Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2012 , Vol 16 , Num 2
Protective effects of Nigella sativa against hypertension-induced oxidative stress and cardiovascular dysfunction in rats
Nur Taşar1, Özer Şehirli1, Ömer Yiğiner2, Selami Süleymanoğlu2, Meral Yüksel3, Berrak Yeğen4, Göksel Şener1
1Marmara University School of Pharmacy, Department of Pharmacology, Istanbul, Türkiye
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

Animals
All 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 measurements
As 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 1: Systolic blood pressures (BP) recorded before the surgical procedure (t1) and at the 3rd (t2) and 9th (t3) weeks after the surgery in the sham-operated control, Nigella sativa (NS)-treated control, vehicle-treated renovascular hypertension (RVH) and NS-treated RVH (RVH+NS) groups. Each group consists of 8 animals.

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.

TABLE 2: Transthoracic echocardiographic measurements recorded at the 9th week in the sham-operated control, Nigella sativa (NS)-treated control, vehicletreated renovascular hypertension (RVH) and NS-treated RVH (RVH+NS) groups. Each group consists of 8 animals.


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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).


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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).

TABLE 3: Effects of Nigella sativa (NS) treatment on tissue luminol and lucigenin chemiluminescence (CL; rlu/mg) levels in the sham-operated control, NS-treated control, vehicle-treated renovascular hypertension (RVH) and NS-treated RVH (RVH+NS) groups. Each group consists of 8 animals.


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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.


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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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