liver transplantation. Therefore, it is concluded that herbimycin A has protective effect on hepatic reperfusion injury in liver transplantation via protection of both hepatocytes and vascular endothelial cells. REFERENCES 1. Maeda K, Kimura M, Hayashi S: Br J Pharmacol 108:1077, 1993 2. Rao GN, Berk BC: Circ Res 70:593, 1992 3. Buttke TM, Sandstrom PA: Immunol Today 15:7, 1994 4. Ueda N, Shah SV: J Clin Invest 90:2593, 1992 5. Hansson M, Asea A, Ersson U, et al: J Immunol 156:42, 1996 6. Sandstrom PA, Mannie MD, Buttke TM: J Leukoc Biol 55:221, 1994 7. Watson RWG, Redmond HP, Wang JH, et al: J Immunol 156:3986, 1996 8. Khar A, Pardhasaradhi BV, Varalakshmi C, et al: Immunol Lett 46:165, 1995 9. Watson RWG, Rotstein OD, Nathens AB, et al: Surgery 120:150, 1996 10. Nakano H, Boudjema K, Alexandre E, et al: Hepatology 22:539, 1995 11. Nakano H, Monden M, Umeshita K, et al: Surgery 116:883, 1994 12. Matsumoto F, Sakai H, Yamaguchi M, et al: Eur Surg Res 29:429, 1997 13. Shimura H, Yamaguchi M, Kuzume M, et al: Eur Surg Res (in press)