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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 28, No 6, November/December 2017
390
AFRICA
form a toxic oxygen metabolite, peroxynitrite (ONOO
-
), which
causes damage in the tissues.
15,16
Therefore, elucidation of the
changes in NO levels caused by the administration of ILO and
SIL, which were used for the reperfusion of tissues, could help
explain the mechanisms underlying this process.
Atpresent,sildenafilisemployedtocorrecterectiledysfunction.
12
Since it acts as a vasodilator, it can serve as a therapeutic agent
during ischaemia.
14
Vasodilatation enhances oxygenation and
therefore mediates in the elimination of ischaemia and increases
adenosine triphosphate (ATP) formation. It is established that
aerobic ATP formation is blocked in hypoxic states. Therefore,
ischaemia leads to a decrease in ATP production.
17
Another
molecule that causes a reduction in ATP levels is irisin.
18,19
By
increasing the amount of uncoupling proteins, this molecule leads
to the release of heat rather than ATP from molecules.
18
Since ILO,
7
used in ischaemic peripheral artery disease, and
SIL,
12
used in erectile dysfunction, increase oxygenation through
vasodilatation, the tissues recovered from ischaemia would
theoretically be expected to have elevated ATP levels. On the
other hand, in the presence of irisin, heat production would
increase through uncoupling of proteins and cause a decrease
in ATP production.
18,19
Therefore there seems to be an obvious
correlation between the treatment of ischaemic tissue with ILO
and SIL, and irisin levels.
Furthermore, myocardial ischaemia does not only affect
heart tissue. It was reported in previous studies that myocardial
ischaemia could directly impact on kidney tissue,
20
which is an
excretory organ, and the liver,
21
where glycogenesis takes place.
In addition, there is an increased need for energy (glucose)
during ischaemic conditions. It was reported that irisin inhibited
glycogenesis, or impeded the production of glucose.
22
Therefore the aim of this study was to determine the change in
irisin level in tissues with increased energy needs under ischaemic
conditions. Our principal objectives were to explore (1) whether
ILO and SIL played a part in recovery after myocardial injury
and how they changed irisin expression in experimentally induced
myocardial ischaemia–reperfusion; (2) whether ILO, SIL, or
a combination of both were more efficient in the treatment of
ischaemic injury; (3) how NO levels were altered in response
to these therapeutic agents; (4) whether irisin, which causes
metabolisation of ATP, was down- or upregulated in tissues with
an increased need for ATP, as in the case of ischaemia; and (5)
how ILO and SIL treatment affected irisin expression in heart,
liver and kidney tissues under ischaemic conditions.
Methods
All protocols of the animal experiments were approved (date
5.2.2014, decision no: 35) by the Animal Ethics Committee
(FUAEC) in accordance with the policy of the European
convention for the protection of vertebrate animals. The study
included adult male Sprague-Dawley rats aged 10 months and
weighing between 250 and 280 g. The rats were randomly divided
into the following groups: control group (sham: no procedure to
be applied, only physiological serum administered), ILO, SIL,
ILO
+
SIL, myocardial ischaemia (MI), MI
+
ILO, MI
+
SIL and
MI
+
ILO
+
SIL. Each group contained five rats.
Ischaemia was induced by left coronary artery ligation,
as described previously.
23
In rat experiments, sildenafil citrate
(Viagra) is usually used in the 1–2.5-mg/kg dose range,
24,25
and
ILO in the 0.2–2-
μ
g/kg range.
26,27
In this study, 2 mg/kg sildenafil
citrate was administered to the SIL group and 1
μ
g/kg to the
ILO group via the intraperitoneal route before the induction of
ischaemia–reperfusion, as described previously by Harada
et al
.
28
A 30-minute occlusion was then induced using a plastic ligature,
as described previously.
29
After the ligature was released, blood
flow was visually confirmed. All rats were sacrificed at 24 hours
following the reperfusion procedure.
Blood samples were collected as described for previous
experiments,
30
centrifuged at 4 000 rpm and stored at –80°C until the
irisin analysis. Glucose, creatine kinase (CK), creatine kinase MB
(CKMB) and troponin I on the other hand, were analysed without
delay on an auto-analyser. Heart, liver and kidney tissue was fixed
in 10% formaldehyde solution and stored for immunohistochemical
analysis. The remaining heart, liver and kidney tissue, after the wet
weight was determined, were homogenised and the supernatants
were stored at –80°C for NO analysis.
As its half life is short, it is difficult to directly analyse NO. For
NO measurements, its stable end-products, nitrite and nitrate,
are quantified in tissues with a spectrophotometric method. This
method is based on the principle of measuring the absorbance at
545 nm of the complex formed when nitrate is reduced to nitrite
in the presence of nitrate reductase enzyme, and the resulting
nitrite reacts with sulfanylamide and N-ethylendiamin.
31
Serum irisin levels were determined using the ELISA method,
following the catalogue guidelines provided by the manufacturing
firm.
32
The kit was reported to have a minimum irisin detection
limit of 1.29 ng/ml and minimal cross-reactivity (~9%) with
fibronectin type III domain-containing protein 5 (FNDC5). In
our laboratory results, we found an intra-assay value of 8% and
inter-assay value of 10%.
Histopathological examinations were carried out using the
triphenyl tetrazolium chloride method to identify the damage
to the myocardium and other tissues, as described previously.
23
Myocardial injurywas assessed according to the semi-quantitative
method of Miller
et al
.
33
The Abc immunohistochemical method
of Hsu
et al.
34
was used to determine the distribution of irisin
expression in the myocardium and other tissues.
34
Statistical analysis
The extent of the damage in the myocardium and other tissues
was determined using the Student’s
t
-test. SPSS 22 software
was employed in all statistical analyses. Level of statistical
significance was determined at a
p
-value of 0.05.
Results
Masson’s trichrome staining results under light microscopy
showed that the heart tissue of the control group had a normal
appearance (Fig. 1A). The MI group, however, showed an
increase in inflammatory cells (black arrow), congestion (red
arrow), impairment of tissue integrity and oedema (Fig. 1B).
Data from the statistical analysis of histopathological changes in
the MI group are given in Table 1.
Evaluationunderthelightmicroscopeof immunohistochemical
staining revealed irisin immunoreactivity in the muscle cells of
the cardiac tissue (black arrow). The control (Fig. 2A), ILO
(Fig. 2B), SIL (Fig. 2C) and ILO
+
SIL (Fig. 2D) groups had
similar irisin immunoreactivity. Compared to the control group,