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HEPATOPROTECTIVE EFFECT OF NICORANDIL AGAINST CARBON
TETRACHLORIDE-INDUCED HEPATOTOXICITY IN RATS
By
Ashraf Taye*, Mohamed A. El-Moselhy*, Mohamed A. Morsy**
and Gamal A. El-Sherbiny***
Departments of Pharmacology and Toxicology, Faculties of Pharmacy,
*
Minia and ***Beni-Suef Universities; and **Department of Pharmacology,
Faculty of Minia Medicine
ABSTRACT:
Nicorandil (NIC), an anti-anginal drug, was reported to act as an adenosine
triphosphate-sensitive potassium (KATP) channel opener, a nitric oxide (NO) donor
and also has an antioxidant action. Based on these pharmacological properties, an
attempt has been made to evaluate the potential hepatoprotective effect of NIC, and to
elucidate its possible mechanism(s) of action in carbon tetrachloride (CCl4)-induced
hepatotoxicity in rats. The rats were randomized into 5 groups to receive either
vehicle, CCl4, NIC, glibenclamide (GLB, an antagonist of KATP channels), or a
combination of NIC and glibenclamide for 5 weeks. NIC administration under CCl4induced intoxication resulted in hepatoprotective effect as evident by significant
decrease in serum enzymatic activities of alanine aminotransferase (ALT) and
aspartate aminotransferase (AST), hepatic triglycerides, total cholesterol, nitric oxide
(NO), and malondialdehyde (MDA) contents, with a concurrent increase in serum
albumin level and hepatic catalase activity as compared to CCl4 group. The
hepatoprotective effect of NIC was confirmed by histopathological examination of the
liver tissues. On the other hand, the protective effect of NIC was maintained in the
presence of GLB. In conclusion, NIC has a hepatoprotective effect on CCl4-induced
hepatotoxicity in rats may be through, at least in part, its antioxidant activity but not
likely to be coupled with activation of KATP channels.
KEY WORDS:
Nicorandil
Glibenclamide
Hepatoprotective
Carbon tetrachloride
et al., 2004), ameliorates lung allograft
reperfusion injury through activation of
KATP channels (Yamashita, Ogawa,
and Akaike, 1996), has anti-apoptotic
effects in neurons mediated by KATP
channels (Teshima et al., 2003), and
protects against gastric ulcer in rats by
KATP channel opening and free radical
scavenging (Ismail et al., 2007).
INTRODUCTION:
Nicorandil, an efficacious drug
for the treatment of ischemic heart
disease,
exerts
multiple
pharmacological actions that involve
activation of KATP channels, generation
of NO, and scavenging of free radicals
(Miura and Miki, 2003). These
pharmacological multiactions offer the
possibility of interacting with various
experimentally-induced pathological
conditions usually through one or more
of these actions. For examples, NIC
improves diabetes and rat islet β cell
damage via a radical scavenging effect
but not KATP channel opening (Kasono
Liver disease is a serious health
problem. There is scanty information
concerning both the effect of NIC on
liver injury and the mechanism(s)
involved in this effect. The preventive
action of CCl4-induced liver damage
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has been widely used as an indicator of
liver protective activity of drugs
(Weber et al., 2003). Therefore, the
present study was undertaken to
evaluate the putative hepatoprotective
effect of NIC against CCl4-induced
hepatotoxicity as well as to reveal the
mechanism(s) implicated in this
protective effect. In addition, to
determine whether KATP channel
opening activity contributes to the
likelihood of NIC's hepatoprotection,
the effect of NIC alone and in the
presence of the KATP channel blocker
GLB was studied.
treated
with
the
combined
administration of CCl4 (1 ml/kg) and
GLB (3 mg/kg). The fifth group was
treated with combined administration
of CCl4 (1 ml/kg) and GLB (3 mg/kg)
in addition to NIC (3 mg/kg). After
completion of 5 weeks, animals were
kept starved overnight before being
decapitated, blood was collected from
orbital sinuous of the eye and the
serum
obtained
was
analyzed.
Afterwards,
liver
tissues
were
immediately dissected out and
weighed, fixed in 10% formalin for
histopathology or snap frozen in liquid
nitrogen and stored at -80°C.
Thereafter, the liver tissue was
homogenized in 0.25 M sucrose, 10
mM Tris-HCl, 1 mM EDTA medium,
pH 7.4, centrifuged at 10,000g for 10
min and supernatants used for
biochemical analysis.
MATERIALS:
Drugs and chemicals
Carbon
tertrachloride
(BDH
Chemicals,
England);
Nicorandil
(Torrent, India); Glibenclamide (a kind
gift from Aventis Company, Egypt).
Animals
Adult male Sprague-Dawley
rats (150–180 g) were used throughout
the experiments. Rats were fed a
standard diet of commercial rat chow
and tap water ad libitum and left to
acclimatize to the environment for at
least one week prior to inclusion in the
experiments.
Experiments
were
conducted in accordance with the
guidelines for animal care of the
United States Naval Medical Research
Centre, Unit No. 3, Abbaseya, Cairo,
Egypt, accredited by the Association
for Assessment and Accreditation of
Laboratory Animal Care international
(AAALAC international). Experimental animals randomly divided into
5 groups (n = 8–10 per group: the first
group constituted hepato-intoxicated
rats, was i.p. injected CCl4-olive
oil(1:1,1 ml/kg)
(Sur-Altiner and
Yenice, 2000) twice weekly for five
weeks, whereas the second served as
vehicle control and was given 1 ml/kg,
i.p. olive oil in saline, third group was
received NIC daily at doses (3 mg/kg,
i.p.) for 5 weeks, the fourth group was
METHODS:
Assessment
of
serum
aminotransferases and albumin
Serum ALT, AST and albumin levels
were determined using spectrophotometric assay kits purchased from
(Randox, Laboratory. Ltd., UK).
Estimation of hepatic triglycerides,
total cholesterol, catalase, malondialdehyde and nitrite/nitrate levels
Liver triglycerides contents were
assayed spectrophotometrically as
described by Roeschlau et al. (1974)
using a commercially available kit
(Spectrum, Egypt). Hepatic total
cholesterol level was determined also
using a commercially available kit
(Spectrum, Egypt). Catalase activity in
liver homogenates was determined
spectrophotometrically as described by
Aebi (1984) using commercially
available kit (Biodiagnostic, Egypt).
Lipid peroxidation was determined in
liver homogenates as thiobarbituric
acid reactive species (TBARS;
sometimes refereed to as MDA) as a
marker of oxidative stress according to
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the previously described method
(Buege and Aust, 1978). The total of
nitrate/nitrite in the samples was
assayed as nitrite after reduction of
nitrate into nitrite using the cadmium
reduction method as described by
Sastry and his colleges (Sastry et al.,
2002). All parameters measured in
hepatic tissues were normalized to the
protein content of the homogenate.
KATP potassium channel blocker, GLB.
It is observed that administration of
GLB does not block the protective
effect of NIC.
Effect of various treatments on
hepatic triglycerides, total cholesterol, catalase, malondialdehyde and
nitrite/nitrate levels
As shown in Figs. 4 and 5,
CCl4 administration exhibited a
marked increased hepatic MDA levels
(p<0.01) (Fig. 4), whereas the hepatic
catalase activity significantly (p<0.01)
decreased compared to those of control
(Fig. 5). NIC had the opposite effect,
where it markedly inhibited MDA
level and increased the catalase activity
nearly to that of control. Compared to
control, CCl4 exerted a significant
(p<0.001) elevation in hepatic NO
level (reflected by total nitrate and
nitrite), whereas NIC administration
markedly reduced NO production as
shown in (Fig. 6). On the other hand,
GLB could not reverse the protective
effect of NIC on the antioxidant
enzyme activities of CCl4-induced liver
injury. CCl4 administration resulted in
a dramatic elevation of hepatic
triglyceride and total cholesterol
contents compared to control group
(p<0.01 and p<0.001, respectively).
Simultaneous administration of NIC
with CCl4 reduced the lipid profile
indices near to those of control.
However, GLB administration could
not inhibit the protective effect of NIC
on lipid profile (Figs. 7 and 8).
Histopathological examination
Excised liver tissues from each
rat were fixed in 100 ml/L neutral
formalin, embedded in paraffin, and
stained with hematoxylin-eosin (HE)
and
masson's
trichrome.
The
evaluation of hepatic fibrosis was
determined by a semi-quantitative
method to assess the degree of
histological injury in chronic hepatic
fibrosis (Constandinou, Henderson,
and Iredale, 2005).
Statistical analysis
Data are presented as means ±
S.E.M. Group comparison were
performed (ANOVA) for repeated
measures followed by Bonferroni's
test.
Results
were
considered
statistically significant at p<0.05.
RESULTS:
Effect of various treatments on ALT,
AST and albumin serum levels
In the CCl4-induced liver
injury, the serum levels of ALT and
AST were markedly (p<0.01) raised
compared to control group, indicating
the severity of hepatic injury caused by
CCl4. Daily administration of NIC
effectively inhibited the elevated
hepatic enzymes ALT and AST (Figs.
1 and 2). Moreover, albumin levels of
CCl4-treated group were significantly
(p<0.01) reduced. Similarly, NIC
administration restores the reduced
albumin to its normal level (Fig. 3). To
gain insight into the mechanism of
hepatoprotective effect of NIC, we
examined its effect in the presence of
Histopathological studies
Hematoxylin
and
eosin
histological slides were evaluated by a
blinded investigator and analyzed
using a semiquantitative score for
steatosis and inflammation.
In control group, there are no
pathological changes in healthy control
livers which showed normal lobular
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architecture with central vein and
radiating hepatic cords (Fig. 9)
The lesions in the liver of CCL4
+ NIC-group group were completely
disappeared except for congestion of
some portal blood vessels (Fig.11A)
and mild and focal vacuolations of
some hepatocytes with necrosis of
individual hepatocytes (Fig. 11B). The
aforementioned lesions were only
detected in the liver of 2 rats; meanwhile, the livers of the others were
normal. To investigate role of KATP
channels in the protective of NIC, we
treated the CCl4 with GLB prior to
NIC administration. Surprisingly,
similar lesions were seen to that
described in CCL4 + NIC-group. The
portal areas showed congestion of the
portal
veins
and
lymphocytic
infiltration (Fig. 11C). Ballooning
degeneration and rarely fatty change
were also seen (Fig. 11D).
Consistent with serum ALT and
AST levels, histological examination
of the liver showed distorted
architecture with distorted central vein
and multifocal areas of coagulative
necrosis along the hepatic parenchyma
in CCl4-treated group (Fig.10A).
Severe and diffuse fatty change,
ballooning degeneration of hepatocytes, mononuclear cell infiltration, hemorrhages and Kupffer cell hyperplasia were detected (Fig. 10B and
10C). The portal triad showed fibrous
portal expansion with moderate
fibrosis, biliary hyperplasia with
formation newly formed bile ductules
and moderate round cell infiltration in
the portal areas (Fig. 10D). There was
marked centrilobular congestion and
hemosiderosis.
ALT Levels
(IU/l)
150
p<0.01
a
p<0.05
b
100
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
0
Fig. 1: Effect of various treatments on serum alanine aminotransferase (ALT)
level. Values are mean ± S.E.M.; (a) versus control group at p< 0.01 and (b) versus
(a) at p<0.05.
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p<0.01
a
p<0.01 p<0.01
a
100
AST levels
(IU/l)
ALT Levels
(IU/l)
75
b
150
p<0.05
b
50
100
25
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
0
Fig. 2: Effect of various treatments on serum aspartate aminotransferase (AST)
level. Values are mean ± S.E.M.; (a) versus control group at p<0.01 and (b) versus (a)
0
at p<0.01.
Albumin levels
(g/dl)
ALT Levels
(IU/l)
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
150
p<0.01 p<0.05
b
p<0.05 a
a
p<0.05
b
100
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
Fig. 3: Effect of various treatments on serum albumin level. Values are mean ±
S.E.M.; (a) versus control
0 group at p<0.01 and (b) versus (a) p<0.05.
p<0.01
a p<0.01
10.0
150
a
p<0.05
b
7.5
ALT Levels
(IU/l)
Malondialdehyde
MDA
(nmol/g
tisuue)
(nmol/ml)
12.5
5.0
p<0.05
b
100
2.5
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
0.0
Fig. 4: Effect of various treatmentsMDA
on hepatic malondialdehyde level. Values are
0 control group at p<0.01 and (b) versus (a) at p<0.05.
mean ± S.E.M.; (a) versus
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p< 0.01
p<0.01b
a
100
150
75
ALT Levels
(IU/l)
Catalase activity
(U/g tissue)
125
50
25
0
100
p< 0.01
a
p<0.05
b
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
Fig. 5: Effect of various treatments on hepatic catalase activity. Values are mean ±
S.E.M.; (a) versus control group at p< 0.01 and (b) versus (a) at p<0.01.
0
p<0.001
a
p<0.01
2000
ALT Levels
(IU/l)
NO2/ NO3 ratio
(nmol/g tisuue)
150
1000
a
p<0.01
b p<0.05
100
b
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
0
Fig. 6: Effect of various treatments
on hepatic nitric oxide level. Values are mean
0
± S.E.M.; (a) versus control group at p< 0.001 and (b) versus (a) at p<0.01.
200
150
p<0.001p<0.01
a
a
p<0.01
b
150
100
50
ALT Levels
(IU/l)
Triglyceride contents
(mg /g tissue)
250
p<0.05
b
100
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
0
Fig. 7: Effect of various treatments
on hepatic triglyceride level. Values are mean
0
± S.E.M.; (a) versus control group at p< 0.001 and (b) versus (a) at p<0.01.
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25
150
a p<0.01
a
20
15
ALT Levels
(IU/l)
Total cholestrol contents
(mg/g tisuue)
p<0.01
p<0.05
p<0.01
b
b
100
10
5
Control
CCl4
CCl4+GLB
CCl4+NIC
CCl4+NIC+GLB
50
0
Fig. 8: Effect of various treatments
on hepatic total cholesterol level. Values are
0
mean ± S.E.M.; (a) versus control group at p< 0.01 and (b) versus (a) at p<0.01.
Fig. 9: Liver of healthy control group showing normal lobular architecture with
central vein and radiating hepatic cords, H&E.x300.
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A
C
B
D
Fig. 10: A) Liver of CCl4 showing an area of coagulative necrosis (arrow),
H&E.x500; B) Liver of CCl4 showing diffuse fatty change (arrowheads) and
mononuclear cell infiltration (arrow), H&E.x300; C) Liver of CCl4 showing
diffuse fatty change (arrowheads) and hemorrhage (arrow), H&E.x500; and D)
Liver of CCl4 showing portal fibrosis (arrows), biliary hyperplasia (arrow-heads)
and moderate round cell infiltration in the portal areas, H&E.x100.
A
B
D
C
Fig. 11: A) Liver of CCl4 + NIC showing congestion of the portal blood vessels
(arrows), H&E.x300; B) Liver of CCl4 + NIC showing focal vacuolations of some
hepatocytes (arrows) and necrosis of individual hepatocytes (arrowheads),
H&E.x500; C) Liver of CCl4 + GLB + NIC showing congestion of the portal
veins (arrows) and lymphocytic infiltration (arrowheads), H&E.x500; and D)
Liver of CCl4+ GLB + NIC showing congestion of the portal veins (arrows) and
ballooning degeneration in the hepatocytes (arrowheads), H&E.x500.
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DISCUSSION:
CCl4 has been widely used to
induce experimental hepatic damage as
it induces liver cell necrosis and
apoptosis, and can be used to induce
hepatic fibrosis or cirrhosis by its
repetitive administration (Constandinou et al., 2005). The changes
induced by CCl4 are quite similar to
that of acute viral hepatitis. We
verified the hepatotoxicity of CCl4 by
the increased serum amino-transferases
and
decreased
albumin
levels
compared to that of the control groups.
Raised serum enzyme levels in CCL4injected rats can be attributed to the
damaged hepatocellular structural
integrity (Xu, et al., 2006). Several
reports investigated effects of various
agents that protect against liver toxicity
induced by CCl4 (Abraldes and Bosch,
2007; Guven, et al., 2003; Tan et al.,
2000). Here, we try to emphasize the
putative hepatoprotective effect of NIC
and the underlying mechanism of this
protection.
Noteworthy,
NIC
administration not only normalize the
serum aminotransferases to their
normal levels but also serum albumin
level as well.
accelerate several metabolic pathways
(Stoyanovsky and Cederbaum, 1999).
These radicals appear to affect the
adjacent lipids in the tissues and induce
lipid peroxidation. We suggest that
increased MDA level in the CCl4treated group is attributed to increased
lipid peroxidation-associated tissue
damage. Our results showed that NIC
appears to play a key role in the
attenuation of CCl4-induced liver
injury. In accordance, NIC appeared to
have a protective effect on the lipid
peroxidation, as evident by scavenging
free radical and increasing the
antioxidant catalase enzyme activity.
The reduction of lipid peroxidation by
NIC might be important because
increased oxidative stress is a feature
of the CCl4-induced liver injury and
significant protection was obtained
with the use of antioxidants (Jaeschke,
1990). Moreover, our results showed
that CCl4 administration significantly
increased liver triglycerides, and total
cholesterol, these observations are in
accordance with that of Venukumar
and Latha (2002). Herein, decreased
liver triglycerides, and total cholesterol
contents and reduced lipid peroxidation
by NIC introduced a more satisfactory
explanation for the protective action of
NIC against both fatty degeneration
and hepatomegaly induced by CCl4
and elucidated that hepatomegaly
possibly is induced by an increase in
all lipid forms at the expense of protein
content; NIC efficiently prevented
deterioration of liver function and this
may account for its protection against
the deleterious effect of CCl4 on fatty
liver.
Reactive oxygen species, such
as superoxide anions, hydrogen
peroxide, and hydroxyl radical,
produced by the partial reduction of
oxygen, are highly unstable and
extremely reactive. The short half-lives
of many of these species make them
highly toxic to tissues (Noyan et al.,
2006). Increased lipid peroxidation is
generally believed to be an important
underlying cause of the initiation of
oxidative stress related to various
tissue injuries, cell death, and the
progression of many acute and chronic
diseases (Halliwell, 1997). During the
initial phase of CCl4 toxicity following
its administration, a large amount of
CCl4 is converted to trichloromethyl
radical or other radicals, which in turn
Interestingly,
the
present
biochemical findings were reflected on
the structural changes of the liver as
evident by the hepatic necrosis, diffuse
fatty changes, portal fibrosis, biliary
and
Kupffer
cell
hyperplasia,
haemorrhage and mononuclear cell
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infiltration. Such lesions were induced
via the formation of free radicals,
especially the reactive oxygen species.
The hepatotoxicity of CCl4 results
from its metabolism by the cytochrome
P450 enzyme system into the reactive
intermediates, which leads to oxidative
stress and causes hepatic necrosis. In
consistent with the present findings,
the hepatotoxicity effect of CCl4 was
subsided and reversed with complete
disappearance of hepatic damage after
treatment with NIC. Thus, antioxidant
effect of NIC appears to play a key role
in the attenuation of inflammation, and
then preserve the structural integrity of
the hepatocellular membrane resulting
in amelioration of liver function.
GLB to reverse the protective effect
NIC in CCl4-induced hepatotoxicity.
In conclusion, NIC have a
potential hepatoprotectve effect and the
possible mechanism of hepatoprotective action of NIC may be due to
its antioxidant activity as indicated by
protection against increased lipid
peroxidation and maintained catalase
contents. The rest of the biochemical
and
histpathological
parameters
studied indicate the status of structural
and functional integrity of the cells and
provide further support to the
suggestive mechanism of action.
Acknowledgment
To Dr. Mohamed Hamed Mohamed.
Department of Pathology, Faculty of
Veterinary
Medicine,
Zagazig
University for his kind help in the
histopathology in this work.
On the other hand, it has been
reported that liver injury induced by
CCl4 is associated by elevated NO
level
due
to
increased
the
proinflammatory cytokines tumor
necrosis factors- (TNF-) which
induce NO expression (Dang et al.,
2007). NIC has been shown in rat
myocardial mitochondria to produce
NO (Sakai et al., 2000), that plays an
important
role
in
ischemic
preconditioning of rat livers (Peralta et
al., 1997). The present study revealed
that concurrent administration of NIC
with CCl4 restored NO to its normal
level. This can be explained by that the
released amount of NO from NIC has
been consumed in the scavenging of
free radicals. Additionally, NIC may
also inhibit the inducible NO synthase
via its inhibitory effect on TNF-
(Zhan et al., 2003). To verify
the
hepatoprotective mechanism of NIC,
we treated rats with GLB as a KATP
sensitive channel blocker. Although
NIC acts as a KATP-channel opener
(Miura and Miki, 2003; Ohta et al.,
1991), it is unlikely that the
hepatoprotective effect of NIC is via
KATP receptors as evident by failure of
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‫التأثير الواقي للنيكورانديل ضد التسمم الكبدي المستحدث بمادة رابع كلوريد‬
‫الكربون في الجرذان‬
‫أشرف أبو الوفا طايع*‪ ،‬محمد أحمد المصيلحي*‪ ،‬محمد علي مرسي**‪،‬‬
‫جمال أحمد الشربيني***‬
‫*قسم األدوية والسموم – كلية الصيدلة – جامعة المنيا ‪* ،‬قسم األدوية – كلية الطب – جامعة‬
‫المنيا ‪*** ،‬قسم األدوية والسموم – كلية الصيدلة – جامعة بني سويف‬
‫يعتبر عقار النيكورانديل المضاد للذبحة الصدرية واحدًا من محفزات قنوات البوتاسيوم الحساسة‬
‫تأثيرا مانعًا لألكسدة‪ .‬واعتمادًا‬
‫لألدينوسين ثالثي الفوسفات‪ ،‬ومولدًا ألكسيد النيتريك‪ ،‬كما أن له‬
‫ً‬
‫على هذه الخصائص فقد أجريت محاولة لتقدير تأثير النيكورانديل الواقي للكبد ولتوضيح آليات‬
‫هذه التأثيرات الوقائية في حاالت التسمم الكبدي المستحدث بمادة رابع كلوريد الكربون في‬
‫الجرذان‪ .‬قسمت الجرذان إلى خمس مجموعات‪ :‬مجموعة أعطيت المذيب ومجموعة أعطيت‬
‫رابع كلوريد الكربون فقط وأعطيت المجموعات الثالث المتبقية رابع كلوريد الكربون مع‬
‫إعطاء مجموعة منهم النيكورانديل والثانية أعطيت الجاليبنكالميد والثالثة أعطيت النيكورانديل‬
‫والجاليبنكالميد معا ً‪ .‬وقد اتضح تأثير النيكورانديل الواقي على الكبد من خالل النقص الواضح‬
‫في نشاط إنزيم األالنين أمينوترانسفيريز واألسبارتات أمينوترانسفيريز في مصل الدم‪ ،‬وكذلك‬
‫نقص معدل الدهون الثالثية في الكبد والكوليستيرول الكلي وأكسيد النيتريك ومحتوى الكبد من‬
‫نواتج تأكسد الدهون (المالون ثنائي األلدهيد)‪ ،‬باإلضافة إلى زيادة تركيز األلبيومين في مصل‬
‫الدم وزيادة نشاط إنزيم الكتاليز في الكبد مقارنة بالمجموعة التي أعطيت رابع كلوريد الكربون‬
‫فقط‪ .‬وقد أ ُ ّكدَت التأثيرات الوقائية للنيكورانديل بتحليل أنسجة الكبد‪ .‬ومما يسترعي االنتباه أن‬
‫تأثيرات النيكورانديل الواقية للكبد قد بقيت حتى في وجود الجاليبنكالميد‪ .‬ونخلص من ذلك كله‬
‫بأن النيكورانديل له تاثير وقائي على الكبد ضد التسمم الكبدي المستحدث برابع كلوريد‬
‫الكربون‪ ،‬وأن هذا التأثير قد يُعزى بشكل جزئي إلى خصائص النيكورانديل المانعة للتأكسد‬
‫وليس إلى تحفيز قنوات البوتاسيوم الحساسة لألدينوسين ثالثي الفوسفات‪.‬‬
‫‪323‬‬