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P38 mitogen-activated protein kinase inhibition attenuates ischemia-reperfusion injury of the rat liver
P38 mitogen-activated protein kinase inhibition attenuates ischemia-reperfusion injury of the rat liver Mitsunobu Kobayashi, MD, Izumi Takeyoshi, MD, Daisuke Yoshinari, MD, Koshi Matsumoto, MD, and Yasuo Morishita, MD, Maebashi and Kawasaki, Japan
Background. Several studies have implicated the mitogen-activated protein kinase (MAPK) signal pathway in non-hepatic organ ischemia-reperfusion injury. However, the role of p38 MAPK in hepatic ischemia-reperfusion injury remains unclear. This study investigated the role of p38 MAPK in hepatic ischemia-reperfusion injury. Methods. Male Sprague-Dawley rats were divided into 4 groups (sham, FR-only, control, and FR-treated groups). The animals in the control and FR-treated groups were subjected to 30 minutes of warm ischemia with congestion of the gut. The FR-only and FR-treated groups received FR167653 (FR), which is a novel p38 MAPK inhibitor. The serum levels of aspartate transaminase, alanine transaminase, lactate dehydrogenase, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were measured (each, n = 6). Liver tissue blood flow was measured at pre-ischemia, end-ischemia, and 30, 60, 90, and 120 minutes after reperfusion (each, n = 4). The liver tissues in the control and FR-treated groups were excised for p38 MAPK and c-Jun N-terminal kinase (JNK) analyses and histopathology (each, n = 4). Results. Serum levels of aspartate transaminase, alanine transaminase, lactate dehydrogenase, TNF-α, and IL-1β were significantly lower in the FR-treated group than in the control group, and liver tissue blood flow was significantly higher in the FR-treated group than in the control group. Histopathologically, tissue damage was milder in the FR-treated group than in the control group. Both p38 MAPK and JNK were markedly phosphorylated after 30 minutes of reperfusion, and FR inhibited the phosphorylation of p38 MAPK without affecting the JNK. Conclusions. FR decreased serum TNF-α and IL-1β levels and liver injury associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate warm ischemia-reperfusion injury of the liver. (Surgery 2002;131:344-9.) From the Second Department of Surgery, Gunma University School of Medicine, Department of Pathology, Nippon Medical School Second Hospital, Kawasaki, Japan
THE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) family is reported to play an important role in intracellular signal transduction in response to extracellular stimuli.1-8 Among mammalian MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK) are reported to be activated by a variety of cellular stresses, such as ischemia-reperfusion.9-11 In the kidney, the induction of tumor necrosis factor (TNF) gene transcription after ischemiareperfusion is due to direct activation of p38 MAPK This work was supported in part by a fund from Fujisawa Pharmaceutical Co Ltd, Osaka, Japan. Accepted for publication September 22, 2001. Reprint requests: Izumi Takeyoshi, Second Department of Surgery, Gunma University School of Medicine, 3-39-15, Showa Machi, Maebashi, Gunma 371-8511, Japan. Copyright © 2002 by Mosby, Inc. 0039-6060/2002/$35.00 + 0 11/56/121097 doi:10.1067/msy.2002.121097
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by locally formed reactive oxygen species.12 In addition, several studies showed that the inhibition of p38 MAPK decreased ischemia reperfusion– induced apoptosis of the cardiomyocyte and improved cardiac function.13-15 Only a few studies have reported on the activation of MAPKs in the liver during warm ischemia and reperfusion.16-18 Thus, the role of p38 MAPK activation in response to hepatic ischemia-reperfusion remains unclear. FR167653 (FR; Fujisawa Pharmaceutical, Osaka, Japan) is a newly synthesized organic compound that was originally characterized as a potent suppresser of interleukin-1β (IL)-1β and TNF-α production.19,20 The activation of p38 MAPK is required for lipopolysaccharide-induced production of IL-1β and TNF-α in monocytes.21 Previously, we found that FR inhibited cytokine production through the inhibition of p38 MAPK activation in a rat endotoxemia model.22 This experimental study was designed to examine the
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activation of p38 MAPK and JNK in the hepatic ischemia-reperfusion process, and to evaluate the effect of p38 MAPK inhibition with FR in hepatic ischemia-reperfusion injury. MATERIAL AND METHODS Animals. Adult male Sprague-Dawley rats weighing 280 to 340 g were used in this experiment. Animals were allowed free access to food and water in a constant temperature environment with a 14hour to 10-hour light-dark cycle. All animals were maintained in accordance with the guidelines described in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health publication 85-23, revised 1985). This study was performed with the approval of the Animal Care and Experimentation Committee, Gunma University, Showa Campus. Animal model of hepatic ischemia and reperfusion injury. Animals were anesthetized with light ether inhalation, and then cannulated in the right jugular vein with a PE-50 polyethylene tube (Becton Dickinson, Sparks, Md) for the administration of a saline vehicle at a rate of 5 mL/kg/h with or without FR. After a midline laparotomy, total liver ischemia, including splanchnic congestion, was induced for 30 minutes by occluding the hepatoduodenal ligament with a microvascular clip. Reperfusion was initiated by releasing the vascular clip. Experimental design. The animals were divided into 4 groups. Animals in the sham-operated group were infused with normal saline solution with no hepatic ischemia. The control group received normal saline solution, and was subjected to 30 minutes of warm ischemia by total inflow occlusion with congestion of the gut. The FR-treated group received FR, which is a novel p38 MAPK inhibitor (0.1 mg/kg/hr), from 30 minutes before ischemia to 2 hours after reperfusion. The FR-only group received FR for 3 hours with no hepatic ischemia. Blood analysis. Blood samples were collected from the suprahepatic vena cava 2 hours after reperfusion. The serum was collected and stored at –80°C until assayed as described later. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured to investigate the degree of liver injury. AST, ALT, and LDH activities were determined with an autoanalyzer (Hitachi 736-60, Hitachi, Tokyo) with an ultraviolet rate assay. Serum levels of TNF-α and IL-1β were measured with a “sandwich” enzyme-linked immunosorbent assay (ELISA) with TNF-α and IL-1β ELISA kits for rats (BioSource International,
Fig 1. Liver tissue blood flow expressed as percentage of pre-clamp flow level. Blood flow after 60 and 90 minutes of reperfusion was significantly higher in FR-treated group than in control group. Data are expressed as mean ± SEM. *P < .05 versus sham-operated group. +P < .05 versus control group. Pre, Pre-ischemia; end, end-ischemia; 30, 30 minutes after reperfusion; 60, 60 minutes after reperfusion; 90, 90 minutes after reperfusion; 120, 120 minutes after reperfusion.
Camarillo, Calif), according to the manufacturer’s instructions. In brief, a rabbit polyclonal anti-rat TNF-α antibody was used as both the capture antibody coating the wells, and as the detecting antibody for TNF-α assays. For the IL-1β assay, a murine monoclonal anti-rat IL-1β antibody was used as the capture antibody, and a rabbit polyclonal anti-rat IL-1β antibody was used as the detecting antibody. Each cytokine level in rat samples was calculated from a standard curve generated from recombinant rat TNF-α or IL-1β. Six rats were assigned to each group. Tissue blood flow in the liver. The liver tissue flow was measured with a laser Doppler flowmeter (MBF 3; Moor Instruments, Devon, England) at pre-ischemia, end-ischemia, and at 30, 60, 90, and 120 minutes after reperfusion. The laser probe was always placed on the left lateral lobe of the liver. The blood flow was expressed as a percentage of the pre-clamp level. Four rats were assigned to each group. Histologic study. After 2 and 6 hours of reperfusion, liver specimens were collected and fixed in 10% formalin. Tissues were dehydrated, embedded in paraffin, cut into 5 µm sections, and mounted. After deparaffinization, tissues were stained with hematoxylin and eosin for histologic study.
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A
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B Fig 2. Histopathologic findings in the control (A) and FR-treated (B) groups (2 hours after reperfusion). In the control group, hepatocellular vacuolization was seen in midzonal area. On the other hand, in FR-treated group, no significant hepatocellular damage was observed.
Fig 3. Western blot analysis of ischemia-reperfusion– induced phosphorylation of p38 MAPK and JNK in the liver tissue. Both JNK and p38 MAPK in liver tissue were not phosphorylated at end-ischemia compared with preischemia in the control group. Both JNK and p38 MAPK in the control group were markedly phosphorylated 30 minutes after reperfusion, then the expression of phosphorylated JNK and p38 MAPK attenuated after 2 hours of reperfusion (left side). After 30 minutes of reperfusion, the expression of phosphorylated JNK did not differ between the control and FR-treated groups. Meanwhile, the expression of phosphorylated p38 MAPK after 30 minutes of reperfusion was dramatically suppressed in the FR-treated group compared with the control group (right side). Rp, reperfusion; Phospho-p38, phosphorylated p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase.
Four rats each were assigned to the control and FR-treated groups. Western blot analysis for p38 MAP kinase and JNK. Before ischemia, at end-ischemia, at 30 minutes after, and at 2 hours after reperfusion, small pieces of the left lateral lobe of the liver were collected and
frozen at –80°C until homogenization. The extraction buffer contained 10 mmol/L of Tris-hydrochloric acid (pH 7.5), 0.25 mol/L of sucrose, 5 mmol/L of ethylenediamine tetraacetic acid, 50 mmol/L of sodium chloride, 30 mmol/L of sodium pyrophosphate, 50 mmol/L of sodium fluoride, 100 µmol/L of sodium orthovanadate, 1 µ/mL of pepstatin A, and 2 µ/mL of leupeptin. Phenylmethylsulfonyl fluoride (PMSF, 1 mmol/L) was added just before use. Samples were homogenized in 1000 µL of extraction buffer on ice with a micro-homogenizer (NS-310E; Microtec, Chiba, Japan). All debris and nuclei were removed by centrifugation at 900g at 4°C for 10 minutes, and the supernatant obtained was used for Western blot analysis. Protein concentrations were determined with bovine serum albumin as the reference standard with Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, Calif). Eighty micrograms of protein were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), by means of the LaemmLi method in 10% (weight/volume) gels containing acrylamide and N,N´-methylene-bis-acrylamide in a ratio of 37:1 in the presence of 0.1% (weight/ volume) SDS, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass). The PVDF membranes were first incubated with 5% skim milk in Tris-buffered saline (TBS) solution and washed 3 times in 0.05% Tween 20-TBS (TBST). The membranes were incubated with either a rabbit polyclonal anti-phosphorylated JNK antibody or a rabbit polyclonal anti-phosphorylated p38 MAPK antibody, diluted with TBST at a ratio of 1:1000, at 37°C for 3 hours. After being washed 3 times in TBST, the membranes were incubated with an anti-rabbit IgG antibody conjugated
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Surgery Volume 131, Number 3 Table. Two hours after reperfusion Group Sham FR-only Control FR-treated
AST (U/L) 80.6 ± 6.5 80.3 ± 3.0 1623 ± 269* 530 ± 77*†
ALT (U/L) 35.2 ± 31.0 ± 1270 ± 403 ±
2.8 0.7 259* 88*†
LDH (U/L) 435.4 ± 36.5 408.7 ± 43.8 25590 ± 5209* 9166 ± 1173*†
TFN-α (pg/mL) 4±0 4±0 32.0 ± 9.2* 4.0 ± 0.0†
IL-1β (pg/mL) 5.9 ± 3.8 ± 149.3 ± 7.9 ±
1.9 1.4 63.1 3.6
Values are mean SEM. *P < .05 versus sham-operated group. †P < .05 versus control group.
to horseradish peroxidase, which was diluted with TBST at a ratio of 1:2000, at 37°C for 1 hour. After being washed 3 times in TBST, the membranes were then incubated with ECL reagent (Amersham International PLC, Buckinghamshire, England) for 20 seconds and exposed to x-ray film. PhosphoPlus p38 MAP Kinase Antibody and PhosphoPlus JNK Antibody kits were purchased from New England BioLabs (Beverly, Mass). Four rats each were assigned to the control and FR-treated groups. Statistical analysis. The data are expressed as the mean ± SE of the mean. Differences in blood analysis among groups were analyzed with the 1way analysis of variance (ANOVA). A Scheffé’s post hoc test was used to perform multiple comparisons. Repeated-measures ANOVA was performed to evaluate the tissue blood flow. If the repeated-measures ANOVA showed a significant interaction, statistical significance among 4 groups at each time point was determined by the use of Scheffé’s post hoc test. Statistical significance was defined as P < .05. RESULTS After 2 hours of reperfusion, serum levels of AST, ALT, LDH, and TNF-α in the FR-treated group were significantly lower than those in the control group. Serum levels of IL-1β in the FRtreated group were lower than those in the control group; however, there was no significant difference. Serum levels of TNF-α and IL-1β in the FRtreated group were not significantly different from those in the sham-operated and FR-only groups. Serum levels of AST, ALT, and LDH in the FR-only group were not significantly different from those in the sham-operated group (Table). The liver tissue blood flow decreased to approximately 10% of the preischemic level and increased after reperfusion; liver tissue blood flow after 60 and 90 minutes of reperfusion was significantly higher in the FR-treated group than in the control group (Fig 1). The liver tissue blood flow was stable throughout the experiment in the sham-operated and FR-only groups.
Histopathologically, hepatocellular vacuolization in the midzonal area and partial collapse were observed after 2 hours of reperfusion in the control group. After 6 hours of reperfusion, acidophyllic change and condensation of nuclei were observed in the control group. In the FR-treated group, no significant hepatocellular deterioration was observed after both 2 and 6 hours of reperfusion (Fig 2). P38 MAPK and JNK in liver tissue were examined by Western blot analysis (n = 4 each). Typical examples are shown in Fig 3. Both JNK and p38 MAPK in liver tissue were not phosphorylated at end-ischemia compared with pre-ischemia in the control group. Both JNK and p38 MAPK in the control group were markedly phosphorylated 30 minutes after reperfusion, then the expression of phosphorylated JNK and p38 MAPK attenuated after 2 hours of reperfusion. After 30 minutes of reperfusion, the expression of phosphorylated JNK did not differ between the control and FR-treated groups. Meanwhile, the expression of phosphorylated p38 MAPK after 30 minutes of reperfusion was dramatically suppressed in the FR-treated group compared with the control group. DISCUSSION TNF-α and IL-β derived from activated Kupffer’s cells play an important role in the pathogenesis of hepatic ischemia-reperfusion injury. These cytokines are capable of up-regulating adhesion molecules and cause polymorphonuclear neutrophils to adhere to endothelial cells. This causes microcirculation disturbance, which is thought to be a major mechanism of ischemia-reperfusion injury, including the “no-flow phenomenon.”23 MAPK family members are activated by dual phosphorylation on tyrosine and threonine in response to extracellular stimuli.1-8 Once activated, these kinases are translocated to the nucleus, where they phosphorylate and activate different transcription factors and transactivate target genes. In this group of kinases, classical extracellular signal– regulated kinase is mainly stimulated by growth fac-
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tors, and is associated with proliferation. On the other hand, p38 MAPK and JNK are reported to be activated by a variety of cellular stresses, such as inflammatory cytokines, lipopolysaccharides, heat shock, osmotic stress, and ischemia-reperfusion.9-11 Pyridinyl-imidazole compounds, which are considered cytokine–suppressive anti-inflammatory drugs, are reported to block p38 MAPK activity and inhibit cytokine biosynthesis.21,24 The specificity of these compounds is related to their ability to bind the ATP pocket within p38 MAPK.25 Cuenda et al26 reported that SB203580, a related compound, did not prevent the activation of other MAPK pathways in vitro. In contrast, Clerk and Sugden27 recently reported that 2 JNK isoforms activated in perfused hearts were inhibited by SB203580. The ischemia-reperfusion process differentially activates MAPKs. In renal ischemia-reperfusion, p38 MAPK is activated by ischemia, whereas JNK is not activated by ischemia alone but is activated by reperfusion after ischemia.28 A similar activation pattern has been demonstrated in an isolated perfused rat heart model.11 In our study, 30-minute total ischemia alone did not induce significant phosphorylation of p38 MAPK and JNK. This difference may reflect differences in tissue specificity or the ischemic period. Ma et al15 reported that the administration of SB203580 decreased myocardial apoptosis and improved cardiac function after myocardial ischemia-reperfusion by inhibiting p38 MAPK. In addition, Donahoo et al12 suggest that anti-TNF therapy, including p38 MAPK inhibition, may be a part of the strategy to prevent renal ischemia-reperfusion injury. Only one article has reported on the inactivation of p38 MAPK in ischemia-reperfusion injury.29 Bradham et al29 reported that reperfusion after liver transplantation activated 3 different members of the MAPK family differently. In their study, JNK activation was marked and sustained after 60 minutes of reperfusion, whereas p38 MAPK activation was unchanged during cold storage and the reperfusion process. In our study, both JNK and p38 MAPK were markedly phosphorylated 30 minutes after reperfusion following 30 minutes of global warm ischemia of the liver. One possible cause of this inconsistency is the difference in protocol for analysis of MAPK activity. Bradham et al29 examined the activation of MAPKs only in nuclear extract, whereas we examined it in the whole cell lysate of the liver tissue. In heart tissue, it has been reported that p38 MAPK and p54 JNK are activated in cytosolic fraction and not in nuclear fraction after 4 minutes of warm ischemia and a subsequent 5 minutes of reperfusion, whereas p46 JNK is acti-
Surgery March 2002 vated in both fractions.30 P38 MAPK has also been shown to be exported from nucleus to cytoplasm after phosphorylation of MAPK activated protein kinase-2, which is a substrate of p38 MAPK itself.31 Thus, the main localization of activated p38 MAPK in cells may be not in the nucleus, which is different from that of JNK. Both TNF-α and IL-1 are reported to be strong activators of p38 MAPK in vitro.32 In addition, several reports also suggest the role of p38 MAPK phosphorylation in regulating TNF-α and IL-1 production.21,24 Our results suggest that the phosphorylation of p38 MAPK plays an important role in inflammatory cytokine biosynthesis and increased cytotoxic potential after warm ischemia-reperfusion of the liver. CONCLUSION In this warm ischemic model, both p38 MAPK and JNK were phosphorylated during the reperfusion process after ischemia. FR decreased serum TNF-α and IL-1β levels, liver injury associated with the inhibition of p38 MAPK activation, and the noninhibited phosphorylation of JNK. These results suggest that inhibiting the activation of p38 MAPK may attenuate warm ischemia-reperfusion injury of the liver.
REFERENCES 1. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 1993;268:14553-6. 2. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinase on apoptosis. Science 1995;270:1326-31. 3. Koibuchi Y, Iino Y, Uchida T, Nagasawa M, Morishita Y. Effects of estrogen and tamoxifen on the MAP kinase cascade in experimental rat breast cancer. Int J Oncol 1997;11:583-9. 4. Bogoyevitch MA, Ketterman AJ, Sugden PH. Cellular stresses differentially activated c-Jun N-terminal protein kinase and extracellular signal-regulated protein kinase in cultured ventricular myocyte. J Biol Chem 1995;270:29710-7. 5. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, et al. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 1994;369:156-60. 6. Wang X, Martindale JL, Liu Y, Holbrook NJ. The cellular response to oxidative stress: influence of mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 1995;270:7420-6. 7. Seth A, Gonzalez FA, Gupta S, Raden DL, Davis RJ. Signal transduction within the nucleus by mitogen-activated protein kinase. J Biol Chem 1992;267:24796-804. 8. Han J, Lee JD, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 1994;265:808-11. 9. Clerk A, Fuller SJ, Michael A, Sudgen PH. Stimulation of “stress-regulated” mitogen-activated protein kinases (stressactivated protein kinase/c-Jun N-terminal kinases and p38mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stress. J Biol Chem 1998;273:7228-34.
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Surgery Volume 131, Number 3 10. Silva HS, Cioff C, Yin T, Sandhu G, Webb RL, Whelan J. Identification of a novel stress activated kinase in kidney and heart. Biochem Biophys Res Commun 1998;250:647-52. 11. Yin T, Sandhu G, Wolfgang CD, Burrier A, Webb RL, Rigel DF, et al. Tissue specific pattern of stress kinase activation in ischemic/reperfused heart and kidney. J Biol Chem 1997;272:19943-50. 12. Donahoo KK, Shames SD, Harken AH, Meldrum DR. Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury. J Urol 1999;162:196-203. 13. Cain BS, Meldrum DR, Meng X, Dinarello CA, Shames BD, Banerjee A, et al. P38 MAPK inhibition decreases TNFalpha production and enhances postischemic human myocardial function. J Surg Res 1999:83:7-12. 14. Mackey K, Rosen DM. An inhibitor of p38 mitogenactivated protein kinase protects neonatal cardiac myocytes from ischemia. J Biol Chem 1999;274:6272-9. 15. Ma XL, Kumar S, Gao F, Louden CS, Bernard LL, Christopher TA, et al. Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion. Circulation 1999;1685-91. 16. Zwacka RM, Zhang Y, Zhou W, Halldorson J, Engelhardt JF. Ischemia/reperfusion injury in the liver of BALB/c mice activates AP-1 and nuclear factor kappaB independently of IkappaB degradation. Hepatology 1998;28:1022. 17. Bendinelli P, Piccoletti R, Maroni P, Bernelli-Zazzera A. The MAP kinase cascades are activated during post-ischemic liver transplantation. FEBS Lett 1996;398:193. 18. Onishi I, Tani T, Hashimoto T, Shimuzu K, Yagi M, Yamamoto K, et al. Activation of c-Jun N-terminal kinase during ischemia and reperfusion in mouse liver. FEBS Lett 1997;420:201. 19. Yamamoto N, Sakai F, Yamazaki H, Sato N, Nakahara K, Okuhara M. FR167653, a dual inhibitor of interleukin-1 and tumor necrosis factor-α, ameliorates endotoxin-induced shock. Eur J Pharmacol 1997;327:169-75. 20. Yamamoto N, Sakai F, Yamazaki H, Sato N, Nakahara K, Okuhara M. Effect of FR167653, a cytokine suppressive agent, on endotoxin-induced disseminated intravascular coagulation. Eur J Pharmacol 1996;314:137-42. 21. Lee JC, Layson JT, McDonnel PC, Gallagher TF, Kumar S, Green D, et al. A protein kinase involved in the regulation
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of inflammatory cytokine biosynthesis. Nature 1994; 372:739-46. Yoshinari D, Takeyoshi I, Koibuchi Y, Matsumoto K, Kawashima Y, Koyama T, et al. Effect of a dual inhibitor of TNF-α and IL-1 on lipopolysaccahide-induced lung injury in rats: Involvement of the p38 mitogen-activated protein kinase pathway. Crit Care Med 2001;29:628-34. Andus T, Bauer J, Gerock W. Effects of cytokines on the liver. Hepatology 1991;13:364-75. Lee JC, Young PR. Role of CSBP/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J Leukoc Biol 1996;59:152-7. Tong L, Pav S, White DM, Rogers S, Crane KM, Cywin CL, et al. A highly specific inhibitor of human p38 kinase binds in the ATP pocket. Nat Struct Biol 1997;4:311-6. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995;364:229-33. Clerk A, Sugden PH. The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinase/c-Jun N-terminal kinases (SAPKs/JNKs). FEBS Lett 1998;426:93-6. Bogoyevitch MA, Brown JG, Ketterman AJ, Fuller SJ, Levy RB, Ashworth A, et al. Stimulation of the stress-activated mitogen activated protein kinase subfamilies in perfused heart. Circ Res 1996;79:162-73. Bradham CA, Stachlewitz RF, Gao W, Qian T, Jayadev S, Jenkins G, et al. Hepatology 1997;25:1128-35. Ping P, Zhang J, Huang S, Cao X, Tang XL, Li RC, et al. PKC-dependent activation of p46/54 JNKs during ischemic preconditioning in conscious rabbits. Am J Physiol 1999;277:H1771-85. Ben-Levy R, Hooper S, Wilson R, Paterson HF, Marshall CJ. Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr Biol 1998;8:1049-57. Raingeaud J, Cupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 1995;270:7420-6.
Background. Several studies have implicated the mitogen-activated protein kinase (MAPK) signal pathway in non-hepatic organ ischemia-reperfusion injury. However, the role of p38 MAPK in hepatic ischemia-reperfusion injury remains unclear. This study investigated the role of p38 MAPK in hepatic ischemia-reperfusion injury. Methods. Male Sprague-Dawley rats were divided into 4 groups (sham, FR-only, control, and FR-treated groups). The animals in the control and FR-treated groups were subjected to 30 minutes of warm ischemia with congestion of the gut. The FR-only and FR-treated groups received FR167653 (FR), which is a novel p38 MAPK inhibitor. The serum levels of aspartate transaminase, alanine transaminase, lactate dehydrogenase, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were measured (each, n = 6). Liver tissue blood flow was measured at pre-ischemia, end-ischemia, and 30, 60, 90, and 120 minutes after reperfusion (each, n = 4). The liver tissues in the control and FR-treated groups were excised for p38 MAPK and c-Jun N-terminal kinase (JNK) analyses and histopathology (each, n = 4). Results. Serum levels of aspartate transaminase, alanine transaminase, lactate dehydrogenase, TNF-α, and IL-1β were significantly lower in the FR-treated group than in the control group, and liver tissue blood flow was significantly higher in the FR-treated group than in the control group. Histopathologically, tissue damage was milder in the FR-treated group than in the control group. Both p38 MAPK and JNK were markedly phosphorylated after 30 minutes of reperfusion, and FR inhibited the phosphorylation of p38 MAPK without affecting the JNK. Conclusions. FR decreased serum TNF-α and IL-1β levels and liver injury associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate warm ischemia-reperfusion injury of the liver. (Surgery 2002;131:344-9.) From the Second Department of Surgery, Gunma University School of Medicine, Department of Pathology, Nippon Medical School Second Hospital, Kawasaki, Japan
THE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) family is reported to play an important role in intracellular signal transduction in response to extracellular stimuli.1-8 Among mammalian MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK) are reported to be activated by a variety of cellular stresses, such as ischemia-reperfusion.9-11 In the kidney, the induction of tumor necrosis factor (TNF) gene transcription after ischemiareperfusion is due to direct activation of p38 MAPK This work was supported in part by a fund from Fujisawa Pharmaceutical Co Ltd, Osaka, Japan. Accepted for publication September 22, 2001. Reprint requests: Izumi Takeyoshi, Second Department of Surgery, Gunma University School of Medicine, 3-39-15, Showa Machi, Maebashi, Gunma 371-8511, Japan. Copyright © 2002 by Mosby, Inc. 0039-6060/2002/$35.00 + 0 11/56/121097 doi:10.1067/msy.2002.121097
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by locally formed reactive oxygen species.12 In addition, several studies showed that the inhibition of p38 MAPK decreased ischemia reperfusion– induced apoptosis of the cardiomyocyte and improved cardiac function.13-15 Only a few studies have reported on the activation of MAPKs in the liver during warm ischemia and reperfusion.16-18 Thus, the role of p38 MAPK activation in response to hepatic ischemia-reperfusion remains unclear. FR167653 (FR; Fujisawa Pharmaceutical, Osaka, Japan) is a newly synthesized organic compound that was originally characterized as a potent suppresser of interleukin-1β (IL)-1β and TNF-α production.19,20 The activation of p38 MAPK is required for lipopolysaccharide-induced production of IL-1β and TNF-α in monocytes.21 Previously, we found that FR inhibited cytokine production through the inhibition of p38 MAPK activation in a rat endotoxemia model.22 This experimental study was designed to examine the
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activation of p38 MAPK and JNK in the hepatic ischemia-reperfusion process, and to evaluate the effect of p38 MAPK inhibition with FR in hepatic ischemia-reperfusion injury. MATERIAL AND METHODS Animals. Adult male Sprague-Dawley rats weighing 280 to 340 g were used in this experiment. Animals were allowed free access to food and water in a constant temperature environment with a 14hour to 10-hour light-dark cycle. All animals were maintained in accordance with the guidelines described in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health publication 85-23, revised 1985). This study was performed with the approval of the Animal Care and Experimentation Committee, Gunma University, Showa Campus. Animal model of hepatic ischemia and reperfusion injury. Animals were anesthetized with light ether inhalation, and then cannulated in the right jugular vein with a PE-50 polyethylene tube (Becton Dickinson, Sparks, Md) for the administration of a saline vehicle at a rate of 5 mL/kg/h with or without FR. After a midline laparotomy, total liver ischemia, including splanchnic congestion, was induced for 30 minutes by occluding the hepatoduodenal ligament with a microvascular clip. Reperfusion was initiated by releasing the vascular clip. Experimental design. The animals were divided into 4 groups. Animals in the sham-operated group were infused with normal saline solution with no hepatic ischemia. The control group received normal saline solution, and was subjected to 30 minutes of warm ischemia by total inflow occlusion with congestion of the gut. The FR-treated group received FR, which is a novel p38 MAPK inhibitor (0.1 mg/kg/hr), from 30 minutes before ischemia to 2 hours after reperfusion. The FR-only group received FR for 3 hours with no hepatic ischemia. Blood analysis. Blood samples were collected from the suprahepatic vena cava 2 hours after reperfusion. The serum was collected and stored at –80°C until assayed as described later. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured to investigate the degree of liver injury. AST, ALT, and LDH activities were determined with an autoanalyzer (Hitachi 736-60, Hitachi, Tokyo) with an ultraviolet rate assay. Serum levels of TNF-α and IL-1β were measured with a “sandwich” enzyme-linked immunosorbent assay (ELISA) with TNF-α and IL-1β ELISA kits for rats (BioSource International,
Fig 1. Liver tissue blood flow expressed as percentage of pre-clamp flow level. Blood flow after 60 and 90 minutes of reperfusion was significantly higher in FR-treated group than in control group. Data are expressed as mean ± SEM. *P < .05 versus sham-operated group. +P < .05 versus control group. Pre, Pre-ischemia; end, end-ischemia; 30, 30 minutes after reperfusion; 60, 60 minutes after reperfusion; 90, 90 minutes after reperfusion; 120, 120 minutes after reperfusion.
Camarillo, Calif), according to the manufacturer’s instructions. In brief, a rabbit polyclonal anti-rat TNF-α antibody was used as both the capture antibody coating the wells, and as the detecting antibody for TNF-α assays. For the IL-1β assay, a murine monoclonal anti-rat IL-1β antibody was used as the capture antibody, and a rabbit polyclonal anti-rat IL-1β antibody was used as the detecting antibody. Each cytokine level in rat samples was calculated from a standard curve generated from recombinant rat TNF-α or IL-1β. Six rats were assigned to each group. Tissue blood flow in the liver. The liver tissue flow was measured with a laser Doppler flowmeter (MBF 3; Moor Instruments, Devon, England) at pre-ischemia, end-ischemia, and at 30, 60, 90, and 120 minutes after reperfusion. The laser probe was always placed on the left lateral lobe of the liver. The blood flow was expressed as a percentage of the pre-clamp level. Four rats were assigned to each group. Histologic study. After 2 and 6 hours of reperfusion, liver specimens were collected and fixed in 10% formalin. Tissues were dehydrated, embedded in paraffin, cut into 5 µm sections, and mounted. After deparaffinization, tissues were stained with hematoxylin and eosin for histologic study.
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A
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B Fig 2. Histopathologic findings in the control (A) and FR-treated (B) groups (2 hours after reperfusion). In the control group, hepatocellular vacuolization was seen in midzonal area. On the other hand, in FR-treated group, no significant hepatocellular damage was observed.
Fig 3. Western blot analysis of ischemia-reperfusion– induced phosphorylation of p38 MAPK and JNK in the liver tissue. Both JNK and p38 MAPK in liver tissue were not phosphorylated at end-ischemia compared with preischemia in the control group. Both JNK and p38 MAPK in the control group were markedly phosphorylated 30 minutes after reperfusion, then the expression of phosphorylated JNK and p38 MAPK attenuated after 2 hours of reperfusion (left side). After 30 minutes of reperfusion, the expression of phosphorylated JNK did not differ between the control and FR-treated groups. Meanwhile, the expression of phosphorylated p38 MAPK after 30 minutes of reperfusion was dramatically suppressed in the FR-treated group compared with the control group (right side). Rp, reperfusion; Phospho-p38, phosphorylated p38 mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase.
Four rats each were assigned to the control and FR-treated groups. Western blot analysis for p38 MAP kinase and JNK. Before ischemia, at end-ischemia, at 30 minutes after, and at 2 hours after reperfusion, small pieces of the left lateral lobe of the liver were collected and
frozen at –80°C until homogenization. The extraction buffer contained 10 mmol/L of Tris-hydrochloric acid (pH 7.5), 0.25 mol/L of sucrose, 5 mmol/L of ethylenediamine tetraacetic acid, 50 mmol/L of sodium chloride, 30 mmol/L of sodium pyrophosphate, 50 mmol/L of sodium fluoride, 100 µmol/L of sodium orthovanadate, 1 µ/mL of pepstatin A, and 2 µ/mL of leupeptin. Phenylmethylsulfonyl fluoride (PMSF, 1 mmol/L) was added just before use. Samples were homogenized in 1000 µL of extraction buffer on ice with a micro-homogenizer (NS-310E; Microtec, Chiba, Japan). All debris and nuclei were removed by centrifugation at 900g at 4°C for 10 minutes, and the supernatant obtained was used for Western blot analysis. Protein concentrations were determined with bovine serum albumin as the reference standard with Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, Calif). Eighty micrograms of protein were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), by means of the LaemmLi method in 10% (weight/volume) gels containing acrylamide and N,N´-methylene-bis-acrylamide in a ratio of 37:1 in the presence of 0.1% (weight/ volume) SDS, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, Mass). The PVDF membranes were first incubated with 5% skim milk in Tris-buffered saline (TBS) solution and washed 3 times in 0.05% Tween 20-TBS (TBST). The membranes were incubated with either a rabbit polyclonal anti-phosphorylated JNK antibody or a rabbit polyclonal anti-phosphorylated p38 MAPK antibody, diluted with TBST at a ratio of 1:1000, at 37°C for 3 hours. After being washed 3 times in TBST, the membranes were incubated with an anti-rabbit IgG antibody conjugated
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Surgery Volume 131, Number 3 Table. Two hours after reperfusion Group Sham FR-only Control FR-treated
AST (U/L) 80.6 ± 6.5 80.3 ± 3.0 1623 ± 269* 530 ± 77*†
ALT (U/L) 35.2 ± 31.0 ± 1270 ± 403 ±
2.8 0.7 259* 88*†
LDH (U/L) 435.4 ± 36.5 408.7 ± 43.8 25590 ± 5209* 9166 ± 1173*†
TFN-α (pg/mL) 4±0 4±0 32.0 ± 9.2* 4.0 ± 0.0†
IL-1β (pg/mL) 5.9 ± 3.8 ± 149.3 ± 7.9 ±
1.9 1.4 63.1 3.6
Values are mean SEM. *P < .05 versus sham-operated group. †P < .05 versus control group.
to horseradish peroxidase, which was diluted with TBST at a ratio of 1:2000, at 37°C for 1 hour. After being washed 3 times in TBST, the membranes were then incubated with ECL reagent (Amersham International PLC, Buckinghamshire, England) for 20 seconds and exposed to x-ray film. PhosphoPlus p38 MAP Kinase Antibody and PhosphoPlus JNK Antibody kits were purchased from New England BioLabs (Beverly, Mass). Four rats each were assigned to the control and FR-treated groups. Statistical analysis. The data are expressed as the mean ± SE of the mean. Differences in blood analysis among groups were analyzed with the 1way analysis of variance (ANOVA). A Scheffé’s post hoc test was used to perform multiple comparisons. Repeated-measures ANOVA was performed to evaluate the tissue blood flow. If the repeated-measures ANOVA showed a significant interaction, statistical significance among 4 groups at each time point was determined by the use of Scheffé’s post hoc test. Statistical significance was defined as P < .05. RESULTS After 2 hours of reperfusion, serum levels of AST, ALT, LDH, and TNF-α in the FR-treated group were significantly lower than those in the control group. Serum levels of IL-1β in the FRtreated group were lower than those in the control group; however, there was no significant difference. Serum levels of TNF-α and IL-1β in the FRtreated group were not significantly different from those in the sham-operated and FR-only groups. Serum levels of AST, ALT, and LDH in the FR-only group were not significantly different from those in the sham-operated group (Table). The liver tissue blood flow decreased to approximately 10% of the preischemic level and increased after reperfusion; liver tissue blood flow after 60 and 90 minutes of reperfusion was significantly higher in the FR-treated group than in the control group (Fig 1). The liver tissue blood flow was stable throughout the experiment in the sham-operated and FR-only groups.
Histopathologically, hepatocellular vacuolization in the midzonal area and partial collapse were observed after 2 hours of reperfusion in the control group. After 6 hours of reperfusion, acidophyllic change and condensation of nuclei were observed in the control group. In the FR-treated group, no significant hepatocellular deterioration was observed after both 2 and 6 hours of reperfusion (Fig 2). P38 MAPK and JNK in liver tissue were examined by Western blot analysis (n = 4 each). Typical examples are shown in Fig 3. Both JNK and p38 MAPK in liver tissue were not phosphorylated at end-ischemia compared with pre-ischemia in the control group. Both JNK and p38 MAPK in the control group were markedly phosphorylated 30 minutes after reperfusion, then the expression of phosphorylated JNK and p38 MAPK attenuated after 2 hours of reperfusion. After 30 minutes of reperfusion, the expression of phosphorylated JNK did not differ between the control and FR-treated groups. Meanwhile, the expression of phosphorylated p38 MAPK after 30 minutes of reperfusion was dramatically suppressed in the FR-treated group compared with the control group. DISCUSSION TNF-α and IL-β derived from activated Kupffer’s cells play an important role in the pathogenesis of hepatic ischemia-reperfusion injury. These cytokines are capable of up-regulating adhesion molecules and cause polymorphonuclear neutrophils to adhere to endothelial cells. This causes microcirculation disturbance, which is thought to be a major mechanism of ischemia-reperfusion injury, including the “no-flow phenomenon.”23 MAPK family members are activated by dual phosphorylation on tyrosine and threonine in response to extracellular stimuli.1-8 Once activated, these kinases are translocated to the nucleus, where they phosphorylate and activate different transcription factors and transactivate target genes. In this group of kinases, classical extracellular signal– regulated kinase is mainly stimulated by growth fac-
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tors, and is associated with proliferation. On the other hand, p38 MAPK and JNK are reported to be activated by a variety of cellular stresses, such as inflammatory cytokines, lipopolysaccharides, heat shock, osmotic stress, and ischemia-reperfusion.9-11 Pyridinyl-imidazole compounds, which are considered cytokine–suppressive anti-inflammatory drugs, are reported to block p38 MAPK activity and inhibit cytokine biosynthesis.21,24 The specificity of these compounds is related to their ability to bind the ATP pocket within p38 MAPK.25 Cuenda et al26 reported that SB203580, a related compound, did not prevent the activation of other MAPK pathways in vitro. In contrast, Clerk and Sugden27 recently reported that 2 JNK isoforms activated in perfused hearts were inhibited by SB203580. The ischemia-reperfusion process differentially activates MAPKs. In renal ischemia-reperfusion, p38 MAPK is activated by ischemia, whereas JNK is not activated by ischemia alone but is activated by reperfusion after ischemia.28 A similar activation pattern has been demonstrated in an isolated perfused rat heart model.11 In our study, 30-minute total ischemia alone did not induce significant phosphorylation of p38 MAPK and JNK. This difference may reflect differences in tissue specificity or the ischemic period. Ma et al15 reported that the administration of SB203580 decreased myocardial apoptosis and improved cardiac function after myocardial ischemia-reperfusion by inhibiting p38 MAPK. In addition, Donahoo et al12 suggest that anti-TNF therapy, including p38 MAPK inhibition, may be a part of the strategy to prevent renal ischemia-reperfusion injury. Only one article has reported on the inactivation of p38 MAPK in ischemia-reperfusion injury.29 Bradham et al29 reported that reperfusion after liver transplantation activated 3 different members of the MAPK family differently. In their study, JNK activation was marked and sustained after 60 minutes of reperfusion, whereas p38 MAPK activation was unchanged during cold storage and the reperfusion process. In our study, both JNK and p38 MAPK were markedly phosphorylated 30 minutes after reperfusion following 30 minutes of global warm ischemia of the liver. One possible cause of this inconsistency is the difference in protocol for analysis of MAPK activity. Bradham et al29 examined the activation of MAPKs only in nuclear extract, whereas we examined it in the whole cell lysate of the liver tissue. In heart tissue, it has been reported that p38 MAPK and p54 JNK are activated in cytosolic fraction and not in nuclear fraction after 4 minutes of warm ischemia and a subsequent 5 minutes of reperfusion, whereas p46 JNK is acti-
Surgery March 2002 vated in both fractions.30 P38 MAPK has also been shown to be exported from nucleus to cytoplasm after phosphorylation of MAPK activated protein kinase-2, which is a substrate of p38 MAPK itself.31 Thus, the main localization of activated p38 MAPK in cells may be not in the nucleus, which is different from that of JNK. Both TNF-α and IL-1 are reported to be strong activators of p38 MAPK in vitro.32 In addition, several reports also suggest the role of p38 MAPK phosphorylation in regulating TNF-α and IL-1 production.21,24 Our results suggest that the phosphorylation of p38 MAPK plays an important role in inflammatory cytokine biosynthesis and increased cytotoxic potential after warm ischemia-reperfusion of the liver. CONCLUSION In this warm ischemic model, both p38 MAPK and JNK were phosphorylated during the reperfusion process after ischemia. FR decreased serum TNF-α and IL-1β levels, liver injury associated with the inhibition of p38 MAPK activation, and the noninhibited phosphorylation of JNK. These results suggest that inhibiting the activation of p38 MAPK may attenuate warm ischemia-reperfusion injury of the liver.
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