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[d -Ala2, d -Leu5] enkephalin (DADLE) protects liver against ischemia-reperfusion injury in the rat
Journal of Surgical Research 114, 72–77 (2003) doi:10.1016/S0022-4804(03)00196-3
[D-Ala 2, D-Leu 5] enkephalin (DADLE) Protects Liver against Ischemia-Reperfusion Injury in the Rat Kousyou Yamanouchi, M.D., 1 Katsuhiko Yanaga, M.D., Ph.D., Sadayuki Okudaira, M.D., Susumu Eguchi, M.D., Ph.D., Junichiro Furui, M.D., Ph.D., and Takashi Kanematsu, M.D., Ph.D. Department of Transplantation and Digestive Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Submitted for publication February 9, 2003
Key Words: liver; ischemia-reperfusion injury; [D-Ala 2, D-Leu 5] enkephalin (DADLE); malondialdehyde (MDA).
Background. [D-Ala 2, D-Leu 5] enkephalin (DADLE) is a synthetic delta class of opioid and is reported to induce hibernation as well as hibernation induction trigger (HIT) in the serum of hibernating mammals. DADLE and HIT have been demonstrated to protect the heart, lung, and jejunum against ischemiareperfusion (I-R) injury. In the present study, we examined the effect of DADLE on I-R injury of the liver in rats. Materials and methods. After administration of DADLE (DADLE group) or normal saline as a vehicle (Control group), partial hepatic ischemia was induced by occluding the vessels supplying 92% of the liver for 45 min, followed by declamping the vessels and resection of the non-ischemic lobe. After 120 min of reperfusion, serum glutamic-pyruvic transaminase (GPT), hyaluronic acid (HA) levels, and concentrations of malondialdehyde (MDA) of the liver tissue were measured. Additionally, bile output from the ischemic lobes was measured after reperfusion. Results. GPT levels were significantly lower in the DADLE group as compared to those of the Control group (P < 0.05), but the serum levels of HA were not different between the two groups. The concentrations of MDA of the liver tissue were significantly lower in the DADLE group than in the Control group (P < 0.01). The bile output after reperfusion was not significantly different between the two groups. Conclusion. DADLE protects against I-R injury in hepatocytes, but not in the sinusoidal endothelial cells of the liver in rats. An anti-oxidative effect is suggested to be responsible for this effect. © 2003 Elsevier Inc.
INTRODUCTION
Hepatic ischemia is often encountered in a number of clinical settings, such as hepatic resection, liver transplantation, and hemorrhagic shock [1, 2]. The reperfusion subjects the ischemic liver to further insults and aggravates ischemic injury, which is referred to as ischemia-reperfusion (I-R) injury [3, 4]. Many investigations have been performed to clarify the pathogenesis of I-R injury of the liver, and various factors have D een suggested to play a role in I-R injury, which includes a complex network of chemical mediators including cytokines [5, 6], reactive oxygen species (ROS) [7], and neutrophils [7, 8]. Hibernation induction trigger (HIT), an 88-kDa peptide, has been demonstrated to exist in the serum of winter hibernating mammals, including ground squirrels, woodchucks, brown cave bats, and black bears [9, 10]. HIT was found to induce hibernation via stimulation of the delta opioid receptors [11]. [D-Ala 2, D-Leu 5] enkephalin (DADLE) is a synthetic delta class of opioid which is also able to induce hibernation [11]. Both HIT and DADLE have been reported to improve the viability of various canine organs preserved en bloc in multiorgan autoperfusion systems at 32°C [12, 13]. Since then, DADLE as well as HIT has been demonstrated to protect various organs against I-R [14 –20]. Several mechanisms for the attenuation of I-R injury by HIT and DADLE have been proposed, which include promoting the intracellular signaling pathways [17, 21, 22], and suppressing the activation and adhesion of neutrophils [23].
All rights reserved.
1 To whom correspondence and reprint requests should be addressed at Department of Transplantation and Digestive Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 852-8501, Japan. E-mail: [email protected].
0022-4804/03 $35.00 © 2003 Elsevier Inc. All rights reserved.
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YAMANOUCHI ET AL.: EFFECT OF DADLE ON HEPATIC ISCHEMIA-REPERFUSION
In the present study, the effect of DADLE on normothermic I-R injury of the rat liver was investigated. MATERIALS AND METHODS Animal Experiments Male Wistar rats (SLC, Shizuoka, Japan) weighing 200 to 250 g were used in this study. The animals were housed in a temperaturecontrolled environment with a 12-h light/dark cycle, and were given standard rat chow and water ad libitum. All animals were fasted overnight before the experiments, but were allowed free access to water. All experiments were performed according to the Guidelines for Animal Experimentation at Nagasaki University. Each experiment was started between 8:00 and 12:00 a.m. to avoid the effect of diurnal variation. After the animals were anesthetized with ether inhalation for induction and intraperitoneal injections of pentobarbital sodium (50 mg/kg body weight) for maintenance, a midline laparotomy was made and the liver was skeletonized. To determine the dose of DADLE (Sigma, St. Louis, MO), we performed a preliminary experiment, in which DADLE at a dose of 1, 3, 5, 10, and 20 mg/kg body weight (n ⫽ 5 each) or vehicle only (control, n ⫽ 5) was given 15 min before induction of hepatic ischemia [22]. At 120 min after reperfusion, the serum glutamic-pyruvic transaminase (GPT) values were as follows: 483.2 ⫾ 180, 338.2 ⫾ 93, 345.6 ⫾ 53, 520.2 ⫾ 216, 404.8 ⫾ 196, and 500.6 ⫾ 196 IU/l. Since 5 mg/kg body weight of DADLE exhibited significantly lower values of serum GPT than those of the control (P ⬍ 0.05), this dose was chosen for the experiments. The animals were divided into two groups. DADLE at a dose of 5 mg/kg body weight (DADLE group) or normal saline as a vehicle (Control group) was administered intravenously into the inferior vena cava. Fifteen minutes later, partial hepatic ischemia was induced by occluding the inflow vessels, i.e., the hepatic arteries and the portal veins, supplying 92% of the liver, i.e., the left lateral, median, and right lateral lobes with small vascular clamps. Splanchnic congestion was not observed grossly during the ischemia period due to portal decompression through the caudate lobe. After 45 min of ischemia, the vessels were reperfused and the non-ischemic lobe (caudate lobe) was then resected, leaving only ischemic lobes in place. After the operation, access to water and rat chow was unlimited. Blood and liver samples for analysis were collected and the animals were killed at 120 min after reperfusion. Blood samples were drawn from the inferior vena cava and centrifuged to obtain the serum. The liver specimens were immersed in liquid nitrogen immediately after sampling. The serum and liver samples were stored at – 80 degrees C until analysis. The remaining liver samples were fixed with buffered formalin for histological examinations.
Biochemical Analysis The GPT was determined using commercially available kits (Wako Pure Chemicals, Osaka, Japan). The serum hyaluronic acid (HA) levels were measured as a marker of sinusoidal endothelial cell damage with a sandwich binding protein assay kit (Chugai Diagnostics Science CO., LTD, Tokyo, Japan).
Levels of Lipid Peroxidation in the Liver Tissue The levels of malondialdehyde (MDA) in the liver tissue were estimated as an index of lipid peroxidation by the thiobarbituric acid method described by Uchiyama and Mihara [24]. The tissue levels of protein were determined by the method of Lowry et al. [25].
Bile Output In separate experiments, the bile output was measured from the ischemic lobes after reperfusion for 120 min through a choledochotomy tube under general anesthesia.
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Histological Examination Formalin-fixed specimens were embedded in paraffin. Sections of 5 m thickness were stained with hematoxylin and eosin and examined histologically. The numbers of neutrophils infiltrating into the liver were counted under 20 high-power fields (HPF, ⫻400), selected at random in each section.
Statistical Analysis The results were expressed as a mean ⫾ standard deviation (SD). The Mann–Whitney’s U test was used for the comparison of the two groups. A P value less than 0.05 was considered statistically significant.
RESULTS Biochemical Analysis
After 120 min of reperfusion, the serum GPT levels were significantly lower in the DADLE group than in the Control group (P ⬍ 0.05). However, the serum HA levels were not different between the two groups (Fig. 1). Levels of Lipid Peroxidation in the Liver Tissue Figure 2 shows the levels of MDA in the liver tissue 120 min after reperfusion. In the DADLE group, the value was significantly lower than that of the Control group (P ⬍ 0.01). Bile Output There were no differences in bile output for 120 min after reperfusion between the two groups (Fig. 3). Histological Examination At 120 min after reperfusion, the liver of the DADLE group exhibited much less sinusoidal dilation or structural derangement around the central vein as compared to the Control group (Fig. 4). The accumulation of neutrophils in the sinusoid was more prominent in the livers of the Control group than those of the DADLE group, although there were no statistical differences (Fig. 5). DISCUSSION
I-R is an inevitable process in resection and transplantation of the liver. The mechanisms of hepatic I-R injury have not been completely clarified, and are considered multifactorial [1–3]. ROS is one of the important mediators responsible for tissue damage after ischemia, and the Kupffer cells are regarded as one of the major sources of ROS [7]. In addition, the production and release of pro-inflammatory cytokines from the Kupffer cells, such as tumor necrosis factor alpha and interleukin 1 beta, lead to the expression of adhesion molecules on endothelial cells and neutrophils [1,
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FIG. 1. Serum GPT and HA levels at 120 min after reperfusion. The serum GPT levels of the DADLE group were significantly lower than those of the Control group (P ⬍ 0.05). However, the serum HA levels were not different between the two groups (n ⫽ 10 each). GPT, glutamic-pyruvic transaminase; HA, hyaluronic acid.
5, 6]. Consequently, neutrophils infiltrate into the hepatic parenchyma and injure hepatocytes by releasing proteases and ROS [7, 8]. Oxygen radical scavengers such as superoxide dismutase (SOD) and glutathione (GSH) were demonstrated to suppress I-R injury of the rat liver [26, 27]. Suzuki et al. [8] reported that the administration of tacrolimus reduced the number of infiltrating neutrophils and oxidative damage, i.e., lipid peroxidation, in the liver tissue, which was connected to improved survival after I-R injury.
Hibernation is a fascinating phenomenon in which various physiological changes occur, including hypothermia, bradycardia, respiratory depression, and generalized metabolic suppression. It is amazing that no organs of animals aroused from hibernation exhibit signs of injury due to I-R after hibernation, which usually lasts several months in a hypoxic condition [12]. Thus, HIT and even DADLE have been considered to possess the ability to improve organ survival. Chien et al. [12, 13] reported that HIT and DADLE extended the survival time of canine en block organs, which consisted of heart, lungs, jejunum, liver, and kidneys, preserved in a multiorgan autoperfusion system. In this setting, the possible protective mechanisms of HIT and DADLE for various organs are proposed as follows: 1) reduced tissue metabolism, 2) decreased or elimi-
FIG. 2. MDA in the liver tissue at 120 min after reperfusion. The values of the DADLE group were significantly lower than those of the Control group (P ⬍ 0.01, n ⫽ 10 each). MDA, malondialdehyde.
FIG. 3. Bile output from the ischemic lobes for 120 min after reperfusion. There were no differences in bile output between the two groups at each time point. Control, 䊐; DADLE, f.
YAMANOUCHI ET AL.: EFFECT OF DADLE ON HEPATIC ISCHEMIA-REPERFUSION
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FIG. 4. Light micrographs of the liver at 120 min after reperfusion. (a) Control group, sinusoidal dilation and structural derangement around the central vein were observed. (b) DADLE group, such changes were suppressed.
nated liver congestion, 3) improved tissue perfusion, and 4) reduced platelet/leukocyte aggregation. DADLE has also been demonstrated to protect from I-R injury of the central nervous system [14] and various organs, such as the heart [15–18], the lung [19], and the jejunum [20]. In particular, many investigators have reported the beneficial effect of DADLE in reducing both normothermic and cold I-R injury of the heart. In addition to DADLE, morphine has been demonstrated to reduce the I-R injury of the heart [17, 28]. Although morphine is primarily a mu class of opioid, morphine-induced cardioprotection has been demonstrated to be the result of activation of the delta opioid receptors, but not the mu opioid receptors; namely, crosstalk between the mu and delta opioid receptors could occur [17, 29]. Cardioprotection via the stimulation of the delta opioid receptors has been demon-
FIG. 5. Neutrophils in the liver tissue at 120 min after reperfusion. Infiltration of the neutrophils in the liver parenchyma of the Control group was more prominent than in the DADLE group, but a statistically significant difference was not achieved (n ⫽ 8 each).
strated to participate in intracellular signaling pathways via G proteins and protein kinase C (PKC), followed by activation of membranous ATP dependent potassium (K ATP) channels [21, 22] and mitogenactivated protein kinase (MAPK) [30]. Recently, another mechanism has been reported. Wang and coworkers [23] demonstrated the cardioprotective effect of morphine by attenuating the expression of adhesion molecules of endothelial cells, and, consequently, suppressing the activation and infiltration of neutrophils. In the present study, we demonstrated that pretreatment of DADLE significantly decreased serum GPT levels, which indicated that DADLE has a protective effect on hepatocytes against I-R injury. However, DADLE did not affect I-R injury of the sinusoidal endothelial cells assessed by serum HA levels after reperfusion. We also showed that the reduction of lipid peroxidation as expressed by tissue MDA levels might contribute to hepatocyte protection after I-R. Contrary to the report for the heart [23], hepatic I-R injury was not associated with the reduction of neutrophil infiltration by pre-treatment of DADLE. This suggests that lipid peroxidation is not due to ROS from the neutrophils infiltrating the liver, but is caused by other mechanisms. For instance, DADLE might have the ability to decrease the generation of ROS from other elements, such as the Kupffer cells, or to make hepatocytes acquire a tolerance to ROS. In isolated rat hepatocytes, an intracellular signaling pathway involving G proteins, PKC, and p38 MAPK is proposed to be responsible for protecting the hepatocyte from hypoxia [31]. The genes for the delta opioid receptors are expressed in rat livers [32], and delta opioid receptors are coupled with G proteins [33]. Therefore, stimulation of the delta opioid receptors might induce the activation of the signaling pathway, and consequently hepatocytes acquire an ability to maintain integrity of the cell membrane. During cardiac ischemia, DADLE is suggested to preserve adenosine 5⬘-triphosphate (ATP) [16]. Bile
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production after ischemia is well associated with ATP levels in the liver tissue [34]. In the present study, bile output from the ischemic liver during 120 min after reperfusion was not different between the two groups, suggesting that DADLE-induced protection of the liver from I-R injury might be independent of preservation of ATP or rapid recovery of hepatic ATP. In our preliminary experiment, the protective effect of DADLE on hepatocytes was not dose-dependent. In an isolated rat heart model, Aitchison et al. [18] reported that, while DADLE at a low concentration decreased infarct size via stimulation of delta opioid receptors, DADLE at a high concentration, however, was demonstrated to increase infarct size. The absence of dose-dependent cardioprotective effect of DADLE may be related to the fact that DADLE at a high concentration also binds to kappa opioid receptors. That same mechanism may apply to the effect of DADLE on the liver. In conclusion, pre-treatment of DADLE ameliorated hepatocyte injury but not sinusoidal cell injury early after reperfusion. Our results suggest that an antioxidative ability might be responsible for this effect.
opioid receptor ligand selectively induced hibernation in summer-active ground squirrels. Life Sci. 43: 1565, 1988. 12.
Chien, S., Oeltgen, P. R., Diana, J. N., Shi, X., Nilekani, S. P., and Salley, R. Two-day preservation of major organs with autoperfusion multiorgan preparation and hibernation induction trigger. J. Thorac. Cardiovasc. Surg. 102: 224, 1991.
13.
Chien, S., Oeltgen, P. R., Diana, J. N., and Salley, R. K. Extension of tissue survival time in multiorgan block preparation with a delta opioid DADLE ([D-Ala 2, D-Leu 5] enkephalin). J. Thorac. Cardiovasc. Surg. 107: 964, 1994.
14.
Borlongan, C. V., Su, T. P., and Wang, Y. Treatment with delta opioid peptide enhances in vitro and in vivo survival of rat dopaminergic neurons. Neuroreport 11: 923, 2000.
15.
Bolling, S. F., Tramontini, N. L., Kilgore, K. S., Su, T. P., Oeltgen, P. R., and Harlow, H. H. Use of “natural” hibernation induction triggers for myocardial protection. Ann. Thorac. Surg. 64: 623, 1997.
16.
Bolling, S. F., Su, T. P., Childs, K. F., Ning, X. H., Horton, N., Kilgore, K., and Oeltgen, P. R. The use of hibernation induction triggers for cardiac transplant preservation. Transplantation 63: 326, 1997.
17.
Schultz, J. J., Hsu, A. K., and Gross, G. J. Ischemic preconditioning and morphine-induced cardioprotection involve the delta (␦)-opioid receptor in the intact rat heart. J. Mol. Cell. Cardiol. 29: 2187, 1997.
18.
Aitchison, K. A., Baxter, G. F., Awan, M. M., Smith, R. M., Yellon, D. M., and Opie, L. H. Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning. Basic. Res. Cardiol. 95: 1, 2000.
19.
Wu, G., Zhang, F., Salley, R. K., Diana, J. N., Su, T. P., and Chien, S. F. Delta opioid extends hypothermic preservation time of the lung. J. Thorac. Cardiovasc. Surg. 111: 259, 1996.
20.
Tubbs, R. J., Porcaro, W. A., Lee, W. J., Blehar, D. J., Carraway, R. E., Przyklenk, K., and Dickson, E. W. Delta opiates increase ischemic tolerance in isolated rabbit jejunum. Acad. Emerg. Med. 9: 555, 2002.
21.
Schultz, Jel-J, Hsu, A. K., Nagase, H., and Gross, G. J. TAN-67, a delta 1-opioid receptor agonist, reduces infarct size via activation of G i/o proteins and K ATP channels. Am. J. Physiol. 274: H909, 1998.
22.
Fryer, R. M., Wang, Y., Hsu, A. K., and Gross, G. J. Essential activation of PKC-delta in opioid-initiated cardioprotection. Am. J. Physiol. Heart. Circ. Physiol. 280: H1346, 2001.
23.
Wang, T. L., Chang, H., Hung, C. R., and Tseng, Y. Z. Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction. Cardiovasc. Res. 40: 557, 1998.
24.
Mihara, M., and Uchiyama, M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Ann. Biochem. 86: 271, 1978.
REFERENCES 1.
Lentsch, A. B., Kato, A., Yoshidome, H., McMasters, K. M., and Edwards, M. J. Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury. Hepatology 32: 169, 2000.
2.
Bilzer, M., and Gerbes, A. L. Preservation injury of the liver: mechanisms and novel therapeutic strategies. J. Hepatol. 32: 508, 2000.
3.
Serracino-Inglott, F., Habib, N. A., and Mathie, R. T. Hepatic ischemia-reperfusion injury. Am. J. Surg. 181: 160, 2001.
4.
Bilzer, M., Witthaut, R., Paumgartner, G., and Gerbes, A. L. Prevention of ischemia/reperfusion injury in the rat liver by atrium natriuretic peptide. Gastroenterology 106: 143, 1994.
5.
Wanner, G. A., Ertel, W., Muller, P., Hofer, Y., Leiderer, R., Menger, M. D., and Messmer, K. Liver ischemia and reperfusion induces a systemic inflammatory response through Kupffer cell activation. Shock 5: 34, 1996.
6.
Colletti, L. M., Kunkel, S. L., Walz, A., Burdick, M. D., Kunkel, R. G., Wilke, C. A., and Strieter, R. M. The role of cytokine networks in the local liver injury following hepatic ischemia/ reperfusion in the rat. Hepatology 23: 506, 1996.
7.
Jaeschke, H., and Farhood, A. Neutrophil and Kupffer cellinduced oxidant stress and ischemia-reperfusion injury in rat liver. Am. J. Physiol. 260: G355, 1991.
25.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurements with folin phenol reagent. J. Biol. Chem. 193: 265, 1951.
8.
Suzuki, S., Toledo-Pereyra, L. H., Rodriguez, F. J., and Cejalvo, D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulation effects of FK506 and cyclosporine. Transplantation 55: 1265, 1993.
26.
Kondo, S., Segawa, T., Tanaka, K., Izawa, K., Hashida, M., and Kanematsu, T. Mannosylated superoxide dismutase inhibits hepatic reperfusion injury in rats. J. Surg. Res. 60: 36, 1996.
27.
9.
Dawe, A. R., and Spurrier, W. A. Hibernation induced in ground squirrels by blood transfusion. Science 163: 298, 1969.
Bilzer, M., Paumgartner, G., and Gerbes, A. L. Glutathione protects the rat liver against reperfusion injury after hypothermic preservation. Gastroenterology 117: 200, 1999.
10.
Oeltgen, P. R., Bergmann, L. C., Spurrier, W. A., and Jones, S. B. Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks. Prep. Biochem. 8: 171, 1978.
28.
11.
Oeltgen, P. R., Nilekani, S. P., Nuchols, P. A., Spurrier, W. A., and Su, T. P. Further studies on opioids and hibernation: delta
Schultz, J. E., Hsu, A. K., and Gross, G. J. Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ. Res. 78: 1100, 1996.
29.
Traynor, J. R., and Elliott, J. Delta-opioid receptor subtypes
YAMANOUCHI ET AL.: EFFECT OF DADLE ON HEPATIC ISCHEMIA-REPERFUSION and cross-talk with mu-receptors. Trends. Pharmacol. Sci. 14: 84, 1993. 30. Gutstein, H. B., Rubie, E. A., Mansour, A., Akil, H., and Woodgett, J. R. Opioid effects on mitogen-activated protein kinase signaling cascades. Anesthesiology 87: 1118, 1997. 31. Carini, R., De Cesaris, M. G., Splendore, R., Vay, D., Domenicotti, C., Nitti, M. P., Paola, D., Pronzato, M. A., and Albano, E. Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes. Hepatology 33: 131, 2001. 32. Wittert, G., Hope, P., and Pyle, D. Tissue distribution of opioid
77
receptor gene expression in the rat. Biochem. Biophys. Res. Commun. 218: 877, 1996. 33.
Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C. G. The delta opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc. Natl. Acad. Sci. USA 89: 12048, 1992.
34.
Kamiike, W., Nakahara, M., Nakao, K., Koseki, M., Nishida, T., Kawashima, Y., Watanabe, F., and Tagawa, K. Correlation between cellular ATP level and bile excretion in the rat liver. Transplantation 39: 50, 1985.
[D-Ala 2, D-Leu 5] enkephalin (DADLE) Protects Liver against Ischemia-Reperfusion Injury in the Rat Kousyou Yamanouchi, M.D., 1 Katsuhiko Yanaga, M.D., Ph.D., Sadayuki Okudaira, M.D., Susumu Eguchi, M.D., Ph.D., Junichiro Furui, M.D., Ph.D., and Takashi Kanematsu, M.D., Ph.D. Department of Transplantation and Digestive Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Submitted for publication February 9, 2003
Key Words: liver; ischemia-reperfusion injury; [D-Ala 2, D-Leu 5] enkephalin (DADLE); malondialdehyde (MDA).
Background. [D-Ala 2, D-Leu 5] enkephalin (DADLE) is a synthetic delta class of opioid and is reported to induce hibernation as well as hibernation induction trigger (HIT) in the serum of hibernating mammals. DADLE and HIT have been demonstrated to protect the heart, lung, and jejunum against ischemiareperfusion (I-R) injury. In the present study, we examined the effect of DADLE on I-R injury of the liver in rats. Materials and methods. After administration of DADLE (DADLE group) or normal saline as a vehicle (Control group), partial hepatic ischemia was induced by occluding the vessels supplying 92% of the liver for 45 min, followed by declamping the vessels and resection of the non-ischemic lobe. After 120 min of reperfusion, serum glutamic-pyruvic transaminase (GPT), hyaluronic acid (HA) levels, and concentrations of malondialdehyde (MDA) of the liver tissue were measured. Additionally, bile output from the ischemic lobes was measured after reperfusion. Results. GPT levels were significantly lower in the DADLE group as compared to those of the Control group (P < 0.05), but the serum levels of HA were not different between the two groups. The concentrations of MDA of the liver tissue were significantly lower in the DADLE group than in the Control group (P < 0.01). The bile output after reperfusion was not significantly different between the two groups. Conclusion. DADLE protects against I-R injury in hepatocytes, but not in the sinusoidal endothelial cells of the liver in rats. An anti-oxidative effect is suggested to be responsible for this effect. © 2003 Elsevier Inc.
INTRODUCTION
Hepatic ischemia is often encountered in a number of clinical settings, such as hepatic resection, liver transplantation, and hemorrhagic shock [1, 2]. The reperfusion subjects the ischemic liver to further insults and aggravates ischemic injury, which is referred to as ischemia-reperfusion (I-R) injury [3, 4]. Many investigations have been performed to clarify the pathogenesis of I-R injury of the liver, and various factors have D een suggested to play a role in I-R injury, which includes a complex network of chemical mediators including cytokines [5, 6], reactive oxygen species (ROS) [7], and neutrophils [7, 8]. Hibernation induction trigger (HIT), an 88-kDa peptide, has been demonstrated to exist in the serum of winter hibernating mammals, including ground squirrels, woodchucks, brown cave bats, and black bears [9, 10]. HIT was found to induce hibernation via stimulation of the delta opioid receptors [11]. [D-Ala 2, D-Leu 5] enkephalin (DADLE) is a synthetic delta class of opioid which is also able to induce hibernation [11]. Both HIT and DADLE have been reported to improve the viability of various canine organs preserved en bloc in multiorgan autoperfusion systems at 32°C [12, 13]. Since then, DADLE as well as HIT has been demonstrated to protect various organs against I-R [14 –20]. Several mechanisms for the attenuation of I-R injury by HIT and DADLE have been proposed, which include promoting the intracellular signaling pathways [17, 21, 22], and suppressing the activation and adhesion of neutrophils [23].
All rights reserved.
1 To whom correspondence and reprint requests should be addressed at Department of Transplantation and Digestive Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 852-8501, Japan. E-mail: [email protected].
0022-4804/03 $35.00 © 2003 Elsevier Inc. All rights reserved.
72
YAMANOUCHI ET AL.: EFFECT OF DADLE ON HEPATIC ISCHEMIA-REPERFUSION
In the present study, the effect of DADLE on normothermic I-R injury of the rat liver was investigated. MATERIALS AND METHODS Animal Experiments Male Wistar rats (SLC, Shizuoka, Japan) weighing 200 to 250 g were used in this study. The animals were housed in a temperaturecontrolled environment with a 12-h light/dark cycle, and were given standard rat chow and water ad libitum. All animals were fasted overnight before the experiments, but were allowed free access to water. All experiments were performed according to the Guidelines for Animal Experimentation at Nagasaki University. Each experiment was started between 8:00 and 12:00 a.m. to avoid the effect of diurnal variation. After the animals were anesthetized with ether inhalation for induction and intraperitoneal injections of pentobarbital sodium (50 mg/kg body weight) for maintenance, a midline laparotomy was made and the liver was skeletonized. To determine the dose of DADLE (Sigma, St. Louis, MO), we performed a preliminary experiment, in which DADLE at a dose of 1, 3, 5, 10, and 20 mg/kg body weight (n ⫽ 5 each) or vehicle only (control, n ⫽ 5) was given 15 min before induction of hepatic ischemia [22]. At 120 min after reperfusion, the serum glutamic-pyruvic transaminase (GPT) values were as follows: 483.2 ⫾ 180, 338.2 ⫾ 93, 345.6 ⫾ 53, 520.2 ⫾ 216, 404.8 ⫾ 196, and 500.6 ⫾ 196 IU/l. Since 5 mg/kg body weight of DADLE exhibited significantly lower values of serum GPT than those of the control (P ⬍ 0.05), this dose was chosen for the experiments. The animals were divided into two groups. DADLE at a dose of 5 mg/kg body weight (DADLE group) or normal saline as a vehicle (Control group) was administered intravenously into the inferior vena cava. Fifteen minutes later, partial hepatic ischemia was induced by occluding the inflow vessels, i.e., the hepatic arteries and the portal veins, supplying 92% of the liver, i.e., the left lateral, median, and right lateral lobes with small vascular clamps. Splanchnic congestion was not observed grossly during the ischemia period due to portal decompression through the caudate lobe. After 45 min of ischemia, the vessels were reperfused and the non-ischemic lobe (caudate lobe) was then resected, leaving only ischemic lobes in place. After the operation, access to water and rat chow was unlimited. Blood and liver samples for analysis were collected and the animals were killed at 120 min after reperfusion. Blood samples were drawn from the inferior vena cava and centrifuged to obtain the serum. The liver specimens were immersed in liquid nitrogen immediately after sampling. The serum and liver samples were stored at – 80 degrees C until analysis. The remaining liver samples were fixed with buffered formalin for histological examinations.
Biochemical Analysis The GPT was determined using commercially available kits (Wako Pure Chemicals, Osaka, Japan). The serum hyaluronic acid (HA) levels were measured as a marker of sinusoidal endothelial cell damage with a sandwich binding protein assay kit (Chugai Diagnostics Science CO., LTD, Tokyo, Japan).
Levels of Lipid Peroxidation in the Liver Tissue The levels of malondialdehyde (MDA) in the liver tissue were estimated as an index of lipid peroxidation by the thiobarbituric acid method described by Uchiyama and Mihara [24]. The tissue levels of protein were determined by the method of Lowry et al. [25].
Bile Output In separate experiments, the bile output was measured from the ischemic lobes after reperfusion for 120 min through a choledochotomy tube under general anesthesia.
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Histological Examination Formalin-fixed specimens were embedded in paraffin. Sections of 5 m thickness were stained with hematoxylin and eosin and examined histologically. The numbers of neutrophils infiltrating into the liver were counted under 20 high-power fields (HPF, ⫻400), selected at random in each section.
Statistical Analysis The results were expressed as a mean ⫾ standard deviation (SD). The Mann–Whitney’s U test was used for the comparison of the two groups. A P value less than 0.05 was considered statistically significant.
RESULTS Biochemical Analysis
After 120 min of reperfusion, the serum GPT levels were significantly lower in the DADLE group than in the Control group (P ⬍ 0.05). However, the serum HA levels were not different between the two groups (Fig. 1). Levels of Lipid Peroxidation in the Liver Tissue Figure 2 shows the levels of MDA in the liver tissue 120 min after reperfusion. In the DADLE group, the value was significantly lower than that of the Control group (P ⬍ 0.01). Bile Output There were no differences in bile output for 120 min after reperfusion between the two groups (Fig. 3). Histological Examination At 120 min after reperfusion, the liver of the DADLE group exhibited much less sinusoidal dilation or structural derangement around the central vein as compared to the Control group (Fig. 4). The accumulation of neutrophils in the sinusoid was more prominent in the livers of the Control group than those of the DADLE group, although there were no statistical differences (Fig. 5). DISCUSSION
I-R is an inevitable process in resection and transplantation of the liver. The mechanisms of hepatic I-R injury have not been completely clarified, and are considered multifactorial [1–3]. ROS is one of the important mediators responsible for tissue damage after ischemia, and the Kupffer cells are regarded as one of the major sources of ROS [7]. In addition, the production and release of pro-inflammatory cytokines from the Kupffer cells, such as tumor necrosis factor alpha and interleukin 1 beta, lead to the expression of adhesion molecules on endothelial cells and neutrophils [1,
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FIG. 1. Serum GPT and HA levels at 120 min after reperfusion. The serum GPT levels of the DADLE group were significantly lower than those of the Control group (P ⬍ 0.05). However, the serum HA levels were not different between the two groups (n ⫽ 10 each). GPT, glutamic-pyruvic transaminase; HA, hyaluronic acid.
5, 6]. Consequently, neutrophils infiltrate into the hepatic parenchyma and injure hepatocytes by releasing proteases and ROS [7, 8]. Oxygen radical scavengers such as superoxide dismutase (SOD) and glutathione (GSH) were demonstrated to suppress I-R injury of the rat liver [26, 27]. Suzuki et al. [8] reported that the administration of tacrolimus reduced the number of infiltrating neutrophils and oxidative damage, i.e., lipid peroxidation, in the liver tissue, which was connected to improved survival after I-R injury.
Hibernation is a fascinating phenomenon in which various physiological changes occur, including hypothermia, bradycardia, respiratory depression, and generalized metabolic suppression. It is amazing that no organs of animals aroused from hibernation exhibit signs of injury due to I-R after hibernation, which usually lasts several months in a hypoxic condition [12]. Thus, HIT and even DADLE have been considered to possess the ability to improve organ survival. Chien et al. [12, 13] reported that HIT and DADLE extended the survival time of canine en block organs, which consisted of heart, lungs, jejunum, liver, and kidneys, preserved in a multiorgan autoperfusion system. In this setting, the possible protective mechanisms of HIT and DADLE for various organs are proposed as follows: 1) reduced tissue metabolism, 2) decreased or elimi-
FIG. 2. MDA in the liver tissue at 120 min after reperfusion. The values of the DADLE group were significantly lower than those of the Control group (P ⬍ 0.01, n ⫽ 10 each). MDA, malondialdehyde.
FIG. 3. Bile output from the ischemic lobes for 120 min after reperfusion. There were no differences in bile output between the two groups at each time point. Control, 䊐; DADLE, f.
YAMANOUCHI ET AL.: EFFECT OF DADLE ON HEPATIC ISCHEMIA-REPERFUSION
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FIG. 4. Light micrographs of the liver at 120 min after reperfusion. (a) Control group, sinusoidal dilation and structural derangement around the central vein were observed. (b) DADLE group, such changes were suppressed.
nated liver congestion, 3) improved tissue perfusion, and 4) reduced platelet/leukocyte aggregation. DADLE has also been demonstrated to protect from I-R injury of the central nervous system [14] and various organs, such as the heart [15–18], the lung [19], and the jejunum [20]. In particular, many investigators have reported the beneficial effect of DADLE in reducing both normothermic and cold I-R injury of the heart. In addition to DADLE, morphine has been demonstrated to reduce the I-R injury of the heart [17, 28]. Although morphine is primarily a mu class of opioid, morphine-induced cardioprotection has been demonstrated to be the result of activation of the delta opioid receptors, but not the mu opioid receptors; namely, crosstalk between the mu and delta opioid receptors could occur [17, 29]. Cardioprotection via the stimulation of the delta opioid receptors has been demon-
FIG. 5. Neutrophils in the liver tissue at 120 min after reperfusion. Infiltration of the neutrophils in the liver parenchyma of the Control group was more prominent than in the DADLE group, but a statistically significant difference was not achieved (n ⫽ 8 each).
strated to participate in intracellular signaling pathways via G proteins and protein kinase C (PKC), followed by activation of membranous ATP dependent potassium (K ATP) channels [21, 22] and mitogenactivated protein kinase (MAPK) [30]. Recently, another mechanism has been reported. Wang and coworkers [23] demonstrated the cardioprotective effect of morphine by attenuating the expression of adhesion molecules of endothelial cells, and, consequently, suppressing the activation and infiltration of neutrophils. In the present study, we demonstrated that pretreatment of DADLE significantly decreased serum GPT levels, which indicated that DADLE has a protective effect on hepatocytes against I-R injury. However, DADLE did not affect I-R injury of the sinusoidal endothelial cells assessed by serum HA levels after reperfusion. We also showed that the reduction of lipid peroxidation as expressed by tissue MDA levels might contribute to hepatocyte protection after I-R. Contrary to the report for the heart [23], hepatic I-R injury was not associated with the reduction of neutrophil infiltration by pre-treatment of DADLE. This suggests that lipid peroxidation is not due to ROS from the neutrophils infiltrating the liver, but is caused by other mechanisms. For instance, DADLE might have the ability to decrease the generation of ROS from other elements, such as the Kupffer cells, or to make hepatocytes acquire a tolerance to ROS. In isolated rat hepatocytes, an intracellular signaling pathway involving G proteins, PKC, and p38 MAPK is proposed to be responsible for protecting the hepatocyte from hypoxia [31]. The genes for the delta opioid receptors are expressed in rat livers [32], and delta opioid receptors are coupled with G proteins [33]. Therefore, stimulation of the delta opioid receptors might induce the activation of the signaling pathway, and consequently hepatocytes acquire an ability to maintain integrity of the cell membrane. During cardiac ischemia, DADLE is suggested to preserve adenosine 5⬘-triphosphate (ATP) [16]. Bile
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production after ischemia is well associated with ATP levels in the liver tissue [34]. In the present study, bile output from the ischemic liver during 120 min after reperfusion was not different between the two groups, suggesting that DADLE-induced protection of the liver from I-R injury might be independent of preservation of ATP or rapid recovery of hepatic ATP. In our preliminary experiment, the protective effect of DADLE on hepatocytes was not dose-dependent. In an isolated rat heart model, Aitchison et al. [18] reported that, while DADLE at a low concentration decreased infarct size via stimulation of delta opioid receptors, DADLE at a high concentration, however, was demonstrated to increase infarct size. The absence of dose-dependent cardioprotective effect of DADLE may be related to the fact that DADLE at a high concentration also binds to kappa opioid receptors. That same mechanism may apply to the effect of DADLE on the liver. In conclusion, pre-treatment of DADLE ameliorated hepatocyte injury but not sinusoidal cell injury early after reperfusion. Our results suggest that an antioxidative ability might be responsible for this effect.
opioid receptor ligand selectively induced hibernation in summer-active ground squirrels. Life Sci. 43: 1565, 1988. 12.
Chien, S., Oeltgen, P. R., Diana, J. N., Shi, X., Nilekani, S. P., and Salley, R. Two-day preservation of major organs with autoperfusion multiorgan preparation and hibernation induction trigger. J. Thorac. Cardiovasc. Surg. 102: 224, 1991.
13.
Chien, S., Oeltgen, P. R., Diana, J. N., and Salley, R. K. Extension of tissue survival time in multiorgan block preparation with a delta opioid DADLE ([D-Ala 2, D-Leu 5] enkephalin). J. Thorac. Cardiovasc. Surg. 107: 964, 1994.
14.
Borlongan, C. V., Su, T. P., and Wang, Y. Treatment with delta opioid peptide enhances in vitro and in vivo survival of rat dopaminergic neurons. Neuroreport 11: 923, 2000.
15.
Bolling, S. F., Tramontini, N. L., Kilgore, K. S., Su, T. P., Oeltgen, P. R., and Harlow, H. H. Use of “natural” hibernation induction triggers for myocardial protection. Ann. Thorac. Surg. 64: 623, 1997.
16.
Bolling, S. F., Su, T. P., Childs, K. F., Ning, X. H., Horton, N., Kilgore, K., and Oeltgen, P. R. The use of hibernation induction triggers for cardiac transplant preservation. Transplantation 63: 326, 1997.
17.
Schultz, J. J., Hsu, A. K., and Gross, G. J. Ischemic preconditioning and morphine-induced cardioprotection involve the delta (␦)-opioid receptor in the intact rat heart. J. Mol. Cell. Cardiol. 29: 2187, 1997.
18.
Aitchison, K. A., Baxter, G. F., Awan, M. M., Smith, R. M., Yellon, D. M., and Opie, L. H. Opposing effects on infarction of delta and kappa opioid receptor activation in the isolated rat heart: implications for ischemic preconditioning. Basic. Res. Cardiol. 95: 1, 2000.
19.
Wu, G., Zhang, F., Salley, R. K., Diana, J. N., Su, T. P., and Chien, S. F. Delta opioid extends hypothermic preservation time of the lung. J. Thorac. Cardiovasc. Surg. 111: 259, 1996.
20.
Tubbs, R. J., Porcaro, W. A., Lee, W. J., Blehar, D. J., Carraway, R. E., Przyklenk, K., and Dickson, E. W. Delta opiates increase ischemic tolerance in isolated rabbit jejunum. Acad. Emerg. Med. 9: 555, 2002.
21.
Schultz, Jel-J, Hsu, A. K., Nagase, H., and Gross, G. J. TAN-67, a delta 1-opioid receptor agonist, reduces infarct size via activation of G i/o proteins and K ATP channels. Am. J. Physiol. 274: H909, 1998.
22.
Fryer, R. M., Wang, Y., Hsu, A. K., and Gross, G. J. Essential activation of PKC-delta in opioid-initiated cardioprotection. Am. J. Physiol. Heart. Circ. Physiol. 280: H1346, 2001.
23.
Wang, T. L., Chang, H., Hung, C. R., and Tseng, Y. Z. Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction. Cardiovasc. Res. 40: 557, 1998.
24.
Mihara, M., and Uchiyama, M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Ann. Biochem. 86: 271, 1978.
REFERENCES 1.
Lentsch, A. B., Kato, A., Yoshidome, H., McMasters, K. M., and Edwards, M. J. Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury. Hepatology 32: 169, 2000.
2.
Bilzer, M., and Gerbes, A. L. Preservation injury of the liver: mechanisms and novel therapeutic strategies. J. Hepatol. 32: 508, 2000.
3.
Serracino-Inglott, F., Habib, N. A., and Mathie, R. T. Hepatic ischemia-reperfusion injury. Am. J. Surg. 181: 160, 2001.
4.
Bilzer, M., Witthaut, R., Paumgartner, G., and Gerbes, A. L. Prevention of ischemia/reperfusion injury in the rat liver by atrium natriuretic peptide. Gastroenterology 106: 143, 1994.
5.
Wanner, G. A., Ertel, W., Muller, P., Hofer, Y., Leiderer, R., Menger, M. D., and Messmer, K. Liver ischemia and reperfusion induces a systemic inflammatory response through Kupffer cell activation. Shock 5: 34, 1996.
6.
Colletti, L. M., Kunkel, S. L., Walz, A., Burdick, M. D., Kunkel, R. G., Wilke, C. A., and Strieter, R. M. The role of cytokine networks in the local liver injury following hepatic ischemia/ reperfusion in the rat. Hepatology 23: 506, 1996.
7.
Jaeschke, H., and Farhood, A. Neutrophil and Kupffer cellinduced oxidant stress and ischemia-reperfusion injury in rat liver. Am. J. Physiol. 260: G355, 1991.
25.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurements with folin phenol reagent. J. Biol. Chem. 193: 265, 1951.
8.
Suzuki, S., Toledo-Pereyra, L. H., Rodriguez, F. J., and Cejalvo, D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulation effects of FK506 and cyclosporine. Transplantation 55: 1265, 1993.
26.
Kondo, S., Segawa, T., Tanaka, K., Izawa, K., Hashida, M., and Kanematsu, T. Mannosylated superoxide dismutase inhibits hepatic reperfusion injury in rats. J. Surg. Res. 60: 36, 1996.
27.
9.
Dawe, A. R., and Spurrier, W. A. Hibernation induced in ground squirrels by blood transfusion. Science 163: 298, 1969.
Bilzer, M., Paumgartner, G., and Gerbes, A. L. Glutathione protects the rat liver against reperfusion injury after hypothermic preservation. Gastroenterology 117: 200, 1999.
10.
Oeltgen, P. R., Bergmann, L. C., Spurrier, W. A., and Jones, S. B. Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks. Prep. Biochem. 8: 171, 1978.
28.
11.
Oeltgen, P. R., Nilekani, S. P., Nuchols, P. A., Spurrier, W. A., and Su, T. P. Further studies on opioids and hibernation: delta
Schultz, J. E., Hsu, A. K., and Gross, G. J. Morphine mimics the cardioprotective effect of ischemic preconditioning via a glibenclamide-sensitive mechanism in the rat heart. Circ. Res. 78: 1100, 1996.
29.
Traynor, J. R., and Elliott, J. Delta-opioid receptor subtypes
YAMANOUCHI ET AL.: EFFECT OF DADLE ON HEPATIC ISCHEMIA-REPERFUSION and cross-talk with mu-receptors. Trends. Pharmacol. Sci. 14: 84, 1993. 30. Gutstein, H. B., Rubie, E. A., Mansour, A., Akil, H., and Woodgett, J. R. Opioid effects on mitogen-activated protein kinase signaling cascades. Anesthesiology 87: 1118, 1997. 31. Carini, R., De Cesaris, M. G., Splendore, R., Vay, D., Domenicotti, C., Nitti, M. P., Paola, D., Pronzato, M. A., and Albano, E. Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes. Hepatology 33: 131, 2001. 32. Wittert, G., Hope, P., and Pyle, D. Tissue distribution of opioid
77
receptor gene expression in the rat. Biochem. Biophys. Res. Commun. 218: 877, 1996. 33.
Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C. G. The delta opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc. Natl. Acad. Sci. USA 89: 12048, 1992.
34.
Kamiike, W., Nakahara, M., Nakao, K., Koseki, M., Nishida, T., Kawashima, Y., Watanabe, F., and Tagawa, K. Correlation between cellular ATP level and bile excretion in the rat liver. Transplantation 39: 50, 1985.