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Protective Effect of Hydroxytyrosol Against

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Induced Myocardial Infarction in Rat

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Protective Effect of Hydroxytyrosol Against
Cardiac Remodeling After Isoproterenol-
Induced Myocardial Infarction in Rat

Kais Mnafgui, Raouf Hajji, Fatma

Derbali, Ines Khlif, Ftouma Kraiem,
Hedi Ellefi, Abdelfattah Elfeki,
Noureddine Allouche, et al.
Cardiovascular Toxicology

ISSN 1530-7905

Cardiovasc Toxicol
DOI 10.1007/s12012-015-9323-1

1 23
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1 23
 Author's personal copy
Cardiovasc Toxicol
DOI 10.1007/s12012-015-9323-1

Protective Effect of Hydroxytyrosol Against Cardiac Remodeling

After Isoproterenol-Induced Myocardial Infarction in Rat
Kais Mnafgui1,2 • Raouf Hajji2 • Fatma Derbali2 • Ines Khlif3 • Ftouma Kraiem4 •

Hedi Ellefi5 • Abdelfattah Elfeki1 • Noureddine Allouche3 • Neji Gharsallah6

Ó Springer Science+Business Media New York 2015

Abstract The present study aimed to investigate the car-
dioprotective effect of hydroxytyrosol (HT) against isopro-
terenol-induced myocardial infarction in rats. Male rats were
randomly divided into four groups, control, isoproterenol
(Isop) and pretreated animals with HT in two different doses
(2 and 5 mg/kg) orally for 7 days and intoxicated with
isoproterenol (Isop ? HT1) and (Isop ? HT2) groups.
Myocardial infarction in rats was induced subcutaneously by
isoproterenol (100 mg/kg, s.c.) at an interval of 24 h on 6th
and 7th day. On 8th day, electrocardiographic (ECG) pat-
tern, gravimetric and biochemical parameters were assessed.
Isoproterenol exhibited changes in ECG pattern, including
significant ST-segment elevation and increase in the serum
troponin-T level by 317 % as compared to control rats.
Moreover, cardiac injury markers (creatine kinase-MB,
lactate dehydrogenase, alanine aminotransferase) underwent
a notable rise in serum of infarcted animals. Else, a distur-
bance in lipids profile and significant increase in lipase and
angiotensin-converting enzyme (ACE) activities and heart
weight ratio were observed in isoproterenol group. However,
pre- and co-treatment with HT (2 and 5 mg/kg) improved
the myocardium injury, restored the hemodynamic function
and inhibited the ACE activity that prevent cardiac hyper-
trophy and remodeling. Overall, these findings demonstrated
that HT exerted a potent cardioprotective effect against
isoproterenol-induced myocardial infarction.
Keywords ACE
ECG
Hydroxytyrosol
Myocardial
infarction
Troponin-T

Introduction
Raouf Hajji and Fatma Derbali have contributed equally to this work.

& Kais Mnafgui

Acute myocardial infarction is an important ischemic heart
mnafgui_kais@yahoo.fr disease and a major cause of morbidity and mortality
worldwide [1]. It is the acute condition of myocardial
1
Laboratory of Animal Physiology, Faculty of Sciences of necrosis that occurs as a result of imbalance between
Sfax, University of Sfax, P.O. Box 95, 3052 Sfax, Tunisia
coronary blood supply and myocardial demand. Clinical
2
Department of Internal Medicine, Hospital of Sidi Bouzid, markers of myocardial necrosis and dysfunction include
9100 Sidi Bouzid, Tunisia
blood pressure, heart rate and electrocardiographic (ECG)
3
Laboratory of Chemistry of Natural Products, Faculty of changes and left ventricular (LV) dysfunction accompanied
Sciences of Sfax, University of Sfax, B.P. 1171, 3000 Sfax,
Tunisia
with increased serum levels of cardiac-specific proteins.
4
Cardiac troponins are frequently associated with myocar-
Faculty of Medicine of Monastir, Avenue Avicenne,
5019 Monastir, Tunisia
dial infarction and inflammation-related proteins at
5
elevated levels with heart attack [2]. The animal model of
Department of Cardiology, Centre Hospitalier
isoproterenol-induced myocardial infarction presents an
Intercommunal Robert Ballanger,
93600 Boulevard Robert Ballanger, Aulnay-Sous-Bois, important endorsed technique for studying the effects of
France several potential cardioprotective bioactive compounds [3].
6
Laboratory of Plant Biotechnology, Faculty of Sciences of Isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropy-
Sfax, University of Sfax, B.P. 1171, 3000 Sfax, Tunisia laminoethanol hydrochloride] is a synthetic catecholamine

123
 Author's personal copy
and b-adrenergic agonist used in inframaximal doses to
regulate heart function. However, the administration of
isoproterenol in surpramaximal doses could induce severe
stress in the myocardium through the depletion of the car-
diomyocytes energy reserve which is resulting in irreversible
cellular injury and ultimately infract like necrosis [3, 4].
Many synthetic drugs are used for the management of heart
attack but they are not free from side effects. Hence, several
studies have focused on identifying new therapeutic ap-
proaches to prevent myocardial infarction.
Recently, greater attention has been focused on the
phenols as effective bioactive compounds that protect cells
or molecules from myocardial damage. In this regard, hy-
droxytyrosol (HT), a natural phenol present in large
amounts in all parts of olive trees (Olea europaea), has
been found to be a potent antioxidant and hypocholes-
terolemic agent in various animal models of disease in-
cluding dyslipidemia, atherosclerosis and diabetes [4–6].
Polyphenols are excellent cardioprotectors. Hence, we
suggested that HT may have a cardioprotective effect. The
purpose of the current study was designed to investigate the
protective effect of the oral pretreatment of HT on ex-
perimentally induced myocardial infarction in Wistar rats.
Materials and Methods
Chemicals
Lipase kit was purchased from Biolabo reagents France,
HT [2-(3,4-dihydroxyphenyl)ethanol] was obtained from
Cayman Chemical (Ann Arbor, MI, USA); the purity was
C98 %. Isoproterenol hydrochloride powder was obtained
from Sigma-Aldrich, St. Louis, USA. Angiotensin-con-
verting enzyme (ACE) kit was purchased from Trinity, UK.
The remaining chemicals used were of analytical grade.
Animals
The essays of this study were conducted on adult male Wistar
rats, weighting 260 ± 10 g, which were obtained from the local
Central Pharmacy, Tunisia. All rats were kept in an environ-
mentally controlled breeding room (temperature: 20 ± 2 °C;
humidity: 60 ± 5 %; 12-h dark/light cycle) where they had
standard diets and free access to tap water. The experimental
protocols were conducted in accordance with the guide for the
care and use of laboratory animals issued by the University of
Sfax, Tunisia, and approved by the Committee of Animal Ethics.
Induction of Experimental Myocardial Infarction
Isoproterenol was dissolved in normal saline and injected
to rats (100 mg/kg body weight) and injected
123
Cardiovasc Toxicol
subcutaneously at an interval of 24 h for 2 days to induce
experimental myocardial infarction [7]. Animals were kil-
led 48 h after the first dose of isoproterenol.
Experimental Protocols
After acclimatization, the animals were randomly divided
into the following groups consisting of eight rats each:
Group I: (Control) rats received standard laboratory diet
and drinking saline water ad libitum and served as a control;
Group II: (Isop) rats received saline water for 7 days and
at the 6th day subcutaneously injected with isoproterenol
(100 mg/kg, subcutaneously injected, once at an interval
of 24 h for two consecutive days);
Group III: (Isop ? HT1) rats pretreated with hydroxyty-
rosol (2 mg/kg bw; gastric gavages, respectively) for
7 days and at the 6th day subcutaneously injected with
isoproterenol (100 mg/kg bw) for two consecutive days;
Group IV: (Isop ? HT2) rats received HT (5 mg/kg bw)
for 7 days and were injected subcutaneously with
isoproterenol (100 mg/kg bw) on day 6th and 7th. All
rats are fasted overnight but had free access to water at
the last administration of the drug.
After the 7-days induction, the animals were weighted
and killed by decapitation in order to minimize the han-
dling stress, and the trunk blood collected. The serum was
prepared by centrifugation (15009g, 15 min, 4 °C), frozen
and stored at -20 °C until analysis. Immediately after
killing, the heart was excised out, washed with saline and
fixed in fixed in a Bouin solution for 24 h and then em-
bedded in paraffin. Sections of 5 lm thickness were stained
with hematoxylin–eosin. The slides were photographed
with an Olympus U-TU1X-2 camera connected to an
Olympus CX41 microscope (Tokyo, Japan).
Measurement of Blood Pressure by Noninvasive
Method
Twenty-four hours after the second injection of isopro-
terenol, blood pressures were measured using noninvasive
blood pressure Monitor (CODATM Surgical Monitor, USA)
which consists of placing a cuff on the animal’s tail to
occlude the blood flow. The pressure was raised and then
slowly released. The cuff pressure when the pulse signal
reappears is intended as the systolic pressure. The cuff
pressure when the pulse signal level recovers its initial
level is intended as diastolic pressure.
Electrocardiography
Twenty-four hours after the second dose of isoproterenol,
rats of all the groups were anesthetized with ketamine
 Author's personal copy
Cardiovasc Toxicol
hydrochloride (100 mg/kg bw) intraperitoneally. Needle
electrodes were inserted under the skin of the animals in
lead II position. ECG recordings were made using veteri-
nary electrocardiograph (ECG VET 110, Biocare, China).
Biochemical Analysis
After killing, the heart was dissected out, immediately
washed in ice-cold saline and a homogenate was prepared in
0.1 M Tris–HCl buffer (pH 7.4). Homogenate was cen-
trifuged, and supernatant was used for the assay of marker
enzymes. The collected serum was used for the determina-
tion of serum activity of lipase and ACE, the cardiac marker
enzymes as creatinine kinase-MB (CK-MB), lactate dehy-
drogenase (LDH), alanine transaminase (ALT) and tropon-
in-I rates were measured in frozen aliquots of serum by
standardized enzymatic procedures using commercial kits
from (Biolabo, France) on an automatic biochemistry
analyzer (Vitalab Flexor E, USA) using commercially
available standard enzymatic kits (Biolabo, France). Serum
lipids level of triglycerides (TG), total cholesterol (T-Ch),
high density lipoprotein-cholesterol (HDL-c) and low den-
sity lipoprotein-cholesterol (LDL-c) were measured using
the corresponding commercial kits (Biolabo, France) on an
automatic biochemistry analyzer (Kenza, France) at the
clinic pathological laboratory of Sidi Bouzid Hospital.
Serum LDL-cholesterol concentration was determined ac-
cording to formula: LDL_cholesterol = Total choles-
terol_(Triglycerides/5) - (HDL_cholesterol) performed by
Friedewal et al. [8].
Statistical Analysis
Data are presented as mean ± standard deviation (SD).
Determinations were performed from eight animals per
group, and differences were examined by a one-way ana-
lysis of variance (ANOVA) followed by the Fisher test
(Stat View). *P \ 0.05 was considered statistically
significant.
Results
Effect of Hydroxytyrosol (HT) on Heart Weight
to Body Weight Ration of Experimental Rats
As shown in Table 1, there was no significant difference in
the body weight between the groups observed. Isopro-
terenol-induced myocardial infarcted rats exhibited sig-
nificant increases in heart weight and the heart weight to
body weight ration as compared to control rats (P \ 0.05).
However, in response to HT1 and HT2, the infarcted rats
showed significant (P \ 0.05) decrease in the heart weight
by 17 and 37 %, respectively, as compared to untreated
myocardial infarcted rats.
Electrocardiogram Patterns of Normal
and Experimental Animals
Control animals showed normal ECG pattern with normal
heart rate (375 ± 13.92 bpm). While infarcted rats evi-
denced a significant (P \ 0.05) increase in heart rate
(430 ± 11.65 bpm) which became irregular, remarkable
elevation of ST-segment (wave of Paradee) and QT inter-
val along with no identifiable P wave as compared to the
normal animals (Figs. 1, 2). However, oral pre- and co-
treatment of isoproterenol-induced rats with 2 mg/kg of
HT showed acceleration in heart rate (430 ± 12.34 bpm),
remaining regular sinus with the appearance of a discrete
ST-elevation. Else, an aspect of electrical alternating was
observed (alternating between QRS ample and QRS less
ample). Though, the HT2 group exhibited sinus rhythm not
accelerated relative to healthy control (375 ± 10.31 bpm),
with no ST-segment elevation, no alternating electric sinus
and rare atrial premature as compared to groups II and III.
Effect of Hydroxytyrosol on Hemodynamic Function
As shown in Table 2, significant decreases in systolic, di-
astolic and mean arterial blood pressure were observed in
isoproterenol-induced rats as compared to control group
(P \ 0.05). The pre- and co-treatment of infarcted rats with
HT at 2 and 5 mg/kg significantly restored arterial pressure
in a dose-dependent pattern.
Effect of Hydroxytyrosol on Serum Cardiac
Markers
Table 3 indicated the variation of CK-MB and LDH both
in serum and heart tissues and ALT, troponin-T in serum of
control and experimental rats. The rats induced with iso-
proterenol evidenced a significant (P \ 0.05) increase in
the serum levels CK-MB, ALT, LDH and troponin-T by
66, 112, 70 and 317 %, respectively, as compared to con-
trol rats associated with significant decrease in myocardi-
um LDH and CK-MB, whereas pre- and co-treatment with
HT (5 mg/kg bw) daily for 7 days significantly (P \ 0.05)
decreased the levels of serum cardiac markers better than
the administrated dose of 2 mg/kg of HT in isoproterenol-
induced rats compared to untreated infarcted rats.
Histopathological Examination of Cardiac Tissues
The histopathological examination of heart tissues showed
that control group of rats evidenced a normal myofibrillar
structure without any infarction edema and inflammatory
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Cardiovasc Toxicol

Table 1 Effect of hydroxytyrosol pre- and co-treatment on heart weight, body weight and heart weight/body weight ratio in isoproterenol-
induced myocardial infarction in rats
Parameters Control Isop Isop ? HT1 Isop ? HT2

Body weight (g) 269.20 ± 9.83 271.20 ± 10.28 273.80 ± 10.44 268.21 ± 8.70
,#
Heart weight (g) 0.95 ± 0.10 1.50 ± 0.09* 1.24 ± 0.13* 0.96 ± 0.11#,@
,#
Heart weight/body weight ratio 0.35 ± 0.02 0.55 ± 0.02* 0.45 ± 0.04* 0.36 ± 0.03#,@
Values are given as mean ± SD for groups of eight animals each
HT1, 2 mg/kg bw of hydroxytyrosol; HT2, 5 mg/kg bw of hydroxytyrosol
Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared
to isoproterenol group of rats. @P \ 0.05 significant differences compared to HT1 group

0.5
79 %, respectively, with notable decrease in serum HDL-c
* by 26 % when compared to normal control animals
0.45
(Table 4), whereas the administration of HT at 2 and 5 mg/
0.4
kg to infarcted rats induced a significant decrease in pan-
0.35 creatic lipase activity by 35 and 42 %, respectively, ac-
ST-Segment (mV)

0.3 companied with remarkable decrease in T-Ch, TG, LDL-c

0.25 by (13, 23 and 24 %) and (21, 35 and 39 %), respectively,
0.2 with considerable increase in HDL-c by 19 and 29 %, re-
0.15 spectively, as compared to isoproterenol-induced rats alone
*# (P \ 0.05).
0.1 #@

0.05
Effect of Hydroxytyrosol on Serum ACE Activity
0
Control Isop Isop+HT1 Isop+HT2
Groups Figure 5 showed that the ACE activity in serum of un-
treated infarcted rats underwent a significant increase by
Fig. 1 Effect of hydroxytyrosol on ST-segment elevation (mV) in the 67 % as compared to normal control animals (P \ 0.05).
ECG (recorded from limb lead II) in normal control, isoproterenol
However, treatment with HT at doses of 2 and 5 mg/kg
alone injected and treated rats. Values are given as mean ± SD for
group of eight rats. Statistically, values are represented as follows: induced a remarkable decrease of ACE activity by 28 and
*P \ 0.05 significant differences compared to controls. #P \ 0.05 36 %, respectively, in serum of isoproterenol-induced
significant differences compared to isoproterenol group. @P \ 0.05 myocardial infarction in rats.
significant differences compared to isoproterenol-treated group with
2 mg/kg of HT

cells (Fig. 3a). However, tissues from isoproterenol-treated
rats revealed widespread myocardial structure disorder and
subendocardial necrosis with loss of transverse striation
and obvious leukocyte infiltration (Fig. 3b) as compared to
control group, whereas pretreatment with HT (2 and 5 mg/
kg, respectively) exhibited marked improvement in iso-
proterenol-induced myocardial degeneration and inflam-
matory cells infiltration (Fig. 3c, d).
Effect of Hydroxytyrosol on Serum Lipase Activity
and Lipids Profile
As shown in Fig. 4, the isoproterenol-induced group un-
derwent a significant (P \ 0.05) increase in serum pan-
creatic lipase activity by 89 % which led to remarkable rise
in serum levels of T-Ch, TG and LDL-c by 30, 67 and
Discussion
HT (3,4-dihydroxyphenylethanol) is an amphipathic
molecule that is derived from the hydrolysis of oleuropein.
Oleuropein hydrolysis takes places during olive matura-
tion, as well as during olive oil storage [9, 10]. This phe-
nolic bioactive compound has been proved to be endowed
with several pharmaceutical properties such as antioxidant,
anti-inflammatory, antithrombotic, antidiabetic and hy-
polipidemic effects [11–14]. In the current study, we ex-
plored, for the first time, the therapeutic efficacy of HT in
rats with myocardial infarction. A subcutaneous injection
of a supramaximal dose of isoproterenol was a conse-
quence of electrocardiographic (ECG), biochemical and
structural changes in the heart as mimetic to that occurs in
patients with myocardial infarction. Changes in ECG pat-
tern are commonly used for the diagnosis of acute

123
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Cardiovasc Toxicol

Fig. 2 Effect of hydroxytyrosol on electrocardiographic (ECG)
pattern in normal and experimental rats. ECG pattern of normal
control group showing normal cardiograph. ECG pattern of isopro-
terenol (100 mg/kg)-induced group rats showing pathological
changes such as ST-segment elevation. Electrocardiogram pattern
of HT (2 mg/kg)-treated isoproterenol-induced group rats showing
minimized ST-segment elevation with aspect of electrical alternating.
ECG pattern of HT (5 mg/kg) pre- and co-treated isoproterenol-
induced group rats showing almost normal cardiograph without any
elevation in ST-segment

Table 2 Hemodynamic Parameters Control Isop Isop ? HT1 Isop ? HT2

parameters in different
experimental groups SAP (mmHg) 128 ± 5.56 88.20 ± 4.76* 104.40 ± 4.03*,# 122 ± 5.24#,@
DAP (mmHg) 83.40 ± 3.04 56 ± 3.80* 69.80 ± 4.81*,# 79.60 ± 4.15#,@
MAP (mmHg) 105.70 ± 4.13 72.12 ± 1.51* 87.10 ± 2.82*,# 100.80 ± 4.57#,@
HR (bpm) 375 ± 13.92 430 ± 11.65* 430 ± 11.65* 375 ± 10.31#,@
SAP systolic arterial blood pressure, DAP diastolic arterial blood pressure, MAP mean arterial blood
pressure, HR heart rate
Values are given as mean ± SD for group of eight animals each. Values are statistically presented as
follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences com-
pared to isoproterenol group. @P \ 0.05 significant differences to diabetic rats treated with HT1

Table 3 Effect of Groups Control Isop Isop ? HT1 Isop ? HT2

hydroxytyrosol (HT) on cardiac
markers in serum and heart Serum
tissues
ALT (UI/L) 41.4 ± 4.27 88 ± 4.01* 62.40 ± 6.24*,# 54.60 ± 9.65*,#
,#
LDH (UI/L) 519.8 ± 37.31 883.2 ± 40.61* 671.6 ± 63.38* 543.2 ± 65.72#,@
CK-MB (UI/L) 21.70 ± 0.96 36.16 ± 1.21* 25.65 ± 0.71*,# 22.72 ± 1.40#,@
,#
Troponin-T (ng/mL) 0.57 ± 0.06 2.38 ± 0.33* 0.76 ± 0.13* 0.53 ± 0.05#,@
Heart
LDH (lKat/mg) 7.31 ± 0.53 4.28 ± 0.21* 5.67 ± 0.47*,# 6.87 ± 0.32#,@
,#
CK-MB (lKat/mg) 2.45 ± 0.14 0.97 ± 0.16* 1.45 ± 0.13* 2.07 ± 0.24#,@
Alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB) and
serum troponin-T level of control and experimental groups of rats. CK-MB and LDH in the heart tissue are
expressed in lkat/mg of proteins. Values are given as mean ± SD for group of eight animals each
Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls.
#
P \ 0.05 significant differences compared to isoproterenol group @P \ 0.05 significant differences to
diabetic rats treated with HT1

123
 Author's personal copy
Fig. 3 Pathological changes of myocardial tissue (H&E 9500). a Control
group showing normal myocardial histology, clear transverse striations
and no inflammatory cell infiltration. b Isoproterenol group showing
myocardial cells necrosis, separation of cardiac myofibrillar and large
180
*
160
Serum lipase activity (UI/L)
140
*#
120
100
80
60
40
20
0
Control Isop Isop+HT1
Groups
Fig. 4 Effect of hydroxytyrosol on serum lipase activity in normal and
experimental rats. Values are given as mean ± SD for group of eight
rats. Statistically, values are represented as follows: *P \ 0.05 significant
differences compared to controls. #P \ 0.05 significant differences
compared to isoproterenol group. @P \ 0.05 significant differences
compared to isoproterenol-treated group with 2 mg/kg of HT
myocardial infarction. Rats treated with isoproterenol
showed a marked elevation of ST-segment with remarkable
decrease in P wave indicative of myocardial ischemia and
infarction in accordance with previous study [15]. The al-
teration of ECG pattern could be related to the consecutive
loss of integrity of cell membrane in injured myocardium
[16]. The supramaximal dose of isoproterenol also injected
increased heart rate (tachycardia) due to involvement of
baroreceptor reflex-mediated changes in autonomic nerve
activity [17], whereas pre- and co-treatment with HT
123
Cardiovasc Toxicol
inflammatory cells infiltration. c Isop ? HT1 (2 mg/kg)-treated group
showing few inflammatory cell infiltration and improvement of my-
ocardium necrosis. d Isop ? HT2 (5 mg/kg) showing normal myocardial
arrangement, clear transverse striations and little few inflammatory cells
(5 mg/kg) significantly reduced the abnormalities observed
in the ECG of isoproterenol-induced rats. Accordingly, it
has been demonstrated that HT improved the cardiac dis-
turbances enhanced by doxorubicin by significantly re-
*#@
ducing the percentage of altered mitochondria also
enhancing the mitochondrial electron transport chain and
antioxidant defense system [18]. Several previous studies
have demonstrated that the myocardium injury induced by
isoproterenol in experimental rats is strongly associated
with deterioration of hemodynamic and LV contractile
Isop+HT2 function [14–17]. While, HT pre- and co-treatment showed
significant improvement in systolic and diastolic arterial
pressure (SAP and DAP, respectively) and subsequent in-
crease in mean arterial pressure (MAP), a marker of after
load.
Serum cardiac troponins and cytosolic enzyme activities
such as CK-MB, LDH and AST are very sensitive and
specific diagnostic markers of acute myocardial infarction
[19, 20]. Our results confirmed previous studies reporting
that isoproterenol induced significant increases in serum
troponin-T level and CK-MB, LDH and AST activities
which reflected the severe damage in myocardium tissues
[20, 21]. HT pre- and co-treatment reduced the activity of
the marker enzymes in serum of isoproterenol-induced rats.
It confirmed that HT could maintain membrane integrity,
so restricting the leakage of these enzymes.
Previous study clearly evidenced that administration of
HT to normal rats showed no toxicity effect in tolerant
dose, and there was no significant difference between
 Author's personal copy
Cardiovasc Toxicol

Table 4 Effect of Groups Control Isop Isop ? HT1 Isop ? HT2

hydroxytyrosol (HT) on serum
lipids profile T-Ch 1.81 ± 0.12 2.35 ± 0.10* 2.04 ± 0.16*,# 1.86 ± 0.05#,@
,#
TG 0.72 ± 0.05 1.20 ± 0.10* 0.92 ± 0.11* 0.77 ± 0.11#,@
,#
LDL-c 0.84 ± 0.06 1.50 ± 0.14* 1.13 ± 0.18* 0.92 ± 0.03*,#,@
,#
HDL-c 0.82 ± 0.05 0.61 ± 0.05* 0.72 ± 0.04* 0.79 ± 0.02#,@
Total cholesterol (T-ch), triglycerides (TG), low density lipoprotein-cholesterol (LDL-c) and high density
lipoprotein-cholesterol (HDL-c) in serum. Values are given as mean ± SD for group of eight rats
Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls.
#
P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences
compared to isoproterenol-treated group with 2 mg/kg of HT

considerable rise in serum T-Ch, TG and LDL-c and re-

90
markable decreased in HDL-c in isoproterenol-induced
*
80 myocardial infarcted rats. These changes in lipids profile
ACE activity in serum (UI/L)

70 could be attributed to cardiac cyclic adenosine

*#
60
50
40
30
20
10
0
Control Isop Isop+HT1
Groups
Fig. 5 ACE activity in serum of normal and experimental rats.
Values are given as mean ± SD for group of eight rats. Statistically,
values are represented as follows: *P \ 0.05 significant differences
compared to controls. #P \ 0.05 significant differences compared to
isoproterenol group. @P \ 0.05 significant differences compared to
isoproterenol-treated group with 2 mg/kg of HT
normal rats treated with HT and untreated normal group in
all tested parameters such as heart weight, ratio, cardiac
enzymes markers, oxidative stress statute and myocardium
histology [18].
Our histopathological findings of the isoproterenol-in-
duced myocardial infarction showed large infarcted zone
with edema, necrosis and obvious separation of cardiac
myofibrillar and inflammatory cells which explained the
ST-segment elevation observed in the ECG findings. The
encroachment of the injured myocardium by neutrophils
during ischemia could be considered as the major source of
free radicals [22]. However, HT pre- and co-treatment (2
and 5 mg/kg, respectively) showed clear protective effects
in isoproterenol-treated groups. Furthermore, lipids have a
major role in cardiovascular diseases through the devel-
opment of hyperlipidemia, hence increasing atherosclerosis
risks. Else, it could lead to modify the composition,
structure and stability of the cellular membranes.
In the present study, a significant increase in serum
pancreatic lipase activity was observed which led to
monophosphate that enhanced lipid biosynthesis during the
#@
hypoxic condition in isoproterenol-induced myocardial
infarcted rats [23]. In fact, lipase is secreted from the
pancreas, transported to small intestine and hydrolyzes
triglycerides nonabsorbable into absorbable monoglyc-
erides and free fatty acids absorbable by small intestine.
Hence, inhibition of lipase activity by HT explains the
significant reduced uptake of triglycerides from the circu-
Isop+HT2
lation of infarcted rats which is confirmed in our previous
study [24]. Queenthy and John [7] revealed the positive
correlation between the risk of developing ischemic heart
disease and serum LDL-c level. In this regard, previous
investigations have demonstrated that HT can correct
dyslipidemia, improve triglyceride metabolism, enhance
endothelial function, increase antioxidant potential and
decrease low density lipoprotein-cholesterol (LDL-c),
which is an important cardiovascular diseases risk factor
due to its role in atherosclerosis [25, 26].
Accumulating evidence suggests that the cardiac ren-
nin–angiotensin system (RAS) is activated during the LV
remodeling process after acute myocardial infarction [27,
28]. Our data evidenced that serum ACE activity of iso-
proterenol-induced rats underwent a significant increase
accompanied with elevated heart weight ratio (hypertro-
phy) indicative of ventricular remodeling process. It in-
cludes infarct expansion as well as compensatory reactive
hypertrophy and dilation of the noninfarcted LV [28]. Pre-
and co-treatment of isoproterenol-induced myocardial in-
farction in rat at moderate dose of HT caused a significant
decrease of serum ACE activity with marked reduce in
heart weight ratio indicative of cardioprotective effect
preventing the increased risk of infarct expansion and LV
remodeling that follows myocardial infarction. Actually,
there is evidence from several experimental studies that the
cardiac tissue-type rennin–angiotensin system is activated
after myocardial infarction and failure [28, 29]. However,
ACE inhibition has been revealed to prevent cardiac

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 Author's personal copy
remodeling and to prolong survival in experimental my-
ocardial infarction. This process has been related to the
reduced levels of circulating and tissue angiotensin II
which is involved in the development of vascular and
ventricular remodeling. Hence, ACE inhibitor increases
tissue bradykinin accumulation, and the bradykinin has
antigrowth effects and reduces vasomotor [30].
In conclusion, the present study provides experimental
evidence that oral pre- and co-treatment with HT (2 and
5 mg/kg) evidence cardioprotective effect in isoproterenol-
induced myocardial infarction in rats by improving heart
weight, lipids profile and cardiac dysfunctional markers as
well as demonstrating the beneficial role of HT as ACE
inhibitor to prevent cardiac remodeling and failure.
Acknowledgments This research was supported by the Tunisian
Ministry of Higher Education and Scientific Research and the Tu-
nisian Ministry of Public Health. Authors wish to thank Mrs. Sadok
Slema, Mbarek Nasri and Chedly Tmar for their assistance and
cooperation.
Conflict of interests The authors declare no conflict of interest.
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