- Email: [email protected]
A novel prostaglandin E receptor subtype agonist, 0N0-4819, attenuates acute experimental liver injury in rats
Hepatology Research 21 (2001) 252– 260
www.elsevier.com/locate/ihepcom
A novel prostaglandin E receptor subtype agonist, 0N0-4819, attenuates acute experimental liver injury in rats Kazuhiro Kasai *, Shin-ichiro Sato, Kazuyuki Suzuki First Department of Internal Medicine, Iwate Medical Uni6ersity School of Medicine, Uchimaru 19 -1, Morioka 020 -8505, Japan Received 22 January 2001; received in revised form 7 March 2001; accepted 16 March 2001
Abstract We evaluated the efficacy of ONO-4819, a newly developed agonist of a prostaglandin receptor subtype (EP4), on experimental model of acute liver injury in rats. Acute liver injury was induced by simultaneous intraperitoneal (i.p.) administration of D-galactosamine (GalN, 1 g/kg body weight) and lipopolysaccharide (LPS, 100 mg/kg body weight). The rats received a single intraperitoneal injection of ONO-4819 (0.2 mg/kg body weight) or physiological saline immediately after GalN/LPS administration. Submassive hepatic necrosis with marked elevation of serum total bilirubin, serum aspartate aminotransferase and serum alanine aminotransferase levels developed 24 h after GalN/ LPS administration. The administration of ONO-4819 significantly inhibited the development of submassive hepatic necrosis and inhibited the elevation in levels of biochemical markers that indicate liver function. In addition, the apoptotic index of hepatocytes assessed by the TUNEL method was significantly lower in rats treated with ONO-4819 than in the control. Although serum levels of tumor necrosis factor-a (TNF-a), interferon-g (IFN-g) and interleukin-8 (IL-8) were markedly elevated after GalN/LPS administration, ONO-4819 significantly inhibited the elevation of those of TNF-a and IFN-g but not that of IL-8. The beneficial effect of ONO-4819 for acute liver injury was similar at doses of 0.1, 0.05 and 0.01 mg/kg body weight. These results suggest that the EP4 agonist, ONO-4819, may have a protective effect against experimental liver injury in rats through the suppression of inflammatory cytokines. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Acute liver injury; Fulminant hepatic failure; Prostaglandin E; EP4 agonist; Tumor necrosis factor-a; Interferon-g; Interleukin-8
1. Introduction Fulminant hepatic failure (FHF) including fulminant hepatitis (FH) indicate poor prognosis [1,2]. In Japan, FH in 90% of the patients is * Corresponding author. Tel.: + 81-19-6515111; fax: +8119-6526664. E-mail address: [email protected] (K. Kasai).
caused by hepatitis viruses, mainly hepatitis A, hepatitis B and hepatitis nonA-nonB viruses and 10% by drug intoxication [3]. On the other hand, the pathogenesis of massive hepatic necrosis which is characteristic of FHF still remains unclarified. Among the factors known to be related to the mechanism, hyperimmunoreaction [4,5], apoptosis of hepatocytes [6–9] and microcirculatory disturbances of the hepatic sinusoidal space
1386-6346/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 1 3 8 6 - 6 3 4 6 ( 0 1 ) 0 0 1 0 3 - 6
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
[10,11] have been studied. In addition, inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1 and interferon-g (INF-g), may play an important role in apoptosis and massive hepatic necrosis [12–14]. Furthermore, interleukin-8 (IL-8) participates as a neutrophil chemotactic factor and an activator of adhesion molecules on neutrophils in these processes [11,15]. Prostaglandins (PGs) are cyclooxygenase metabolites of arachidonate and act as local mediators under various inflammatory conditions [16]. Among PGs, PGE1 and PGE2 have been considered as potential therapeutic agents in the treatment of FHF based on their cytoprotective effects [17 –26]. However, its clinical application has not been realized in Japan. The actions of PGE1 and PGE2 are mediated by specific receptors on target cells. There are four subtypes of PGE receptors; EP1, EP2, EP3 and EP4. These are encoded by different genes and differ in their tissue expression [27]. Recently, a novel EP4 agonist, ONO-4819, which acts on PGE receptors has been developed in Japan [28]. This compound inhibits the production of cytokines such as TNF-a and IFN-g in vitro [28]. In the present study, we attempted to evaluate the effectiveness of ONO-4819 for experimental acute liver injury induced by GalN/LPS in rats
253
which shows that TNF-a and IFN-g play a crucial role in this model [8,9,12–14].
2. Materials and methods
2.1. Animals and reagents Nine-week-old male Wistar rats (CLEA JAPAN, Inc., Tokyo, Japan) weighing 200–250 g, were provided with food and water ad libitum and fasted for 24 h prior to the experiments. All the procedures in the present study were done according to the animal treatment rules of The Institute of Experimental Animals in Iwate Medical University. Hydrochloric acid, D-galactosamine (GalN; Wako Pure Chemical Industries, Ltd., Tokyo, Japan) was dissolved in physiological saline to prepare a 200 mg/ml solution. The pH of the solution was adjusted to 7.0 with 1 N sodium hydroxide. Lipopolysaccharide (LPS from Escherichia coli, 0111:B4; Sigma Chemical Co., St. Louis, MO, USA) was dissolved in physiological saline to prepare a 40 mg/ml solution. The EP4 agonist, ONO-4819, a gift from Ono Pharmaceutical Co., Ltd. (Osaka, Japan), was dissolved in physiological saline to prepare a 0.04 mg/ml solution.
Table 1 Serial changes of serum markers of liver and renal function on saline and ONO-4819-treated groups after administration of GalN/LPSa Time after GalN/LPS administration
T.Bil. (mg/dl) AST (IU/l) ALT (IU/l) BUN (mg/dl) CRNN (mg/dl)
1h
3h
6h
12 h
24 h
0.1 9 0.0 0.19 0.0 151.7945.6 114.89 6.4 27.39 3.6 23.39 1.2 16.2 91.4 13.39 1.4 0.3 90.1 0.39 0.1
0.1 9 0.0 0.1 9 0.0 261.89 57.0 214.5 9 19.6 129.7 9 60.3 70.29 12.4 20.5 9 2.1 28.29 4.6 0.4 9 0.1 0.6 9 0.1
0.2 9 0.1 0.1 9 0.0 1876.39 272.7* 826.5 9 113.9* 1411.79 144.1 858.8 9 173.2 17.9 91.9 14.8 9 1.1 0.4 9 0.1 0.3 9 0.0
0.7 90.5** 0.1 9 0.0** 12 215.3 9 7277.1*** 1384.8 9219.9*** 13 165.5 9 7912.9** 1984.7 9253.0** 20.6 9 3.6 16.8 97.9 0.3 9 0.1 0.3 9 0.1
2.8 91.0** 0.5 90.3** 24 690.8 95257.6*** 12 383.3 94206.0*** 32 877.7 97838.6*** 14 645.2 97633.8*** 47.7 919.9 31.7 915.4 0.7 90.3 0.4 90.2
a Values represent mean 9 S.D. of six rats in each time. Upper and lower data indicated saline-treated group and ONO-4819treated group, respectively. *PB0.05, **PB0.01, ***PB0.001.
254
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
abdominal aorta of rats under pentobarbital sodium anesthesia 1, 3, 6, 12 or 24 h following the drug administration, and liver tissue samples were removed each time for histological examination. In the second set of experiments, to determine the effect of dose at ONO-4819, different doses (0.1, 0.05 and 0.01 mg/kg body weight) was injected to rats treated with GalN/LPS (n= 24). Blood and liver tissue samples were collected 24 h following the drug administration. We measured levels serum of total bilirubin(T. Bil.), serum aspartate aminotransferase(AST), serum alanine aminotransferase (ALT), blood urea nitrogen (BUN) and serum creatinine (CRNN) using a conventional autoanalyzer (TBA-80FR NEO; Toshiba, Tokyo, Japan). Serum levels of TNF-a, IFN-g and IL-8 were also measured by enzyme-linked immunoassay using commercially available kits (Rat TNF-a measurement kit; Genzyme, Cambridge, MA, USA, Rat IFN-g measurement kit; Genzyme, Minneapolis, MN, USA, Rat IL-8 measurement kit; Panafarni Laboratories, Kumamoto, Japan).
2.3. Histopathological examination The liver tissue samples were fixed in 10% neutral buffered formalin, and embedded in paraffin. Sections were prepared and stained with hematoxylin-eosin (HE) for histopathological examination. Fig. 1. Effects of ONO-4819 on T.Bil., AST and ALT levels in the serum 24 h after the administration of GalN/LPS. Each column and bar represents the mean 9 S.D. of six rats.
2.2. Experimental method Acute liver injury was induced by simultaneous intraperitoneal injection of GalN (1 g/kg body weight) and LPS (100 mg/kg body weight). In the first set of experiments, ONO-4819 was administered intraperitoneally at a dose of 0.2 mg/kg to the rats immediately after the administration of GalN/LPS (n=30; ONO-4819-treated group). The same volume of physiological saline was administered as the control (n =30; saline-treated group). Blood samples were collected from the
2.4. Detection of apoptosis A portion of the liver tissue samples was fixed with 4% periodate-lysine-paraformaldehyde solution for the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. TUNEL was performed using a commercially available kit (Apoptag; Intergen, Purchase, NY, USA). The number of TUNEL-positive hepatocytes among 1000 hepatocytes was counted at 400× magnification and the percentage of TUNEL-positive hepatocytes was used as the apoptotic index.
2.5. Statistical analyses The results were expressed as mean9 Standard deviation (S.D.). Fisher’s protected least signifi-
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
cant difference test and Student’s t-test were used for comparisons between the groups. The differences were regarded as significant when P B 0.05.
3. Results
3.1. Serial changes in le6els of biochemical markers In the saline-treated group, serum levels of T. Bil., AST and ALT were significantly elevated 12 and 24 h after GalN/LPS administration (Table
255
1). In the ONO-4819-treated group, on the contrary, T. Bil., AST and ALT levels were significantly lower 12 and 24 h after GalN/LPS administration than those in the saline-treated group. Serum levels of BUN and CRNN slightly increased following the administration of GalN/ LPS in both groups, although there was no significant difference between the two groups. The beneficial effect of ONO-4819 was apparent even at a concentration as low as 0.01 mg/kg. Although the serum T. Bil. levels showed a tendency to decrease at a high dosage (0.2 mg/kg) of ONO-4819, the serum AST and ALT levels did
Fig. 2. Serial changes in TNF-a, IFN-g, and IL-8 levels in the serum of the ONO-4819-treated group and saline-treated group after the administration of GalN/LPS.
256
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
Fig. 3. Histological findings 24 h after administration of GalN/LPS (HE, ×100); (a) saline-treated group. (b) ONO-4819-treated group. Submassive hemorrhagic necrosis and inflammatory cell infiltration were observed in the saline-treated group. Hepatic damage in the ONO-4819-treated group apparently was suppressed compared with the saline treated group.
not show any significant differences among the different ONO-4819 dosages (Fig. 1).
3.2. Serial changes in serum le6els of TNF-h, IFN-k and IL-8 In the saline-treated group, levels of TNF-a and IFN-g in the serum increased in a biphasic manner after the administration of GalN/LPS (Fig. 2). In contrast, in the ONO-4819-treated group, serum levels of TNF-a and IFN-g
were significantly decreased compared with those in the saline-treated group (TNF-a: 36.79 12.7 vs. 87.89 30.8 pg/ml at 3 h, 34.89 15.0 vs. 295.79 149.5 pg/ml at 6 h; and IFN-g: 345.09 153.1 vs. 875.09 230.9 pg/ml at 1 h, 97.79 59.2 vs. 784.09 290.1 pg/ml at 6 h, respectively). On the other hand, there were no differences in serum IL-8 levels between the ONO-4819-treated and saline-treated groups at any time point following the administration of GalN/LPS.
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
3.3. Histopathological examination In the saline-treated group, condensation and fragmentation of the hepatocyte nucleus and inflammatory cell infiltration were first observed 3 h after the administration of GalN/LPS. The extent of these changes increased with time, and submassive hepatic necrosis with hemorrhage was observed 24 h after the administration (Fig. 3a). On
257
the other hand, in the ONO-4819-treated group, the histological changes were mild and showed focal to confluent necrosis 24 h after the administration of GalN/LPS (Fig. 3b). In the test using different doses of ONO-4819, the degree of hepatic necrosis was milder than that observed in the saline-treated group and was not significantly different among rats treated at different doses.
3.4. Detection of apoptosis The number of TUNEL-positive hepatocytes were increased 6 h after the drug administration in both groups, and peaked at 12 h in the salinetreated group and at 24 h in the ONO-4819treated group. The apoptosis index was significantly lower in the ONO-4819-treated group than in the saline-treated group at 6 and 12 h (Fig. 4, Table 2).
4. Discussion
Fig. 4. TUNEL positivity in the liver 12 h after administration of GalN/LPS (×100); (a) saline-treated group. (b) ONO4819-treated group. The number of TUNEL-positive hepatocytes in the ONO-4819-treated group was significantly less than that in the saline-treated group.
Sinclair et al. first reported the beneficial effect of PGE1 and PGE2 intravenously administered to patients with fulminant hepatitis in 1989 [24]. In Japan, the therapeutic efficacy of PGE1 for fulminant and acute severe hepatitis was also investigated in several studies [25,26]. However, clinical application of these drugs has not been realized. PGE1 and PGE2 have been known to attenuate hepatic damage in experimental models of acute liver injury induced by Propionibacterium acnes (P. acnes) and LPS, carbon tetrachloride, aflatoxin B1, galactosamine or ischemia-reperfusion [18–21,29,30]. Concerning the possible mechanism of attenuation of the liver injury by PGE1 and PGE2, Mizoguchi et al. demonstrated that PGE1 suppressed the production of a hepatocytotoxic factor which is induced in rats following the administration of P. acnes and LPS [19]. Karck et al. showed that PGE2 suppressed the release of TNF-a from rat Kupffer cells treated with LPS and recombinant IFN-g [31]. Masaki et al. also showed that PGE1 and PGE2 attenuated lipid peroxidation-dependent cell killing in cultured rat hepatocytes and reduced the extent of increased plasma membrane microviscosity of these cells
258
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
Table 2 Comparison of apoptotic index between the ONO-4819 and the saline-treated groupsa Time after GalN/LPS administration
Saline-treated group ONO-4819-treated group
1h
3h
6h
12 h
24 h
0.479 0.23 0.20 9 0.07
0.55 90.22 0.41 9 0.18
1.96 90.49* 1.279 0.43*
7.56 9 0.83** 0.96 90.21**
4.34 90.07 3.24 90.07
a Data are expressed as percentage of TUNEL-positive hepatocytes that accounted for the total hepatocyte population, *PB0.05, **PB0.001.
[21]. Furthermore, Natori et al., using the ischemia-reperfusion model, reported that PGE1 protects cells from liver injury by reducing leukocyte-endothelial cell adhesion via down modulation of adhesion molecule expression on the endothelium [30]. On the other hand, PGE receptors are divided into four subtypes, EP1, EP2, EP3 and EP4. These subtypes differ in their organ location and have different signal transduction mechanisms [16]. Although we did not examine the expression level of EP4 messenger RNA (mRNA) in the liver tissue, EP3 and EP4 are expressed in the rat liver [32]. Kastuyama et al. revealed that EP2 and EP4 mRNAs are expressed in a mouse macrophagelike cell line and these expression are increased by the addition of LPS in a dose-dependent manner [33]. Furthermore, PGEs increase the proliferation rate of hepatocytes through EP3 [34]. Studies concerning PGE receptor agonists and antagonists and their functions are ongoing and advanced, because PGE1 and PGE2 have various side effects when administered in patients with FHF and severe type of acute hepatitis [24,25]. An EP4 agonist, ONO-4819, that was recently developed in Japan, inhibits production of TNF-a and IFN-g in the blood [28]. These findings suggest that ONO-4819 instead of PGE1 and PGE2 may be applied for the treatment of FHF. However, its efficacy has not yet been tested. We, therefore, investigated the effect of ONO-4819 on a model of acute liver injury induced by GalN/ LPS. Laboratory test results indicate that the administration of ONO-4819 significantly improved the liver function in terms of the serum TNF-a and IFN-g levels and histological damages in the liver.
Furthermore, a dose-independent improvement of serum transaminase levels and histological changes were observed. Earlier studies indicate that TNF-a and IFN-g play a crucial role in the pathogenesis of GalN/LPS-induced acute liver injury model [8,9,12–14]. In clinical studies, Muto et al. reported that the rates of TNF-a and interleukin-1 production in circulating peripheral monocytes are high in patients with FHF [35]. Suzuki et al. demonstrated that serum TNF-a levels are significantly increased in patients with acute hepatitis and FHF, indicating their positive correlation with the soluble Fas ligand concentration [36]. Therefore, our results indicate that ONO-4819 may be effective against acute liver injury. However, in the present study, we administered ONO-4819 and GalN/LPS simultaneously. Since patients with FHF already show severe liver dysfunction when they were admitted into the hospital, drugs for improvement of liver function are usually administered after the occurrence of liver injury. Accordingly, in order to estimate the effects of ONO-4819 for clinical application, further studies regarding the precise dose and timing of administration are needed. On the other hand, although we showed that the serum TNF-a and IFN-g levels are significantly decreased in ONO-4819-treated group compared with those of saline-treated group, the serum IL-8 levels did not change. IL-8, which is a major cytokine associated with neutrophil infiltration of the liver tissue, is produced in endothelial and Kupffer cells [37]. Although the reasons why the serum IL-8 levels were not affected by ONO4819 is unknown, our earlier study[15] showed that multiple-interval injection of cyclosporin A significantly inhibited the elevation of serum IL-8
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
levels in the same models. Further studies are needed to clarify the production of IL-8 by cells and its role in the massive hepatic necrosis. In the rats model of liver injury induced by GalN/LPS, it is considered that apoptosis of hepatocytes occurs in the early stage and then massive hepatic necrosis develops due to microcirculatory disturbance of the hepatic sinusoidal space [8]. Although the TUNEL method and agarose gel electrophoresis are widely used for detection of cell apoptosis, we only used the TUNEL method. Our study showed that the number of TUNEL -positive hepatocytes serially decreased in the ONO-4819 treated group indicating that ONO-4819 may inhibit apoptosis, which may be considered as an initial event of the process in hepatic cell necrosis. To clarify whether ONO-4819 inhibits apoptosis and liver injury, further experimental studies are required in the future. In conclusion, a novel PGE receptor subtype agonist, ONO-4819 attenuates the acute liver injury in rats induced by GalN/LPS. We suggest that a possible mechanism of attenuation by ONO-4819 may be the suppression of production of inflammatory cytokines such as TNF-a.
Acknowledgements The authors thank Dr Shunichi Sato (vice-president, Iwate Medical University) for advice on this manuscript and Dr Tadashi Kawakami for excellent technical assistance. The authors wish to express there thanks to the Ono Pharmaceutical Co., Ltd., Osaka, Japan for the supply of ONO4819.
References [1] Sama C, Mastroianni A, Danesi G, et al. Fulminant hepatic failure. Gastroenterol Int 1999;12:83 – 7. [2] Riordan SM, Williams R. Cause and prognosis in acute liver failure. Liver Transplant Surg 1999;5:86 – 9. [3] Sato S, Suzuki K, Takikawa Y, et al. Physiopathology and therapy of fulminant hepatitis. Nippon NaikaGakkaiZasshi 1999;88:1783 –8 In Japanese.
259
[4] Almeida JD, Waterson AP. Immune complexes in hepatitis. Lancet 1969;II:983 – 6. [5] Dudley FJ, Fox RA, Sherlock S. Cellular immunity and hepatitis-associated Australia antigen liver disease. Lancet 1972;I:723 – 6. [6] Ogasawara J, Watanabe-Fukunaga R, Adachi M, et al. Lethal effect of the anti-Fas antibody in mice. Nature 1993;364:806 – 9. [7] Alido K, Moriyama T, Guidotti LG, et al. Mechanism of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541 – 54. [8] Morikawa A, Sugiyama T, Kato Y, et al. Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model. Infect Immun 1996;64:734 – 8. [9] Leist M, Gantner F, Bohlinger I, et al. Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J Pathol 1995;146:1220 – 34. [10] Mochida S, Ohno A, Arai M, Fujiwara K. Role of adhesion between activated macrophages and endothelial cells in the development of two types of massive hepatic necrosis in rats. J Gastroenterol Hepatol 1995;10:S38 – 42. [11] Ohira H, Ueno T, Torimura T, Tanikawa K, Kasukawa R. Leukocyte adhesion molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver injury. Scand J Gastroenterol 1995;30:1027 – 35. [12] Nagaki M, Tanaka M, Sugiyama A, et al. Interleukin-10 inhibits hepatic injury and tumor necrosis factor-a and interferon-g mRNA expression induced by staphylococcal enterotoxin B or lipopolysaccharide in galactosamine-sensitized mice. J Hepatol 1999;31:815 – 24. [13] Hishinuma I, Nagakawa J, Hirota K, et al. Involvement of tumor necrosis factor-a in development of hepatic injury in galactosamine-sensitized mice. Hepatology 1990;12:1187 – 91. [14] Bruce DC, Vicki ME, Bruno S, et al. Interferon-g receptor deficient mice are resistant to endotoxic shock. J Exp Med 1994;179:1437 – 44. [15] Kawakami T, Sato S, Suzuki K, et al. Beneficial effect of cyclosporin A on acute hepatic injury induced by galactosamine and lipopolysaccharide in rats. Hepatol Res 2000;18:284 – 97. [16] Narumiya S, Sugimoto Y, Ushikubi F, et al. Prostanoid receptors: properties and functions. Physiol Rev 1999;79:1193 – 226. [17] Robert A, Nezamis JE, Lancaster C, Hanchar AJ. Cytoprotection by prostaglandins in rats. Gastroenterology 1979;77:433 – 43. [18] Stachura J, Tarnawski A, Ivey KJ, et al. Prostaglandin protection of carbon tetrachloride induced liver cell necrosis in the rat. Gastroenterology 1981;81:211 – 7. [19] Mizoguchi Y, Tsutsui H, Miyajima K, et al. The protective effects of prostaglandin E1 in an experimental massive hepatic cell necrosis model. Hepatology 1987;7:1184 – 8.
260
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
[20] Rush BD, Wilkinson KF, Nichols NM, et al. Hepatic protection by 16, 16%-dimethyl prostaglandin E2 (DMPG) against acute afratoxin B1-induced injury in the rat. Prostaglandins 1989;37:683 –93. [21] Masaki N, Ohta Y, Shirataki H, et al. Hepatocyte membrane stabilization by prostaglandins El and E2: favorable effects on rat liver injury. Gastroenterology 1992;102:572 – 6. [22] Kikuchi E, Fukui H, Matsumoto M. Effect of prostaglandin E1on experimental acute hepatic failure in rabbits: prostaglandin E1 prevents the development of multiple-organ failure. J Hepatol 1994;20:478 – 82. [23] Abecassis M, Falk R, Blendis L, et al. Treatment of fluminant hepatic failure with a continuous infusion of prostin VT (PGE1). Hepatology 1987;7:1104A. [24] Sinclair SB, Greig PD, Blendis LM, et al. Biochemical and clinical response of fluminant viral hepatitis to administration of prostaglandin E. J Clin Invest 1989;84:1063 – 9. [25] Oda T, Sato S, Muto Y, et al. A double-blind comparative study on G-511 (PGE1) in severe type of acute hepatitis. J Clin Med 1994;10:2299 – 322 In Japanese. [26] Sasaki M. A study on the clinical efficacy and underlying mechanism of prostaglandin E1 in patients with acute severe hepatitis. Acta Sch Med Univ Gifu 1996;44:653 – 68 In Japanese. [27] Coleman RA, Smith WL, Narumiya S. International union of pharmacology classification of prostanoid receptors properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 1994;46:205 – 29. [28] Sakata K, Maruyama T, Seki A et al. The roles of PGE2 receptor EP4 subtype on cytokine regniation and its related disease. 11th international conference on advances in prostaglandin and leukotriene research, Florence, Italy, June 4– 8, 2000; abstract book: 51. [29] Stachura J, Tarnawski A, Szczudrawa J, et al. Cytopro-
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
tective effect of 16, 16%-dimethyl prostaglandin E2 and some drugs on an acute galactsamine induced liver damage in rat. Folia Histochem Cytochem 1980;18:311 – 8. Natori S, Fuji Y, Kurosawa H, et al. Prostaglandin El protects against ischemia reperfusion injury of the liver by inhibition of neutrophil adherence to endothelial cells. Transplantation 1997;64:1514 – 20. Karck U, Peters T, Decker K. The release of tumor necrosis factor from endotoxin stimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone. J Hepatol 1988;7:352 – 61. Bhattacharya M, Pen K, Ribeiro-da-Sliva A, et al. Localizationof functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. J Biol Chem 1999;274:15719 – 24. Katsuyama M, Ikegami R, Karahashi H, et al. Characterization of the LPS- stimulated expression of EP2 and EP4 prostaglandin E receptors in mouse macrophage-like cell line J774. I. Biochem Biophys Res Commun 1998;251:727 – 31. Boie Y, Stocco R, Sawyer N, et al. Molecular cloning and characterization of four rat prostaglandin E2 prostanoid receptor subtypes. Eur J Pharmacol 1997;340:227 – 41. Muto Y, Noun-Aria KT, Meager A, Alexander GJM, Eddleston ALWF, Williams R. Enhanced tumor necrosis factor and interleukin-1 in fulminant hepatic failure. Lancet 1988;II:72 – 4. Suzuki K, Endo R, Nakamura N, et al. Serum levels of soluble Fas ligandin patients with acute and fulminant hepatitis: relationship with soluble Fas, tumor necrosis factor-a and TNF-receptor-1. Hepatol Res 2000;17:19 – 30. Ohkubo K, Matsumoto T, Horuke N, et al. Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells. J Gastroenterol Hepatol 1998;13:696 – 702.
www.elsevier.com/locate/ihepcom
A novel prostaglandin E receptor subtype agonist, 0N0-4819, attenuates acute experimental liver injury in rats Kazuhiro Kasai *, Shin-ichiro Sato, Kazuyuki Suzuki First Department of Internal Medicine, Iwate Medical Uni6ersity School of Medicine, Uchimaru 19 -1, Morioka 020 -8505, Japan Received 22 January 2001; received in revised form 7 March 2001; accepted 16 March 2001
Abstract We evaluated the efficacy of ONO-4819, a newly developed agonist of a prostaglandin receptor subtype (EP4), on experimental model of acute liver injury in rats. Acute liver injury was induced by simultaneous intraperitoneal (i.p.) administration of D-galactosamine (GalN, 1 g/kg body weight) and lipopolysaccharide (LPS, 100 mg/kg body weight). The rats received a single intraperitoneal injection of ONO-4819 (0.2 mg/kg body weight) or physiological saline immediately after GalN/LPS administration. Submassive hepatic necrosis with marked elevation of serum total bilirubin, serum aspartate aminotransferase and serum alanine aminotransferase levels developed 24 h after GalN/ LPS administration. The administration of ONO-4819 significantly inhibited the development of submassive hepatic necrosis and inhibited the elevation in levels of biochemical markers that indicate liver function. In addition, the apoptotic index of hepatocytes assessed by the TUNEL method was significantly lower in rats treated with ONO-4819 than in the control. Although serum levels of tumor necrosis factor-a (TNF-a), interferon-g (IFN-g) and interleukin-8 (IL-8) were markedly elevated after GalN/LPS administration, ONO-4819 significantly inhibited the elevation of those of TNF-a and IFN-g but not that of IL-8. The beneficial effect of ONO-4819 for acute liver injury was similar at doses of 0.1, 0.05 and 0.01 mg/kg body weight. These results suggest that the EP4 agonist, ONO-4819, may have a protective effect against experimental liver injury in rats through the suppression of inflammatory cytokines. © 2001 Published by Elsevier Science Ireland Ltd. Keywords: Acute liver injury; Fulminant hepatic failure; Prostaglandin E; EP4 agonist; Tumor necrosis factor-a; Interferon-g; Interleukin-8
1. Introduction Fulminant hepatic failure (FHF) including fulminant hepatitis (FH) indicate poor prognosis [1,2]. In Japan, FH in 90% of the patients is * Corresponding author. Tel.: + 81-19-6515111; fax: +8119-6526664. E-mail address: [email protected] (K. Kasai).
caused by hepatitis viruses, mainly hepatitis A, hepatitis B and hepatitis nonA-nonB viruses and 10% by drug intoxication [3]. On the other hand, the pathogenesis of massive hepatic necrosis which is characteristic of FHF still remains unclarified. Among the factors known to be related to the mechanism, hyperimmunoreaction [4,5], apoptosis of hepatocytes [6–9] and microcirculatory disturbances of the hepatic sinusoidal space
1386-6346/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. PII: S 1 3 8 6 - 6 3 4 6 ( 0 1 ) 0 0 1 0 3 - 6
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
[10,11] have been studied. In addition, inflammatory cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-1 and interferon-g (INF-g), may play an important role in apoptosis and massive hepatic necrosis [12–14]. Furthermore, interleukin-8 (IL-8) participates as a neutrophil chemotactic factor and an activator of adhesion molecules on neutrophils in these processes [11,15]. Prostaglandins (PGs) are cyclooxygenase metabolites of arachidonate and act as local mediators under various inflammatory conditions [16]. Among PGs, PGE1 and PGE2 have been considered as potential therapeutic agents in the treatment of FHF based on their cytoprotective effects [17 –26]. However, its clinical application has not been realized in Japan. The actions of PGE1 and PGE2 are mediated by specific receptors on target cells. There are four subtypes of PGE receptors; EP1, EP2, EP3 and EP4. These are encoded by different genes and differ in their tissue expression [27]. Recently, a novel EP4 agonist, ONO-4819, which acts on PGE receptors has been developed in Japan [28]. This compound inhibits the production of cytokines such as TNF-a and IFN-g in vitro [28]. In the present study, we attempted to evaluate the effectiveness of ONO-4819 for experimental acute liver injury induced by GalN/LPS in rats
253
which shows that TNF-a and IFN-g play a crucial role in this model [8,9,12–14].
2. Materials and methods
2.1. Animals and reagents Nine-week-old male Wistar rats (CLEA JAPAN, Inc., Tokyo, Japan) weighing 200–250 g, were provided with food and water ad libitum and fasted for 24 h prior to the experiments. All the procedures in the present study were done according to the animal treatment rules of The Institute of Experimental Animals in Iwate Medical University. Hydrochloric acid, D-galactosamine (GalN; Wako Pure Chemical Industries, Ltd., Tokyo, Japan) was dissolved in physiological saline to prepare a 200 mg/ml solution. The pH of the solution was adjusted to 7.0 with 1 N sodium hydroxide. Lipopolysaccharide (LPS from Escherichia coli, 0111:B4; Sigma Chemical Co., St. Louis, MO, USA) was dissolved in physiological saline to prepare a 40 mg/ml solution. The EP4 agonist, ONO-4819, a gift from Ono Pharmaceutical Co., Ltd. (Osaka, Japan), was dissolved in physiological saline to prepare a 0.04 mg/ml solution.
Table 1 Serial changes of serum markers of liver and renal function on saline and ONO-4819-treated groups after administration of GalN/LPSa Time after GalN/LPS administration
T.Bil. (mg/dl) AST (IU/l) ALT (IU/l) BUN (mg/dl) CRNN (mg/dl)
1h
3h
6h
12 h
24 h
0.1 9 0.0 0.19 0.0 151.7945.6 114.89 6.4 27.39 3.6 23.39 1.2 16.2 91.4 13.39 1.4 0.3 90.1 0.39 0.1
0.1 9 0.0 0.1 9 0.0 261.89 57.0 214.5 9 19.6 129.7 9 60.3 70.29 12.4 20.5 9 2.1 28.29 4.6 0.4 9 0.1 0.6 9 0.1
0.2 9 0.1 0.1 9 0.0 1876.39 272.7* 826.5 9 113.9* 1411.79 144.1 858.8 9 173.2 17.9 91.9 14.8 9 1.1 0.4 9 0.1 0.3 9 0.0
0.7 90.5** 0.1 9 0.0** 12 215.3 9 7277.1*** 1384.8 9219.9*** 13 165.5 9 7912.9** 1984.7 9253.0** 20.6 9 3.6 16.8 97.9 0.3 9 0.1 0.3 9 0.1
2.8 91.0** 0.5 90.3** 24 690.8 95257.6*** 12 383.3 94206.0*** 32 877.7 97838.6*** 14 645.2 97633.8*** 47.7 919.9 31.7 915.4 0.7 90.3 0.4 90.2
a Values represent mean 9 S.D. of six rats in each time. Upper and lower data indicated saline-treated group and ONO-4819treated group, respectively. *PB0.05, **PB0.01, ***PB0.001.
254
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
abdominal aorta of rats under pentobarbital sodium anesthesia 1, 3, 6, 12 or 24 h following the drug administration, and liver tissue samples were removed each time for histological examination. In the second set of experiments, to determine the effect of dose at ONO-4819, different doses (0.1, 0.05 and 0.01 mg/kg body weight) was injected to rats treated with GalN/LPS (n= 24). Blood and liver tissue samples were collected 24 h following the drug administration. We measured levels serum of total bilirubin(T. Bil.), serum aspartate aminotransferase(AST), serum alanine aminotransferase (ALT), blood urea nitrogen (BUN) and serum creatinine (CRNN) using a conventional autoanalyzer (TBA-80FR NEO; Toshiba, Tokyo, Japan). Serum levels of TNF-a, IFN-g and IL-8 were also measured by enzyme-linked immunoassay using commercially available kits (Rat TNF-a measurement kit; Genzyme, Cambridge, MA, USA, Rat IFN-g measurement kit; Genzyme, Minneapolis, MN, USA, Rat IL-8 measurement kit; Panafarni Laboratories, Kumamoto, Japan).
2.3. Histopathological examination The liver tissue samples were fixed in 10% neutral buffered formalin, and embedded in paraffin. Sections were prepared and stained with hematoxylin-eosin (HE) for histopathological examination. Fig. 1. Effects of ONO-4819 on T.Bil., AST and ALT levels in the serum 24 h after the administration of GalN/LPS. Each column and bar represents the mean 9 S.D. of six rats.
2.2. Experimental method Acute liver injury was induced by simultaneous intraperitoneal injection of GalN (1 g/kg body weight) and LPS (100 mg/kg body weight). In the first set of experiments, ONO-4819 was administered intraperitoneally at a dose of 0.2 mg/kg to the rats immediately after the administration of GalN/LPS (n=30; ONO-4819-treated group). The same volume of physiological saline was administered as the control (n =30; saline-treated group). Blood samples were collected from the
2.4. Detection of apoptosis A portion of the liver tissue samples was fixed with 4% periodate-lysine-paraformaldehyde solution for the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. TUNEL was performed using a commercially available kit (Apoptag; Intergen, Purchase, NY, USA). The number of TUNEL-positive hepatocytes among 1000 hepatocytes was counted at 400× magnification and the percentage of TUNEL-positive hepatocytes was used as the apoptotic index.
2.5. Statistical analyses The results were expressed as mean9 Standard deviation (S.D.). Fisher’s protected least signifi-
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
cant difference test and Student’s t-test were used for comparisons between the groups. The differences were regarded as significant when P B 0.05.
3. Results
3.1. Serial changes in le6els of biochemical markers In the saline-treated group, serum levels of T. Bil., AST and ALT were significantly elevated 12 and 24 h after GalN/LPS administration (Table
255
1). In the ONO-4819-treated group, on the contrary, T. Bil., AST and ALT levels were significantly lower 12 and 24 h after GalN/LPS administration than those in the saline-treated group. Serum levels of BUN and CRNN slightly increased following the administration of GalN/ LPS in both groups, although there was no significant difference between the two groups. The beneficial effect of ONO-4819 was apparent even at a concentration as low as 0.01 mg/kg. Although the serum T. Bil. levels showed a tendency to decrease at a high dosage (0.2 mg/kg) of ONO-4819, the serum AST and ALT levels did
Fig. 2. Serial changes in TNF-a, IFN-g, and IL-8 levels in the serum of the ONO-4819-treated group and saline-treated group after the administration of GalN/LPS.
256
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
Fig. 3. Histological findings 24 h after administration of GalN/LPS (HE, ×100); (a) saline-treated group. (b) ONO-4819-treated group. Submassive hemorrhagic necrosis and inflammatory cell infiltration were observed in the saline-treated group. Hepatic damage in the ONO-4819-treated group apparently was suppressed compared with the saline treated group.
not show any significant differences among the different ONO-4819 dosages (Fig. 1).
3.2. Serial changes in serum le6els of TNF-h, IFN-k and IL-8 In the saline-treated group, levels of TNF-a and IFN-g in the serum increased in a biphasic manner after the administration of GalN/LPS (Fig. 2). In contrast, in the ONO-4819-treated group, serum levels of TNF-a and IFN-g
were significantly decreased compared with those in the saline-treated group (TNF-a: 36.79 12.7 vs. 87.89 30.8 pg/ml at 3 h, 34.89 15.0 vs. 295.79 149.5 pg/ml at 6 h; and IFN-g: 345.09 153.1 vs. 875.09 230.9 pg/ml at 1 h, 97.79 59.2 vs. 784.09 290.1 pg/ml at 6 h, respectively). On the other hand, there were no differences in serum IL-8 levels between the ONO-4819-treated and saline-treated groups at any time point following the administration of GalN/LPS.
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
3.3. Histopathological examination In the saline-treated group, condensation and fragmentation of the hepatocyte nucleus and inflammatory cell infiltration were first observed 3 h after the administration of GalN/LPS. The extent of these changes increased with time, and submassive hepatic necrosis with hemorrhage was observed 24 h after the administration (Fig. 3a). On
257
the other hand, in the ONO-4819-treated group, the histological changes were mild and showed focal to confluent necrosis 24 h after the administration of GalN/LPS (Fig. 3b). In the test using different doses of ONO-4819, the degree of hepatic necrosis was milder than that observed in the saline-treated group and was not significantly different among rats treated at different doses.
3.4. Detection of apoptosis The number of TUNEL-positive hepatocytes were increased 6 h after the drug administration in both groups, and peaked at 12 h in the salinetreated group and at 24 h in the ONO-4819treated group. The apoptosis index was significantly lower in the ONO-4819-treated group than in the saline-treated group at 6 and 12 h (Fig. 4, Table 2).
4. Discussion
Fig. 4. TUNEL positivity in the liver 12 h after administration of GalN/LPS (×100); (a) saline-treated group. (b) ONO4819-treated group. The number of TUNEL-positive hepatocytes in the ONO-4819-treated group was significantly less than that in the saline-treated group.
Sinclair et al. first reported the beneficial effect of PGE1 and PGE2 intravenously administered to patients with fulminant hepatitis in 1989 [24]. In Japan, the therapeutic efficacy of PGE1 for fulminant and acute severe hepatitis was also investigated in several studies [25,26]. However, clinical application of these drugs has not been realized. PGE1 and PGE2 have been known to attenuate hepatic damage in experimental models of acute liver injury induced by Propionibacterium acnes (P. acnes) and LPS, carbon tetrachloride, aflatoxin B1, galactosamine or ischemia-reperfusion [18–21,29,30]. Concerning the possible mechanism of attenuation of the liver injury by PGE1 and PGE2, Mizoguchi et al. demonstrated that PGE1 suppressed the production of a hepatocytotoxic factor which is induced in rats following the administration of P. acnes and LPS [19]. Karck et al. showed that PGE2 suppressed the release of TNF-a from rat Kupffer cells treated with LPS and recombinant IFN-g [31]. Masaki et al. also showed that PGE1 and PGE2 attenuated lipid peroxidation-dependent cell killing in cultured rat hepatocytes and reduced the extent of increased plasma membrane microviscosity of these cells
258
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
Table 2 Comparison of apoptotic index between the ONO-4819 and the saline-treated groupsa Time after GalN/LPS administration
Saline-treated group ONO-4819-treated group
1h
3h
6h
12 h
24 h
0.479 0.23 0.20 9 0.07
0.55 90.22 0.41 9 0.18
1.96 90.49* 1.279 0.43*
7.56 9 0.83** 0.96 90.21**
4.34 90.07 3.24 90.07
a Data are expressed as percentage of TUNEL-positive hepatocytes that accounted for the total hepatocyte population, *PB0.05, **PB0.001.
[21]. Furthermore, Natori et al., using the ischemia-reperfusion model, reported that PGE1 protects cells from liver injury by reducing leukocyte-endothelial cell adhesion via down modulation of adhesion molecule expression on the endothelium [30]. On the other hand, PGE receptors are divided into four subtypes, EP1, EP2, EP3 and EP4. These subtypes differ in their organ location and have different signal transduction mechanisms [16]. Although we did not examine the expression level of EP4 messenger RNA (mRNA) in the liver tissue, EP3 and EP4 are expressed in the rat liver [32]. Kastuyama et al. revealed that EP2 and EP4 mRNAs are expressed in a mouse macrophagelike cell line and these expression are increased by the addition of LPS in a dose-dependent manner [33]. Furthermore, PGEs increase the proliferation rate of hepatocytes through EP3 [34]. Studies concerning PGE receptor agonists and antagonists and their functions are ongoing and advanced, because PGE1 and PGE2 have various side effects when administered in patients with FHF and severe type of acute hepatitis [24,25]. An EP4 agonist, ONO-4819, that was recently developed in Japan, inhibits production of TNF-a and IFN-g in the blood [28]. These findings suggest that ONO-4819 instead of PGE1 and PGE2 may be applied for the treatment of FHF. However, its efficacy has not yet been tested. We, therefore, investigated the effect of ONO-4819 on a model of acute liver injury induced by GalN/ LPS. Laboratory test results indicate that the administration of ONO-4819 significantly improved the liver function in terms of the serum TNF-a and IFN-g levels and histological damages in the liver.
Furthermore, a dose-independent improvement of serum transaminase levels and histological changes were observed. Earlier studies indicate that TNF-a and IFN-g play a crucial role in the pathogenesis of GalN/LPS-induced acute liver injury model [8,9,12–14]. In clinical studies, Muto et al. reported that the rates of TNF-a and interleukin-1 production in circulating peripheral monocytes are high in patients with FHF [35]. Suzuki et al. demonstrated that serum TNF-a levels are significantly increased in patients with acute hepatitis and FHF, indicating their positive correlation with the soluble Fas ligand concentration [36]. Therefore, our results indicate that ONO-4819 may be effective against acute liver injury. However, in the present study, we administered ONO-4819 and GalN/LPS simultaneously. Since patients with FHF already show severe liver dysfunction when they were admitted into the hospital, drugs for improvement of liver function are usually administered after the occurrence of liver injury. Accordingly, in order to estimate the effects of ONO-4819 for clinical application, further studies regarding the precise dose and timing of administration are needed. On the other hand, although we showed that the serum TNF-a and IFN-g levels are significantly decreased in ONO-4819-treated group compared with those of saline-treated group, the serum IL-8 levels did not change. IL-8, which is a major cytokine associated with neutrophil infiltration of the liver tissue, is produced in endothelial and Kupffer cells [37]. Although the reasons why the serum IL-8 levels were not affected by ONO4819 is unknown, our earlier study[15] showed that multiple-interval injection of cyclosporin A significantly inhibited the elevation of serum IL-8
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
levels in the same models. Further studies are needed to clarify the production of IL-8 by cells and its role in the massive hepatic necrosis. In the rats model of liver injury induced by GalN/LPS, it is considered that apoptosis of hepatocytes occurs in the early stage and then massive hepatic necrosis develops due to microcirculatory disturbance of the hepatic sinusoidal space [8]. Although the TUNEL method and agarose gel electrophoresis are widely used for detection of cell apoptosis, we only used the TUNEL method. Our study showed that the number of TUNEL -positive hepatocytes serially decreased in the ONO-4819 treated group indicating that ONO-4819 may inhibit apoptosis, which may be considered as an initial event of the process in hepatic cell necrosis. To clarify whether ONO-4819 inhibits apoptosis and liver injury, further experimental studies are required in the future. In conclusion, a novel PGE receptor subtype agonist, ONO-4819 attenuates the acute liver injury in rats induced by GalN/LPS. We suggest that a possible mechanism of attenuation by ONO-4819 may be the suppression of production of inflammatory cytokines such as TNF-a.
Acknowledgements The authors thank Dr Shunichi Sato (vice-president, Iwate Medical University) for advice on this manuscript and Dr Tadashi Kawakami for excellent technical assistance. The authors wish to express there thanks to the Ono Pharmaceutical Co., Ltd., Osaka, Japan for the supply of ONO4819.
References [1] Sama C, Mastroianni A, Danesi G, et al. Fulminant hepatic failure. Gastroenterol Int 1999;12:83 – 7. [2] Riordan SM, Williams R. Cause and prognosis in acute liver failure. Liver Transplant Surg 1999;5:86 – 9. [3] Sato S, Suzuki K, Takikawa Y, et al. Physiopathology and therapy of fulminant hepatitis. Nippon NaikaGakkaiZasshi 1999;88:1783 –8 In Japanese.
259
[4] Almeida JD, Waterson AP. Immune complexes in hepatitis. Lancet 1969;II:983 – 6. [5] Dudley FJ, Fox RA, Sherlock S. Cellular immunity and hepatitis-associated Australia antigen liver disease. Lancet 1972;I:723 – 6. [6] Ogasawara J, Watanabe-Fukunaga R, Adachi M, et al. Lethal effect of the anti-Fas antibody in mice. Nature 1993;364:806 – 9. [7] Alido K, Moriyama T, Guidotti LG, et al. Mechanism of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541 – 54. [8] Morikawa A, Sugiyama T, Kato Y, et al. Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model. Infect Immun 1996;64:734 – 8. [9] Leist M, Gantner F, Bohlinger I, et al. Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J Pathol 1995;146:1220 – 34. [10] Mochida S, Ohno A, Arai M, Fujiwara K. Role of adhesion between activated macrophages and endothelial cells in the development of two types of massive hepatic necrosis in rats. J Gastroenterol Hepatol 1995;10:S38 – 42. [11] Ohira H, Ueno T, Torimura T, Tanikawa K, Kasukawa R. Leukocyte adhesion molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver injury. Scand J Gastroenterol 1995;30:1027 – 35. [12] Nagaki M, Tanaka M, Sugiyama A, et al. Interleukin-10 inhibits hepatic injury and tumor necrosis factor-a and interferon-g mRNA expression induced by staphylococcal enterotoxin B or lipopolysaccharide in galactosamine-sensitized mice. J Hepatol 1999;31:815 – 24. [13] Hishinuma I, Nagakawa J, Hirota K, et al. Involvement of tumor necrosis factor-a in development of hepatic injury in galactosamine-sensitized mice. Hepatology 1990;12:1187 – 91. [14] Bruce DC, Vicki ME, Bruno S, et al. Interferon-g receptor deficient mice are resistant to endotoxic shock. J Exp Med 1994;179:1437 – 44. [15] Kawakami T, Sato S, Suzuki K, et al. Beneficial effect of cyclosporin A on acute hepatic injury induced by galactosamine and lipopolysaccharide in rats. Hepatol Res 2000;18:284 – 97. [16] Narumiya S, Sugimoto Y, Ushikubi F, et al. Prostanoid receptors: properties and functions. Physiol Rev 1999;79:1193 – 226. [17] Robert A, Nezamis JE, Lancaster C, Hanchar AJ. Cytoprotection by prostaglandins in rats. Gastroenterology 1979;77:433 – 43. [18] Stachura J, Tarnawski A, Ivey KJ, et al. Prostaglandin protection of carbon tetrachloride induced liver cell necrosis in the rat. Gastroenterology 1981;81:211 – 7. [19] Mizoguchi Y, Tsutsui H, Miyajima K, et al. The protective effects of prostaglandin E1 in an experimental massive hepatic cell necrosis model. Hepatology 1987;7:1184 – 8.
260
Y. Narahara et al. / Hepatology Research 21 (2001) 252–260
[20] Rush BD, Wilkinson KF, Nichols NM, et al. Hepatic protection by 16, 16%-dimethyl prostaglandin E2 (DMPG) against acute afratoxin B1-induced injury in the rat. Prostaglandins 1989;37:683 –93. [21] Masaki N, Ohta Y, Shirataki H, et al. Hepatocyte membrane stabilization by prostaglandins El and E2: favorable effects on rat liver injury. Gastroenterology 1992;102:572 – 6. [22] Kikuchi E, Fukui H, Matsumoto M. Effect of prostaglandin E1on experimental acute hepatic failure in rabbits: prostaglandin E1 prevents the development of multiple-organ failure. J Hepatol 1994;20:478 – 82. [23] Abecassis M, Falk R, Blendis L, et al. Treatment of fluminant hepatic failure with a continuous infusion of prostin VT (PGE1). Hepatology 1987;7:1104A. [24] Sinclair SB, Greig PD, Blendis LM, et al. Biochemical and clinical response of fluminant viral hepatitis to administration of prostaglandin E. J Clin Invest 1989;84:1063 – 9. [25] Oda T, Sato S, Muto Y, et al. A double-blind comparative study on G-511 (PGE1) in severe type of acute hepatitis. J Clin Med 1994;10:2299 – 322 In Japanese. [26] Sasaki M. A study on the clinical efficacy and underlying mechanism of prostaglandin E1 in patients with acute severe hepatitis. Acta Sch Med Univ Gifu 1996;44:653 – 68 In Japanese. [27] Coleman RA, Smith WL, Narumiya S. International union of pharmacology classification of prostanoid receptors properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 1994;46:205 – 29. [28] Sakata K, Maruyama T, Seki A et al. The roles of PGE2 receptor EP4 subtype on cytokine regniation and its related disease. 11th international conference on advances in prostaglandin and leukotriene research, Florence, Italy, June 4– 8, 2000; abstract book: 51. [29] Stachura J, Tarnawski A, Szczudrawa J, et al. Cytopro-
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
tective effect of 16, 16%-dimethyl prostaglandin E2 and some drugs on an acute galactsamine induced liver damage in rat. Folia Histochem Cytochem 1980;18:311 – 8. Natori S, Fuji Y, Kurosawa H, et al. Prostaglandin El protects against ischemia reperfusion injury of the liver by inhibition of neutrophil adherence to endothelial cells. Transplantation 1997;64:1514 – 20. Karck U, Peters T, Decker K. The release of tumor necrosis factor from endotoxin stimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone. J Hepatol 1988;7:352 – 61. Bhattacharya M, Pen K, Ribeiro-da-Sliva A, et al. Localizationof functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. J Biol Chem 1999;274:15719 – 24. Katsuyama M, Ikegami R, Karahashi H, et al. Characterization of the LPS- stimulated expression of EP2 and EP4 prostaglandin E receptors in mouse macrophage-like cell line J774. I. Biochem Biophys Res Commun 1998;251:727 – 31. Boie Y, Stocco R, Sawyer N, et al. Molecular cloning and characterization of four rat prostaglandin E2 prostanoid receptor subtypes. Eur J Pharmacol 1997;340:227 – 41. Muto Y, Noun-Aria KT, Meager A, Alexander GJM, Eddleston ALWF, Williams R. Enhanced tumor necrosis factor and interleukin-1 in fulminant hepatic failure. Lancet 1988;II:72 – 4. Suzuki K, Endo R, Nakamura N, et al. Serum levels of soluble Fas ligandin patients with acute and fulminant hepatitis: relationship with soluble Fas, tumor necrosis factor-a and TNF-receptor-1. Hepatol Res 2000;17:19 – 30. Ohkubo K, Matsumoto T, Horuke N, et al. Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells. J Gastroenterol Hepatol 1998;13:696 – 702.