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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 28, No 3, May/June 2017
142
AFRICA
or function of renal dopaminergic receptors, however the
mechanism is not clear.
11
The exact role of oxidative stress in the
development of HT via NOS inhibition and the regulatory effect
of the anti-oxidant system in this process remains unresolved.
Resveratrol (3,4
′
,5-trihydroxy-trans-stilbene) (RSV) is a type
of natural phenol found in red grapes, peanuts, red wine and other
polyphenol-rich food. Anti-proliferative,
3,4
anti-inflammatory,
5
anti-oxidant
5-7,12-14
and cardioprotective
15
effects of resveratrol
have been shown in different experimental models so far.
Human studies established that acute administration of
RSV generated dose-dependent improvement of endothelium-
dependent vasodilatation.
16
Aminopiridin-sensitive potassium
channels play a role in that process and a potassium-independent
pathway (propably related to voltage-dependent calcium
channels) is also thought to be responsible for the vasodilatory
efects of RSV.
17,18
Furthermore, it was shown that aortic
vasodilation, with a low dose of RSV, was generated via the
endothelial NOS effect.
19
In our study, we aimed to investigate the effect of resveratrol
on blood pressure in rats that become hypertensive via NOS
inhibition with the application of L-NNA in doses that cause
mild hypertension.
20
Changes in parameters related to water–salt
balance and renal functions were also analysed.
Methods
Male Sprague-Dawley rats (230–260 g) from Harlan were housed
under standard conditions with a 12-hour light–dark cycle in
standard cages in a room with a controlled humidity of 40% and
a temperature of 22°C. They had
ad libitum
access to food and
water for 10 days.
Experimental procedures were in agreement with institutional
and legislator regulations and approved by the local ethics
committee for animal experimentation.
The rats (
n
=
35) were randomly divided into five groups (
n
=
7 in each group): control [intraperitoneal (i.p.) 1 ml 0.9% serum
physiological solution applied for 10 days], L-NNA (15 mg/100
ml L-NNA given with drinking water for 10 days), RSV-E [1 ml
resveratrol eluent (20% ethanol) i.p. applied for 10 days], RSV50
(50 mg/kg resveratrol i.p. applied for 10 days) and L-NNA
+
RSV50 (15 mg/100 ml L-NNA given with drinking water and 50
mg/kg resveratrol i.p. applied for 10 days).
The amount of consumed water was quantified every day and
all applications were performed at the same time of day. The dose
of L-NNA was calculated from the amount of consumed water
and the drinking water of all groups was refreshed every day.
Each subject was placed in a separate box in a quiet area. A
tail-cuff pletysmograph (MAY BPHR 9610-PC TAIL-CUFF
Indirect Blood Pressure Recorder, Ankara, Turkey) and its
sensor were fixed to their tails, which were warmed up to
37–38°C for 10–20 minutes, until it picked up regular signals
and obtained pulses. Systolic blood pressure and heart rate were
measured with the indirect tail-cuff method on the first, seventh
and 10th days of the study by investigators who were blinded
to the study protocol. An average of three measurements was
recorded on each occasion.
All rats were put into metabolic cages at the end of study
protocol. The total water intake and urine output were
determined for 24 hours. We added 0.1 ml 6N HCl to the urine
containers and kept the samples in the dark. Urine samples were
put into Eppendorf tubes and stored at –80°C (Sanyo Ultra Low
Temperature Freezer MDF-U4086S).
At the end of the experiment, the animals were anesthetised
with 20% urethane (1 g/kg, i.p.). Blood samples were collected
by heart puncture, and serum samples were obtained after
centrifugation of the blood at 5 400 rpm for 10 minutes and
stored at –80°C. We measured urea, creatinine and sodium
levels in the blood and urine samples with a Roche Cobas 6000
autoanalyser (Mannheim, Germany).
Fluid balance, sodium clearance rate (C
Na
), glomerular
filtration rate (GFR) and fractional sodium excretion (%FE
Na
)
were calculated using the following formulae:
Fluid balance
=
water intake – urine volume
UFR (urine flow rate in min)
=
24-hour urine volume
_______________
1 440
C
Na
=
urine sodium × UFR
_______________
plasma sodium
GFR
=
(urine creatinine × plasma creatinine)
__________________________
UFR
%FE
Na
=
(plasma creatinine × urine sodium)
_________________________
(plasma sodium × urine creatinine) × 100
Statistical analysis
All statistical analyses were performed with IBM SPSS Statistics
16 software (SPSS Inc, Chicago, IL, USA). Data are expressed as
mean
±
standard error. Blood pressure values were compared with
the Student’s
t
-test and biochemical values via one-way analysis
of varience (ANOVA) with
post hoc
Bonferroni comparison.
All
p
-values were two-tailed and
p
<
0.05 was considered to be
statistically significant.
Results
The body weight gains of all groups were similar and are shown
in Table 1. The first measured blood pressure values (before the
protocol) were similar between the groups (Table 2, Fig. 1). At
the end of the study protocol, blood pressures were higher in the
L-NNA (117.8
±
3.5 vs 149.5
±
2.1 mmHg;
p
<
0.05) and L-NNA
+
RSV50 (122.8
±
7.3 vs 155.4
±
4.4 mmHg;
p
<
0.05) groups
(Table 2, Fig. 1).
Table 1.Weight gain in the study groups
Groups (
n
=
7)
First day (g)
Last day (g)
Control
154
±
5.5
199.4
±
7.2
L-NNA
155
±
3.3
209.0
±
3.3
RSV50
151
±
2.8
188.5
±
4.2
RSV-E
188
±
5.6
214.8
±
16.9
L-NNA
+
RSV50
186
±
6.3
194.3
±
5.2
Table 2. Blood pressure measurements of the study groups
at the beginning and end of the study
Groups (
n
=
7) First measured (mmHg) Last measured (mmHg)
Control
123.1
±
5.5
121.1
±
3.5
L-NNA
117.8
±
3.5
149.5
±
2.1*
βγ
RSV50
122.4
±
3.8
124.2
±
2.4
RSV-E
126.6
±
6.4
121.7
±
7.8
L-NNA
+
RSV50
122.8
±
7.3
155.4
±
4.4*
βγ
*
Compared to the control group,
p
<
0.05;
β
compared to the RSV50
group,
p
<
0.05;
γ
compared to the RSV-E group,
p
<
0.05.