Cardiovasc Toxicol DOI 10.1007/s12012-014-9248-0
Effect of Nigella sativa Supplementation to Exercise Training in a Novel Model of Physiological Cardiac Hypertrophy L. I. Al-Asoom • B. A. Al-Shaikh • A. O. Bamosa M. N. El-Bahai
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Ó Springer Science+Business Media New York 2014
Abstract Exercise training is employed as supplementary therapy to patients with heart failure due to its multiple beneficial cardiac effects including physiological remodeling of the heart. However, precautions might be taken for the concomitant high oxidant release. Nigella sativa (NS) has been found to induce cardiac hypertrophy and enhance cardiac function. Combination of NS supplementation and exercise training might induce a safer model of cardiac hypertrophy. Our aim was to study biomarkers associated with cardiac hypertrophy induced by NS supplementation of exercise-trained rats. Forty-five adult male Wistar rats (body weight 150–220 g) were divided equally into three groups: control, exercise-trained (ET) and NS-treated– exercise-trained (NSET) groups. Daily 800 mg/kg NS was administered orally to NSET group for 8 weeks. Rats of the ET and NSET groups were subjected to treadmill running sessions for 2 h/day for 8 weeks. By the end of the experiment, the following were recorded: body, heart and left ventricular weights (BW, HW, LVW), cardiomyocyte diameter, serum growth hormone, insulin growth factor-I (IGF-I), thyroid hormones, catecholamines, total nitrate, ICAM and antioxidant capacity. A homogenous cardiac hypertrophy was evidenced by increased HW/BW, LVW/ BW ratios and cardiomyocyte diameter in the two groups of exercise-trained compared with control rats. Rats of ET group had higher growth hormone. Those of NSET group developed higher IGF-I and total antioxidant capacity, as well as lower serum thyroxin level. Simultaneous NS
L. I. Al-Asoom (&) B. A. Al-Shaikh A. O. Bamosa M. N. El-Bahai Physiology Department, College of Medicine, University of Dammam, P.O. Box 2114, Ad Damma¯m 31451, Saudi Arabia e-mail: [email protected]; [email protected]
supplementation to an exercise training program preserves and augments exercise-induced physiological cardiac hypertrophy with step-forward adaptive signs of increased IGF-I and reduced thyroxin level, and with an added advantage of elevation of total serum antioxidant capacity. Thus, the novel model of NSET-induced cardiac hypertrophy might be introduced as a new therapeutic strategy for the treatment of heart failure with superior advantages to exercise training alone. Keywords Cardiac hypertrophy Exercise training Nigella sativa Growth hormone Insulin-like growth factor (IGF)-I Serum total antioxidant capacity
Introduction Physical activity is associated with a reduction in the incidence of cardiovascular diseases worldwide, owing to favorable physiological adaptations in the heart and the blood vessels induced by exercise training [1–3]. Furthermore, due to its ability to induce physiological cardiac hypertrophy with enhanced cardiac function, exercise training is implemented in the treatment of heart failure [4–6]. Systematic studies of the mechanisms of exerciseinduced cardiac hypertrophy revealed that its intracellular signaling pathways are distinguished from those involved in development of a pathological cardiac hypertrophy [7]. Growth hormone/insulin growth factor-I (GH/IGF-1) is the main mechanism involved in exercise-induced cardiac hypertrophy [8, 9], whereas the activation of G proteincoupled receptor (GPCR) and its multiple mediators (e.g., angiotensin-II, endothelin-1 and catecholamines) precipitate the development of pathological cardiac hypertrophy [10].
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On the other hand, exercise training generates a tremendous amount of reactive oxygen species (ROS) [11]. Oxygen free radicals are part of deleterious signaling pathways mediating heart failure and inducing cellular necrosis and apoptosis [12, 13]. This, consequently, makes it urgent to find a better therapeutic model for the increasing incidence of heart failure. This model must attain most of the benefits of physical training and yet lack its possible side effects. Nigella sativa (NS, the black seed) is commonly used as a remedy for multiple illnesses and diseases [14]. Multiple studies have revealed a wide range of actions of NS including anti-inflammatory [15], anti-oxidant [16], immune-modulatory [17], hypo-glycemic [18] and hypolipidemic [19]. El-Bahai et al. [20] studied the effect of 2 months oral administration of NS to normal rats on the intrinsic properties of their isolated hearts in vitro. In Langendorff’s preparation of the hearts extracted from NS fed rats, there was an increment in the baseline peak tension, rate of tension development, as well as heart rate and myocardial flow rate. Cardiac hypertrophy was demonstrated by an increase in the heart weight, left ventricular weight, heart weight/body weight and left ventricular weight/body weight ratios. Another study [21], performed by the same authors, revealed a positive inotropic effects of NS supplementation with an increased cardiac b-adrenergic responsiveness. These were evidenced by the increase maximal peak tension of the isolated hearts upon infusion with incremental doses of isoproterenol. Accordingly, it was suggested that NS-induced cardiac hypertrophy might share some of the features of exercise-induced physiological cardiac hypertrophy. Furthermore, it was proposed that supplementation of exercise training programs with NS might produce a better model of physiological cardiac hypertrophy. The current study aimed to develop an experimental model of physiological cardiac hypertrophy by combining NS supplementation to an exercise training program and to identify the associated biological biomarkers that characterize the profile of this pattern of cardiac hypertrophy.
Materials and Methods Animals Forty-five adult male Wistar rats (body weight 150–220 g) were obtained from the animal house of Dammam University. All animals were housed individually in labeled cages with adequate ventilation and illumination. Normal laboratory chow and tap water were available ad libitum. Experimental protocol was approved by the ethical
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committee of animal research of the deanship of scientific research of Dammam University, Saudi Arabia. Experimental Protocol Rats were randomly divided into three equal groups: a control (untrained), an exercise-trained (ET) and a NS-treated– exercise-trained (NSET) groups. The exercise regime involved a treadmill sessions (IITC life science, 5-lane rat treadmill) for 8 weeks. Speed, grade and duration were progressively increased during the first week until the final protocol was achieved with 18 m/min speed, 32° inclination, for a 2 h/session. An electric grid at the rear of the belt was used as the running stimulus. Rats of the NSET group were given 800 mg/kg NS daily for 8 weeks. Oral administration was done via a feeding needle. A suspension of NS was prepared by dissolving 10 g of freshly ground NS in 100 ml distilled water. Concomitantly, rats of the other groups were given an equivalent volume of water. At the end of the 8-week protocol, rats were anesthetized with a ketamine cocktail (60 % ketamine, 40 % xylazine). A dose of 0.8 ml/kg body weight was injected intra-peritoneally. Collection of Samples Blood was collected from the abdominal aorta in plane vacutainers centrifuged and stored at -80 °C. Hearts were extracted, soaked and washed by cold Ringer’s solution, blotted dry and weighed. The left ventricles were dissected, blotted dry and weighed. Periodic acid–Schiff (PAS)stained slides were prepared from the free wall of the left ventricles. These slides were used for the determination of cardiomyocyte diameter. Photomicrographs of these slides were captured by digital microscope (Coolscope) and analyzed by image analyzer (Able image analyzer). Immunoassay Several ELISA tests were performed following the instruction of the manufacturer. These tests include serum growth hormone by kit from SPI bio bertin (A05104), T3 and T4 ELISA kit (RE55231 IBL, RE55241, IBL). Epinephrine and norepinephrine ELISA kits (RE59251, IBL) and (RE59261, IBL), IGF-I rat/mouse IGF-I ELISA (AC18F1, IDS), ICAM-1 ELISA kit (BE59001, IBL), total nitrite/nitrate colorimetric assay kit (9780001, Cayman Chemical Company), and total antioxidant capacity (Cayman Chemical Company, catalog number 709001). Statistical Analysis All data were analyzed using SPSS software version 11.5, expressed as mean ± SEM. ANOVA with LSD post hoc
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test was used to compare all data among the groups. Correlation between heart weight, left ventricular weight and their indices, and the serum level of different biomarkers was performed using Pearson’s correlation and linear regression. Level of significance was set at p \ 0.05.
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Cardiac weight indices, heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/ BW) ratios were significantly higher in all trained rats compared with the untrained (control) group (p \ 0.01 for both the ET and NSET groups). However, there was no significant change in left ventricular weight/heart weight ratio (LVW/HW) in all the rat groups (Fig. 1). The mean diameter of the cardiomyocytes measured in PAS sections and expressed in microns was significantly larger in ET (144 ± 6 lm), and NSET (159 ± 5 lm) groups compared to the control group (105 ± 4 lm), p \ 0.001.
0 IGF I
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Fig. 2 Comparison of serum growth hormone GH (ng/ml) and serum IGF-I (ng/ml) in all experimental groups (control, ET exercisetrained, NSET Nigella sativa-treated–exercise-trained groups) at the end of 8 weeks. Note IGF-I values appear in the graph is original serum IGF-I values/10. *p \ 0.5; **p \ 0.01 compared with the control group 12
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Serum growth hormone was significantly higher in the ET group compared with the control group (p \ 0.01, Fig. 2). Serum IGF-I was significantly higher in NSET group when compared with the control and ET groups (p \ 0.01, Fig. 2). Rats of the NSET group had a significantly lower serum T4 when compared with the other two groups HW/BW (mg/g)
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Fig. 3 Comparison of thyroid hormones (T3 ng/ml and T4 pg/ml) and catecholamines (epinephrine and norepinephrine ng/ml) in all experimental groups (control, ET exercise-trained, NSET Nigella sativatreated–exercise-trained groups) at the end of 8 weeks. **p \ 0.01 compared to the control group
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(p \ 0.05, Fig. 3). There was no significant difference among all the groups in regard to the levels of serum catecholamines (Fig. 3).
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Fig. 1 Cardiac weight indices: heart weight/body weight (HW/BW), left ventricular weight/body weight (LVW/BW), left ventricular weight/heart weight (LVW/HW) in all rat groups (control, ET exercise-trained, NSET Nigella sativa-treated–exercise-trained groups) at the end of 8 weeks. *p \ 0.5; **p \ 0.01 compared with the control group
Serum Nitrate, ICAM-I and Total Serum Antioxidant Capacity Rats of NSET group had significantly higher serum total antioxidant capacity when compared to the control and ET groups (p \ 0.001, Fig. 4). There was no significant difference among the groups in regard to the level of ICAM-I and total nitrate. Pearson Correlation and Linear Regression There was a significant correlation between serum IGF-I and the heart weight in rats involved in the exercise
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Fig. 4 Comparison of serum total nitrate, ICAM-I and total antioxidant capacity among the experimental groups (control, ET exercisetrained, NSET Nigella sativa-treated–exercise-trained groups) at the end of 8 weeks. Note Total antioxidant capacity is the actual value of total antioxidant capacity/100. **p \ 0.001 compared with the control and exercise-trained group
training sessions. Linear regression revealed R2 = 0.50 (p \ 0.01) and 0.55(p \ 0.05) for ET and NSET groups, respectively, Fig. 5. A similar relationship was found between serum IGF-I and left ventricular weight in these rats; R2 = 0.52 (p \ 0.01) and 0.48 (p \ 0.05) for ET and NSET groups, respectively, Fig. 6. Interestingly, no significant correlations were found among these variables in the untrained rats (control group).
GH/IGF-1 axis are suggested to attribute to the underlying physiological hypertrophy in the ET and NSET groups of rats. The involvement of GH/IGF-I axis in mediating cardiac hypertrophy induced by exercise training alone or with the co-administration of NS is further supported by the significant correlations found in this study between serum IGF-I and both the heart weight and left ventricular weight in rats of the ET and NSET groups, and not in the untrained (control) group. We propose that the exercise protocol of our experiment was sufficient to increase serum GH but not serum IGF-I, in rats of the ET group. However, NS supplementation, in rats of the NSET group, produced a synergistic effect to exercise training and facilitated the increment in serum IGF-I. In support to this hypothesis, serum GH level was lower in rats of the NSET group, compared to those of ET group, which could be explained by the negative feedback effect of the high serum IGF-I on GH secretion [22]. A lot of evidence in the literature supports the involvement of the GH/IGF-I axis in exercise training. Some clinical studies [23] have demonstrated an increase of both serum GH and total IGF-I in middle-aged cyclists when compared to age-matched sedentary volunteers. Similarly, a 3-week training of professional cyclists culminated in an increment in serum total IGF-I [24]. Other investigators proposed that the response of GH/IGF-I axis parameters depends on the exercise intensity and degree of adaptation [25, 26]. Exercise training of short duration (B1 week) would induce a reduction of serum IGF-I, due to an inhibitory influence of cytokines [27]; a moderate training duration (2–12 weeks) preserves the same level of IGF-I; and longer training courses (C12 weeks) precipitate an incremental increase of serum IGF-I [28].
Discussion A homogenous cardiac hypertrophy was successfully induced in adult rats by exercise training alone and by coadministration of NS and exercise training. This hypertrophy was evidenced by the increased heart weight/body weight (HW/BW), the left ventricular weight/body weight (LVW/BW) ratios and the larger cardiomyocyte diameter compared with the untrained (control) rats. GH/IGF-I Axis Might be the Main Pathway for Physiological Cardiac Hypertrophy Exercise-trained rats (ET group) showed significant increase in serum growth hormone but no change in serum IGF-I as compared to the untrained (control) group. Rats of the NSET group exhibited significantly higher serum IGF-I but no significant increase in serum growth hormone when compared to the control group. These reported changes in
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Thyroid Hormones’ Role in Signaling the NSET Model of Cardiac Hypertrophy Exercise training sessions in this study showed no effect on plasma levels of T3 and T4; however, the co-administration of NS induced a reduction of plasma T4 compared with the untrained (control) rats. In our study, rats of the ET and NSET groups underwent the same exercise protocol, yet NS supplementation demonstrated lower serum T4 in comparison with the exercise-trained group. Therefore, we postulate the existence of an additive effect of NS administration on the thyroid function that appeared in conjunction with exercise training. Changes in thyroid hormone levels associated with exercise training are a matter of controversy. Two previous studies involving elite weight lifters who underwent strength training for different periods showed no effect on serum T3 and T4 [29, 30]. However, other studies revealed
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T4 and thyroid peroxidase enzyme with no change in serum T3 [32]. On the other hand, some studies using different training protocols in sedentary volunteers and athletes revealed significant reduction in both serum T4 and T3 [33, 34]. The inconsistency of the results of thyroid function response to exercise training in different studies could be explained by the wide range of adopted training protocols, with a possible assumption that the reduction of T4 might indicate an advanced adaptive stage to exercise training [32].
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Exercise training, either alone or supplemented by NS, was not associated significant changes in plasma catecholamines levels. The normal serum catecholamine level in ET and NSET rats could be considered a favorable feature of physiological cardiac hypertrophy that differentiates it from the various patterns of pathological hypertrophy. Similarly, other investigators reported no difference in plasma norepinephrine levels between athletes and sedentary matched controls [35]. In contrast, others [36] revealed a reduction in plasma norepinephrine level, but not in cardiac norepinephrine content, in young healthy volunteers trained on a bicycle for 1 month. They attributed the reduction in total plasma norepinephrine to the reduction in renal norepinephrine but not to cardiac sympathetic activity [36].
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Serum IGF I Fig. 5 Linear regression of serum IGF-I and heart weight. a Control, b ET exercise-trained, c NSET Nigella sativa-treated–exercise-trained groups
that exercise training can reduce the thyroid hormone level. On one hand, a reduction in serum T4 without a change in serum T3 was demonstrated in healthy volunteers after 24 weeks of training [31]. Similarly, male rats trained by different exercise intensities showed a reduction in serum
Both experimental groups in this study showed no significant increase in the plasma level of circulating soluble adhesion molecule-I (ICAM-I) level when compared to the control group. Other previous studies demonstrated no effect of upper- and lower-limb exercise or resistance training on plasma levels of adhesion molecules [37]. ICAM-I, as one of the inflammatory markers, was reported to be elevated in certain pathological conditions including hypertensive cardiac hypertrophy and congestive heart failure [38, 39]. Exercise training was reported to reduce the elevated levels of some adhesion molecules (e.g., ICAM-1, VCAM-1 and p-selectin) in patients with chronic heart failure [40], diabetes [41] or in hypercholesterolemic rabbits [42]. The maintained normal ICAM-I levels demonstrated in our experimental groups may represent a common feature of cardiac hypertrophy induced by either exercise training or co-administration of NS and exercise training. It may
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Serum total nitrate level showed no significant difference in rats of the experimental groups when compared to the control group. Similar data were reported in experimental rats, trained on treadmill for 12 weeks [44] and clinical studies involving strength trained and endurance trained young volunteers [45]. On the other hand, total nitrate level was found reduced in hypertensive cardiac hypertrophy and heart failure [46]. Some reports revealed that exercise training may elevate serum nitrate level in these conditions to maintain the original level. Kohno et al. [47] showed that voluntary wheel running for 10 weeks increases plasma nitrate level in spontaneously hypertensive rats. Accordingly, cardiac hypertrophy induced in the current study by exercise training and NS administration to exercise training differs from pathological cardiac hypertrophy since both experimental interventions were associated with normal level of serum total nitrate.
NSET Model is Uniquely Associated with Increased Antioxidant Capacity
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Serum IGF I Fig. 6 Linear regression of serum IGF-I and left ventricular weight. a Control, b ET exercise-trained, c NSET Nigella sativa-treated– exercise-trained groups
also differentiate these patterns of cardiac hypertrophy from the pathological cardiac hypertrophy. Serum total nitrate level is an indicator of the level of nitric oxide (NO) signaling molecule, since it is the final metabolite of NO [43].
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Though physical exercise is a known source of ROS that increase with the increment in exercise intensity [11], these free radicals induce an upregulation of antioxidant enzymes [48]. Thus, at moderate exercise intensity, this may match the increase in the oxidants and yield a balance between oxidants and antioxidants [49]. On the other hand, increased ROS were described in the literature as one of the main factors mediating pathological cardiac hypertrophy [50, 51]. In this study, the total antioxidant capacity of the blood showed no significant difference in ET rat group compared with the control group, but the NSET rat group exhibited a preferably higher total antioxidant capacity when compared to the control and ET groups. The antioxidant effect of NS has been repeatedly described previously. Thymoquinone, the effective component of NS, was shown to protect the heart against doxorubicin-induced cardiotoxicity in rats [52] by a superoxide scavenging effect that was responsible for the reduction of cardiac enzymes indicating cardiac injury. Similarly, other authors [53, 54] have demonstrated the antioxidant property of NS through upregulation of the activity of antioxidant enzymes (e.g., superoxide dismutase, catalyze and glutathione peroxidase). Therefore, NS supplementation was superior to exercise training in regard to its ability to enhance total antioxidant capacity, and this preferable effect is maintained when NS was administered to exercise training.
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Conclusion In the present work, we have demonstrated that the dietary supplementation of NS to exercise training results in moderate homogenous cardiac hypertrophy manifested by an increase HW/BW, LVW/BW ratios and cardiomyocyte diameter. This model of cardiac hypertrophy possesses multiple features of physiological cardiac hypertrophy, including normal serum catecholamines, ICAM-I and total plasma nitrate level. We believe that this model of cardiac hypertrophy induced by the coadministration of Nigella sativa and exercise training is superior to that induced by only exercise training, due the revealed multiple synergistic effects of both interventions resulting in a better antioxidant capacity, and the step-forward cardiac adaptation to exercise training manifested in the lower thyroid hormones, and stronger GH/IGF-I axis response. This cardiac model may present a future promise to the emerging of a new strategy for either the prevention of pathological cardiac hypertrophy or the treatment of heart failure. Acknowledgments The authors acknowledge the deanship of scientific research in University of Dammam for the Grant No. 8086 that allowed the execution of this research. Conflict of interest All authors declare that there was no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three (3) years of beginning the work submitted that could inappropriately influence (bias) their work.
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peripheral markers of inflammation in patients with chronic heart failure. European Heart Journal, 22(9), 791–797. Zoppini, G., Targher, G., Zamboni, C., Venturi, C., Cacciatori, V., Moghetti, P., et al. (2006). Effects of moderate-intensity exercise training on plasma biomarkers of inflammation and endothelial dysfunction in older patients with type 2 diabetes. Nutrition, Metabolism, and Cardiovascular Diseases, 16(8), 543–549. Yang, A. L., & Chen, H. I. (2003). Chronic exercise reduces adhesion molecules/iNOS expression and partially reverses vascular responsiveness in hypercholesterolemic rabbit aortae. Atherosclerosis, 169(1), 11–17. Poveda, J. J., Riestra, A., Salas, E., Cagigas, M. L., Lopez-Somoza, C., Amado, J. A., et al. (1997). Contribution of nitric oxide to exercise-induced changes in healthy volunteers: Effects of acute exercise and long-term physical training. European Journal of Clinical Investigation, 27(11), 967–971. de Moraes, C., Davel, A. P., Rossoni, L. V., Antunes, E., & Zanesco, A. (2008). Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats. BMC Physiology, 8, 12. Otsuki, T., Maeda, S., Iemitsu, M., Saito, Y., Tanimura, Y., Ajisaka, R., et al. (2007). Vascular endothelium-derived factors and arterial stiffness in strength- and endurance-trained men. American Journal of Physiology Heart and Circulatory Physiology, 292(2), H786–H791. Garcia, J. A., & Incerpi, E. K. (2008). Factors and mechanisms involved in left ventricular hypertrophy and the anti-hypertrophic role of nitric oxide. Arquivos Brasileiros de Cardiologia, 90(6), 409–416. Kohno, H., Furukawa, S., Naito, H., Minamitani, K., Ohmori, D., & Yamakura, F. (2002). Contribution of nitric oxide, angiotensin II and superoxide dismutase to exercise-induced attenuation of blood pressure elevation in spontaneously hypertensive rats. Japanese Heart Journal, 43(1), 25–34. Gomez-Cabrera, M. C., Domenech, E., & Vina, J. (2008). Moderate exercise is an antioxidant: Upregulation of antioxidant genes by training. Free Radical Biology & Medicine, 44(2), 126–131. Powers, S. K., Quindry, J. C., & Kavazis, A. N. (2008). Exerciseinduced cardioprotection against myocardial ischemia-reperfusion injury. Free Radical Biology & Medicine, 44(2), 193–201. Finkel, T. (1999). Signal transduction by reactive oxygen species in non-phagocytic cells. Journal of Leukocyte Biology, 65(3), 337–340. Nakagami, H., Takemoto, M., & Liao, J. K. (2003). NADPH oxidase-derived superoxide anion mediates angiotensin IIinduced cardiac hypertrophy. Journal of Molecular and Cellular Cardiology, 35(7), 851–859. Nagi, M. N., & Mansour, M. A. (2000). Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: A possible mechanism of protection. Pharmacological Research, 41(3), 283–289. Abdel-Wahhab, M. A., & Aly, S. E. (2005). Antioxidant property of Nigella sativa (black cumin) and Syzygium aromaticum (clove) in rats during aflatoxicosis. Journal of Applied Toxicology, 25(3), 218–223. Kanter, M., Coskun, O., Korkmaz, A., & Oter, S. (2004). Effects of Nigella sativa on oxidative stress and beta-cell damage in streptozotocin-induced diabetic rats. The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology, 279(1), 685–691.
Effect of Nigella sativa Supplementation to Exercise Training in a Novel Model of Physiological Cardiac Hypertrophy L. I. Al-Asoom • B. A. Al-Shaikh • A. O. Bamosa M. N. El-Bahai
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Ó Springer Science+Business Media New York 2014
Abstract Exercise training is employed as supplementary therapy to patients with heart failure due to its multiple beneficial cardiac effects including physiological remodeling of the heart. However, precautions might be taken for the concomitant high oxidant release. Nigella sativa (NS) has been found to induce cardiac hypertrophy and enhance cardiac function. Combination of NS supplementation and exercise training might induce a safer model of cardiac hypertrophy. Our aim was to study biomarkers associated with cardiac hypertrophy induced by NS supplementation of exercise-trained rats. Forty-five adult male Wistar rats (body weight 150–220 g) were divided equally into three groups: control, exercise-trained (ET) and NS-treated– exercise-trained (NSET) groups. Daily 800 mg/kg NS was administered orally to NSET group for 8 weeks. Rats of the ET and NSET groups were subjected to treadmill running sessions for 2 h/day for 8 weeks. By the end of the experiment, the following were recorded: body, heart and left ventricular weights (BW, HW, LVW), cardiomyocyte diameter, serum growth hormone, insulin growth factor-I (IGF-I), thyroid hormones, catecholamines, total nitrate, ICAM and antioxidant capacity. A homogenous cardiac hypertrophy was evidenced by increased HW/BW, LVW/ BW ratios and cardiomyocyte diameter in the two groups of exercise-trained compared with control rats. Rats of ET group had higher growth hormone. Those of NSET group developed higher IGF-I and total antioxidant capacity, as well as lower serum thyroxin level. Simultaneous NS
L. I. Al-Asoom (&) B. A. Al-Shaikh A. O. Bamosa M. N. El-Bahai Physiology Department, College of Medicine, University of Dammam, P.O. Box 2114, Ad Damma¯m 31451, Saudi Arabia e-mail: [email protected]; [email protected]
supplementation to an exercise training program preserves and augments exercise-induced physiological cardiac hypertrophy with step-forward adaptive signs of increased IGF-I and reduced thyroxin level, and with an added advantage of elevation of total serum antioxidant capacity. Thus, the novel model of NSET-induced cardiac hypertrophy might be introduced as a new therapeutic strategy for the treatment of heart failure with superior advantages to exercise training alone. Keywords Cardiac hypertrophy Exercise training Nigella sativa Growth hormone Insulin-like growth factor (IGF)-I Serum total antioxidant capacity
Introduction Physical activity is associated with a reduction in the incidence of cardiovascular diseases worldwide, owing to favorable physiological adaptations in the heart and the blood vessels induced by exercise training [1–3]. Furthermore, due to its ability to induce physiological cardiac hypertrophy with enhanced cardiac function, exercise training is implemented in the treatment of heart failure [4–6]. Systematic studies of the mechanisms of exerciseinduced cardiac hypertrophy revealed that its intracellular signaling pathways are distinguished from those involved in development of a pathological cardiac hypertrophy [7]. Growth hormone/insulin growth factor-I (GH/IGF-1) is the main mechanism involved in exercise-induced cardiac hypertrophy [8, 9], whereas the activation of G proteincoupled receptor (GPCR) and its multiple mediators (e.g., angiotensin-II, endothelin-1 and catecholamines) precipitate the development of pathological cardiac hypertrophy [10].
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On the other hand, exercise training generates a tremendous amount of reactive oxygen species (ROS) [11]. Oxygen free radicals are part of deleterious signaling pathways mediating heart failure and inducing cellular necrosis and apoptosis [12, 13]. This, consequently, makes it urgent to find a better therapeutic model for the increasing incidence of heart failure. This model must attain most of the benefits of physical training and yet lack its possible side effects. Nigella sativa (NS, the black seed) is commonly used as a remedy for multiple illnesses and diseases [14]. Multiple studies have revealed a wide range of actions of NS including anti-inflammatory [15], anti-oxidant [16], immune-modulatory [17], hypo-glycemic [18] and hypolipidemic [19]. El-Bahai et al. [20] studied the effect of 2 months oral administration of NS to normal rats on the intrinsic properties of their isolated hearts in vitro. In Langendorff’s preparation of the hearts extracted from NS fed rats, there was an increment in the baseline peak tension, rate of tension development, as well as heart rate and myocardial flow rate. Cardiac hypertrophy was demonstrated by an increase in the heart weight, left ventricular weight, heart weight/body weight and left ventricular weight/body weight ratios. Another study [21], performed by the same authors, revealed a positive inotropic effects of NS supplementation with an increased cardiac b-adrenergic responsiveness. These were evidenced by the increase maximal peak tension of the isolated hearts upon infusion with incremental doses of isoproterenol. Accordingly, it was suggested that NS-induced cardiac hypertrophy might share some of the features of exercise-induced physiological cardiac hypertrophy. Furthermore, it was proposed that supplementation of exercise training programs with NS might produce a better model of physiological cardiac hypertrophy. The current study aimed to develop an experimental model of physiological cardiac hypertrophy by combining NS supplementation to an exercise training program and to identify the associated biological biomarkers that characterize the profile of this pattern of cardiac hypertrophy.
Materials and Methods Animals Forty-five adult male Wistar rats (body weight 150–220 g) were obtained from the animal house of Dammam University. All animals were housed individually in labeled cages with adequate ventilation and illumination. Normal laboratory chow and tap water were available ad libitum. Experimental protocol was approved by the ethical
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committee of animal research of the deanship of scientific research of Dammam University, Saudi Arabia. Experimental Protocol Rats were randomly divided into three equal groups: a control (untrained), an exercise-trained (ET) and a NS-treated– exercise-trained (NSET) groups. The exercise regime involved a treadmill sessions (IITC life science, 5-lane rat treadmill) for 8 weeks. Speed, grade and duration were progressively increased during the first week until the final protocol was achieved with 18 m/min speed, 32° inclination, for a 2 h/session. An electric grid at the rear of the belt was used as the running stimulus. Rats of the NSET group were given 800 mg/kg NS daily for 8 weeks. Oral administration was done via a feeding needle. A suspension of NS was prepared by dissolving 10 g of freshly ground NS in 100 ml distilled water. Concomitantly, rats of the other groups were given an equivalent volume of water. At the end of the 8-week protocol, rats were anesthetized with a ketamine cocktail (60 % ketamine, 40 % xylazine). A dose of 0.8 ml/kg body weight was injected intra-peritoneally. Collection of Samples Blood was collected from the abdominal aorta in plane vacutainers centrifuged and stored at -80 °C. Hearts were extracted, soaked and washed by cold Ringer’s solution, blotted dry and weighed. The left ventricles were dissected, blotted dry and weighed. Periodic acid–Schiff (PAS)stained slides were prepared from the free wall of the left ventricles. These slides were used for the determination of cardiomyocyte diameter. Photomicrographs of these slides were captured by digital microscope (Coolscope) and analyzed by image analyzer (Able image analyzer). Immunoassay Several ELISA tests were performed following the instruction of the manufacturer. These tests include serum growth hormone by kit from SPI bio bertin (A05104), T3 and T4 ELISA kit (RE55231 IBL, RE55241, IBL). Epinephrine and norepinephrine ELISA kits (RE59251, IBL) and (RE59261, IBL), IGF-I rat/mouse IGF-I ELISA (AC18F1, IDS), ICAM-1 ELISA kit (BE59001, IBL), total nitrite/nitrate colorimetric assay kit (9780001, Cayman Chemical Company), and total antioxidant capacity (Cayman Chemical Company, catalog number 709001). Statistical Analysis All data were analyzed using SPSS software version 11.5, expressed as mean ± SEM. ANOVA with LSD post hoc
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test was used to compare all data among the groups. Correlation between heart weight, left ventricular weight and their indices, and the serum level of different biomarkers was performed using Pearson’s correlation and linear regression. Level of significance was set at p \ 0.05.
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Cardiac weight indices, heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/ BW) ratios were significantly higher in all trained rats compared with the untrained (control) group (p \ 0.01 for both the ET and NSET groups). However, there was no significant change in left ventricular weight/heart weight ratio (LVW/HW) in all the rat groups (Fig. 1). The mean diameter of the cardiomyocytes measured in PAS sections and expressed in microns was significantly larger in ET (144 ± 6 lm), and NSET (159 ± 5 lm) groups compared to the control group (105 ± 4 lm), p \ 0.001.
0 IGF I
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Fig. 2 Comparison of serum growth hormone GH (ng/ml) and serum IGF-I (ng/ml) in all experimental groups (control, ET exercisetrained, NSET Nigella sativa-treated–exercise-trained groups) at the end of 8 weeks. Note IGF-I values appear in the graph is original serum IGF-I values/10. *p \ 0.5; **p \ 0.01 compared with the control group 12
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Serum growth hormone was significantly higher in the ET group compared with the control group (p \ 0.01, Fig. 2). Serum IGF-I was significantly higher in NSET group when compared with the control and ET groups (p \ 0.01, Fig. 2). Rats of the NSET group had a significantly lower serum T4 when compared with the other two groups HW/BW (mg/g)
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Fig. 3 Comparison of thyroid hormones (T3 ng/ml and T4 pg/ml) and catecholamines (epinephrine and norepinephrine ng/ml) in all experimental groups (control, ET exercise-trained, NSET Nigella sativatreated–exercise-trained groups) at the end of 8 weeks. **p \ 0.01 compared to the control group
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(p \ 0.05, Fig. 3). There was no significant difference among all the groups in regard to the levels of serum catecholamines (Fig. 3).
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Fig. 1 Cardiac weight indices: heart weight/body weight (HW/BW), left ventricular weight/body weight (LVW/BW), left ventricular weight/heart weight (LVW/HW) in all rat groups (control, ET exercise-trained, NSET Nigella sativa-treated–exercise-trained groups) at the end of 8 weeks. *p \ 0.5; **p \ 0.01 compared with the control group
Serum Nitrate, ICAM-I and Total Serum Antioxidant Capacity Rats of NSET group had significantly higher serum total antioxidant capacity when compared to the control and ET groups (p \ 0.001, Fig. 4). There was no significant difference among the groups in regard to the level of ICAM-I and total nitrate. Pearson Correlation and Linear Regression There was a significant correlation between serum IGF-I and the heart weight in rats involved in the exercise
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Fig. 4 Comparison of serum total nitrate, ICAM-I and total antioxidant capacity among the experimental groups (control, ET exercisetrained, NSET Nigella sativa-treated–exercise-trained groups) at the end of 8 weeks. Note Total antioxidant capacity is the actual value of total antioxidant capacity/100. **p \ 0.001 compared with the control and exercise-trained group
training sessions. Linear regression revealed R2 = 0.50 (p \ 0.01) and 0.55(p \ 0.05) for ET and NSET groups, respectively, Fig. 5. A similar relationship was found between serum IGF-I and left ventricular weight in these rats; R2 = 0.52 (p \ 0.01) and 0.48 (p \ 0.05) for ET and NSET groups, respectively, Fig. 6. Interestingly, no significant correlations were found among these variables in the untrained rats (control group).
GH/IGF-1 axis are suggested to attribute to the underlying physiological hypertrophy in the ET and NSET groups of rats. The involvement of GH/IGF-I axis in mediating cardiac hypertrophy induced by exercise training alone or with the co-administration of NS is further supported by the significant correlations found in this study between serum IGF-I and both the heart weight and left ventricular weight in rats of the ET and NSET groups, and not in the untrained (control) group. We propose that the exercise protocol of our experiment was sufficient to increase serum GH but not serum IGF-I, in rats of the ET group. However, NS supplementation, in rats of the NSET group, produced a synergistic effect to exercise training and facilitated the increment in serum IGF-I. In support to this hypothesis, serum GH level was lower in rats of the NSET group, compared to those of ET group, which could be explained by the negative feedback effect of the high serum IGF-I on GH secretion [22]. A lot of evidence in the literature supports the involvement of the GH/IGF-I axis in exercise training. Some clinical studies [23] have demonstrated an increase of both serum GH and total IGF-I in middle-aged cyclists when compared to age-matched sedentary volunteers. Similarly, a 3-week training of professional cyclists culminated in an increment in serum total IGF-I [24]. Other investigators proposed that the response of GH/IGF-I axis parameters depends on the exercise intensity and degree of adaptation [25, 26]. Exercise training of short duration (B1 week) would induce a reduction of serum IGF-I, due to an inhibitory influence of cytokines [27]; a moderate training duration (2–12 weeks) preserves the same level of IGF-I; and longer training courses (C12 weeks) precipitate an incremental increase of serum IGF-I [28].
Discussion A homogenous cardiac hypertrophy was successfully induced in adult rats by exercise training alone and by coadministration of NS and exercise training. This hypertrophy was evidenced by the increased heart weight/body weight (HW/BW), the left ventricular weight/body weight (LVW/BW) ratios and the larger cardiomyocyte diameter compared with the untrained (control) rats. GH/IGF-I Axis Might be the Main Pathway for Physiological Cardiac Hypertrophy Exercise-trained rats (ET group) showed significant increase in serum growth hormone but no change in serum IGF-I as compared to the untrained (control) group. Rats of the NSET group exhibited significantly higher serum IGF-I but no significant increase in serum growth hormone when compared to the control group. These reported changes in
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Thyroid Hormones’ Role in Signaling the NSET Model of Cardiac Hypertrophy Exercise training sessions in this study showed no effect on plasma levels of T3 and T4; however, the co-administration of NS induced a reduction of plasma T4 compared with the untrained (control) rats. In our study, rats of the ET and NSET groups underwent the same exercise protocol, yet NS supplementation demonstrated lower serum T4 in comparison with the exercise-trained group. Therefore, we postulate the existence of an additive effect of NS administration on the thyroid function that appeared in conjunction with exercise training. Changes in thyroid hormone levels associated with exercise training are a matter of controversy. Two previous studies involving elite weight lifters who underwent strength training for different periods showed no effect on serum T3 and T4 [29, 30]. However, other studies revealed
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T4 and thyroid peroxidase enzyme with no change in serum T3 [32]. On the other hand, some studies using different training protocols in sedentary volunteers and athletes revealed significant reduction in both serum T4 and T3 [33, 34]. The inconsistency of the results of thyroid function response to exercise training in different studies could be explained by the wide range of adopted training protocols, with a possible assumption that the reduction of T4 might indicate an advanced adaptive stage to exercise training [32].
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Exercise training, either alone or supplemented by NS, was not associated significant changes in plasma catecholamines levels. The normal serum catecholamine level in ET and NSET rats could be considered a favorable feature of physiological cardiac hypertrophy that differentiates it from the various patterns of pathological hypertrophy. Similarly, other investigators reported no difference in plasma norepinephrine levels between athletes and sedentary matched controls [35]. In contrast, others [36] revealed a reduction in plasma norepinephrine level, but not in cardiac norepinephrine content, in young healthy volunteers trained on a bicycle for 1 month. They attributed the reduction in total plasma norepinephrine to the reduction in renal norepinephrine but not to cardiac sympathetic activity [36].
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Serum IGF I Fig. 5 Linear regression of serum IGF-I and heart weight. a Control, b ET exercise-trained, c NSET Nigella sativa-treated–exercise-trained groups
that exercise training can reduce the thyroid hormone level. On one hand, a reduction in serum T4 without a change in serum T3 was demonstrated in healthy volunteers after 24 weeks of training [31]. Similarly, male rats trained by different exercise intensities showed a reduction in serum
Both experimental groups in this study showed no significant increase in the plasma level of circulating soluble adhesion molecule-I (ICAM-I) level when compared to the control group. Other previous studies demonstrated no effect of upper- and lower-limb exercise or resistance training on plasma levels of adhesion molecules [37]. ICAM-I, as one of the inflammatory markers, was reported to be elevated in certain pathological conditions including hypertensive cardiac hypertrophy and congestive heart failure [38, 39]. Exercise training was reported to reduce the elevated levels of some adhesion molecules (e.g., ICAM-1, VCAM-1 and p-selectin) in patients with chronic heart failure [40], diabetes [41] or in hypercholesterolemic rabbits [42]. The maintained normal ICAM-I levels demonstrated in our experimental groups may represent a common feature of cardiac hypertrophy induced by either exercise training or co-administration of NS and exercise training. It may
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Serum total nitrate level showed no significant difference in rats of the experimental groups when compared to the control group. Similar data were reported in experimental rats, trained on treadmill for 12 weeks [44] and clinical studies involving strength trained and endurance trained young volunteers [45]. On the other hand, total nitrate level was found reduced in hypertensive cardiac hypertrophy and heart failure [46]. Some reports revealed that exercise training may elevate serum nitrate level in these conditions to maintain the original level. Kohno et al. [47] showed that voluntary wheel running for 10 weeks increases plasma nitrate level in spontaneously hypertensive rats. Accordingly, cardiac hypertrophy induced in the current study by exercise training and NS administration to exercise training differs from pathological cardiac hypertrophy since both experimental interventions were associated with normal level of serum total nitrate.
NSET Model is Uniquely Associated with Increased Antioxidant Capacity
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also differentiate these patterns of cardiac hypertrophy from the pathological cardiac hypertrophy. Serum total nitrate level is an indicator of the level of nitric oxide (NO) signaling molecule, since it is the final metabolite of NO [43].
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Though physical exercise is a known source of ROS that increase with the increment in exercise intensity [11], these free radicals induce an upregulation of antioxidant enzymes [48]. Thus, at moderate exercise intensity, this may match the increase in the oxidants and yield a balance between oxidants and antioxidants [49]. On the other hand, increased ROS were described in the literature as one of the main factors mediating pathological cardiac hypertrophy [50, 51]. In this study, the total antioxidant capacity of the blood showed no significant difference in ET rat group compared with the control group, but the NSET rat group exhibited a preferably higher total antioxidant capacity when compared to the control and ET groups. The antioxidant effect of NS has been repeatedly described previously. Thymoquinone, the effective component of NS, was shown to protect the heart against doxorubicin-induced cardiotoxicity in rats [52] by a superoxide scavenging effect that was responsible for the reduction of cardiac enzymes indicating cardiac injury. Similarly, other authors [53, 54] have demonstrated the antioxidant property of NS through upregulation of the activity of antioxidant enzymes (e.g., superoxide dismutase, catalyze and glutathione peroxidase). Therefore, NS supplementation was superior to exercise training in regard to its ability to enhance total antioxidant capacity, and this preferable effect is maintained when NS was administered to exercise training.
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Conclusion In the present work, we have demonstrated that the dietary supplementation of NS to exercise training results in moderate homogenous cardiac hypertrophy manifested by an increase HW/BW, LVW/BW ratios and cardiomyocyte diameter. This model of cardiac hypertrophy possesses multiple features of physiological cardiac hypertrophy, including normal serum catecholamines, ICAM-I and total plasma nitrate level. We believe that this model of cardiac hypertrophy induced by the coadministration of Nigella sativa and exercise training is superior to that induced by only exercise training, due the revealed multiple synergistic effects of both interventions resulting in a better antioxidant capacity, and the step-forward cardiac adaptation to exercise training manifested in the lower thyroid hormones, and stronger GH/IGF-I axis response. This cardiac model may present a future promise to the emerging of a new strategy for either the prevention of pathological cardiac hypertrophy or the treatment of heart failure. Acknowledgments The authors acknowledge the deanship of scientific research in University of Dammam for the Grant No. 8086 that allowed the execution of this research. Conflict of interest All authors declare that there was no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three (3) years of beginning the work submitted that could inappropriately influence (bias) their work.
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