© 2008 The Authors

Doi: 10.1111/j.1742-7843.2008.00219.x

Journal compilation

© 2008 Nordic Pharmacological Society

. Basic & Clinical Pharmacology & Toxicology

,

102

, 394–398

Blackwell Publishing Ltd

Suppression of Ischaemia-Induced Cytokine Release by Dimaprit and Amelioration of Liver Injury in Rats

Atsuko Motoki

1

, Naoto Adachi

1,2,3

, Keyue Liu

2

, Hideo K. Takahashi

2

, Masahiro Nishibori

2

, Toshihiro Yorozuya

1

, Tatsuru Arai

1

and Takumi Nagaro

1

1

Department of Anaesthesiology and Resuscitology, Ehime University Graduate School of Medicine, Shitsukawa, Touon-shi, Ehime, Japan,

2

Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho, Okayama-shi, Okayama, Japan, and

3

Medical Division, Mabuchi Clinic, Kakimoto-cho, Kyoto-shi, Kyoto, Japan

(Received July 27, 2007; Accepted October 12, 2007)

Abstract:

Inflammatory reactions play an important role in ischaemia/reperfusion injury in various organs. Since histamineH

4

action has been shown to prevent the development of ischaemia/reperfusion liver injury, we examined the effects ofdimaprit, a histamine H

2

/H

4

receptor agonist, on ischaemia-induced cytokine release and liver damage. Male Wistar rats(300 g) were subjected to warm ischaemia for 30 min. by occlusion of the left portal vein and hepatic artery under halothaneanaesthesia. Saline or dimaprit (20 mg/kg, subcutaneously) was injected immediately after reperfusion of blood flow.Transient ischaemia provoked severe liver damage 24 hr after reperfusion, and the plasma concentrations of alaninetransaminase and aspartate transaminase were 4600 IU/l and 13,200 IU/l, respectively. The values in the dimaprit groupwere 55% and 46% of those in control animals, respectively. Dimaprit also reduced the infarct size to 50%. Liver ischaemiamarkedly increased interleukin-12 levels 2–24 hr after reperfusion. The dimaprit treatment depressed the values to 40–64%of those in the corresponding control group 4–24 hr after reperfusion. Since interleukin-12 facilitates cell-mediated

cytotoxicity, the protective effect of dimaprit may be attributed to regulation of cytokine release during reperfusion.

Although the principal therapy against ischaemia-inducedorgan injury is the early restoration of blood flow, there aremany factors in tissue damage during reperfusion, such asformation of oxygen free radicals, inflammatory cell infiltra-tion, and intracellular Ca

2+

overload. In the liver, activationof cytotoxic immune responses may play a pivotal role inreperfusion injury in the liver, because Kupffer cells areliver-resident macrophages and readily activated by ischaemia[1–3]. Prevention of reperfusion injury also contributes tosurvival of allografts in liver transplantation, because theliver is exposed to ischaemia during the organ harvest.

In our previous study, we demonstrated prevention ofischaemia/reperfusion liver damage by post-ischaemicadministration of histamine [4]. The preventive effect wasobserved in animals treated with dimaprit, a histamine H

2

/H

4

receptor agonist, to an extent similar to that observed inhistamine-treated animals. The amelioration by these agentswas reversed by thioperamide, a histamine H

3

/H

4

receptorantagonist, but not by histamine H

2

receptor antagonists.Concerning the difference in localization between H

3

and H

4

receptors, histamine H

3

receptors are found mainly in thebrain, whereas histamine H

4

receptors are primarily expressedon peripheral tissues, particularly the haematopoieticlineage, such as bone marrow, spleen, thymus and leucocytes

[5–8]. Taken together with these findings, the protectiveeffect may be mediated by histamine H

4

receptors.Recently, histamine H

4

receptors have been shown tomediate immune responses and inflammatory cell recruitment.While stimulation of histamine H

4

receptors induceschemotaxis of mast cells and eosinophils [9,10], histamineH

4

action suppresses interleukin (IL)-12, which is a keyinducer of cell-mediated immunity [11]. Because histamineH

4

action suppresses IL-12 and ameliorates ischaemia-induced live injury, it is likely that the protective effect ofdimaprit is related to suppression of IL-12. In the presentstudy, therefore, we evaluated effects of dimaprit on ischaemia-induced release of cytokines and correlated them withchanges in functional and morphologic outcomes.

Materials and Methods

Drugs and chemicals.

Dimaprit dihydrochloride was purchased fromSigma-Aldrich (St. Louis, MO, USA). 2,3,5-Triphenyltetrazoliumchloride was purchased from Research Organics (Cleveland, OH,USA). The dose of agents was expressed as the weight of the free base.

Animals.

This study was approved by the Committee on AnimalExperimentation at Ehime University Graduate School ofMedicine, Ehime, Japan. All animals were cared for in compliancewith the Principles of Laboratory Animal Care formulated byEhime University Graduate School of Medicine. Male Wistar rats(Charles River, Yokohama, Japan) weighting approximately 300 gwere kept in groups in a room controlled at 23 ± 2

°

and maintainedunder an alternating 12-hr light:dark cycle (lights on at 6 a.m.).Food and water were provided

ad libitum

.

Author for correspondence: Atsuko Motoki, Department ofAnesthesiology and Resuscitology, Ehime University GraduateSchool of Medicine, Shitsukawa, Touon-shi, Ehime 791-0295,Japan (fax +81-89-960-5386, e-mail atsumotoki@hotmail.com).

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Sixty-one rats were allocated to one of eight groups, and eachanimal was subjected to transient liver ischaemia for 30 min. Afterreperfusion of blood flow, saline or dimaprit (20 mg/kg, sub-cutaneously) was injected. Then, animals in the saline and dimapritgroups were killed 2 hr (n = 7 and 9), 4 hr (n = 8 and 8), 8 hr(n = 5 and 6), and 24 hr (n = 9 and 9) after reperfusion, respectively.Another set of 10 rats served as an intact group.

Liver ischaemia.

Anaesthesia was induced by inhalation with 2%halothane in a gas mixture of oxygen and nitrous oxide (50:50).After induction, the halothane concentration was reduced to 1–1.5%for surgical preparation. After an abdominal incision along theanterior transverse line, the left portal vein and hepatic artery wereexposed. A thermocouple probe was inserted into the rectum, andthe rectal temperature was maintained at 37.5

°

with a heating lamp.Heparin (100 IU, subcutaneously) was injected, and the left portalvein and hepatic artery were clamped with a temporary vascularclip. This method produces ischaemic infarcts in most liver lobes,except for the two right lateral lobes [12]. After ischaemia for30 min. at 37.5

°

under anaesthesia, the clip was removed, and thesurgical incision was sutured. Saline or dimaprit (20 mg/kg,subcutaneously) was injected immediately after reperfusion ofblood flow. The rectal temperature was maintained until the animalwas allowed to recover from anaesthesia. Then, the animal wasbrought to its cage in a room maintained at constant temperature,and allowed access to food and water

ad libitum

.

Blood analysis.

Blood samples were obtained from the left ventricleunder halothane anaesthesia 2, 4, 8 or 24 hr after reperfusion. Then,each animal was killed and the liver was dissected at each corre-sponding time-point. The plasma concentrations of alaninetransaminase (ALT) and aspartate transaminase (AST) weredetermined by routine laboratory procedures.

The plasma concentrations of IL-12 and interferon (IFN)-

γ

weredetermined by ELISA (BioSource International, Camarillo, CA,USA).

The plasma concentrations of histamine were determined byion-pair high-performance liquid chromatography coupled withpost-column fluorescent derivatization [13].

Tissue analysis.

After collecting blood samples, a median lobe ofthe liver was removed and quickly frozen in liquid nitrogen. The livertissues were homogenized in 4 ml 0.4 M perchloric acid and centrifuged.The adenosine 5

′

-triphosphate (ATP) concentrations in supernatantswere determined by high-performance liquid chromatography [14].

Another median lobe of the liver was removed and cut into slices.The slices, 5-mm thick, were incubated for 20 min. with 1% 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer (pH 7.4) at37

°

. As 2,3,5-triphenyltetrazolium chloride reacts with dehydrogenaseenzymes and NADH in viable tissue to form a red formazanpigment, liver tissue that did not stain was presumed to be necrotic.The ratio of the necrotic area to the section for each slice wasdetermined by an investigator who was unaware of the particulartreatment group using computer-aided planimetry.

Statistical analysis.

All data were analysed using unpaired t-tests.

Results

All animals regained consciousness and the righting reflexwithin 15 min. after the halothane anaesthesia was stopped.No animal died during the experimental period.

Transaminases.

The plasma concentrations of ALT and AST in intact ratswere 29 and 56 IU/l, respectively. Severe liver damagebased on enzyme levels was found in animals subjected to

transient liver ischaemia at normothermia for 30 min. Theplasma concentrations of ALT and AST increased markedly2–24 hr after liver ischaemia (fig. 1). There were no remarkabledifferences in transaminase levels between the dimaprit andcorresponding saline-injected control groups 2–8 hr afterreperfusion. However, the post-ischaemic treatment withdimaprit (20 mg/kg) decreased the ALT and AST levels24 hr after liver ischaemia. The values were 55% and 46% ofthose in control animals, respectively.

Cytokines.

The plasma concentration of IL-12 increased markedly 2 hrafter liver ischaemia, the value being 790% of that in theintact group (fig. 2A). The post-ischaemic treatment withdimaprit depressed the IL-12 level 4–24 hr after reperfusion,and the value was 40% of that in the saline-injected ischaemicgroup after 8 hr. Liver ischaemia did not affect the IFN-

γ

level markedly, and there were no differences in the IFN-

γ

value between the dimaprit and control groups (fig. 2B).

Fig. 1. Effects of post-ischaemic administration of dimaprit onischaemia-induced liver injury. The plasma concentrations ofalanine transaminase (ALT) (A) and aspartate transaminase (AST)(B) were determined after 2, 4, 8 and 24 hr following 30 min. ofischaemia. Each value represents the mean ± S.D. for 5–9 animals.*P < 0.05, **P < 0.01 as compared to the saline-injected controlgroup. Values in intact animals were 29 ± 7 IU/l (n = 10) and56 ± 7 IU/l (n = 10), respectively. Sal, saline; Dim, dimaprit.

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Histamine.

The plasma concentrations of histamine were almostconstant 2–24 hr after reperfusion in both the saline anddimaprit groups, and the values were lower than that inintact animals (fig. 3).

ATP.

The tissue concentration of ATP decreased gradually afterliver ischaemia in the saline-injected groups (fig. 4). Thepost-ischaemic treatment with dimaprit suppressed thedecrease in the ATP level. The value in the dimaprit-treatedgroup was 164% of that in saline-injected animals after24 hr.

Infarct size.

Morphologic damage observed after 2,3,5-triphenyltetrazoliumchloride stain was not marked 2–8 hr after 30 min. of warm

ischaemia in either saline- or dimaprit-injected group (fig. 5).However, liver sections obtained from saline-injected animalsafter 24 hr showed atrophy and necrosis. Post-ischaemicadministration of dimaprit markedly ameliorated liver injury,and the infarct area was 50% of that in the saline group24 hr after liver ischaemia. The magnitude of morphologiclesion was consistent with that assessed by the plasma con-centrations of transaminases and the tissue concentrationof ATP.

Fig. 2. Effects of post-ischaemic administration of dimaprit on thecytokine levels. The plasma concentrations of interleukin (IL)-12(A) and interferon (IFN)-γ (B) were determined after 2, 4, 8 and24 hr following 30 min. of ischaemia. Each value represents themean ± S.D. for 4–9 animals. *P < 0.05, **P < 0.01 as comparedto the saline-injected group. Values in intact animals were218 ± 71 pg/ml (n = 8) and 7.3 ± 6.8 pg/ml (n = 8), respectively.Sal, saline; Dim, dimaprit.

Fig. 3. Effects of post-ischaemic administration of dimaprit on theplasma concentration of histamine. The plasma concentration ofhistamine was determined after 2, 4, 8 and 24 hr following 30 min.of ischaemia. Each value represents the mean ± S.D. for 5–9animals. The value in intact animals was 0.65 ± 0.10 µM (n = 10).Sal, saline; Dim, dimaprit.

Fig. 4. Effects of post-ischaemic administration of dimaprit on theATP level in the liver tissue. The ATP level was determined after 2,4, 8 and 24 hr following 30 min. of ischaemia. Each valuerepresents the mean ± S.D. for 5–9 animals. *P < 0.05 as comparedwith the saline-injected group. The value in intact animals was0.9 ± 0.5 µmol/g (n = 10). Sal, saline; Dim, dimaprit.

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Discussion

In the present study, the post-ischaemic administration ofdimaprit decreased the IL-12 level after reperfusion andameliorated ischaemia-induced liver damage.

The function of mitochondria is very sensitive to ischaemia,and depletion of ATP is the initial factor in the developmentof organ damage by warm ischaemia. Since energy failureprecedes cellular oedema, structural degeneration andnecrosis, the tissue concentration of ATP is presumed to bea predicting factor in the development of liver injury [15]. Inthe present study, however, the ATP concentrations graduallydecreased in both the dimaprit and saline-injected controlgroups 2–8 hr after reperfusion, and there were no remarkabledifferences in the ATP level between the corresponding twogroups within 8 hr. Despite similar ATP levels within 8 hrafter reperfusion, the magnitude of tissue necrosis after24 hr was more marked in the control group than in thedimaprit group. Another mechanism besides energydepletion may be related to the development of reperfusioninjury.

In animal studies on acetaminophen-induced liverdamage, elimination of hepatic macrophages by an injectionof liposomes containing dichloromethylene diphosphonateprior to administration of acetaminophen resulted in lesssevere liver damage in mice [16]. The results suggest thatmacrophages play an important role in progression of liverdamage. Similar findings have been reported in ethanol-induced liver damage in rats and mice [17,18]. Furthermore,suppression of immune responses by cyclosporine or tacrolimushas been shown to alleviate ischaemia-induced liver injury[12,19]. Because macrophages are activated by ischaemia aswell as toxic substances [1–3], cell-mediated cytotoxicity is alikely mechanism underlying reperfusion injury.

Naive CD4

+

T cells differentiate into either T helper type1 (Th1) cells or T helper type 2 (Th2) cells, both of whichplay important roles in the pathogenesis of inflammatorydiseases and allergy. Th1 cells produce IFN-

γ

and tumournecrosis factor-alpha, which promote cell-mediated immunity,while Th2 cells activate humoural immunity. When macro-phages are activated, they secrete IL-12, which stimulatesthe differentiation of CD4

+

helper T cells into IFN-

γ

producing Th1 cells. Secreted IFN-

γ

further facilitates IL-12production in macrophages, thus, forming a positiveamplification loop [20]. In contrast, IL-10 functions todown-regulate all reactions induced by IL-12, because IL-10inhibits IL-12 production and suppresses macrophageactivation [21]. In the present study, the plasma concentra-tion of IL-12 increased markedly after liver ischaemia, andthe post-ischaemic treatment with dimaprit depressed theincrease in the IL-12 level 4–24 hr after liver ischaemia.Because histamine H

4

receptor stimulation blocks IL-12production of stimulated monocyte-derived dendritic cells[11], dimaprit may have depressed cell-mediated cytotoxicityduring reperfusion through histamine H

4

receptors bydecreasing the Th1/Th2 balance.

In our previous study, the protective effect of dimapritwas more prominent than that in the present study [4]. TheALT and AST values in the dimaprit group were 31% and18%, respectively, of those in the control group in ourprevious study, whereas those in the present study were 55%and 46%, respectively. The difference may be caused by thedifference in the duration of dimaprit action. In theprevious study, dimaprit was injected twice (10 mg/kg each),immediately and 6 hr after reperfusion, while we administereddimaprit (20 mg/kg) once immediately after reperfusion inthe present study. Suppression of macrophage/Kupffer cellactivation for a long duration may be useful in preventingreperfusion injury.

The concentration of circulating histamine was lower inboth the control and dimaprit groups than that in the intactgroup. Considering that the concentration of endogenoushistamine is depressed after reperfusion, stimulation ofhistamine H

4

receptors by exogenously administered agentsmay provide a remarkable benefit.

Dimaprit prevented the development of reperfusioninjury, even when administered after ischaemic events. Ourfindings may be a new strategy for liver protection afterischaemia or liver transplantation, and further studies arerequired to determine the precise mechanism of protection.

AcknowledgementThis work was supported by a Grant-in-Aid for Scientific

Research 18791083 from the Ministry of Education, Culture,Sports, Science and Technology, Japan.

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