1. Doi: 10.1111/j.1742-7843.2008.00219.x
© 2008 The Authors
Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 102, 394–398
Suppression of Ischaemia-Induced Cytokine Release by Dimaprit and
Blackwell Publishing Ltd
Amelioration of Liver Injury in Rats
Atsuko Motoki1, Naoto Adachi1,2,3, Keyue Liu2, Hideo K. Takahashi2, Masahiro Nishibori2, Toshihiro Yorozuya1,
Tatsuru Arai1 and Takumi Nagaro1
1
Department of Anaesthesiology and Resuscitology, Ehime University Graduate School of Medicine, Shitsukawa, Touon-shi,
Ehime, Japan, 2Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, Shikata-cho, Okayama-shi, Okayama, Japan, and 3Medical 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 histamine
H4 action has been shown to prevent the development of ischaemia/reperfusion liver injury, we examined the effects of
dimaprit, a histamine H2/H4 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 halothane
anaesthesia. 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 alanine
transaminase and aspartate transaminase were 4600 IU/l and 13,200 IU/l, respectively. The values in the dimaprit group
were 55% and 46% of those in control animals, respectively. Dimaprit also reduced the infarct size to 50%. Liver ischaemia
markedly 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-induced [5–8]. Taken together with these findings, the protective
organ injury is the early restoration of blood flow, there are effect may be mediated by histamine H4 receptors.
many factors in tissue damage during reperfusion, such as Recently, histamine H4 receptors have been shown to
formation of oxygen free radicals, inflammatory cell infiltra- mediate immune responses and inflammatory cell recruitment.
tion, and intracellular Ca2+ overload. In the liver, activation While stimulation of histamine H 4 receptors induces
of cytotoxic immune responses may play a pivotal role in chemotaxis of mast cells and eosinophils [9,10], histamine
reperfusion injury in the liver, because Kupffer cells are H4 action suppresses interleukin (IL)-12, which is a key
liver-resident macrophages and readily activated by ischaemia inducer of cell-mediated immunity [11]. Because histamine
[1– 3]. Prevention of reperfusion injury also contributes to H4 action suppresses IL-12 and ameliorates ischaemia-
survival of allografts in liver transplantation, because the induced live injury, it is likely that the protective effect of
liver is exposed to ischaemia during the organ harvest. dimaprit is related to suppression of IL-12. In the present
In our previous study, we demonstrated prevention of study, therefore, we evaluated effects of dimaprit on ischaemia-
ischaemia/reperfusion liver damage by post-ischaemic induced release of cytokines and correlated them with
administration of histamine [4]. The preventive effect was changes in functional and morphologic outcomes.
observed in animals treated with dimaprit, a histamine H2/H4
receptor agonist, to an extent similar to that observed in
Materials and Methods
histamine-treated animals. The amelioration by these agents
was reversed by thioperamide, a histamine H3/H4 receptor
Drugs and chemicals. Dimaprit dihydrochloride was purchased from
antagonist, but not by histamine H2 receptor antagonists. Sigma-Aldrich (St. Louis, MO, USA). 2,3,5-Triphenyltetrazolium
Concerning the difference in localization between H3 and H4 chloride was purchased from Research Organics (Cleveland, OH,
receptors, histamine H3 receptors are found mainly in the USA). The dose of agents was expressed as the weight of the free base.
brain, whereas histamine H4 receptors are primarily expressed
Animals. This study was approved by the Committee on Animal
on peripheral tissues, particularly the haematopoietic
Experimentation at Ehime University Graduate School of
lineage, such as bone marrow, spleen, thymus and leucocytes
Medicine, Ehime, Japan. All animals were cared for in compliance
with the Principles of Laboratory Animal Care formulated by
Ehime University Graduate School of Medicine. Male Wistar rats
Author for correspondence: Atsuko Motoki, Department of (Charles River, Yokohama, Japan) weighting approximately 300 g
were kept in groups in a room controlled at 23 ± 2° and maintained
Anesthesiology and Resuscitology, Ehime University Graduate
School of Medicine, Shitsukawa, Touon-shi, Ehime 791-0295, under an alternating 12-hr light:dark cycle (lights on at 6 a.m.).
Japan (fax +81-89-960-5386, e-mail atsumotoki@hotmail.com). Food and water were provided ad libitum.

2. 395
DIMAPRIT AND LIVER ISCHAEMIA
Sixty-one rats were allocated to one of eight groups, and each
animal was subjected to transient liver ischaemia for 30 min. After
reperfusion of blood flow, saline or dimaprit (20 mg/kg, sub-
cutaneously) was injected. Then, animals in the saline and dimaprit
groups 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 the
anterior transverse line, the left portal vein and hepatic artery were
exposed. A thermocouple probe was inserted into the rectum, and
the rectal temperature was maintained at 37.5° with a heating lamp.
Heparin (100 IU, subcutaneously) was injected, and the left portal
vein and hepatic artery were clamped with a temporary vascular
clip. This method produces ischaemic infarcts in most liver lobes,
except for the two right lateral lobes [12]. After ischaemia for
30 min. at 37.5° under anaesthesia, the clip was removed, and the
surgical incision was sutured. Saline or dimaprit (20 mg/kg,
subcutaneously) was injected immediately after reperfusion of
blood flow. The rectal temperature was maintained until the animal
was allowed to recover from anaesthesia. Then, the animal was
brought 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 ventricle
under 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 alanine
transaminase (ALT) and aspartate transaminase (AST) were
determined by routine laboratory procedures.
The plasma concentrations of IL-12 and interferon (IFN)-γ were
determined by ELISA (BioSource International, Camarillo, CA,
USA).
Fig. 1. Effects of post-ischaemic administration of dimaprit on
The plasma concentrations of histamine were determined by
ischaemia-induced liver injury. The plasma concentrations of
ion-pair high-performance liquid chromatography coupled with
alanine transaminase (ALT) (A) and aspartate transaminase (AST)
post-column fluorescent derivatization [13].
(B) were determined after 2, 4, 8 and 24 hr following 30 min. of
ischaemia. Each value represents the mean ± S.D. for 5 – 9 animals.
Tissue analysis. After collecting blood samples, a median lobe of
*P < 0.05, **P < 0.01 as compared to the saline-injected control
the liver was removed and quickly frozen in liquid nitrogen. The liver
group. Values in intact animals were 29 ± 7 IU/l (n = 10) and
tissues were homogenized in 4 ml 0.4 M perchloric acid and centrifuged.
56 ± 7 IU/l (n = 10), respectively. Sal, saline; Dim, dimaprit.
The adenosine 5′-triphosphate (ATP) concentrations in supernatants
were 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- transient liver ischaemia at normothermia for 30 min. The
triphenyltetrazolium chloride in 0.1 M phosphate buffer (pH 7.4) at
plasma concentrations of ALT and AST increased markedly
37°. As 2,3,5-triphenyltetrazolium chloride reacts with dehydrogenase
2– 24 hr after liver ischaemia (fig. 1). There were no remarkable
enzymes and NADH in viable tissue to form a red formazan
differences in transaminase levels between the dimaprit and
pigment, liver tissue that did not stain was presumed to be necrotic.
The ratio of the necrotic area to the section for each slice was corresponding saline-injected control groups 2–8 hr after
determined by an investigator who was unaware of the particular
reperfusion. However, the post-ischaemic treatment with
treatment group using computer-aided planimetry.
dimaprit (20 mg/kg) decreased the ALT and AST levels
24 hr after liver ischaemia. The values were 55% and 46% of
Statistical analysis. All data were analysed using unpaired t-tests.
those in control animals, respectively.
Results Cytokines.
The plasma concentration of IL-12 increased markedly 2 hr
All animals regained consciousness and the righting reflex
after liver ischaemia, the value being 790% of that in the
within 15 min. after the halothane anaesthesia was stopped.
intact group (fig. 2A). The post-ischaemic treatment with
No animal died during the experimental period.
dimaprit depressed the IL-12 level 4–24 hr after reperfusion,
Transaminases. and the value was 40% of that in the saline-injected ischaemic
group after 8 hr. Liver ischaemia did not affect the IFN-γ
The plasma concentrations of ALT and AST in intact rats
level markedly, and there were no differences in the IFN-γ
were 29 and 56 IU/l, respectively. Severe liver damage
value between the dimaprit and control groups (fig. 2B).
based on enzyme levels was found in animals subjected to
© 2008 The Authors
Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 102, 394–398

3. 396 ATSUKO MOTOKI ET AL.
Fig. 3. Effects of post-ischaemic administration of dimaprit on the
plasma concentration of histamine. The plasma concentration of
histamine was determined after 2, 4, 8 and 24 hr following 30 min.
of ischaemia. Each value represents the mean ± S.D. for 5–9
animals. The value in intact animals was 0.65 ± 0.10 µM (n = 10).
Sal, saline; Dim, dimaprit.
ischaemia in either saline- or dimaprit-injected group (fig. 5).
However, liver sections obtained from saline-injected animals
after 24 hr showed atrophy and necrosis. Post-ischaemic
administration of dimaprit markedly ameliorated liver injury,
and the infarct area was 50% of that in the saline group
24 hr after liver ischaemia. The magnitude of morphologic
lesion was consistent with that assessed by the plasma con-
Fig. 2. Effects of post-ischaemic administration of dimaprit on the centrations of transaminases and the tissue concentration
cytokine levels. The plasma concentrations of interleukin (IL)-12
of ATP.
(A) and interferon (IFN)-γ (B) were determined after 2, 4, 8 and
24 hr following 30 min. of ischaemia. Each value represents the
mean ± S.D. for 4–9 animals. *P < 0.05, **P < 0.01 as compared
to the saline-injected group. Values in intact animals were
218 ± 71 pg/ml (n = 8) and 7.3 ± 6.8 pg/ml (n = 8), respectively.
Sal, saline; Dim, dimaprit.
Histamine.
The plasma concentrations of histamine were almost
constant 2 – 24 hr after reperfusion in both the saline and
dimaprit groups, and the values were lower than that in
intact animals (fig. 3).
ATP.
The tissue concentration of ATP decreased gradually after
liver ischaemia in the saline-injected groups (fig. 4). The
post-ischaemic treatment with dimaprit suppressed the
decrease in the ATP level. The value in the dimaprit-treated
group was 164% of that in saline-injected animals after
Fig. 4. Effects of post-ischaemic administration of dimaprit on the
24 hr. ATP level in the liver tissue. The ATP level was determined after 2,
4, 8 and 24 hr following 30 min. of ischaemia. Each value
Infarct size. represents the mean ± S.D. for 5 – 9 animals. *P < 0.05 as compared
Morphologic damage observed after 2,3,5-triphenyltetrazolium with the saline-injected group. The value in intact animals was
0.9 ± 0.5 µmol/g (n = 10). Sal, saline; Dim, dimaprit.
chloride stain was not marked 2 – 8 hr after 30 min. of warm
© 2008 The Authors
Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 102, 394–398

4. 397
DIMAPRIT AND LIVER ISCHAEMIA
Naive CD4+ T cells differentiate into either T helper type
1 (Th1) cells or T helper type 2 (Th2) cells, both of which
play important roles in the pathogenesis of inflammatory
diseases and allergy. Th1 cells produce IFN-γ and tumour
necrosis factor-alpha, which promote cell-mediated immunity,
while Th2 cells activate humoural immunity. When macro-
phages are activated, they secrete IL-12, which stimulates
the differentiation of CD4 + helper T cells into IFN- γ
producing Th1 cells. Secreted IFN-γ further facilitates IL-12
production in macrophages, thus, forming a positive
amplification loop [20]. In contrast, IL-10 functions to
down-regulate all reactions induced by IL-12, because IL-10
inhibits IL-12 production and suppresses macrophage
activation [21]. In the present study, the plasma concentra-
tion of IL-12 increased markedly after liver ischaemia, and
the post-ischaemic treatment with dimaprit depressed the
Fig. 5. Effects of post-ischaemic administration of dimaprit on the
increase in the IL-12 level 4–24 hr after liver ischaemia.
infarct area in the liver. The percentage of the infarct area was
Because histamine H4 receptor stimulation blocks IL-12
determined after 2, 4, 8 and 24 hr following 30 min. of ischaemia.
production of stimulated monocyte-derived dendritic cells
Each value represents the mean ± S.D. for 5 – 9 animals. *P < 0.01
[11], dimaprit may have depressed cell-mediated cytotoxicity
as compared to the saline-injected control group. Sal, saline; Dim,
dimaprit. during reperfusion through histamine H4 receptors by
decreasing the Th1/Th2 balance.
In our previous study, the protective effect of dimaprit
was more prominent than that in the present study [4]. The
Discussion
ALT and AST values in the dimaprit group were 31% and
In the present study, the post-ischaemic administration of 18%, respectively, of those in the control group in our
dimaprit decreased the IL-12 level after reperfusion and previous study, whereas those in the present study were 55%
ameliorated ischaemia-induced liver damage. and 46%, respectively. The difference may be caused by the
The function of mitochondria is very sensitive to ischaemia, difference in the duration of dimaprit action. In the
and depletion of ATP is the initial factor in the development previous study, dimaprit was injected twice (10 mg/kg each),
of organ damage by warm ischaemia. Since energy failure immediately and 6 hr after reperfusion, while we administered
precedes cellular oedema, structural degeneration and dimaprit (20 mg/kg) once immediately after reperfusion in
necrosis, the tissue concentration of ATP is presumed to be the present study. Suppression of macrophage/Kupffer cell
a predicting factor in the development of liver injury [15]. In activation for a long duration may be useful in preventing
the present study, however, the ATP concentrations gradually reperfusion injury.
decreased in both the dimaprit and saline-injected control The concentration of circulating histamine was lower in
groups 2 – 8 hr after reperfusion, and there were no remarkable both the control and dimaprit groups than that in the intact
differences in the ATP level between the corresponding two group. Considering that the concentration of endogenous
groups within 8 hr. Despite similar ATP levels within 8 hr histamine is depressed after reperfusion, stimulation of
after reperfusion, the magnitude of tissue necrosis after histamine H4 receptors by exogenously administered agents
24 hr was more marked in the control group than in the may provide a remarkable benefit.
dimaprit group. Another mechanism besides energy Dimaprit prevented the development of reperfusion
depletion may be related to the development of reperfusion injury, even when administered after ischaemic events. Our
injury. findings may be a new strategy for liver protection after
In animal studies on acetaminophen-induced liver ischaemia or liver transplantation, and further studies are
damage, elimination of hepatic macrophages by an injection required to determine the precise mechanism of protection.
of liposomes containing dichloromethylene diphosphonate
prior to administration of acetaminophen resulted in less Acknowledgement
severe liver damage in mice [16]. The results suggest that This work was supported by a Grant-in-Aid for Scientific
macrophages play an important role in progression of liver Research 18791083 from the Ministry of Education, Culture,
damage. Similar findings have been reported in ethanol- Sports, Science and Technology, Japan.
induced liver damage in rats and mice [17,18]. Furthermore,
suppression of immune responses by cyclosporine or tacrolimus
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© 2008 The Authors
Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 102, 394–398