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ORIGINAL ARTICLE
Thymoquinone mitigate ischemia-reperfusion-induced liver
injury in rats: a pivotal role of nitric oxide signaling pathway
Mohamed Abd-Elbaset
1
&El-Shaimaa A. Arafa
1,2
&Gamal A. El Sherbiny
3
&
Mohamed S. Abdel-Bakky
4,5
&Abdel Nasser A.M. Elgendy
6
Received: 27 June 2016 /Accepted: 23 September 2016 /Published online: 7 October 2016
#Springer-Verlag Berlin Heidelberg 2016
Abstract Oxidative and nitrosative stress-induced endotheli-
al cell damage play an essential role in the pathogenesis of
hepatic ischemia-reperfusion (IR) injury. IR is associated with
reduced eNOS expression and exacerbated by superimposed
stress. NOSTRIN induces intracellular endothelial nitric oxide
synthase (eNOS) translocation and inducible nitric oxide syn-
thase (iNOS) increases nitric oxide (NO) production. Our aim
was to assess hepatic expression of iNOS, eNOS, and
NOSTRIN in IR with or without N-acetylcysteine (NAC) or
thymoquinone (TQ) pretreatment and to compare their hepa-
toprotective effects. Surgical induction of IR was performed
by occlusion of hepatic pedicle for 30 min with mini-clamp
and reperfused for 30 min. The effects of TQ (20 mg/kg/day)
or NAC (300 mg/kg/day) administered orally for 10 days were
evaluated by serum ALT and AST, oxidative stress parame-
ters, NO production, and histopathological analysis. Also, lo-
calization and expression of iNOS, eNOS, and NOSTRIN
were assessed by immunofluorescence. TQ or NAC pretreat-
ment significantly decreased elevated serum aspartate amino-
transferase (AST), alanine aminotransferase (ALT), and
myeloperoxidase (MPO) activities, malondialdehyde (MDA)
level, and NO production. In addition, they restored the de-
pleted GSH content and alleviated histopathological changes.
Furthermore, they up-regulated eNOS and down-regulated
iNOS and NOSTRIN expressions. TQ exerts its hepatoprotec-
tive effect, at least in part, by nitric oxide signaling pathway
through modulation of iNOS, eNOS, and NOSTRIN expres-
sions as well as suppression of oxidative stress.
Keywords Thymoquinone .Hepatic ischemia–reperfusion .
iNOS .eNOS .NOSTRIN
Introduction
Hepatic ischemia–reperfusion (IR) injury is one of the major
obstacles in different clinical conditions such as liver surgery,
especially in transplantation, hepatic resection, and trauma.
Several relevant factors and mediators such as nitric oxide
(NO) were involved in the ischemic injury of the liver. The
inducible nitric oxide synthase (iNOS) and the endothelial
nitric oxide synthase (eNOS) are two isoforms of nitric oxide
synthase that play an important but opposed role in liver IR
(Trocha et al. 2010). NO derived from iNOS and its oxidative
byproduct peroxynitrite cause increased hepatic injury and
systemic hypotension which reduce liver blood flow
(Jaeschke 2003). On the contrary, NO produced by eNOS
has a physiological protective role by maintaining liver blood
flow, inhibition of leukocyte adhesion, platelet aggregation,
and scavenging of free radicals (Shah and Kamath 2003).
Whereas, eNOS traffic inducer (NOSTRIN)-involved in shut-
tling of eNOS was originally described as an F-BAR domain-
*El-Shaimaa A. Arafa
elshimaa.arafa@pharm.bsu.edu.eg
1
Department of Pharmacology and Toxicology, Faculty of Pharmacy,
Beni-Suef University, Beni-Suef 62514, Egypt
2
Department of Pharmacology and Toxicology, College of Pharmacy
and Health Sciences, Ajman University of Science and Technology,
Ajman, United Arab Emirates
3
Department of Pharmacology and Toxicology, Faculty of Pharmacy,
Kafr El-Sheikh University, Kafr El-Sheikh, Egypt
4
Department of Pharmacology and Toxicology, Faculty of Pharmacy,
Al-Azhar University, Cairo, Egypt
5
Department of Pharmacology, College of pharmacy, Al Jouf
University, Al Jouf, Kingdom of Saudi Arabia
6
Department of Pharmacology, Faculty of Veterinary medicine,
Beni-Suef University, Beni-Suef 62514, Egypt
Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390:69–76
DOI 10.1007/s00210-016-1306-7
containing protein which binds to eNOS and strongly attenu-
ated eNOS-dependent NO production (Zimmermann et al.
2002). Nostrin binds to eNOS causing decrease of eNOS en-
zyme activity. Moreover, caveolin and nostrin can augment
the binding of each other’s to distinct sites on eNOS
(Schilling et al. 2006). Nostrin may work as a scaffold protein
that recruits eNOS, dynamin-2, and Wiskott-Aldrich syn-
drome protein type N (N-WASP) and controls their internali-
zation via caveolar endocytosis (Zimmermann et al. 2002).
Other various mechanisms had been implicated in liver IR
injury such as abundant amounts of reactive oxygen species
(ROS) (Jaeschke 2003), activation of Kupffer cells, and in-
flammatory cytokines release (Montalvo-Jave et al. 2008).
However, the mechanisms that coordinate and integrate this
pathologic response are still unclear. It was evident that oxi-
dative stress plays a major role in tissue injury including IR
(Zimmermann BJ and Granger DN 1994). Thymoquinone
(TQ) is the main constituent of Nigella sativa, with a rich
background in history and religion, used to promote health
and for the treatment of different diseases (El-Ghany et al.
2009). Recent research revealed that TQ possesses a strong
antioxidantproperty(Gali-Muhtasibetal.2006), neuroprotec-
tive effects against transient forebrain ischemia (Al-Majed
et al. 2006), cardioprotective effects against oxidative damage
induced by doxorubicin (NAGI and MANSOUR 2000)and
gastroprotective effects against IR injury (El-Abhar et al.
2003). Moreover, it exerts antitumor, immunomodulatory,
and anti-inflammatory effects (Gali-Muhtasib et al. 2006).
N-acetylcysteine (NAC) is a thiol-containing compound. It
is extensively used in the management of paracetamol over-
dose induced fulminant liver failure (Prescott et al. 1979)and
as a hepatoprotective agent in liver transplantation (Thies et al.
1998). Furthermore, it has a therapeutic value for reducing
endothelial dysfunction, inflammation, and fibrosis
(Zafarullah et al. 2003). Finally, the use of antioxidants for
liver IR is still ongoing looking for any beneficial effects
and their underlying mechanisms. The aim of our study is to
investigate the possible hepatoprotective effect of TQ in IR-
induced liver injury through its endothelial NO-regulatory,
anti-inflammatory, and/or antioxidant activity.
Materials and methods
Chemicals
Thymoquinone (TQ), N-acetylcysteine (NAC), 4,6-diamidino-
2-phenylindole (DAPI), O-dianisidine, thiobarbituric acid, sul-
fanilamide, and 5–5′- dithiobis-(2-nitrobenzoic acid)(DTNB)
were purchased from Sigma-Aldrich, USA. Mouse polyclonal
anti-inducible nitric oxide synthase (anti-iNOS) and mouse
polyclonal anti-endothelial nitric oxide synthase (eNOS) were
purchased from BD-Biosciences, USA. Rabbit polyclonal anti-
eNOS trafficking inducer (anti-NOSTRIN, 506 aa and 58 kDa
variant) was purchased from Proteintech, USA. Rabbit anti-
mouse Alexa Fluor 488 secondary antibody was purchased
from Invitrogen, USA. Goat anti-rabbit Cy3 secondary antibody
was purchased from Jackson ImmunoResearch Laboratories,
USA. Aspartate aminotransferase (AST) reagent kit and
Alanine aminotransferase (ALT) reagent kit were purchased
from Spinreact, Spain. The highest analytical grades were se-
lected for other chemicals.
Animals
Adult male Wister albino rats, weighing 300–350 g, were
randomly selected. The protocol of this study has been ap-
proved by Research Ethics Committee of Faculty of
Pharmacy, Beni-Suef University, Egypt. Rats were housed in
metabolic cages under controlled environmental conditions
(25 °C and a 12 h light/dark cycle), allowed free access to
pulverized standard food pellet and tap water ad libitum and
animals were left for 2 weeks of acclimation to animal house
conditions.
Experimental protocol
Rats were randomly divided into four groups (10 rats each):
group I: sham-operated control group (received normal saline
and 1 % Tween 80 orally) and underwent a surgical procedure
similar to the other groups but the entire hepatic pedicule was
not occluded, group II: IR-induced injury group (received
normal saline and 1 % Tween 80 orally) and subjected to
30-min ischemia followed by 30-min reperfusion (Oguz
et al. 2013), group III: rats were pretreated with NAC freshly
dissolved in normal saline (300 mg/kg/day) (Hemalatha et al.
2013) orally for 10 days before subjection to IR, and group IV:
rats were pretreated with TQ suspended in normal saline and
1 % tween 80 (20 mg/kg/day) (El-Ghanyet al. 2009) orally for
10 days before subjection to IR.
Induction of hepatic ischemia-reperfusion
A complete midline laparotomy was performed under intra-
peritoneal injection of ketamine (50 mg/kg) and xylazine HCl
(10 mg/kg) anesthesia (Ara et al. 2005). Surgical induction of
hepatic ischemia was performed by occlusion of hepatic
pedicule (hepatic artery, portal vein, and bile duct), which
supplied the left and medial lobes, with a mini-clamp for
30 min. The vascular clamp was removed and the liver was
perfused for 30 min followed by collection of blood samples
from the retro-orbital plexus for the assay of serum ALT and
AST, then the rats were sacrificed and the liver was quickly
removed, rinsed in ice cold saline. Some portions fixed in
Davidson-Solution for immunofluorescence assessment; oth-
er portions fixed in 10 % formalin saline for histopathological
70 Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390:69–76
examination or were weighed and homogenized in normal
saline, 25 % w/v. Homogenates were then centrifuged for
15 min at 10,000×gand the supernatant was used for deter-
mination of other biochemical parameters.
Estimation of liver functions
Measurement of serum aspartate aminotransferase (AST) and
alanine aminotransferase (ALT) was determined by the meth-
od described by manufacturer’s instructions using activity col-
orimetric assay kits (Spin react, Spain). The absorbance was
measured spectrophotometrically at 340 nm. The activities
were expressed in U/L.
Estimation of malondialdehyde and reduced glutathione
contents
Malondialdehyde (MDA) was measured by the method of
Mihara and Uchiyama (1978). 1,1,3,3-tetramethoxypropane
used to construct the standard curve. MDA content was cal-
culated and expressed in nmol/g tissue. The difference in ab-
sorbance measured spectrophotometrically at 535 and 520 nm
was used to calculate the content of MDA in each sample.
Glutathione (GSH) content was measured according to the
method described by Beutler et al. (1963). The resulting yel-
low color absorbance was measured spectrophotometrically
within 5 min at 412 nm. GSH content was expressed in mg/
gtissue.
Estimation of myeloperoxidase activity and nitric oxide
production
Myeloperoxidase (MPO) activity was determined in tissue by
the method of Krawisz et al. (1984). The absorbance was
measured spectrophotometrically at 460 nm. MPO activity
was calculated according to the following equation: MPO ac-
tivity (U/g tissue) = ΔA/min × D. F / extinction coefficient,
where dilution factor = reciprocal of the relative sample vol-
ume in the reaction mixture × dilution factor of the homoge-
nate × 1000. Extinction coefficient = 1.13 × 104 Cm
−1
M
−1
.
Nitric oxide was measured in tissue according to the method
described by Miranda et al. (2001). Nitric oxide (NO) produc-
tion was calculated according to the standard curve using
NaNO
3
as a standard and expressed in μM/g tissue. The ab-
sorbance was measured spectrophotometrically at 540 nm.
Immunofluorescence assessment of iNOS, eNOS,
and NOSTRIN expressions
Paraffin embedded tissue sections (4 μm thin) were put in oven at
60 °C for 20 min, then deparaffinized in xylene 100 % two times,
15 min each to dissolve wax. A rehydration step was performed
by using graded ethanol 100 % for 5 min, 90, 70, 50, and 30 %,
5 min each. After rehydration, tissue was washed with distilled
water for 5 min and tissue section was incubated in Dako solution
(citrate buffer, pH 6) in microwave at 500 watt for 20 min. After
cooling of the solution, slides were washed three times by PBST
(0.05 % of tween 20 in phosphate buffer saline (PBS) pH 7.4),
3 min each. Fixation was done using methanol for 30 min. After
washing, slides were blocked by 10 % horse serum in 1 % bovine
serum albumin of PBS blocking solution to block the non-specific
binding of antibodies. Tissue sections were incubated with the
primary antibodies (mouse polyclonal iNOS or eNOS or rabbit
polyclonal NOSTRIN antibodies) in the concentration of 1:50 in
cool place overnight. Bound antibodies were detected by rabbit
anti-mouse Alexa fluor 488 (green) or goat anti-rabbit Cy3 (red)
secondary antibodies for 30 min, respectively. Nuclei were
stained with DAPI and washed by PBST for 30 min. Slides were
mounted with Fluoromount and evaluated by fluorescence mi-
croscopy (Leica DM 5000B).
Histopathological examination
The liver of the animals was removed and fixed in 10 % formalin,
dehydrated with graded ethanol (30, 50, 70, 90 and 100 %), and
cleared the in two changes of xylene. Samples were then steeped
with two times in molten paraffin wax. Paraffin sections (5 μm)
thick were stained with hematoxylin and eosin (H&E) according to
Jones et al. (2008). Stained tissues of sham-operated control and
treated rats were analyzed for alterations in the architecture,inflam-
matory infiltration, congestion of the central vein, pyknosis, and
cytoplasmic degeneration. The zero score was given to normal
tissues with no alteration, + indicates slight histopathological alter-
ations, ++ indicates moderate histopathological alterations, +++
referred to severe histopathological alterations while ++++ direct-
ed to very severe histopathological alterations. All experiments
were conducted in accordance with the guidelines approved by
the University of Beni-Suef Ethical Committee.
Statistical analysis
Statistical analysis was performed using SPSS statistical soft-
ware (version 22.0). Data were expressed as means ± standard
error (S.E.). Statistical evaluation of data was performed using
one-way analysis of variances (ANOVA), with Tukey post
hoc test to compare the means. A P < 0.05 was considered
statistically significant.
Results
Effect of TQ pretreatment on liver enzymes in IR operated
rats
The activities of liver toxicity marker enzymes, AST and ALT,
are represented in Table 1. IR group exhibited significant
Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390:69–76 71
elevation in serum activities of both enzymes to
914.16 ± 46.88 and 846.46 ± 91.92 %, respectively, compared
to the corresponding sham-operated control group. TQ pre-
treatment significantly decreased the elevated liver enzymes
to 52.31 ± 2.39 and 71.72 ± 1.03 %, respectively, similarly IR
operated rats pretreated with NAC also showed lowered AST
and ALT activities to 66.04 ± 2.42 and 67.10 ± 5.22 %, re-
spectively, compared to IR group.
Effect of TQ pretreatment on hepatic lipid peroxidation
and GSH contents in IR operated rats
Climbing to approximately 240.10 ± 24.51 % in IR operated
rats compared to sham-operated rats, Hepatic MDA content
significantly decreased to 79.70 ± 9.82 and 66.76 ± 3.78 % in
NAC and TQ-pretreated rats respectively compared to IR
group. In contrast, from a depletion of 20.24 ± 0.96 % in IR
group compared to sham-operated group, the reduced GSH
content significantly increased to 476.47 ± 23.53 and
388.23 ± 41.18 % in NAC and TQ-pretreated group respec-
tively compared to IR group (Table 1).
Effect of TQ pretreatment on hepatic MPO activity
and NO production in IR operated rats
IR group showed a significant increase in MPO activity and NO
production to 617.65 ± 70.59 and 254.79 ± 6.99 % respectively
compared to sham-operated group. NAC-pretreated rats sharply
declined to 43.81 ± 5.71 and 78.98 ± 4.03 % respectively com-
pared to IR group. Likewise, TQ-pretreated group dramatically
decreased these values to 25.71 ± 1.90 and 69.83 ± 4.28 %,
respectively (Table 1).
Effect of TQ pretreatment on protein expressions of iNOS,
eNOS, and NOSTRIN in IR operated rats
Negative iNOS and NOSTRIN protein expressions were seen in
sham-operated rats. On the other hand, livers of rats subjected to
IR revealed diffused up-regulation of iNOS and NOSTRIN
expression in the pericentral hepatocytes compared to sham-
operated rats. While TQ or NAC-pretreated groups exhibited
decreased expression of iNOS and NOSTRIN in the sinusoidal
areas closed to the central area compared to the IR group. Semi-
quantitative analysis of the immunofluorescence staining
expressed as fluorescence intensity demonstrated that IR oper-
ated group showed a significant increase in the hepatic iNOS
and NOSTRIN expression to approximately 153.10 ± 9.68 and
197.14 ± 6.50 %, respectively, compared to sham-operated rat
values. Conversely, the expression of iNOS and NOSTRIN in
the hepatic tissues of IR pretreated with TQ or NAC significant-
ly decreased to 69.87 ± 2.82 and 53.64 ± 3.84 % or 72.61 ± 3.13
and 55.07 ± 3.18 % respectively compared to IR group (Figs. 1
and 2, respectively).
Sinusoidal eNOS protein expression was seen in sham-
operated rats while liver sections from animals exposed to
IR revealed strong down-regulation of eNOS expression com-
pared to sham-operated rats. A dramatic rise in eNOS expres-
sion was seen in the sinusoidal areas as well as in periportal
and pericentral hepatocytes in TQ and NAC-pretreated IR-
exposed animals compared to the IR group. Fluorescence in-
tensity of eNOS revealed that IR processed group showed a
significant reduction in the hepatic eNOS protein expression
to 51.75 ± 2.29 % compared to sham-operated rats
(26.60 ± 1.18 %). Conversely, the expression of eNOS in
the hepatic tissues of IR pretreated with TQ or NAC group
significantly up-regulated to 174.70 ± 9.10 and
153.27 ± 7.48 %, respectively (Fig. 3).
Histopathological examination of liver sections
TheeffectsofTQorNACinIRoperatedratsonliver
histopathological features are illustrated in Table 2and
presented in Fig. 4. The sham-operated group revealed normal
hepatic architecture with central vein (CV) and radiating cords
of normal hepatocytes with central rounded vesicular nuclei
and prominent nucleoli. Hepatic cords are separated by blood
sinusoids (interstitial spaces) lined with endothelium and
Kupffer cells (KC). In contrast, liver sections of rats subjected
Tabl e 1 Effect of TQ pretreatment on liver enzymes and oxidative stress parameters in IR operated rats
Parameters Groups Sham IR NAC + IR TQ + IR
AST (U/L) 143.00 ± 3.05 1307.25 ± 67.04
*
863.33 ± 29.24
*@
682.33 ± 31.15
*@
ALT (U/L) 127.00 ± 10.15 1075.00 ± 116.74
*
721.33 ± 56.17
*@
771.00 ± 11.06
*@
MDA (nmol/g tissue) 17.73 ± 1.81 42.57 ± 3.60
*
33.93 ± 4.18
*@
28.42 ± 1.61
*@
GSH (mg/g tissue) 0.84 ± 0.04 0.17 ± 0.03
*
0.81 ± 0.04
@
0.66 ± 0.07
*@
MPO (U/g tissue) 0.17 ± 0.01 1.05 ± 0.12
*
0.46 ± 0.06
*@
0.27 ± 0.02
@
NO (μM/g tissue) 583.00 ± 38.31 1485.42 ± 40.74
*
1173.19 ± 59.91
*@
1037.21 ± 63.59
*@
All data were expressed as means ± standard error (S.E.) of six rats per group
*Significantly different from sham-operated group at P < 0.05
@
Significantly different from IR group at P < 0.05
72 Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390:69–76
to IR injury showed massively congested CV and dilated in-
terstitial spaces lined with activated KC, in addition many
hepatocytes showed pyknotic nuclei, cytoplasmic degenera-
tion, and inflammatory cell infiltration. On the other hand,
pretreatment with NAC or TQ resulted in a marked attenua-
tion of liver injury, as the majority of hepatic lobules revealed
normal liver picture while the former was associated with a
slight congestion of CV and interstitial spaces and the latter
was accompanied by just a moderate congestion of CV and a
slight dilation of blood sinusoids.
Discussion
Liver injury induced by IR remains an important clinical prob-
lem during liver transplantation, hemorrhagic shock, and
surgical procedures. The proposed mechanisms in the patho-
physiology of IR injury included massive production of ROS,
NO, and their reaction product of peroxynitrite (Montalvo-
Jave et al. 2008). Moreover, leukocytic infiltration induced
an inflammatory reaction proceeding oxidative stress upon
reperfusion (Sewerynek et al. 1995). It is apparent from the
present study that IR-induced liver injury is confirmed bio-
chemicallyby a considerable elevation in ALT, AST and MPO
activities as well as MDA level while reduced GSH content is
significantly depleted. Also, IR caused a marked histopatho-
logical alteration including pyknosis, degeneration of cyto-
plasm, congestion of CV, and PMNs infiltration in hepato-
cytes. Further and even more importantly, though, it signifi-
cantly increased liver iNOS expression along with NO over-
production. Meanwhile, it obviously increased NOSTRIN ex-
pression in the liver tissues while it was associated with a
Sham IR
NAC+IR TQ+IR
A
B
Fig. 1 Effect of TQ pretreatment on iNOS localization and expression in
hepatic tissues of IR operated rats. aImmunofluorescence staining of
liver sections obtained from control rats, showing negative expression
of iNOS while IR markedly increased expression of iNOS. TQ or NAC
pretreatment reduced the expression level of iNOS. Scale bar = 100 μm. b
Quantitative image analysis of immunofluorescence staining expressed as
fluorescence intensity of iNOS expression of sham-operated control and
IR with or without NAC or TQ pretreatment obtained from eight fields
from each rat’s section (minimally three rats in each group) using ImageJ
software (National Institutes of Health, Bethesda, MD). *Significantly
different from sham-operated group at P < 0.05.
@
Significantly different
from IR group at P<0.05
Sham IR
TQ+IR
NAC+IR
A
B
Fig. 2 Effect of TQ Pretreatment on NOSTRIN Localization and
Expression in Hepatic Tissues of IR Operated Rats. aControl rats,
revealing negligible expression of NOSTRIN while its expression in IR
subjected animals is concentrated in the nuclei of the hepatocytes as the
red fluorescence is co-localized with DAPI stain of the nuclei (arrows).
TQ or NAC pretreatment markedly reduced NOSTRIN expression in the
sinusoidal areas closed to the central area. Scale bar = 50 μm. b
Fluorescence intensity of NOSTRIN expression of sham-operated control
and IR with or without NAC or TQ pretreatment obtained from eight
fields from each rat’s section (minimally three rats in each group) using
ImageJ software (National Institutes of Health, Bethesda, MD).
*Significantly different from sham-operated group at P < 0.05.
@
Significantly different from IR group at P < 0.05
Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390:69–76 73
discernible decline in liver eNOS expression. These findings
are in harmony with our recent study (Abd-Elbaset et al.
2015). In the current study, pretreatment with TQ significantly
alleviated biochemical and histopathological status of the IR-
induced liver damage through a significant decline in ALT and
AST levels and restoration of normal hepatocyte architecture.
In addition, it significantly mitigated inflammatory infiltration
in the rat liver induced by IR as manifested by the reduced
hepatic MPO activity. It has been shown earlier that Nigella
sativa significantly lowers ALT, AST, and MPO activities, in
addition it decreases pathological changes in rats subjected to
IR-induced liver damage (Yildiz et al. 2008). TQ, meanwhile,
significantly decreased lipid peroxidation and enhanced the
antioxidant capacity by restoring reduced GSH content.
These results are comparable with a previous study
demonstrating that TQ pretreatment restored GSH depletion
and decreased MDA elevation in the rat hippocampus subject-
ed to transient forebrain ischemia (Al-Majed et al. 2006). An
interesting finding of our study is the suppression of iNOS
expression and reduction of NO overproduction, hence its
byproduct, peroxynitrite, which were deleterious and might
initiate hepatic inflammatory reaction. El-Mahmoudy et al.
(2002) stated that TQ decreased immunoreactivity of iNOS
in peritoneal macrophages and decreased nitrite production, a
stable product of NO parameter, induced by lipopolysaccha-
ride (LPS). We also found that TQ pretreatment increases the
expression of eNOS in the endothelial cells of hepatic blood
vessels compared to IR alone as revealed by immunofluores-
cence. This may explain the beneficial role of TQ during IR-
induced liver injury. TQ caused a marked increase in eNOS
activity in isolated rabbit aorta subjected to endothelial dys-
function induced by pyrogallol (El-Agamy and Nader 2012).
Moreover, the co-localization of NOSTRIN with DAPI indi-
cated nuclear translocation of NOSTRIN in hepatocytes of
rats subjected to IR alone while TQ decreased its nuclear
expression, indicating no more eNOS translocation from the
plasma membrane to vesicle-like subcellular structures and
leading to augmentation of eNOS-dependent NO production.
Mookerjee et al. (2007) demonstrated that increased
NOSTRIN may partly explain the decreased enzymatic activ-
ity of eNOS in diseased liver. Furthermore, increased gene and
protein expression of NOSTRIN has been observed in liver
samples of alcoholic hepatitis patients, which may contribute
to the reduced eNOS activity and increased intrahepatic resis-
tance in these chronically cirrhotic livers. In our study, we
reported an obvious increase of NO level in IR operated rats.
Additionally, it has been reported that NOSTRIN poses a sig-
nificant inhibition of NO release by direct inhibition of eNOS
activity/modulation of regulatory mechanism such as phos-
phorylation or protein association (Icking et al. 2005). The
increased NOSTRIN protein expression and NO production
along with the reduced eNOS expression in the current work
may be explained by the inhibitory effect of NOSTRIN me-
diating NO release which is mostly due to its influence on
eNOS cellular trafficking. These findings displayed that NO
could be good or bad according to NOS isoforms and the
stimuli triggering its production (Thippeswamy et al. 2006).
Administration of NAC prior to IR significantly decreased
serum AST and ALT activities, MDA level, and MPO activity.
Moreover, it strongly restored GSH content. In consistentwith
our results, Sener et al. (2003) stated that NAC alone or in
combination with melatonin was effective in bringing signif-
icant reduction in AST, ALT, MDA, and MPO as well as
significantly restoring GSH content in a model of hepatic
IR. In addition, histopathological examination of NAC pre-
treatment showed a marked improvement in the liver cell
structure. Similar result has been reported by Attri et al.
(2000) who reported that NAC faithfully maintained normal
Sham IR
NAC+IR TQ+IR
A
B
Fig. 3 Effect of TQ pretreatment with on eNOS localization and
expression in hepatic tissues of IR operated rats. aControl rats,
showing constitutive expression of eNOS and it is highly concentrated
in hepatocytes around the central vein. The expression is obviously
downregulated in the pericentral hepatocytes in IR subjected animals. In
TQ + IR and NAC + IR groups, the expression is concentrated in the
sinusoidal areas as well as in periportal and pericentral hepatocytes as the
green fluorescence is located in the blood vessels endothelial cells. Scale
bar = 100 μm. bFluorescence intensity of eNOS expression of sham-
operated control and IR with or without NAC or TQ pretreatment
obtained from eight fields from each rat’s section (minimally three rats
in each group) using ImageJ software (National Institutes of Health,
Bethesda, MD). *Significantly different from sham-operated group at
P<0.05.
@
Significantly different from IR group at P < 0.05
74 Naunyn-Schmiedeberg's Arch Pharmacol (2017) 390:69–76
morphology in a model of isoniazid and rifampicin induced
hepatic injury. Furthermore, NAC decreased iNOS protein
expression along with reducing NO production. The same
observation was reported by Bergamini et al. (2001)who
demonstrated that NAC administration inhibited iNOS ex-
pression and the massive NO production induced by LPS.
Controversially, NAC restored eNOS protein expression, the
finding which was in harmony with a study demonstrating that
cultured vascular endothelial cell (ECV304) treated withNAC
increased eNOS expression along with NO production
resulting in improved cell viability and reduced apoptosis in-
duced by TNF-α(Xia et al. 2006). Thus, there is no significant
difference between pretreatment with TQ or NAC, which was
used as standard treatment in our study. In conclusion, our
findings highlight the ability of TQ to ameliorate the oxidative
stress, nitrosative stress, and inflammatory responses second-
ary to IR. Therefore, using TQ as a potential candidate as
supplement or adjunct therapy in patients subjected to liver
transplantation or surgery is strongly recommended.
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Tabl e 2 Histopathological
examination of the effect of TQ
pretreatment in IR operated rats
Findings Groups Sham IR NAC + IR TQ + IR
Inflammatory infiltration 0 ++++ 0 0
Congestion of CV 0 ++++ + ++
Sinusoidal (interstitial) dilation 0 +++ + +
Pyknosis 0 ++++ 0 0
Cytoplasmic degeneration 0 +++ + +
IR was performed by (30-min ischemia + 30-min reperfusion). NAC (300 mg/kg, orally) or TQ (20 mg/kg, orally)
was given once daily for ten consecutive days followed by performing IR
0Non histopathological alterations
+Slight histopathological alterations
++Moderate histopathological alterations
+++Severe histopathological alterations
++++Very severe histopathological alterations
A
B-1 B-2
C D
Fig. 4 Histopathological examination of liver sections. ×400, H&E stain.
aPhotomicrograph of sham group shows normal hepatic architecture.b
Photomicrograph of I R operated rats (B-1) shows very severely congested
central vein, hepatocytes (H) are separated by dilated blood sinusoids (S)
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infiltration (encircled). An additional photomicrograph of IR group (B-2)
shows pyknotic nuclei and cytoplasmic degeneration (encircled). c
Photomicrograph of NAC-pretreated group shows normal hepatic
architecture with slightly congested central vein and slightly dilated
blood sinusoids. dPhotomicrograph of TQ-pretreated group shows
normal hepatic architecture with moderately congested central vein and
slightly dilated blood sinusoids
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