Key-words:
Blood pressure - hypertension - SHRsp - Sprague–Dawley - sulforaphane
Introduction
Persistent hypertension is linked to an increased risk of morbidity and mortality.
The escalating health problem, hypertension, increases at an alarming rate. Worldwide,
approximately 1 billion individuals are hypertensive; by 2025, this number is projected
to increase to 29%, 1.56 billion, and more than 50% of Canadians aged from 55 to 74
years old are hypertensive.[[1]] A new therapeutic approach, such as the dietary intervention, could increase the
percentage (5%–23%) of patients with normal blood pressure (BP) (<140/90 mmHg), in
so doing, it will increase the successful rate of the pharmacotherapy and decrease
the number and severity of adverse effects associated with the use of pharmacotherapy.[[2]] Various data support the therapeutic effects of the cruciferous vegetables (mustard
family) of the genus brassica including cauliflower, broccoli, cabbage, and Brussels
sprouts in several diseases.[[3]] In young ([[4]] Small quantities of young crucifer sprouts (e.g., 3-day-old broccoli sprouts) protect
against chemical carcinogens.[[3]] Previously demonstrated in our lab that in SHRsp rats, diet-containing broccoli
sprouts decreases inflammation, oxidative stress, hypertension and ageing-related
degenerative changes in aging population.[[5]] Also, feeding hypertensive rats 200 mg/day of dried broccoli sprouts for 14 weeks,
ameliorates hypertension and atherosclerotic changes.[[6]] Most of the available raw plant extracts contain a mixture of compounds, for instance,
broccoli extracts contain multiple bioactive components such as indole glucosinolates,
a precursor of indole-3-carbinol (enhance tumorigenesis), flavonoids (i.e., quercetin),
selenium, and sulforaphane.[[5]],[[6]] Consequently, the health beneficial effects seen with broccoli consumption in our
previous studies could be due to the synergistic or inhibitory effects in complex
mixtures. Therefore, the aim of this study is to investigate whether the potent inducer
of the cytoprotective phase-2 protein, sulforaphane (independent of other ingredients
in the broccoli sprouts), attenuates hypertension in female SHRsp.[[7]]
Materials and Methods
Animal model
A total of Fourty two 5-week-old female rats, including 21 SHRsp rats and 21 age-matched
Sprague–Dawley (SD) rats (end of the 4th week postnatal), were purchased from Charles
River Laboratories (St. Constant, Quebec Canada). The rats were treated in accordance
with the guidelines of the Canadian Council on Animal Care, and the experimental protocols
were approved by the Animal Care Committee at the University of Saskatchewan. With
free access to water and regular food, the animals were kept under standard 12 h light/12
h dark cycle and humidity condition.
Animal groups
After 1 week of adaptation, the 5-week old female SHRsp and SD rats were divided into
four groups and administered daily (8–10 a. m.) by gavage: (i) corn oil (vehicle)
alone (control, n = 5); (ii) sulforaphane (5 μmol/kg body weight, n = 5) in corn oil;
(iii) sulforaphane (10 μmol/kg body weight, n = 5) in corn oil; and (iv) sulforaphane
(20 μmol/kg body weight, n = 6) in corn oil. Systolic BP was determined weekly (8–10
p. m.) using a standard tail-cuff noninvasive BP measurement system (model 29-SSP;
Harvard Apparatus, St. Laurent, QC, Canada). The gavage treatment lasted for 15 weeks
(20 weeks old). At the end of the treatment period, the animals were euthanized and
perfused with normal saline, and tissues, such as hearts and kidneys, were collected
and stored under −80°C for further analysis.
Statistical methods
All data are expressed as means ± standard error of the mean. Statistical significance
was tested using Student's t-test or one–way analysis of variance followed by a post
hoc analysis. Check test of homogeneity of variances was performed. If equal variances
were determined, the Bonferroni test was used, whereas if equal variances were not
determined, Tamhane's T2 test was used using SPSS software version 14.0 for windows
(SPSS Inc. Chicago, IL, USA). The significance level was set at P < 0.05.
Results
Sulforaphane administration daily for 15 weeks attenuates hypertension in hypertensive
SHRsp rats. Sulforaphane could neither affect rats' body weights nor influence organ
weights.
Organs of Sprague–Dawley and SHRsp female rats
At the end of the study, we collected and weighed the hearts and kidneys of SD and
SHRsp rats (data not shown). Within the strains, SHRSP and SD rats did not have any
significant difference in weights when compared with the controls.
Body weights of Sprague–Dawley and SHRsp female rats
To investigate any effects of sulforaphane on body weights (g), we weekly measured
the body weights of the animals. For 15 weeks, despite the initial (184.34 ± 3.94
vs. 120.14 ± 3.84) and the final body weights of SD rats being significantly higher
(320.08 ± 11.3 vs. 216.2 ± 1.84) than SHRsp rats (strain difference), 15 weeks of
daily sulforaphane administration did not have any effect on body weights [[Figure 1]].
Figure 1: Effect of sulforaphane on initial and final body weight of SHRsp and Sprague-Dawley
rats, *P < 0.05 versus the Sprague-Dawley control at the two respective time points,
n = 5-6 rats per group, means ± standard error of the mean
Blood pressure in SD and SHRsp female rats.
During the 15-weeks study, we measured BP weekly in the morning (8–10 a. m.) using
tail-cuff machine. Sulforaphane administration attenuated BP in SHRsp rats, but it
did not have any effect in normotensive SD rats. Comparing with the SD control, sulforaphane
did not have any effect in SD rats (83.98 ± 4.3 mmHg). However, when compared with
the SHRsp rats (179.9 ± 4.3 mm Hg), 15 weeks of sulforaphane administration significantly
attenuate BP to 157 ± 5.21 (10 μmol/kg body weight), 136.57 ± 1.96 (20 μmol/kg body
weight), and 129.33 ± 6.10 (5 μmol/kg body weight), respectively, in SHRsp rats [[Figure 2]]. Previous studies in our laboratory have shown that higher doses than 5.5 μmol
sulforaphane may have more profound antihypertensive effects.
Figure 2: Effect of sulforaphane on systolic blood pressure (measured by tail-cuff) in SHRsp
and Sprague-Dawley rats. The systolic blood pressure in SHRsp and Sprague-Dawley rats
is significantly different (#P < 0.05). The blood pressure measured in sulforaphane-treated
SHRsp rats is significantly lower than that in corn oil-treated SHRsp rats, *P < 0.05
versus the control of the same age and strain, n = 5-6 rats per group, mean ± standard
error of the mean
Discussion
SHRsp is an adequate experimental model with elevated BP, together with structural
and functional abnormalities.[[8]] As an animal model of hypertension, our laboratory has been using this model to
study the therapeutic effects of broccoli sprouts (containing sulforaphane precursor)
on hypertension. In this study, using an animal model of hypertension such as SHRsp,
we evaluated the antihypertensive effects of pure sulforaphane. Our studies have shown
that chronic oral administration of sulforaphane, a phase-2 protein inducer, (a) had
comparable effects in body weights among all sulforaphane-treated groups throughout
the experiment [[Figure 1]], (b) significantly reduced BP at the end of the 15-week treatment [[Figure 2]], and (c) it did not have any effects on animals of normal redox physiology such
as SD.
The comparable body and organ weights (i.e., hearts and kidneys) among the sulforaphane-treated
and untreated groups suggest that there were no toxicities associated with the chronic
oral administration of sulforaphane. One could easily understand this since sulforaphane
is of the many ingredients in broccoli sprouts, little or no adverse effects would
be expected from sulforaphane administration. These findings are in agreement with
those of Conaway.[[9]] In A/J mice (lung cancer animal model), sulforaphane, phenyl isothiocyanate, and
their N-acetylcysteine conjugate-treated mice had comparable body weights as compared
to untreated groups.
In our study, we have shown that sulforaphane reduced hypertension in SHRsp rats.
Unlike SHRsp rats, the normotensive SD rats which their BP remain constant during
the sulforaphane administration. This means that sulforaphane needs time to produce
therapeutic effects when given daily by gavage to rats. In normal physiology rats
(SD), sulforaphane did not have any effects; this needs more investigations to find
an answer for this outcome. Similarly, sulforaphane reduces hypertension in SHRsp
rats but did not affect SD rats.[[10]] Moreover, glucoraphanin, a sulforaphane precursor, is high in broccoli sprouts.
In SHRsp rats, diet-containing broccoli sprouts high in glucoraphanin reduce oxidative
stress, inflammation, and hypertension.[[11]]
Growing body of evidence supports the protective effects of broccoli (which contains
sulforaphane precursor) in several chronic diseases, which have the oxidative stress
and inflammation components. For example, bothin vitro and in a diabetic rat model,
broccoli offers a protective effect by reducing the oxidative stress load.[[12]] Moreover, in the hypertensive rat model, 14-week administration of rats with 200
mg/day of dried broccoli sprouts that contained glucoraphanin (0.5 and 5.5 μmol sulforaphane
equivalents) attenuates blood pressure.[[6]] Cruciferous vegetables (i.e., broccoli) possess similar chemistry, metabolism,
and protective effects as sulforaphane (e.g., glucobrassicin, gluconasturtiin–phenethyl
isothiocyanate, glucoerucin [sulfide analog of sulforaphane], and glucoiberin-iberin).[[13]] Not only sulforaphane but also broccoli contains multiple bioactive components
such as indole-3-carbinol and flavonoids (i.e., quercetin).
Our study shows that independent of these bioactive components, long term oral administration
of sulforaphane alone reduces BP in hypertensive rats. How does long-term administration
of sulforaphane decrease BP in hypertensive in hypertensive rats? Further experiments
are needed to understand the mechanism(s) by which sulforaphane reduces BP in SHRsp
rats or why sulforaphane did not have any effects in SD rats.
Conclusion
According to this study, (i) a minimal changes in our diet (adding broccoli sprouts
that contain sulforaphane) may have a major impact in our health, (ii) the beneficial
health effects previously seen with consumption of broccoli sprout are due to conversion
of the sulforaphane precursor – glucoraphanin to sulforaphane – a potent phase-2 protein
inducer, and (iii) the health-promoting effects of sulforaphane can be seen after
long-term administration (~3 months).
