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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 25, No 5, September/October 2014
AFRICA
213
It has been reported that inflammatory NFkB expression
increased in the I/R-related infarct area; inflammation was
suppressed when NFkB expression was inhibited, and cardiac
preservation was provided.
10
In this context, caveolin-1 was
shown to regulate eNOS activation consistently with other
signalling molecules such as hsp 90.
11
Interaction of hsp 90 with
eNOS increases eNOS activity, and consequently, NO production
increases.
12,13
Myocardial caveolin-1 content is reported to decrease
following ischaemia–reperfusion.
14
Caveolin-1 deficiency was
noted to aggravate cardiac dysfunction and reduce the survival
rate in mice that had experienced myocardial infarction (MI).
15
Rosuvastatin is a synthetic hydrophilic statin widely used in
the treatment of dyslipidaemia, as it increases levels of high-
density lipoprotein (HDL) cholesterol, and reduces low-density
lipoprotein (LDL) cholesterol and triglyceride levels. Statins
have been reported to have anti-inflammatory, antiproliferative,
antithrombotic, anti-atherogenic and antihypertensive effects
in addition to their cholesterol-lowering effects.
8,16-18
Recent
studies indicate that rosuvastatin decreases levels of ADMA
in hypercholesterolaemia,
19
levels of caveolin,
20
and also NFkB
levels
21
in subaracnoid bleeding.
To our knowledge, the effects of rosuvastatin on ADMA,
rhokinase, caveolin-1, hsp 90 and NFkB levels are not known
in cardiac I/R injury. In this study, we aimed to investigate the
influence of rosuvastatin on oxidative stress-related rhokinase,
NADPH oxidase, ADMA, caveolin-1 and hsp 90 levels in a rat
model of I/R injury.
Methods
Male Sprague Dawley rats weighing 250–300 g were kept in a
quiet, temperature- (21
±
2°C) and humidity- (60
±
5%) controlled
room in which a 12-hour light–dark cycle was maintained. All
experiments were performed between 9:00 and 17:00.
The rats were divided into three groups: control (sham), I/R
+ vehicle (physiological saline) and I/R + rosuvastatin. Vehicle
or rosuvastatin (10 mg/kg) were administered in the afternoon
(17:00) by intraperitoneal injection for 15 days before ischaemia.
I/R protocols were performed in the morning (08:00–12:00).
Measurement of myocardial tissue rhokinase, NADPH
oxidase, caveolin-1, hsp 90, NFkB and ADMA levels were
performed in seven animals in each group. Rosuvastatin (Abdi
Ibrahim Pharmaceutical Co, Istanbul, Turkey) was dissolved in
physiological saline.
All experiments in this study were performed in accordance
with the guidelines for animal research from the National
Institutes of Health and were approved by the local committee
on animal research (FUHADYEK -13.06.2012-76).
Ischaemia–reperfusion procedure
Rats were anesthetised with urethane (1.2–1.4 g/kg) administered
intraperitoneally. The jugular vein and trachea were cannulated
for drug administration and artificial respiration, respectively.
Systemic blood pressure (BP) was monitored via the carotid
artery with a Harvard model 50-8952 transducer (Harvard
Apparatus Inc, Massachusetts, USA) and displayed on a Harvard
Universal pen recorder (Harvard Apparatus, Inc, Massachusetts,
USA) together with a standard 12-lead ECG.
The chest was opened via a left thoracotomy, followed by
sectioning the fourth and fifth ribs, about 2 mm to the left of
the sternum. Positive-pressure artificial respiration was started
immediately with room air, using a volume of 1.5 ml/100 g body
weight at a rate 60 beats/min to maintain normal pCO
2
, pO
2
and
pH parameters.
After the pericardium was incised, the heart was exteriorised
by gentle pressure on the outside of the rib cage. A 6/0 silk
suture attached to a 10-mm micropoint reverse-cutting needle
was quickly placed under the left anterior descending coronary
artery. The heart was then carefully replaced in the chest, and the
animal was allowed to recover for 20 min. Any animal in which
this procedure produced arrhythmias or a sustained decrease in
mean arterial BP to less than 70 mmHg was discarded.
A small plastic snare was threaded through the ligature
and placed in contact with the heart. The artery could then be
occluded by applying tension to the ligature, and reperfusion was
achieved by releasing the tension. At the end of the experimental
period, left ventricle myocardial samples, distal to the left main
coronary artery occlusion, were collected for analysis and
analysed within one month.
Quantitative real-time polymerase chain reaction
analysis (qRT-PCR)
Tissue samples were immersed in RNAlater. After overnight
saturation with RNAlater, the tissues were stored at –80°C.
All protocols were performed according to the manufacturer’s
instructions. Total RNA was extracted from rat heart tissues
using TRizol reagent (Invitrogen, Carlsbad, USA).
To carry out the PCR assay, total RNA from the heart
samples in each experimental group was pooled (3 μg total).
cDNA from the pooled samples was synthesised using a high-
capacity RNA-to-cDNA kit (Invitrogen, Carlsbad, USA).
Relative expression levels of mRNA were determined using a
7500 fast real-time PCR (PE Biosystems, Foster City, CA, USA)
with Taq Man master mix and rat-specific assays for NFkB,
caveolin-1, hsp 90 and GAPDH genes. The relative abundance of
mRNA was calculated after normalisation to GAPDH.
Triplicate assays were performed. PCR reactions were
performed after heating to 50°C for 2 min followed by 40 cycles
of denaturation at 95°C for 10 min, 95°C for 15 sec and 60°C
for 1 min. ADMA, rhokinase and NADPH oxidase levels were
evaluated with ELISA.
Statistical analysis
Data are expressed as arithmetic means
±
SEM. When
p
<
0.05,
the difference was considered to be statistically significant.
Normality of the distribution within the groups was evaluated
with the Shapiro–Wilk test. Multiple comparisons between the
experimental groups were performed by one-way analysis of
variance with the Tukey
post hoc
test.
Results
I/R caused a significant increase in ADMA levels. This increase
was limited although not statistically significantly attenuated in
the rosuvastatin group (Fig. 1).
While NFkB levels increased 2.2-fold with I/R, they
significantly decreased in the rosuvastatin-treated group (Fig.