Objective: To investigate the effect of sildenafil on reducing the impact of hepatic ischemia/reperfusion (HIR) injury established by Pringle maneuver on the heart of rats. Study Design: Forty Wistar albino rats were divided into 4 groups: Sham (laparotomy only), Control (laparotomy following sildenafil application), IR (ischemia/reperfusion injured by HIR), and IR+SIL (injured by HIR following sildenafil application). Ischemia was developed by clamping the hepatoduodenal ligament for 30 minutes; then reperfusion was applied for 30 minutes. Sildenafil (single dose of 50 mg/kg) was administered by oral gavage for 15 minutes before ischemia. Blood samples of rats were collected from Sham and Control groups at 60 minutes and from IR and IR+SIL groups at 30 minutes after initiation of reperfusion for biochemical analysis. Meanwhile, heart tissues were sampled for biochemical analysis. Malondialdehyde (MDA) and total antioxidant capacity (TAC) in serum samples and TAC, total oxidative capacity (TOC), and oxidative stress index in heart tissues were examined biochemically. Results: Serum MDA levels were elevated significantly in the IR and IR+SIL groups as compared to the sham group. Sildenafil treatment inhibited MDA increase considerably in the IR+SIL group as compared to the IR group. Serum TAC levels were elevated significantly in the sildenafil and control groups (compared with sham groups) and in the IR+SIL group (compared with the IR group). TAC levels detected in heart tissue increased significantly in the IR group as compared to the sham group; however, sildenafil treatment had no effect on this increase. Conclusion: Heart tissue was affected by HIR. It was revealed that sildenafil treatment may prevent the oxidative stress via increasing serum TAC levels in both control and IR+SIL groups.

1. 116
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/21/4303-0116/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
OBJECTIVE: To investigate the effect of sildenafil on
reducing the impact of hepatic ischemia/reperfusion
(HIR) injury established by Pringle maneuver on the
heart of rats.
STUDY DESIGN: Forty Wistar albino rats were di-
vided into 4 groups: Sham (laparotomy only), Control
(laparotomy following sildenafil application), IR (isch-
emia/reperfusion injured by HIR), and IR+SIL (injured
by HIR following sildenafil application). Ischemia was
developed by clamping the hepatoduodenal ligament for
30 minutes; then reperfusion was applied for 30 min-
utes. Sildenafil (single dose of 50 mg/kg) was admin-
istered by oral gavage for 15 minutes before ischemia.
Blood samples of rats were collected from Sham and
Control groups at 60 minutes and from IR and IR+SIL
groups at 30 minutes after initiation of reperfusion for
biochemical analysis. Meanwhile, heart tissues were
sampled for biochemical analysis. Malondialdehyde
(MDA) and total antioxidant capacity (TAC) in serum
samples and TAC, total oxidative capacity (TOC), and
oxidative stress index in heart tissues were examined
biochemically.
RESULTS: Serum MDA levels were elevated signifi-
cantly in the IR and IR+SIL groups as compared to
the sham group. Sildenafil treatment inhibited MDA
increase considerably in the IR+SIL group as compared
to the IR group. Serum TAC levels were elevated sig-
nificantly in the sildenafil and control groups (compared
with sham groups) and in the IR+SIL group (compared
with the IR group). TAC levels detected in heart tissue
increased significantly in the IR group as compared to
the sham group; however, sildenafil treatment had no
effect on this increase.
CONCLUSION: Heart tissue was affected by HIR. It
was revealed that sildenafil treatment may prevent the
oxidative stress via increasing serum TAC levels in both
control and IR+SIL groups. (Anal Quant Cytopathol
Histpathol 2021;43:116–120)
Keywords: antioxidants, heart, hepatic ischemia/
reperfusion, malondialdehyde, oxidative stress,
reperfusion injury, sildenafil, total antioxidant
capacity, total oxidative capacity.
Ischemia/reperfusion (IR) injury is defined as the
restoration of blood supply to the organs after an
ischemic period led to the parenchymal damage.1
Oxygen deprivation causes ischemia progres-
sion and the production of reactive oxygen spe-
cies (ROS) during reperfusion, resulting in more
Protective Effect of Sildenafil on the Heart in
Hepatic Ischemia/Reperfusion Injury
Aysun Ekinci, M.D., Abdullah Oğuz, M.D., Fırat Aşır, Ph.D., Cenap Ekinci, M.D., and
Recep Dursun, M.D.
From the Departments of Biochemistry, of General Surgery, and of Histology and Embryology, Faculty of Medicine, Dicle University,
and the Department of Emergency Medicine, Dicle University Medical School, Diyarbakır, Turkey.
Aysun Ekinci is Associate Professor, Department of Biochemistry, Faculty of Medicine, Dicle University.
Abdullah Oğuz is Associate Professor, Department of General Surgery, Faculty of Medicine, Dicle University.
Fırat Aşır is Research Assistant, Department of Histology and Embryology, Faculty of Medicine, Dicle University.
Cenap Ekinci is Associate Professor, Department of Histology and Embryology, Faculty of Medicine, Dicle University.
Recep Dursun is Associate Professor, Department of Emergency Medicine, Faculty of Medicine, Dicle University.
Address correspondence to: Cenap Ekinci, M.D., Department of Histology and Embryology, Faculty of Medicine, Dicle University,
University Street, 21280, Diyarbakır, Turkey (cenapekinci@gmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Volume 43, Number 3/June 2021 117
Protective Effect of Sildenafil on Heart
damage.2 However, ROS are able to initiate the
oxidative stress mechanisms via lipid peroxida-
tion.3 ROS can easily react with the membrane-
associated polyunsaturated fatty acids of lipid
membranes and induce lipid peroxidation.4
Sildenafil is a potent and specific inhibitor of
phosphodiesterase-5 (PDE-5), presenting a cardio­
protective effect against IR injury in the healthy
and isolated heart.5,6 The mechanism of cardiopro-
tective effect has not been understood completely.5
In 1989 the discovery of sildenafil was the result of
a comprehensive study on chemical agents target-
ing PDE-5, which could be potentially beneficial
in coronary heart disease. At the beginning of the
1990s the first clinical studies on sildenafil were
not promising in terms of antianginal potential.
However, the discovery of its anti-impotence ef-
fect resulted in its approval for the treatment of
erectile dysfunction. Recently, the expression of
PDE-5 has been revealed in tissues like the ar-
terial vessels, as well as pulmonary and coronary
arteries, venous vessels, skeletal muscles, platelets,
and visceral and tracheobronchial muscles, allow-
ing new therapeutical indicators of sildenafil.7 By
satisfying findings, sildenafil was also investigated
for a possible relation to persistent pulmonary
hypertension.8 There are several evidences which
have shown whether sildenafil has a pre-protective
effect against IR injury in various tissues such as
heart, lung, kidney, and brain.9-12
In this study we aimed to investigate the poten-
tial antioxidant effect of sildenafil against heart
tissue damage induced by hepatic IR injury.
Materials and Methods
Animals and Surgical Procedure
All experimental procedures were conducted in
accordance with the ethical principles in experi-
mental animals and approved by the Animal Care
and Use Local Ethical committee. A total of 40
female Wistar albino rats (weighing 200–250 g
each) were placed in an air-conditioned room at
a constant temperature (22±2°C) with a 12-hour
light-dark cycle. The rats were caged and provid­
ed standard rat feed and water before the experi-
ments. They were fasted overnight before the
experiment but were allowed to drink water ad
libitum. The rats were anesthetized by intramus­
cular injection of 50 mg/kg ketamine hydrochlo­
ride (Ketalar, Parke Davis, Eczacibasi, Istanbul,
Turkey) and 10 mg/kg xylazine (Rompun, Bayer
AG, Leverkusen, Germany) before surgery. Silde-
nafil citrate (Viagra, Pfizer) was dissolved in saline
(1 mg/mL) and stored in brown glass vials at 4°C.
Experimental Protocol
The rats were divided into 4 groups (n=10 each
group) randomly. The Sham group underwent
laparotomy only. The Control group underwent
laparotomy following administration of 50 mg/kg
sildenafil. The IR group was submitted to ische-
mia for 30 minutes and reperfusion for 30 min-
utes. The IR+SIL group was submitted to ischemia
for 30 minutes, reperfusion for 30 minutes, and
administration of 50 mg/kg sildenafil.
Before surgery the abdomens of the rats were
shaved, an incision was made on the middle line,
and liver, diaphragm, and adjacent organs to the
hepatoduodenal ligament were dissected. The hep-
atoduodenal ligament (v. porta, a. hepatica, and
choledochal duct) was placed in the middle and
clamped by microvascular clamp, and the ischemic
period was initiated. After a 30 minute ischemic
period, the microvascular clamp was declamped
and a 30 minute reperfusion period was initiated.
At the end of this period, blood samples were
collected from the heart and the animals were
sacrificed. Sildenafil 50 mg/kg (according to Tmax)
was administered orally to the Control and IR+SIL
groups at 15 minutes before the experiment. Heart
tissues and blood samples were collected. Serum
and tissue samples were stored at −80°C for further
biochemical analysis.
Biochemical Steps and Analysis
To obtain serum, blood samples were centrifuged
at 3000 rpm for 10 minutes. In determining the
levels of malondialdehyde (MDA) and total anti-
oxidant capacity (TAC), serum samples were used.
Tissues were weighted and cut into small pieces.
Dissected tissues were homogenized in 10 volume
ice-cold phosphate buffer solution (50 mM/L, pH
7.0) by using a homogenizer (Ultra-Turrax T8 dis-
persing homogenizer, Staufen, Germany). To ob-
tain supernatant, the homogenate was centrifuged
at 4°C, 15,000 rpm for 10 minutes. In determining
total oxidative capacity (TOC) and TAC levels,
supernatants were used.
Detecting Malondialdehyde Levels
MDA levels were calculated by the double heating
method described by Draper and Hadley.13 The
principle of the method was spectrophotometric
measurement of the color produced during the

3. 118 Analytical and Quantitative Cytopathology and Histopathology®
Ekinci et al
reaction of Thio barbituric acid (TBA) with MDA.
For this purpose, 2.5 mL trichloroacetic acid (TCA)
solution (10%) was added to 0.5 mL serum in each
centrifuge tube and placed in a boiling water bath
for 15 minutes. After cooling under tap water, the
tubes were centrifuged at 1000 g for 10 minutes,
and 2 mL of the supernatant was added to 1 mL
TBA (6.7 g/L) solution in a test tube and placed in
a boiling water bath for 15 minutes. The solu-
tion was then cooled under tap water, and the
absorbance was measured using a spectropho-
tometer (Shimadzu UV-1208, Japan) at 532 nm.
The concentration of MDA was calculated by the
absorbance coefficient of MDA-TBA complex (the
absorbance coefficient of 1.56×105 cm-1M-1). Serum
MDA levels were expressed in µmol/L Hb.
Measurement of Total Oxidative Capacity
TOC was determined by using a new automat-
ed measurement method developed by Erel.14
Oxidants present in the sample oxidized Fe2+-o-
dianisidine complex into Fe2+ ion. The oxidation
reactions progressed by a plentiful amount of glyc-
erol molecules in reaction media. Ferric ion formed
a colored complex with xylenol orange in acidic
media. The color intensity measured spectrophoto-
metrically was associated with the amount of total
oxidant molecules in samples. The experiment was
calibrated by hydrogen peroxide, and results were
expressed in nmol H2O2 Equiv./mg protein.
Measurement of Total Antioxidant Capacity
TAC of samples was determined by using a new
automated measurement method developed by
Erel.15 In testing, ferric ion solution present in
Reactive 1 was mixed with hydrogen peroxide
present in Reactive 2. Antioxidant efficacy of sam-
ples against strong free radical reactions initiated
by produced hydroxyl radical was measured. Re­
sults were expressed in nmol Trolox Equiv./mg
protein.
Oxidative Stress Index
Oxidative stress index (OSI) is an indicator param-
eter of oxidative stress, and the formulation was
given as:
OSI=(TOC (nmol H2O2 Equiv./mg)/TAC (nmol
Trolox Equiv./mg)).16
Statistical Analysis
Windows 11.5 SPSS Package Program (SPSS Inc.,
Chicago, Illinois, USA) was used for statistical
analysis. Kruskal-Wallis test was used to analyze
variants. Bonferroni corrected Mann-Whitney U
test was utilized for pairwise comparison among
groups. P<0.05 was accepted for statistical signif-
icance.
Results
During the experimental procedures, all animals
survived. Table I presents the levels of biochemical
parameters. Serum MDA and TAC levels altered
significantly among groups. MDA content as lipid
peroxidation index was elevated in the IR (p=
0.006) and IR+SIL (p<0.004) groups following re-
perfusion, as compared with the Sham group. Pre-
treatment with sildenafil considerably prevented
Table I Biochemical Findings of Serum and Heart Tissue
Study group
Sham Control IR IR+SIL
Factor Units (Mean±SD) (Mean±SD) (Mean±SD) (Mean±SD)
TAC in serum nmol Trolox Equiv./L 2.03±0.21 2.39±0.22a 2.80±0.43b 3.08±0.4c,d
MDA in serum µM/L 0.45±0.12 0.42±0.08 0.76±0.20b 0.55±0.03c,e
Heart TAC nmol Trolox Equiv./mg 3.76±0.16 3.94±0.23a 4.12±0.14b 4.11±0.15
Heart TOC nmol H2O2 Equiv./mg 145.64±19.85 119.52±32.19a 149.14±24.61 125.57±31.73
Heart OSI 38.73±4.15 30.14±7.02a 36.19±6.01 30.56±6.89f
IR = ischemia reperfusion, MDA = malondialdehyde, OSI = oxidative stress index, SIL = sildenafil, TAC = total antioxidant capacity, TOC = total oxidative
capacity.
ap<0.05 vs. Sham group.
bp<0.01 vs. Sham group.
cp<0.01 vs. Sham group.
dp<0.05 vs. Sham group.
ep<0.01 vs. IR group.
fp<0.05 vs. IR group.

4. Volume 43, Number 3/June 2021 119
Protective Effect of Sildenafil on Heart
MDA increase in the IR+SIL group in comparison
to the IR group (p=0.005).
Serum TAC levels in the Control group were
found to be significantly higher than those in the
Sham group (p=0.016). Moreover, it was also high-
er in the IR+SIL group than in the IR group (p=
0.019). TAC levels in heart tissue increased signifi-
cantly in the IR group as compared with the Sham
group (p<0.001), but pretreatment with sildenafil
had no effect on this increase.
TOC levels and oxidative stress index values,
presenting oxidative stress parameters of tissue,
increased in heart tissue after IR injury and de-
creased by pretreatment with sildenafil; however,
the difference was found not to be statistically sig-
nificant.
Discussion
Recently, liver IR injury has been studied exten-
sively. Liver IR represents a process affecting the
functions of many distant organs such as lung,
kidney, intestine, pancreas, adrenal glands, and
myocardia. Distant organ injury relatively seems
to be the result of inflammatory response after
oxidative burst and reperfusion.17 Several stud-
ies have shown that the main component of side
effects of IR injury is not the hypoxia but initiates
by reversing the oxygenated blood to the ischemic
tissue.18 Several endogenous entities, including free
oxygen radicals, platelet activating factor, arachi-
donic acid metabolites, and bacterial endotoxins,
have a role in pathogenesis of gastrointestinal and
other tissue reperfusion injuries.19 The amount of
produced ROS is controlled by the antioxidant
defense mechanisms.20 There are studies suggest-
ing the antioxidant effects of sildenafil, reducing
oxidative stress and inflammation.21,22
In the present study, as an indicator of lipid per-
oxidation supporting oxidative stress, the increase
in MDA levels occurred at 30 minutes after reper-
fusion established by clamping the hepatoduode-
nal ligament for 30 minutes in rats, and pretreat-
ment with sildenafil prevented this MDA increase.
Moreover, both groups of sildenafil (control and
IR+SIL) had elevated levels of serum TAC. Our
findings are consistent with the study by Guerra-
Mora et al.23 In another study in which 2 doses of
sildenafil (10 mg/kg and 20 mg/kg) were applied
on burns in rats, a protective effect of sildena-
fil against the oxidative stress was detected, by
which MDA levels in liver and kidney tissues ele-
vated, and TAC levels increased, TOC and oxida-
tive stress index levels decreased in serum.22 Our
study was consistent also with these results. An-
other study investigating sildenafil and vardenafil
as inhibitors of phosphodiesterase type 5 report-
ed that sildenafil ameliorated the epinephrine-
induced MDA increase in heart tissue.24 Sildenafil
as well as tadalafil, a prolonged effective inhibitor
of phosphodiesterase-5, were reported to relieve
the oxidative stress, resulting in potent cardiopro-
tective effects against myocardia IR injury, myo-
cardial infarction, and doxorubicin-induced cardio­
myopathy.25,26
The exact mechanism of cardioprotective effect
of sildenafil has not been understood yet; how-
ever, some reports have suggested that this effect
may be introduced by opening mitochondrial
ATP-sensitive K+ channels (mitoKATP) to reduce
IR injury in a myocardial infarction model.27 A
study on mitochondria of isolated heart in rats
reported that sildenafil depressed H2O2 formation
via mitochondria derived from glutamate/malate,
as well as reduced superoxide radicals produced
in a hypoxanthine/xanthine oxidase system, re-
sulting in preventing ROS production and protect-
ing the mitochondrial function in the heart.5 The
results of the present study suggested that pre-
treatment with sildenafil can increase serum TAC
level and reduce MDA levels, hence preventing
the oxidative stress in serum. In terms of biochem-
ical parameters, we could not reach an antioxidant
effect of sildenafil on heart tissue against hepatic
IR injury.
Limitations of this study were the duration of
IR established in experimental procedure and ad-
ministered dose of sildenafil. In a study by Das
et al, the myocardium was exposed to 2 hours
of reperfusion after 30 minutes of ischemia in an
isolated rat heart model.28 They stated that silde-
nafil doses in a narrow range (0.001 to 0.5 mg/kg)
protected the heart against IR injury, resulting in a
ventricular amelioration, but the lower doses had
no effect while higher doses caused a rise in ven-
tricular fibrillation incidence.28 In another study,
a low dose of acute sildenafil (0.7 mg/kg) pro-
vided benefits in the function of the left ventricle,
while this effect was lost in a high dose (1.4 mg/
kg). Chronic sildenafil administration (0.7 mg/kg
twice a day for 3 weeks) was shown to exacer-
bate the functions of the left ventricle during IR.29
To investigate the preventative role of sildenafil
against heart tissue injury following hepatic IR
injury and to justify the potential usage of sildenafil

5. 120 Analytical and Quantitative Cytopathology and Histopathology®
Ekinci et al
in clinical settings, future studies designed more
studiously are needed.
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