2Gülhane Military Medical Academy, Cardiology, Istanbul, Türkiyez
3Marmara University, Vocational School of Health Related Professions, Istanbul, Türkiye
4Marmara University, School of Medicine, Department of Physiology, Istanbul, Türkiye DOI : 10.12991/201216412
Summary
Bu çalışmada Nigella sativa'nın sıçanlarda renovasküler hipertansiyonun (RVH) neden olduğu kalp ve böbrek dokularındaki oksidan hasara karşı koruyucu etkileri incelendi. RVH Wistar albino sıçanların sol böbrek arterine yerleştirilen klip ile oluşturulurken (iki böbrek tek klip modeli; 2B1K, n=8) taklit grubu sıçanlarda (n=8) klip yerleştirmeksizin cerrahi uygulandı. Cerrahi işlemin 3.haftasından başlayarak 6 hafta süresince sıçanlara Nigella sativa (0.2 mg/kg/gün, intraperitoneal) veya taşıyıcı uygulamaları yapıldı. Çalışmanın başlangıcında, 3.haftada ve 9.haftada kan basıncı (KB) ölçümleri alınırken dekapitasyondan önce tüm hayvanların transtorasik ekokardiografik görüntüleri kaydedildi. Plazma örneklerinde asimetrik dimetilarginin (ADMA), nitrik oksid (NO), kreatin kinaz (KK) ve laktatdehidrogenaz (LDH) düzeyleri ölçüldü. Kalp ve böbrek dokularında reaktif oksidan oluşumu kemilüminesans yöntemi ile ölçüldü. Ayrıca oksidan hasar dokuların belirlenmesiiçin malondialdehit (MDA), glutatyon (GSH) düzeyleri ve Na+,K+- ATPaz aktiviteleri ölçümleri yapıldı. İki böbrek tek klip uygulaması, KB'de artışa ve sol ventrikül fonksiyonlarında bozulmaya neden olurken plazma ADMA, KK ve LDH anlamlı olarak artmış bulundu (p<0.05-0.001). Ayrıca hipertansiyon plazma NO düzeylerini ve doku GSH düzeyleri ile Na+,K+-ATPaz aktivitesini düşürürken MDA düzeyleri artmış bulundu (p<0.05-0.001). Diğer taraftan Nigella sativa uygulaması KB'yı anlamlı derecede düşürdü, oksidan hasarı azalttı ve sol ventrikül fonksiyonlarını düzeltti. Nigella sativa antioksidan ve antihipertansif etkileri ile hipertansiyonun neden olduğu renal ve kardiyak doku hasarına karşı koruyucu olmuştur. Bu bulgular Nigella sativanın RVH'da terapötik potansiyeli olabileceğini düşündürmektedir.Introduction
Renovascular disease remains among the most prevalent and important causes of secondary hypertension and renal dysfunction. Hypertension develops in patients with renovascular disease from a complex set of pressor signals, including activation of the renin-angiotensin system, recruitment of oxidative stress pathways, and sympathoadrenergic activation[1]. Activation of the renin-angiotensin system is the essential component of developing renovascular hypertension during the initial stages and plays an important role in the maintenance of hypertension, as well as in cardiac myocyte growth and oxidative stress[2]. As an important component of the renin- angiotensin system, angiotensin II (Ang II) stimulates nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase enzyme, which can induce oxidative stress in the vasculature via generation of oxygen-free radicals[3]. Experimental evidence also suggests that reactive oxygen species (ROS), which occur as a consequence of renal artery stenosis, enhance the hypertensive and hypertrophic responses to Ang II. Increased Ang II activity results in vasoconstriction, increased endothelin release, vascular remodeling, extracellular matrix deposition, and accelerated atherogenesis and glomerulosclerosis[4]. These effects may contribute to the progression of cardiovascular and renal damage well beyond the effects of high blood pressure per se.It is well known that overproduction of ROS can lead to a damaging cycle of lipid peroxidation, depletion of natural antioxidants, perturbation of nitric oxide production, disruption of normal cellular metabolism and endothelial dysfunction[5]. On the other hand, experimental blockade of the oxidative stress pathway with antioxidants in models of Goldblatt hypertension was shown to decrease renal injury and glomerulosclerosis, while renal hemodynamics was improved[6]. Accordingly, free radical scavengers or antioxidants were proposed to be useful in hypertension-induced multi-organ damage. Recently, plant-based antioxidants used as dietary supplements were found to be effective for the management and prevention of hypertension and stress-related diseases due to their minimal side effects.
Nigella sativa L., commonly known as black seed, is a seed of capsulated plants, and belongs to the Ranunculacceae family. It is used in folk medicine as a natural remedy for a number of diseases and symptoms, such as asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness and gastrointestinal disorders[7]. Furthermore, thymoquinone (TQ), an active ingredient of the black seed oil from the plant Nigella sativa, has been shown to possess potent antioxidant properties[8]. In our previous study we have demonstrated that Nigella sativa oil treatment reduced subarachnoid hemorrhage induced oxidative brain injury and associated neurological symptoms[9]. Based on the above findings, in the present study we aimed to investigate the possible beneficial effects of Nigella sativa treatment on cardiovascular function and oxidative damage in rats with renovascular hypertension.
Methods
AnimalsAll experimental protocols were approved by the Marmara University (MU) Animal Care and Use Committee. Male Wistar albino rats (200-250 g), supplied by the MU Animal Center (DEHAMER), were kept at a constant temperature (22 + 1º C) with 12 h light and dark cycles and fed a standard rat chow.
Surgery and Experimental Design
Two-kidney, one-clip (2K1C) has been studied as an Ang IIdependent
model of renovascular hypertension with elevated
circulating levels of Ang II and high Ang II concentration in
the cortical tissue of the clipped and non-clipped kidneys[10].
Clipping of the left renal artery and sham surgery were performed
as previously described[10]. Briefly, a silver clip (internal
diameter 0.25 mm) was placed around the left renal artery
(n=16) of the rats that were anesthetized with ketamine
(100 mg/kg) and chlorpromazine (0.75 mg/kg) given intraperitoneally
(ip). Half of the group with hypertension was
treated with vehicle (corn oil, 1 ml/kg/day, ip), while the other
half was treated with Nigella sativa oil (NS, at adose of 0.2
ml/kg/day completed with corn oil to 1 ml/kg) starting by
the end of the 3rd week after the clip-placement surgery and
continued for the remaining 6 weeks. The rationale for the selected
dose of NS depends on our previous reports demonstrating
its protective action in other oxidative injury models[9]. In the sham-operated control group (n=8), animals had
similar surgical procedures without clip-placement.
To obtain basal readings, systolic blood pressure recordings were obtained in all rats before the surgical procedures (clip placement or sham-operation), and these measurements were repeated at the end of the 3rd and 9th weeks after the surgery. All rats were decapitated following the 9th-week-measurements. Trunk blood was collected and immediately centrifuged at 3,000 g for 10 min to assay the plasma levels of lactate dehydrogenase (LDH), creatine kinase (CK), asymmetric dimethylarginine (ADMA), and nitric oxide (NO). Heart and kidney samples were taken for the determination of luminol and lucigenin chemiluminescence levels, malondialdehyde (MDA) and glutathione (GSH) levels and Na+,K+-ATPase activities.
Measurement of Blood Pressure
Indirect blood pressure measurement was made by the tail
cuff method (Biopac MP35 Systems, Inc.) before the surgery
and at the end of 3rd and 9th weeks following surgery. Initially,
the rats were placed for 10 min in a chamber heated to 35°C.
Then the rats were placed in individual plastic restrainers and
a cuff with a pneumatic pulse sensor was wrapped around
their tails. Blood pressure recorded during each measurement
period was averaged from at least three consecutive readings
on that occasion obtained from each rat.
Echocardiography
Echocardiographic imaging and calculations were done according
to the guidelines published by the American Society
of Echocardiography[11] using a 12 MHz linear transducer
and 5-8 MHz sector transducer (Vivid 3, General Electric Medical
Systems Ultrasound, Tirat Carmel, Israel). Under ketamine
(50 mg/kg, i.p.) anesthesia, measurements were made
from M-mode and two-dimensional images obtained in the
parasternal long and short axes at the level of the papillary
muscles after observation of at least 6 cardiac cycles. Interventricular
septal thickness (IVS), left ventricular diameter (LVD)
and left ventricular posterior wall thickness (LVPW) were
measured during systole (s) and diastole (d). Ejection fraction
(EF), fractional shortening (FS) and left ventricular mass
(LVM) and relative wall thickness (RWT) were calculated from
the M-mode images using the following formulas: % EF =
[(LVDd)3 – (LVDs)3/(LVDd)3 X 100]; % FS= [LVDd-LVDs/
LVDd X 100]; LVM= [1.04 x ((LVDd+LVPWd+IVSd)3 –
(LVDd)3 ) x 0.8 + 0.14]; RWT = [2 x (LVPWd/LVDd)].
Plasma assays
Plasma levels of LDH and CK were determined spectrophotometrically
using an automated analyzer (Bayer Opera biochemical
analyzer, Germany), while ADMA concentration in
plasma was measured with a highly sensitive ELISA kit (Immunodiagnostic
AG, Bensheim, Germany). The intensity of
the color reaction, measured by reading the optical density at
450 nm with a microtiter plate reader, is known to be inversely
proportional with the amount of ADMA in the sample. NO
metabolites (nitrates and nitrites) were assayed in plasma by
the colorimetric method of Griess after enzymatic conversion
of nitrates to nitrites by nitrate reductase using a colorimetric
assay kit (Cayman Chemical, AnnArbor, MI, USA).
Measurement of tissue luminol and lucigenin
chemiluminescence
To assess the contribution of ROS in renovascular hypertensioninduced
tissue damage, luminol and lucigenin chemiluminescences
(CL) were measured as indicators of radical formation.
Lucigenin (bis-N-methylacridiniumnitrate) and luminol (5- amino-2,3-dihydro-1,4-phthalazinedione) were obtained from
Sigma (St Louis, MO). Measurements were made at room temperature
using Junior LB 9509 luminometer (EG&G Berthold,
Germany). Specimens were put into vials containing PBSHEPES
buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2).
ROS were quantitated after the addition of enhancers such as
lucigenin or luminol for a final concentration of 0.2 mM. Luminol
detects a group of reactive species, i.e. .OH, H2O2, HOCl
radicals and lucigenin is selective for O2[12]. Counts were obtained
at 1 min intervals and the results were given as the area
under curve (AUC) for a counting period of 5 minutes. Counts
were corrected for wet tissue weight (rlu/mg tissue).
Measurement of tissue malondialdehyde (MDA) and
glutathione (GSH) levels
Heart and kidney samples were homogenized with ice-cold
150 mM KCl for the determination of MDA and GSH levels.
The MDA levels were assayed for products of lipid peroxidation
by monitoring thiobarbituric acid reactive substance formation
as described previously[13]. Lipid peroxidation was
expressed in terms of MDA equivalents using an extinction coefficient of 1.56 x 105 M–1 cm –1 and results are expressed as
nmol MDA/g tissue. GSH measurements were performed using
a modification of the Ellman procedure[14]. Briefly, after
centrifugation at 3000 rev/min for 10 min, 0.5 ml of supernatant
was added to 2 ml of 0.3 mol/l Na2HPO4.2H2O solution.
A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium
citrate) was added and the absorbance at 412 nm was
measured immediately after mixing. GSH levels were calculated
using an extinction coefficient of 1.36 x 104 M–1 cm –1.
Results are expressed in μmol GSH/g tissue.
Measurement of Na+,K+-ATPase activity
The activity of Na+,K+-ATPase, a membrane-bound enzyme
required for cellular transport, is very sensitive to free radical
reactions and lipid peroxidation. Accordingly, a reduction in
Na+,K+-ATPase activity indirectly indicates membrane damage.
Measurement of Na+,K+-ATPase activity is based on the
measurement of inorganic phosphate released by ATP hydrolysis
during incubation of homogenates with an appropriate
medium. The total ATPase activity was determined in the
presence of 100 mM NaCl, 5 mM KCl, 6 mM MgCl2, 0.1 mM
EDTA, 30 mM Tris HCl (pH 7.4), while the Mg2+-ATPase activity
was determined in the presence of 1mM ouabain. The difference
between the total and the Mg2+-ATPase activities was
taken as a measure of the Na+,K+-ATPase activity[15]. The reaction
was initiated with the addition of the homogenate (0.1
ml) and a 5-min pre-incubation period at 37º C was allowed.
Following the addition of 3 mM Na2ATP and a 10-min re-incubation
period, the reaction was terminated by the addition of
ice-cold 6 % perchloric acid. The mixture was then centrifuged
at 3500 g, and Pi in the supernatant fraction was determined
by the method of Fiske and Subarrow[16]. The specific activity
of the enzyme was expressed as micronmol Pi mg-1 protein h-1.
The protein concentration of the supernatant was measured by
the Lowry method[17].
Statistics
Statistical analysis was carried out using GraphPad Prism 3.0
(GraphPad Software, San Diego; CA; USA). Each group consisted
of 8 animals. All data were expressed as means ± SEM.
Groups of data were compared with an analysis of variance
(ANOVA) followed by Tukey’s multiple comparison tests.
Values of p<0.05 were regarded as significant.
Results
Blood pressure and echocardiograpic measurementsAs shown in Table 1, in the vehicle-treated RVH group the mean systolic blood pressures were significantly elevated at the 3rd (156 ± 3.2 mmHg; p<0.001) and 9th (185 ± 4.8 mmHg; p<0.001) weeks with respect to the basal values. In the NStreated RVH group, at the 3rd week where treatment was not started yet, mean BP was still elevated (145 ± 2.4 mmHg; p<0.001), while at the 9th weeks BP was significantly reduced (151 ± 3.6 mmHg) with respect to vehicle-treated RVH group (p<0.001).
Table 2 summarizes the transthoracic echocardiographic measurements of the experimental groups recorded at the 9th week. As compared to the control values, in the vehicle-treated RVH group, interventricular septal thickness, LV posterior wall thickness, LV end-diastolic and end-systolic dimensions, as well as relative wall thickness, were increased (p<0.001), while percent fractional shortening and ejection fraction were decreased (p<0.01-0.001; Figure 1). On the other hand, in the NS-treated RVH group echocardiographic measurements were significantly reversed and the values were found to be similar to those of the control group.
Click Here to Zoom |
FIGURE 1: Representative echocardiographic scannings of control group with normal M-mode view (A); vehicle-treated RVH group with increased interventricular septum and left ventricular posterior wall thickness (B); NS-treated RVH group demonstrating normal M-mode view as the control group without hypertrophy (C). |
Biochemical parameters in the plasma
Plasma ADMA, CK and LDH levels were significantly elevated
in the vehicle-treated RVH group (p<0.001), while NO metabolites
were decreased (Figure 2). On the other hand, NStreatment
decreased the plasma ADMA, CK and LDH levels,
while NO metabolites were significantly increased (p<0.05-
0.001).
Click Here to Zoom |
FIGURE 2: Plasma levels of (A) asymmetric dimethylarginine (ADMA), (B) nitric oxide (NO) metabolites, (C) creatinine kinase (CK) and d) lactate dehydrogenase (LDH) in the sham-operated control, Nigella sativa (NS)-treated control, vehicle-treated renovascular hypertension (RVH) and NS-treated RVH groups (RVH+NS). *** p<0.001; compared to control, +p<0.05, ++p<0.01; compared to RVH group. |
Biochemical parameters in the tissues
Luminol and lucigenin CL levels indicating oxygen radical
generation were increased in the cardiac and renal tissues of
the vehicle-treated RVH group as compared to those of the
corresponding tissues from the control group (p<0.001, Table
3). However, in the NS-treated RVH group, elevations in both
of the CL levels were abolished (p<0.01-0.01). In accordance
with these results, the levels of MDA, which is a major degradation
product of lipid peroxidation, were significantly increased
(p<0.001) in the heart and kidney tissues of the vehicle
treated-RVH group, while GSH levels were decreased in both
tissues (p<0.001) as compared to the control group (Figure 3
and 4). On the other hand, NS treatment given to the hypertensive
rats caused marked decreases in the MDA levels of both
tissues (p<0.01) and increases in the GSH levels. Furthermore,
RVH-induced oxidative stress caused significant decreases in
the Na+,K+-ATPase activities of the cardiac and renal tissues
when compared to those of the control group (p<0.001), indicating
impaired transport function and membrane damage in
both tissues (Figure 3c and 4c). On the other hand, 2K1C-induced
reductions in tissue Na+,K+-ATPase activities were prevented
with NS treatment in the RVH group (p<0.05).
Click Here to Zoom |
FIGURE 3: Malondialdehyde (MDA) levels (A), glutathione levels (B) and Na+- K+ ATPase activities (C) in the heart tissues of the sham-operated control, Nigella sativa (NS)-treated control, vehicle-treated renovascular hypertension (RVH) and NS-treated RVH groups (RVH+NS). * p<0.05, ** p<0.01, *** p<0.001; compared to control, ++p<0.01, +++p<0.001; compared to RVH group. |
Click Here to Zoom |
FIGURE 4: Malondialdehyde (MDA) levels (A), glutathione levels (B) and Na+,K+-ATPase activities (C) in the kidney tissues of the sham-operated control, Nigella sativa (NS)-treated control, vehicle-treated renovascular hypertension (RVH) and NS-treated RVH groups (RVH+NS). * p<0.05, ** p<0.01, *** p<0.001; compared to control, ++p<0.01, +++p<0.001; compared to RVH group. |
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