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- Biomedical Research (2016) Volume 27, Issue 1

Thymoquinone protects against hypothyroidism-induced cardiac histopathological changes in rats through a nitric oxide/antioxidant mechanism.

Nasra N Ayuoba,b, Nagla A El-Shitanyc,d*, Mohammed Nabil Alamae
aAnatomy Department, Faculty of Medicine, King Abdulaziz University, Saudi Arabia.
bHistology and Cytology Department, Mansoura University, Egypt.
c,Pharmacology and Toxicology Department, Faculty of Pharmacy, King Abdulaziz University, Saudi Arabia.
dPharmacology and Toxicology Department, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
eCardiology Unit, Department of Medicine, King Abdulaziz University Hospital, Jeddah, Saudi Arabia.
Corresponding Author: Nagla A El-Shitany
Department of Anatomy
Faculty of Medicine
King Abdulaziz University, Saudi Arabia
Accepted: November 26, 2015
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Abstract

Background: The heart is one of the organs which is affected by the thyroid hormones and hence the alterations in the thyroid status influences its function and structure. Objectives: This study aimed to reassess hypothyroidism-induced histopathological cardiac changes and to evaluate the proposed protective role of thymoquinone (TQ) against these changes. In addition, the mechanism of TQ-induced protection is studied regarding the oxidative stress and nitric oxide (NO) pathway. Materials and Methods: A model of propylthiouracil (PTU)-induced hypothyroidism in Wister rats is used in this study. Four groups of rats were used; control, TQ, PTU (hypothyroidism) and PTU+TQ groups. Thyroid hormones, cardiac enzymes, NO and antioxidants were assessed in the blood. Hearts were histopathologically and immunohistochemically examined. Results: A significant increase in plasma cardiac enzymes activity was recorded in hypothyroid rats, which is accompanied by significant histopathological changes in the left ventricle. Treatment of rats with TQ significantly protected the heart muscle against hypothyroidism- induced histopathological and immunohistochemical changes. It also significantly decreased plasma cardiac enzymes activity. TQ caused a reduction in malondialdehyde (MDA) formation, and increased, reduced glutathione (GSH), NO and superoxide dismutase (SOD) production. It also, increased the expression of constitutive nitric oxide synthase (eNOS) activity. Conclusion: hypothyroidism may induce cardiac pathological changes, which is prevented by TQ as it restores thyroid hormones, increases NO formation and eNOS expression, and decreases reactive oxygen species (ROS) production.

Keywords
Heart-Hypothyroidism, Thymoquinone-Nitric oxide, Reactive oxygen species, Histopathology

Introduction
Nigella sativa (NS), the promising medicinal plant, commonly known as black seed, was medicinally used for over 2000 years in many middle eastern and eastern Asian countries [1]. NS seeds or its oil was used in the treatment of many diseases, including asthma, inflammations, hypertension and diabetes [2]. Thymoquinone (TQ), the biologically active substance of NS seeds and its oil, was found to possess powerful antioxidant and antiinflammatory activities [3,4].
Thyroid hormones were considered one of the important modulators of the cardiovascular function. In addition, the heart was the target organ of the thyroid hormones [5]. Hypothyroidism, was a syndrome caused by thyroid hormone deficiency. Recently, Ohga et al. [6], reported left ventricular systolic and diastolic dysfunction in propylthiouracil (PTU)-induced hypothyroidism in rats. Recently, Knapp et al. [7] indicated that hypothyroidism resulted in oxidative stress, and heart diseases. In particular, it was suggested that, hypothyroidism increased the formation of reactive oxygen species (ROS) in the heart and consequently, increased lipid peroxidation [8].
In this study, it is hypothesized that hypothyroidisminduced cardiac pathological changes are caused via oxidative stress mechanism and hence, the use of an antioxidant may protect the heart against these changes. This study aimed to reassess, hypothyroidism-induced cardiac histopathological changes and to evaluate the proposed protective role of thymoquinone (TQ) against these changes. In addition, the mechanism of TQ-induced protection is studied regarding the oxidative stress and nitric oxide (NO) pathway.

Materials and Methods

Chemicals
Thymoquinone from NS (28 mg) was obtained from FRINTON LABORATORIES, INC. It is a yellow powder, which was dissolved in DMSO and diluted to 1:100 in a sterile saline solution (0.9% NaCl) to produce a working stock. PTU was purchased from Sigma Aldrich lnc, USA.

Animals and treatments
This study was approved by the Biomedical Ethics Research Committee at the Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia. Adult male Wister rats (180-200 g) were obtained from the Animal Resources Division of King Fahd Medical Research Center. The rats were housed at 22 ± 3°C and relative humidity of 44%-55% with a 12 h dark/light cycle and were provided with standard laboratory feed and water ad libitum. The animals received care, according to institutional guidelines for the care and use of the laboratory animals in King Fahd Medical Research Center. Rats were randomly divided into 4 groups (n=6 each). The control group: rats received normal saline (6 weeks, 2 ml/kg/day, per oral; po); TQ group: rats received TQ (6 weeks, 10 mg/kg/day po) [9]; PTU (hypothyroidism) group: rats received (6 weeks, 6 mg/kg/day, po) as was previously described [10] and PTU+TQ group: received PTU (6 weeks, the same dose, po) and TQ (10 mg/kg/day, po) [11] starting from the 2nd week to 6th week. Body weight of the rats was assessed at the start and at the end of the experiment.

Sample collection
At the end of the 6th week, the rats were decapitated under light ether anesthesia, blood samples were withdrawn by heart puncture. Blood samples were centrifuged and the serum were kept at -80°C until used for the biochemical analysis. Hearts were dissected out, weighted and processed for histopathological examination.

Measurement of serum triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH)
Serum T3, T4 and TSH concentrations were measured in El-Safwa Laboratory; Tanta, Egypt, using ADVIA Centaur automated competitive chemiluminescence immunoassay (Bayer HealthCare).

Measurement of plasma lactate dehydrogenase (LDH) and creatine phosphokinase CPK)
Total plasma LDH and CPK activities were measured using kits of Biodiagnostic, Egypt. LDH activity was measured according to Pesce [12] and the method of Abbot et al. [13] was adopted to determine the CPK enzyme activity.

Measurement of plasma lipid peroxide (measured as malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), glutathione peroxidase enzyme activity (GPx), superoxide dismutase enzyme activity (SOD) and catalase enzyme activity (CAT)
Plasma MDA was measured using Biodiagnostic kits, Egypt, according to Uchiyama and Mihara [14]. Plasma GSH was quantified using Biodiagnostic kits, Egypt, according to Ellman [15]. Plasma NO was measured using Biodiagnostic kits, Egypt, according to Tarpey et al. [16]. Plasma GPx activity was measured using Biodiagnostic kits, Egypt, according to Paglia and Valentine [17]. Plasma SOD activity was measured using Biodiagnostic kits, Egypt, according to Nishikimi et al. [18]. Plasma CAT activity was measured using Biodiagnostic kits, Egypt, according to Aebi [19].

Histopathological examination of the heart
After processing the heart for histopathological examination, paraffin blocks were obtained and serially sectioned (3-5 µm), then stained with hematoxylin and eosin (H and E) and Masson?s trichrome (MT) stains [20]. The cross sectional area of the cardiomyocytes in the left ventricle was measured in 30 field per animal at magnification 100X objective lens and 10X ocular lens using Pro Plus image analysis software version 6.0. The area percent of the MT-stained collagen fiber (stained blue in color) was assessed using the same software as was described by Afifi and Hanon [21]. The histopathologist who examined the slides was blind to the groups studied.

Immunohistochemical examination of heart sections
Immunohistochemical staining was done using streptavidine?biotin?peroxidase technique according to Bancroft [20]. Alpha smooth muscle actin (ASMA; Dako Cytomation, Heverlee, Belgium, at a dilution 1/1000), desmin (Dako, Trappes, Frances, at a dilution 1/100) and endothelial nitric oxide synthetase (eNOS; Abcam, Cambridge, MA at a dilution 1/50) antibodies were used in this study. The nuclei were counterstained with hematoxylin. Semiquantitative analysis of the extension of the immunoreactivity was determined by assessing the area percentage using Pro Plus image analysis software version 6.0. ASMA, desmin, and NOS area percent was assessed in 30 field per animal at magnification 40X objective lens and 10X ocular lens as described by Leslie et al. [22]. On assessing the area percent of ASMA or MTstained collagen fibers, fields with blood vessels were excluded from assessment.

Statistical Analysis
SPSS program (Version 16) was used to statistically analyze all data. The results were expressed as mean ± SDM. Comparisons between different groups were carried out by one-way analysis of variance (ANOVA) followed by Tukey-Kramer test for the parametric data. The morphometric and image analysis data (non-parametric) were analyzed by Kruskal-Wallis ANOVA followed Dunn's test. Person correlation was made between the non- parametric variable. Statistical significance was accepted at p = 0.05.

Results

The effect of PTU and TQ on body and heart weights, serum total T3, T4 and TSH concentrations
Adminstration of PTU significantly reduced (p=0.03) the rats weight gain while it significantly increased (p=0.02) the relative weight of the heart compared to the control. Adminstration of TQ to PTU-treated rats resulted in significant decrease (p=0.04) in the relative weight of the heart compared to the PTU group (Table 1).
Treatments of rats with PTU caused a significant decrease in both serum T3 and T4 concentrations (15% and 51%, respectively) compared to the control concentrations (p=0.000 and 0.008, respectively) (Table 1). PTU resulted in a significant increase in serum TSH concentration (~ 6 fold) compared to the control concentration (p=0.001) (Table 1). Adminstration of TQ to PTU-treated rats resulted in a significant increase in both serum T3 and T4 concentrations (21% and 82%, respectively) and a significant decrease in serum TSH concentration (87%) compared to PTU injected rats (p=0.01; 0.004 and 0.001 respectively) (Table 1).

Effect of TQ on plasma LDH and CPK activities in PTU- induced hypothyroidism
Treatments of rats with PTU significantly increased both plasma LDH and CPK activities (64% and 73%, respectively) compared to the control activities (p=0.000 and 0.009, respectively) (Table 2). TQ significantly decreased both plasma LDH and CPK activities (42% and 75%, respectively) in PTU=TQ group compared to PTU- treated rats (p=0.045 and 0.008, respectively) (Table 2).

Effect of TQ on plasma MDA, GSH and NO concentrations in PTU-induced hypothyroidism
Treatments of rats with PTU significantly increased both plasma MDA and NO levels (~ 3 and 2 fold, respectively) compared to the control levels (p<0.001 and 0.02, respectively) (Figure 1). On the other hand, treatments of rats with PTU caused a nonsignificant decrease (25%) in plasma GSH concentrations compared to the control rats (p=0.535) (Figure 1). Treatment with TQ after 2 weeks of PTU administration caused a significant decrease (p=0.027) in plasma MDA concentrations (31%) and a significant increase in plasma NO and GSH concentrations (22% and 77%, respectively) compared to PTU treated rats (p=0.016 and 0.035, respectively) (Figure 1).

Effect of TQ on plasma GPx, SOD and CAT activities in PTU-induced hypothyroidism
The results of enzymatic antioxidant analyses were shown in (Table 3). Briefly, the activities of GPX were not changed in all treatment regimens. Treatments of rats with PTU significantly decreased plasma SOD activity (63%) compared to the control rats (p=0.000). Treatment with TQ after 2 weeks of PTU adminstration significantly increased plasma SOD activity (~ 2 fold) compared to PTU injected rats (p=0.035). In addition, treatments of rats with PTU caused significantly increased plasma CAT activity (84%) compared to the control rats (p=0.000). Treatment with TQ after 2 weeks of PTU administration caused a nonsignificant decrease in plasma CAT activity (6%) compared to PTU injected rats (p=0.599).
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Effect of TQ on histopathological changes of hearts in PTU-induced hypothyroidism
Left ventricle of the control rats showed intact branching and anastomosing cardiac muscle fibers (CMFs) which possess acidophilic sarcoplasm with regular striations and oval central nuclei. Left ventricular CMFs of TQ group showed no histological changes compared with that of the control group Figure (2A, B). The left ventricle of PTU group showed some myocytes with hydropic changes in the form of lost striations and pyknotic nuclei. Continuity of the adjacent myocytesas well as lateral alignment of the myofibrils was lost in some areas together with nuclear displacement. Cellular infiltration and hemorrhage were also observed. The cross sectional area of the CMFs of this group was significantly decreased compared with that of the control rats (Figure 2C-E, G). The PTU+TQ group showed intact myocytes in almost all left ventricle with fewer cellular infiltrate and hemorrhages compared to the PTU group and the cross sectional area of the CMFs was insignificantly increased compared with that of the PTU group (Figure 2F). A significant increase in area percent of collagen fibers was observed in the left ventricle of PTU group compared with that of the control group while the PTU+TQ group showed a significant decrease compared with that of PTU group (Figure 3). In addition, significant positive correlations existed between area percent of collagen fibers and blood levels of LDH (r=0.532, p=0.01) and CPK (r=0.496, p=0.01).
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Effect of TQ on PTU-induced immunohistochemical changes in ASMA, desmin and eNOS expression
Blood vessel wall showed strong ASMA expression in the left ventricle of both the control and TQ groups, whereas the CMFs showed negative expression. Some CMFs of PTU group showed moderate ASMA expression while fewer CMFs showed weak expression in the PTU+TQ group. Both groups showed many branched myofibroblasts between the CMFs with a strong ASMA expression. A significant increase (p<0.001) was observed in ASMA expression in PTU group compared with the control group and TQ administration significantly reduced it (p<0.001) (Figure 4). A significant positive correlation (r=0.916, p<0.001) was recorded between area percent of ASMA expression and that of collagen fibers in the left ventricle (Figure 3F). In addition, significant positive correlations existed between area percent of ASMA expression and blood levels of LDH (r=0.580, p<0.003) and CPK (r=0.678, p<0.001).
A strong desmin expression was observed in CMFs of both control and TQ groups. Most of CMFs of PTU and PTU+TQ groups showed a strong desmin expression while few of them showed weak or no expression. A significant decrease (p<0.001) in desmin expression was observed in the left ventricular CMFs of PTU group compared with the control, whereas those in the PTU+TQ group showed a significant increase (p<0.05) compared with the PTU group (Figure 5).
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Concerning eNOS, a moderate expression was observed in the CMFs and the endothelial lining of the blood vessels of the control group. PTU group showed an increased expression that was found to be statistically significant (p=0.04) when compared to the control group. The left ventricle of the PTU+TQ group showed strong eNOS expression that was significantly higher (p=0.01) when compared to those of the PTU group (Figure 5).

Discussion
This study was conducted first to reassess the impact of experimentally-induced hypothyroidism on the adult rat heart. An experimental model of thyroid hormone deficiency was made by the reversible goitrogen PTU which decreased the conversion of T4 to T3. The hypothyroid state was ensured as the serum T3 and T4 levels were significantly lower, whereas the serum TSH level was significantly higher compared to control rats. A significant increase in plasma LDH and CPK enzymes activity was recorded in hypothyroid rats compared to control rats which indicate cardiac muscle affection. This was found to be accompanied by significant histopathological changes in left ventricle included hydropic changes in CMFs, cellular infiltration and multiple hemorrhages. These findings were in line with those of Massoud et al. [23]. In this study the hypothyroid rats weight gain was significantly reduced and this was in line with the previous studies [24]. On the other hand, the relative weight of the heart was significantly increased and this could be attributed to the infiltration of the inflammatory cell observed on the microscopic examination.
In the present study, a significant decrease in the CMFs cross sectional area was observed. In previous studies myocardial atrophy in hypothyroidism and marked reduction in muscle fiber mass in the PTU (hypothyroid) rats myocardium was reported [22]. This myocardial atrophy could be explained in the light of Klein and Danzi findings [25]. They reported that thyroid hormone increased total protein synthesis in the cardiac muscle and controls the transcription myosin heavy chain genes that are important for heart function.
A significant increase in ASMA expression was observed in the left ventricle of hypothyroid rats in this study indicating further fibrosis. The area percent of collagen fibers was positively correlated with ASMA expression in all the studied groups, suggesting that the collagen fibers were derived from cells expressing ASMA. This was in line with a recent study which reported that in myocardial stress, and heart diseases, cardiomyocytes are lost due to the myofibroblasts initiated reparative fibrosis with subsequent increase in ASMA-positive cells [26]. It was described that ASMA, a protein that is present in embryonal/fetal heart muscle cells, but absent in adult cardiomyocytes, is reexpressed in cardiomyocytes, undergo dedifferentiation, during heart hypertrophy induced by cardiac overload [27] and this could explain for ASMA expression observed in some left ventricular myocytes in this study.
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The significant decrease in desmin expression was observed in the left ventricle CMFs of hypothyroid rats, in this study, indicating affection of the structural integrity of these fibers as was reported by Paulin et al. [28]. In addition, Capetanaki et al. [29] reported that mice lacking desmin develop numerous muscle architectural and ultrastructural defects, especially in extensively used muscles such as the heart and among the structural abnormalities observed were loss of lateral alignment of myofibrils and loss of nuclear shape and positioning that was observed in the present study.
The second aim of the present work was to assess the suggested cardioprotective action of TQ against hypothyroidism-induced cardiac affection. In this study, treatment of rats with TQ significantly protects the heart muscle against hypothyroidism-induced histopathological and immunohistochemical changes. It also significantly decreased plasma LDH and CPK enzymes activity compared to PTU group. This study reported a protective effect of TQ against PTU-induced hypothyroidism and the associated cardiac damage and this is supported by Nagi and Mansour [30] who reported a protective role of thymoqinone against adriamycin-induced cardiotoxicity.
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Finally, the mechanism of TQ -induced protection is studied regarding the ROS and NO formation. In the present study, the effect of PTU on T3, T4 and TSH seems to be reversed in rats treated with TQ as the levels of the T3, T4 and TSH tends to be near the normal levels. This finding is in line with that of Shariatifar et al. [31] who reported that NS significantly increases the levels of T3 and T4 and decreases the TSH in mice. The role of thyroid hormones in metabolic pathways and antioxidant enzyme activities was well known in many species [32]. In the present study PTU-induced hypothyroidism caused an increase in the plasma MDA formation (a product of lipid peroxidation) and CAT activity and a decrease in plasma SOD activity while both GSH content and GPX activity were not altered. Increased MDA formation suggested enhancement of oxidative stress in hypothyroidism. This is in line with the results of Yilmaz et al. [8], who reported increased plasma, liver, heart and muscle MDA level in hypothyroid rats. In addition, Chattopadhyay et al. [33] reported increased CAT activity in hypothyroidism, heart, which is suggested to be a reflex mechanism against increased oxidative stress-induced by hypothyroidism. Choudhury et al. [34] reported a fall in SOD and CAT activity in hypothyroid rats, but in addition of T3, only the catalase activity was restored. Chattopadhyay et al. [33] described that, a decreased thyroid function may disturb SOD/CAT ratio and hence imbalance the oxidant/antioxidant state resulting in generation of oxidative stress. Petrulea et al. [35] also reported that GSH didn't differ significantly in serum and different tissues of hypothyroid rats and this was observed also in this study. Thymoquinone effect seems to be mediated through a reduction in MDA formation, and increased GSH and SOD production, hence it restores the oxidant/antioxidant balance. The mechanism underlying TQ protective effect in this study could be attributed to the normalization of T3, T4 and TSH. Thymoquinone acts as a potent free radical scavenger against superoxide, hydroxyl and singlet oxygen radical [36].
NO is an important vasodilator produced by coronary endothelial cells, which exerts a cardioprotective effect. It serves as an oxygen free radical scavenger, hence minimizing the deleterious effects of the oxidative stress [37]. In addition, it inhibits contractile tone and the proliferation of underlying vascular smooth muscle cells and promotes diastolic relaxation [38]. This study hypothesized a role of NO in TQ induced protection against hypothyroidism induced pathological cardiac changes. The present results showed that PTU-induced hypothyroidism increased NO formation, which seems to be a protective mechanism, and TQ treatment resulted in a further increase in NO formation. The immunohistochemical results were in line with this as the eNOS expression was increased in the left ventricle of rats received PTU and further increased in TQ rats. This finding is in concordance with some previous studies which reported an increase in NOS activity in both ventricles of the chronic hypothyroidism models and in the atria, endothelial and smooth muscle of the aorta of the acute model of thyroidectomy [39]. Increased NOS activity was described to be a compensatory mechanism stimulated by decreased plasma T3 levels which plays a protective role in maintaining blood ?ow and reduces cardiac after load that resulted in an increase in the cardiac output and attenuation of pulmonary edema, thus markedly improving survival [39].

Conclusion
This study concluded that hypothyroidism may induce pathological cardiac changes, which were prevented by TQ via increased thyroid hormones and NO, and decreased ROS production.

Conflict of Interest
The authors declare that they have no conflict of interest.

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