treatment with β-sqag9 prevents rat hepatic ischemia-reperfusion injury

5
LIVER Treatment With -SQAG9 Prevents Rat Hepatic Ischemia-Reperfusion Injury H. Shima, T. Tsuruma, H. Sahara, M. Takenouchi, N. Takahashi, Y. Iwayama, A. Yagihashi, N. Watanabe, N. Sato, and K. Hirata ABSTRACT Background. Ischemia-reperfusion (I/R) injury occurs in various situations, including transplantation, trauma, and shock. We previously reported that the synthetic -SQDG (18:0), which was derived from sulfoquinovosyl diacylglycerol of the sea urchin, possessed immunosuppressive effects, such as inhibition of T-cell responses in human allogenic human mixed lymphocyte reactions (MLR) and skin allograft survival in rats. -SQAG9 was synthesized from -SQDG (18:0) to improve structural stability in aqueous solution with the same biological activities to bind to CD62L (L-selectin) and CD62P (P-selectin) in vitro. We hypothesized that -SQAG9 might attenuate leukocyte rolling on the endothelium and neutrophil infiltration in which L-selectin and P-selectin are key molecules. We investigated the protective effect of -SQAG9 against hepatic I/R injury. Methods. Male Lewis rats were divided into 6 groups: sham, control, and treatment. Rats in the control, and the treatment groups were subjected to hepatic ischemia for 30 minutes. They were injected with PBS or -SQAG9 at doses of 5, 10, 25, and 50 mg/kg into the penile vein immediately before reperfusion. To assess the damage to the hepatic parenchyma, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured and histological evaluation was performed at 6 hours after reperfusion. Results. In the group treated with -SQAG9 at a dose of 10 mg/kg, AST, ALT, and LDH were significantly reduced, and the amount of neutrophil infiltration also was significantly reduced. Conclusions. Our data suggest that SQAG-9 (10 mg/kg) reduces the warm hepatic I/R injury. W ARM ischemia causes clinical disorders in transplan- tation, trauma, shock, and liver surgery. 1,2 Surgical interventions of hepatic blood flow occlusion by techniques, such as intermittent clamping, the Pringle maneuver, and total vascular exclusion, are performed to minimize blood loss while dividing the liver parenchyma. 3 However, these procedures are associated with intraoperative or postoper- ative liver damage caused by warm ischemia-reputation (I/R) injury. Massive hepatectomy and bile duct extirpation with extensive liver resections, as well as liver transplanta- tion, more frequently cause clinical disorders of hepatic I/R injury. 4 Recent strategies to protect against liver injury by I/R include surgical interventions such as ischemic precon- From the Department of Surgery (H.S., T.T., Y.I., K.H.), Marine Biomedical Institute (H.S.), Department of Pathology (M.T., N.T., N.S.), and Department of Clinical Medicine (A.Y., N.W.), Sapporo Medical University School of Medicine, Sapporo, Japan. Address reprint requests to T. Tsuruma, Department of Sur- gery, Sapporo Medical University, School of Medicine, S1 W16, Chuoku, Sapporo, Hoskaid 0060-0061, Japan. E-mail: [email protected] © 2005 by Elsevier Inc. All rights reserved. 0041-1345/05/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/j.transproceed.2004.12.240 Transplantation Proceedings, 37, 417– 421 (2005) 417

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Page 1: Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

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LIVER

reatment With �-SQAG9 Prevents Rat Hepatic Ischemia-Reperfusionnjury. Shima, T. Tsuruma, H. Sahara, M. Takenouchi, N. Takahashi, Y. Iwayama, A. Yagihashi,. Watanabe, N. Sato, and K. Hirata

ABSTRACT

Background. Ischemia-reperfusion (I/R) injury occurs in various situations, includingtransplantation, trauma, and shock. We previously reported that the synthetic �-SQDG(18:0), which was derived from sulfoquinovosyl diacylglycerol of the sea urchin, possessedimmunosuppressive effects, such as inhibition of T-cell responses in human allogenichuman mixed lymphocyte reactions (MLR) and skin allograft survival in rats. �-SQAG9was synthesized from �-SQDG (18:0) to improve structural stability in aqueous solutionwith the same biological activities to bind to CD62L (L-selectin) and CD62P (P-selectin)in vitro. We hypothesized that �-SQAG9 might attenuate leukocyte rolling on theendothelium and neutrophil infiltration in which L-selectin and P-selectin are keymolecules. We investigated the protective effect of �-SQAG9 against hepatic I/R injury.Methods. Male Lewis rats were divided into 6 groups: sham, control, and treatment.Rats in the control, and the treatment groups were subjected to hepatic ischemia for 30minutes. They were injected with PBS or �-SQAG9 at doses of 5, 10, 25, and 50 mg/kg intothe penile vein immediately before reperfusion. To assess the damage to the hepaticparenchyma, aspartate aminotransferase (AST), alanine aminotransferase (ALT), andlactate dehydrogenase (LDH) were measured and histological evaluation was performedat 6 hours after reperfusion.Results. In the group treated with �-SQAG9 at a dose of 10 mg/kg, AST, ALT, and LDHwere significantly reduced, and the amount of neutrophil infiltration also was significantlyreduced.Conclusions. Our data suggest that SQAG-9 (10 mg/kg) reduces the warm hepatic I/R

injury.

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ARM ischemia causes clinical disorders in transplan-tation, trauma, shock, and liver surgery.1,2 Surgical

nterventions of hepatic blood flow occlusion by techniques,uch as intermittent clamping, the Pringle maneuver, andotal vascular exclusion, are performed to minimize bloodoss while dividing the liver parenchyma.3 However, theserocedures are associated with intraoperative or postoper-tive liver damage caused by warm ischemia-reputationI/R) injury. Massive hepatectomy and bile duct extirpationith extensive liver resections, as well as liver transplanta-

ion, more frequently cause clinical disorders of hepatic I/R t

2005 by Elsevier Inc. All rights reserved.60 Park Avenue South, New York, NY 10010-1710

ransplantation Proceedings, 37, 417–421 (2005)

njury.4 Recent strategies to protect against liver injury by/R include surgical interventions such as ischemic precon-

From the Department of Surgery (H.S., T.T., Y.I., K.H.), Marineiomedical Institute (H.S.), Department of Pathology (M.T., N.T.,.S.), and Department of Clinical Medicine (A.Y., N.W.), Sapporoedical University School of Medicine, Sapporo, Japan.Address reprint requests to T. Tsuruma, Department of Sur-

ery, Sapporo Medical University, School of Medicine, S1 W16,huoku, Sapporo, Hoskaid 0060-0061, Japan. E-mail:

[email protected]

0041-1345/05/$–see front matterdoi:10.1016/j.transproceed.2004.12.240

417

Page 2: Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

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itioning, pharmacologic agents targeting the microcircula-ion, oxidative stress, proteases, or inflammation, and geneherapy.3,5

We previously reported that sulfolipids extracted fromea urchin intestines possess immunosuppressive effects tonhibit the human mixed lymphocyte reaction (MLR) androlong rat skin allograft survival. Moreover, �-SQAG9,hich shows greater structural stability in solution than theriginal sulfonolipid and possesses equal immunosuppres-ive effects, may bind L-selectin (CD62L) and P-selectinCD62P) molecules in vitro.6–9 L-selectin and P-selectin,hich are expressed on leukocytes and/or endothelialells,10,11 mediate adhesion of leukocytes to endothelialells. Each selectin molecule is a single-chain transmem-rane glycoprotein with a similar modular structure. I/Rlicits endothelial cell injury, which is manifest as swellingr detachment from the underlying basement membrane,hereby compromising barrier function. These events maye accompanied by leukocyte-endothelial cell adhesion,hich is manifested as rolling, firm adhesion and emigrationf leukocytes from the postcapillary venules of the micro-asculature.12 There is a substantial body of evidence thateukocytes and adhesion molecules, such as L-selectin, areey mediators of I/R injury in several splanchnic organs,ncluding the liver.10,13 Thus, we investigated the protectiveffect of �-SQAG9 against hepatic I/R injury, consideringhat the target molecule of �-SQAG9 might be selectin, onef the key molecules in neutrophil activation, which is onef the pathways for I/R injury.

ATERIALS AND METHODSnimal Preparation

ale Lewis rats, weighing approximately 200 to 300 g, wereurchased from Sankyo Labo Service (Sapporo, Japan), andoused in a room maintained at a constant temperature with a2-hour light-dark cycle. The rats were fasted for 12 hours beforeperation, but were allowed to drink water ad libitum. They werenesthetized by the inhalation of diethyl ether (SIGMA-LDRICH, St. Louis, Mo, United States) and/or the intraperito-eal injection of 40 mg/kg sodium pentobarbital (Dainippon Phar-aceutical CO., Ltd., Osaka, Japan). All procedures were

erformed with the animals breathing spontaneously. After anes-hesia, including intraperitoneal injection of sodium pentobarbital,he liver was exposed by a midline incision. All structures (hepaticrtery, portal vein, and bile duct) associated with the left andedian liver lobes (70% of liver mass) were occluded with aicrovascular clamp (Mera CO., Tokyo, Japan) for 30 minutes.his method of partial hepatic ischemia allows for portal decom-ression through the right and caudate lobes to prevent mesentericenous congestion. After 30 minutes of ischemia, reperfusion wasnduced by removing the microvascular clamp. Then the abdominalall was closed in standard fashion with 4-0 ETHILON (Johnsonnd Johnson Medical Pty. Ltd, Tokyo, Japan) and the animals wereeturned to their cages. Rats were humanely killed 6 hours aftereperfusion. Sham operation, in which laparotomy was done with-ut vascular occlusion, also was performed for some animals. Allxperiments were performed in accordance with the Guidelines for

nimal Experimentation of Sapporo Medical University. a

rug Preparation

,2-di-O-acyl-3-O-(-D-sulfoquinovosyl)-glyceride with 2 stearic ac-ds (�-SQAG9) was synthesized as previously reported.14

-SQAG9 was mixed with cholesterol in a 3:1 ratio and dissolved inhloroform:methanol:H2O � 100:10:1 in a test tube. The organicolvent mixture was removed using a rotary flash evaporator undereduced pressure. The residual organic solvent was further re-oved by drying overnight at room temperature in a desiccator

nder vacuum. The dried lipid film was hydrated with phosphate-uffered saline (PBS) (pH 7.4), followed by incubation at 64°C for0 minutes. The test tube was then shaken vigorously on a vortexixer. Vesicles adhered to a filter of 200-nm pore size for

niformity.

xperimental Protocol

o determine the optimal dose for the protective effect of-SQAG9 against rat I/R injury, animals were divided into 6xperimental groups. Control Rats were injected with PBS solutionAKO CO., Carpinteria, Calif, United States. Rats in the treat-ent groups were injected with �-SQAG9 at doses of 5, 10, 25, or

0 mg/kg via the penile vein immediately before reperfusion. Theats in the sham operation group underwent laparotomy withoutcclusion. Each experimental group included 6 rats. The rats wereumanely killed 6 hours after reperfusion. Then, hepatic tissue wasbtained to estimate hepatic damage.

iochemical Measurements

o measure plasma concentrations of established markers ofepatic injury, such as serum aspartate aminotransferase (AST),lanine aminotransferase (ALT), and lactate dehydrogenaseLDH), blood was taken from the vena cava inferior 6 hours aftereperfusion. The blood samples were then centrifuged (3000pm/15 min 4°C) to obtain the plasma supernates for biochemicalnalyses. The serum was stored at �80°C until assay.

istology

ollowing injection into the portal vein and washing of the vesselsn the liver with cold isotonic lactated Ringer’s solution (Otsukaharmaceutical Co., Ltd, Tokyo, Japan), the ischemic lobe of the

iver was obtained, fixed in 10% formalin neutral buffered solutionWako Pure Chemical Industries, Ltd, Osaka, Japan), and thenmbedded in paraffin. The sections were stained with hematoxylinnd eosin (H&E) for light microscop’s to study the number ofnfiltrating neutrophils, which were identified by morphology andounted in 10 high-power fields (HPFs; original magnification �00).

tatistical analysis

ll data were expressed as mean values � SD. Comparisonsetween the control group and the other groups used an unpairedtudent test with P � .05 considered significant.

ESULTSstablishment of the Optimal Dose of �-SQAG9 Liposome,hich Could Induce a Protective Effect Against Hepatic I/R

njury

o assess the magnitude of the hepatic injury at 6 hours

fter I/R, we measured the plasma concentrations of AST,
Page 3: Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

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LT, and LDH. Among rats injected with PBS (control

ig 1. The plasma concentrations of AST (A), ALT (B), and LDHC) in the sham operation group and the treated group with-SQAG9 liposome (5, 10, 25, and 50 mg/kg) and PBS 6 hoursfter reperfusion. The mean plasma concentrations of AST, ALT,nd LDH in the rats injected with �-SQAG9 liposome at a dosef 10 mg/kg were significantly lower than in those injected withBS (control group). Thus, the optimal dose of �-SQAG9 lipo-ome, which could induce the protective effect against hepatic/R injury, was 10 mg/kg in this model. Results are expressed ashe mean � SD. *P � .05, †P � .01, and ‡ P � .001 vs PBSroup at each point.

roup), the values were 1025.8 � 279.3 IU/L, 1134.0 � b

72.4 IU/L, and 2052.1 � 866.0 IU/L, respectively, whichere significantly increased compared with the sham oper-tion group. In contrast, rats injected with �-SQAG9iposome (5 mg/kg or 10 mg/kg) showed a dose-decrease in

ean plasma concentrations compared with the controlroup. At 10 mg/kg, the concentrations were significantlyeduced to 557.8 � 90.1 IU/L (P � .01), 466.0 � 191.6 IU/LP � .001), and 872.3 � 27.1 IU/L (P � .05), respectively.owever, among rats injected with 25 mg/kg �-SQAG9

iposome, the mean plasma concentrations were almost theame or increased compared with those in the controlroup. Thus, the optimal dose of �-SQAG9 liposome tonduce a protective effect against hepatic I/R injury was 10

g/kg (Fig 1).

istological Evaluation of Neutrophils in Liver

he number of neutrophils accumulating in the liver at 6ours after reperfusion was significantly lower among theroup treated with �-SQAG9 liposomes (10 mg/kg) thanhe controls (132 � 26.0 vs 201 � 15.6; P � .01; [Fig 2]).istological damage, such as leukocyte infiltration and

epatocyte necrosis with zonal acidophilic degeneration,as the most severe at 6 hours after reperfusion, whereas

ew histological changes were observed at 12 or 24 hoursfter reperfusion (data not shown).

istological Evaluation of Liver Damage

eutrophil infiltration was mainly accompanied by hepato-yte necrosis evidenced by nuclear degeneration and loss ofistinct cellular borders.15 The degree of neutrophil infil-ration was markedly reduced by treatment with �-SQAG9iposome (10 mg/kg) compared with the control group at 6ours after reperfusion (Fig 3).

ISCUSSION

e previously reported that injection of �-SQAG9 into

ig 2. The number of neutrophils accumulated in the liver 6ours after reperfusion was significantly lower in the treatmentroup with �-SQAG9 liposome at a dose of 10 mg/kg than in theontrol group with PBS (control group). Results are expressed ashe mean � SD. n � 6 rats; †P � .01 vs PBS group.

oth the penile vein and the portal vein at a dose of 50

Page 4: Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

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g/kg reduced rat hepatic I/R injury.8 However, this exper-mental protocol was too complicated to apply in thelinical setting. Therefore, in this study, we evaluated therotective effect of �-SQAG9 by a simpler method ofdministration of �-SQAG9, which was injection into theat penile vein immediately before reperfusion.

We previously reported that hepatic damage induced by

ig 3. H&E sections of livers 6ours after reperfusion. The degreef polymorphonuclear neutrophil in-ltration accompanied by necrosisas markedly reduced by treatmentith �-SQAG9 liposome at a dose of0 mg/kg (B) compared with theontrol group with PBS (A).

0 minutes of ischemia became almost normal 24 hours s

fter reperfusion without treatment.15 In this study, at 24ours after reperfusion, the levels of AST, ALT, and LDHfter 30 minutes of ischemia were reduced to almost normalevels and little difference was observed between ratsreated with PBS and 10 mg/kg of �-SQAG9 at 24 hours.he levels of serum markers were significantly increased athours after reperfusion compared with 24 hours (data not

hown). Furthermore, our histological evaluation suggested

Page 5: Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

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clear contrast to the liver damage at 6 hours aftereperfusion (data not shown). Thus, we evaluated 6 hoursfter reperfusion, including histological evaluation.

This study suggested that the optimal dose of �-SQAG9ight be 10 mg/kg in this rat warm hepatic I/R injuryodel. That is, in this experimental model, the protective

ffect of �-SQAG9 against I/R injury was not dose-depen-ent. It has been reported that the survival rates in a pigepatic I/R injury model were worse after administration of.1 mg/kg of an iNOS inhibitor (ONO-1714) than with 0.05g/kg, although this iNOS inhibitor improved the survival

ates and attenuated hepatic I/R injury.17 The suggestedeason was that the high dose (0.1 mg/kg) might inhibit notnly inducible nitric oxide synthase but also endothelialitric oxide synthase, which is believed to maintain ho-eostasis and have a cytoprotective effect.17 Hicks et al

eported that a low dose of recombinant P-selectin glyco-rotein ligand-1 immunoglobulin (rPSGL-Ig), which canind the murine cytokine-induced neutrophil attractantKC) and inhibit neutrophil migration toward the chemoat-ractant in vitro, showed pronounced antiinflammatoryffects and could reduce leukocyte rolling if given as areatment.18 Thus some drugs that induce protective effectsgainst I/R injury at optimal doses may improve clinicalisorders.In this study, we studied the protective effects of

-SQAG9 against hepatic I/R injury. The administration of-SQAG9 attenuated neutrophil infiltration into the liver.his protective mechanism of �-SQAG9 may be inactiva-

ion of neutrophils via inhibition of rolling, because selectin,hich is a key molecule in rolling, may be the targetolecule of �-SQAG9. However, the mechanism of its

rotective activity against I/R injury remains mysterious.t

hus, it requires further investigation including pharmaco-inetics. In summary, this study demonstrates that-SQAG9 at an optimal dose (10 mg/kg) may display arotective effect against I/R injury.

EFERENCES

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0034. Nakamitsu A, Hiyama E, Imamura Y, et al: Surg Today

1:140, 20015. Martinez-Mier G, Toledo-Pereyra LH, McDuffie E, et al:Surg Res 93:156, 20006. Matsumoto Y, Sahara H, Fujita, et al: Transplantation 74:261,

0027. Yamamoto Y, Sahara H, Takenouchi M, et al: (submitted)8. Tsuruma T, Sahara H, Takenoushi M, et al: Transplant Proc

6:1965, 20049. Matsumoto K, Takenouchi M, Ohata K, et al: Biochem

harmacol 15:2379, 200410. Ala A, Dhillon AP, Hodgson HJ: Int J Exp Pathol 84:1, 200311. Abbas AK, Lichtman AH: In Malley J, Krehling H (eds):

ellular and Molecular Immunology. Philadelphia: Saunders;003, p 12212. Burke J, Zibari GB, Brown MF, et al: Transplantation Proc

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27:832, 199814. Sugawara F, Hanashima S, Mizushina Y, et al: Bull Marine

iomed Inst Sapporo Med Univ 4:7, 199915. Yadav SS, Howell DN, Gao W, et al: Am J Physiol 275:1341, 199816. Araya J, Tsuruma T, Hirata K, et al: Transplantation 73:529,

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ation 73:1439, 200218. Hicks AE, Nolan SL, Ridger VC, et al: Blood 101:3249, 2003