Original research paper

The effects of Nigella sativa extract on hypothyroidism-associated learning and memory impairment during neonatal and juvenile growth in rats Farimah Beheshti 1, Mahmoud Hosseini 1,2 , Mohammad Naser Shafei3, Mohammad Soukhtanloo 4, Simagol Ghasemi 2, Farzaneh Vafaee2, Leila Zarepoor 5 1

Neurocognitive Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 2Pharmacological Research Center of Medicinal Plants, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 3Neurogenic Inflammation Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 4Department of Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 5Human Health and Nutritional Sciences Department, College of Biological Sciences, University of Guelph, Canada Objectives: It has been shown that hypothyroidism-induced oxidative damage in brain tissue is involved in its adverse effects on learning and memory. Nigella sativa (N. sativa) has been suggested to have antioxidant and neuroprotective effects. The objective of this study was to investigate the effects of hydroalcoholic extract of N. sativa on hypothyroidism-associated learning and memory impairment during neonatal and juvenile growth in rats. Methods: Thirty pregnant rats were kept in separate cages. After delivery, the mothers and their offspring were randomly divided into six groups including: (1) control, (2) PTU (propylthiouracil), (3) PTU-NS 100, (4) PTU-NS 200, (5) PTU-NS 400, and (6) PTU-Vit C (vitamin C). All dams except the control group received 0.005% PTU in their drinking water during lactation. Besides PTU, dams in groups 3, 4, 5, and 6 received 100, 200, and 400 mg/kg N. sativa extract, or 100 mg/kg Vit C, respectively. After lactation period, pups continued to receive same experimental treatment for the first 8 weeks of their life. Then, 10 male offspring of each group were randomly selected and assessed for the learning and memory abilities by using Morris water maze (MWM) and passive avoidance (PA) tests. Blood samples were collected for thyroxine assessment, animals were euthanized, and the brain tissues were removed and analyzed for total thiol groups and malondialdehyde (MDA) concentrations. Results: PTU exposure significantly increased the time latency in MWM test, while reduced the time spent in target quadrant, and decreased the latency for entering the dark compartment in PA test. These effects were associated with significant reduction in serum thyroxine levels and brain levels of thiol groups, and significant elevation in hippocampal MDA. Administration of 400 mg/kg N. sativa extract and 100 mg/kg Vit C reduced the time latency, while increased the time spent in target quadrant compared to the PTU group in MWM test. Treatment by 100–400 mg/kg of N. sativa extract and also Vit C significantly increased the time latency for entering the dark compartment in PA test. The serum thyroxine concentrations of the animals treated by all doses of the N. sativa extract as well as by Vit C were higher than that of the PTU group. Two hundred and four hundred milligrams/kilogram of NS extract and 100 mg/kg Vit C decreased the MDA concentration in hippocampal tissues, while increased thiol contents compared to the PTU group. Discussion: The results of this study demonstrate that the hydroalcoholic extract of N. sativa have protective effects on hypothyroidism-associated learning and memory impairment during neonatal and juvenile growth in rats. The effects were comparable to Vit C and might be due to the protective effects of N. sativa extract against brain tissues’ oxidative damage. Keywords: Nigella sativa, Hypothyroidism, Learning, Memory, Oxidative stress

Correspondence to: Mahmoud Hosseini, Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, 9177948564 Iran. Email: [email protected]

© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830514Y.0000000144

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Introduction Thyroid hormones have critical roles in the regulation of energy metabolism, mitochondrial activity, oxygen consumption, and active oxygen metabolism.1,2 They also play an important role in generation of new neurons and development of nervous system.3 It has been shown that deficiency of thyroid hormones during brain development is associated with severe cognitive and neurological impairments.4 Previous studies have demonstrated that the experimental induction of hypothyroidism results in behavioral impairments and structural abnormalities in the nervous system of neonatal rats.5 The adverse effects of prenatal hypothyroidism on learning and memory are partly through reduction in neuronal survival, decreased dendritic spine density, and impaired synaptic function.6,7 In addition, it has been shown that maternal hypothyroidism during lactation adversely influences the development of the brain in offspring.8 The negative effects of hypothyroidism on cognitive function is not limited to early stages of life, as the thyroid hormone deficiency in adulthood is also associated with cognitive dysfunctions, depressed mood, and distressed attention.9,10 Previous studies on adult hypothyroid rats have shown that hypothyroid animals have smaller hippocampus,11 and a reduced number of neurons in their hippocampus compared to normal rats.12,13 In contrast to these findings, there are studies that demonstrated the protective effect of hypothyroidism against neuronal death after transient or focal cerebral ischemia in experimental animals,14–16 probably by reducing lipid peroxidation and increasing superoxide dismutase.17 Findings about the effect of hypothyroidism on oxidative stress are controversial. Some studies showed the protective effect of hypothyroidism on oxidative stress,18 while some studies demonstrated the antioxidant properties of thyroid hormones,19 or showed that hypothyroidism selectively induces oxidative stress in the hippocampus and amygdala.20 There are some natural products, such as Nigella sativa (N. sativa), that showed to be protective in some situations associated with neuronal damage. In traditional medicine, N. sativa used to be consumed as a healing medicine for treatment of diverse diseases such as asthma, headache, dysentery, infections, obesity, back pain, hypertension, and gastrointestinal problems.21–25 The major bioactives of N. sativa seeds are thymoquinone, alkaloids (nigellidine, nigellimine, and nigellicine), vitamin-like thiamine, riboflavin, pyridoxine, niacin, folic acid, minerals, and proteins.26–31 N. sativa oil also has shown beneficial effects on neuronal cell. It has been observed that administration of N. sativa oil to the cerebellar neurons cell culture prior to beta-amyloid protein intoxication improves neuronal cell viability.32

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Furthermore, N. sativa oil has demonstrated antioxidant effects during cerebral ischemia–reperfusion injury in rat hippocampus.33 In another study, the methanolic extract of N. sativa showed to be beneficial in modulation of the neuronal release of amino acid neurotransmitters including gamma-aminobutyric acid (GABA), glycine, aspartate, and glutamate in cultured cortical neurons.34 Also, N. sativa showed beneficial effects on learning, memory, and cognition in both human and animal studies.35–38 The beneficial effects of N. sativa on neuronal health and function may be related to its antioxidative properties.35,39 It has been well demonstrated that the brain tissues’ oxidative damage is critically involved in memory impairments, and antioxidative compounds have potentials to improve learning and memory.40,41 Also, it has been suggested that hypothyroidism has deleterious effects on learning and memory, probably by inducing oxidative damage in brain tissues.20 Thus, the objective of this study was to elucidate the effects of hydroalcoholic extract of N. sativa on hypothyroidism-associated oxidative stress, and learning and memory impairment during neonatal and juvenile growth in rats.

Methods Animals and treatments Thirty pregnant female Wistar rats (12 weeks old and weighing 220–250 g) were kept in separate cages at 22 ± 2°C in a room with a 12 hours light/dark cycle (light on at 7:00 am). Animals were randomly divided into six groups including: (1) control, (2) propylthiouracil (PTU), (3) PTU-NS 100, (4) PTU-NS 200, (5) PTU-NS 400, and (6) PTU-vitamin C (PTU-Vit C). Rats in the control group received normal drinking water, whereas the second group received drinking water supplemented with 0.005% PTU (Sigma Co., St. Louis, USA) to develop hypothyroidism.42 Groups 3, 4, and 5 received drinking water supplemented with 0.005% PTU and 100, 200, and 400 mg hydroalcoholic extract of N. sativa per kilogram body weight, respectively.43,44 The animals in group 6 received 100 mg/kg Vit C45 instead of the N. sativa extract. Dams received the experimental treatments from the first day after delivery through the lactation period, and after that pups continued to receive the experimental treatments in their drinking water for the first 2 months of their life. At day 60 post-delivery, 10 male offspring of each group were randomly selected and examined in the Morris water maze (MWM) and passive avoidance (PA) tests. To confirm the hypothyroidism, the serum thyroxine levels were assessed using the radioimmunoassay method. Animal handling and all related procedures were carried out in accordance

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with the procedures approved by the Mashhad University of Medical Sciences Ethical Committee.

MWM apparatus and procedures A circular black pool (136 cm diameter, 60 cm high, and 30 cm deep) was filled with water (24–26°C). A circular platform (10 cm diameter, 28 cm high) was placed within the pool and was submerged ∼2 cm below the surface of the water in the center of the southwest quadrant. Outside the maze, fixed visual cues (i.e. a computer, hardware, and posters) were present at various locations around the room. Before the experiment, each rat was handled daily for 3 days and habituated to the water maze for 30 seconds without a platform. The animals performed four trials daily, for 5 days. Each trial began with the rat being placed in the pool and released facing the side wall at one of the four positions (the boundaries of the four quadrants, labeled north (N), east (E), south (S), and west (W)). Release positions were randomly predetermined. For each trial, the rat was allowed to swim until it found and remained on the platform for 15 seconds. If an animal was not able to find the platform in 60 seconds, it has being guided to the platform by the experimenter and allowed to stay on the platform for 15 seconds. The rat was then removed from the pool, dried, and placed in its holding bin for 5 minutes. The time latency to reach the platform and the length of the swimming path were recorded by a video tracking system. On the sixth day, the platform was removed, and the animals were allowed to swim for 60 seconds. The time spent in the target quadrant was recorded to compare between groups.46,47 All measurements were performed during the second half of the light cycle.

PA apparatus and procedures A PA learning test based on negative reinforcement was used to examine the memory. The apparatus was consisted of a light and a dark compartment with a grid floor adjoining each other through a small gate. The animals were familiarized to the behavioral apparatus during two consecutive days (5 minutes in each day) before the training session. On the third day, the animals were placed in the light compartment, and the time latency for entering the dark compartment was recorded. In the training phase, the rats were placed in the light compartment facing away from the dark compartment. When the animals were entered completely into the dark compartment, they received an electric shock (2 mA, 2-second duration). The rats were then returned to their home cage. One, twenty-four, and seventy-two hours later (the retention phase), the animals were placed in the light compartment, and the time latency for entering the dark

compartment as well as the time spent by the animals in the dark and light compartments were recorded and defined as the retention trial.48

Biochemical assessment After the last session of the behavioral tests, blood samples were taken from all rats to determine hypothyroidism status. Finally, the animals were sacrificed and the cortical and hippocampal tissues were removed, weighed, and were analyzed for total thiol (SH) groups and malondialdehyde (MDA) concentrations. Total SH groups were measured using 2, 2′ dinitro- 5, 5′ -dithiodibenzoic acid (DTNB), a reagent that reacts with the SH groups and produce a yellow colored complex which has a peak absorbance at 412 nm.49 Briefly, 1 ml Tris-EDTA buffer ( pH = 8.6) was added to 50 μl brain homogenate in 1 ml cuvettes and the absorbance was read at 412 nm against TrisEDTA buffer (A1). Then, 20 μl DTNB reagents (10 mM in methanol) were added to the mixture and after 15 minutes incubation in room temperature, the absorbance was read again (A2). The absorbance of DTNB reagent was also read as a blank (B). Total thiol concentration (mM) was calculated based on an equation previously described by Hosseini et al. 40,41 MDA levels, as an index of lipid peroxidation, were measured in cortical and hippocampal tissues. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance and produces a red colored complex which has a peak absorbance at 535 nm. Two milliliters TBA/trichloroacetic acid/hydrochloric acid reagent was added to 1 ml homogenate and the solution was incubated in a boiling water bath for 40 minutes. After cooling, the whole solutions were centrifuged (1000 g for 10 minutes). The absorbance of supernatant was measured at 535 nm. The MDA concentration (C ) was calculated as follows.40,41   C (m) = Absorbance/ 1.65 × 105

Statistical analysis All data were expressed as means ± SEM. The data related to time and distance during 5 days of MWM were compared using repeated measures analysis of variance (ANOVA) followed by Tukey’s post hoc comparisons test. The data due to probe trail in MWM, the data of PA test, thyroxine level, MDA concentrations, and total thiol groups were compared by one-way ANOVA followed by Tukey’s post hoc comparisons test. Differences were considered statistically significant when P < 0.05.

Results MWM test PTU administration significantly increased the time latency to find the platform compared to the control

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group (47.3 ± 1.16 vs. 40.65 ± 1.03 seconds) (Fig. 1A and B; P < 0.01), while treatment by 400 mg/kg N. sativa extract significantly attenuated this effect of PTU by reducing the time latency (40.05 ± 1.57 seconds) to reach the platform compared to the PTU group (Fig. 1A and B; P < 0.01). In addition, exposure to PTU significantly increased the traveled distance to reach the platform (1401.2 ± 29.01 cm) compared to the control group (1096.7 ± 36.84 cm) (Fig. 2A and B; P < 0.001), while treatment with 200 mg/kg of N. sativa extract reduced the traveled distance (1232.6 ± 44.32 cm) to reach the platform compared to the PTU group (Fig. 2A and B; P < 0.05). The ability of animals to remember the location of the platform was evaluated in probe trial. The results showed that the PTU administration reduced the spent time and traveled distance in target quadrant in PTU group compared to the control group (Fig. 3A and B; P
0.05).

Figure 4 Comparison of time latency and the number of entries to the dark compartment after shock. Comparison of time latency for entering the dark compartment (A) and the number of entries to the dark compartment at 1, 24, and 72 hours after receiving the shock in the experimental groups. Data are presented as mean ± SEM (n = 10). Rats in the control group received tap drinking water, the PTU group 0.005% PTU, PTU-NS 100, PTU-NS 200, and PTU-NS 400 groups 0.005% PTU plus 100, 200, and 400 mg/kg of N. sativa extract, respectively. The animals of PTU-Vit C group received 100 mg/kg of Vit C instead of the extract. The time latencies in the PTU group was lower than the control group at 1, 24, and 72 hours, while the time latency in all PTU-NS 100, PTU-NS 200, PTU-NS 400, and PTU-Vit C groups at 1 hour and of PTU-NS 400 and PTU-Vit C groups at 24 and 72 hours were higher than the PTU group. The number of entries to the dark compartment in the PTU group was lower than the control group, while in all PTU-NS 100, PTU-NS 200, PTU-NS 400, and PTU-Vit C groups was lower than the PTU group at 1 hour. In the PTU-Vit C group, the number of entries was lower than PTU at 24 and 72 hours. One-way ANOVA followed by Tukey’s post hoc was used. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group, +P < 0.05, ++P < 0.01, +++P < 0.001 vs. PTU group.

Treatment of the animals with 100–400 mg/kg of N. sativa extract significantly increased the time latency (189.9 ± 37.71, 180.9 ± 40.23, and 219.3 ± 33.51 seconds, respectively) for entering the dark compartment at 1-hour post-shock (Fig. 4A, P < 0.01 for all doses). In addition, 400 mg/kg of N. sativa extract was able to increase the time latency at 24

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dark compartment at 1 hour after the shock (Fig. 5A, P < 0.05), while there was no significant difference between groups at 24 and 72 hours after the shock. In addition, both 400 mg/kg N. sativa extract and 100 mg/kg Vit C significantly increased the spent time in the light compartment (Fig. 5B, P < 0.05).

Biochemical assessment results

Figure 5 Comparison of time spent in the dark and light compartments after the shock. Comparison of the total time spent in the dark (A) and light (B) compartments at 1, 24, and 72 hours after receiving the shock in the experimental groups. Data are presented as mean ± SEM (n = 10). Rats in the control group received tap drinking water, the PTU group 0.005% PTU, PTU-NS 100, PTU-NS 200, and PTU-NS 400 groups 0.005% PTU plus 100, 200, and 400 mg/kg of N. sativa extract, respectively. The animals of the PTU-Vit C group received 100 mg/kg of Vit C instead of the extract. The time spent in the dark compartment in all PTU-NS 100, PTU-NS 200, PTU-NS 400, and PTU-Vit C groups was lower than the PTU group at 1 hour. The time spent in light compartment in the PTU-Vit C group at 1 and 72 hours and in PTU-NS 400 at 72 hours was higher than that of the PTU group. One-way ANOVA followed by Tukey’s post hoc was used. +P < 0.05 vs. PTU group.

(217 ± 35.04 seconds) and 72 hours (183.4 ± 40.16 seconds) after receiving the shock (Fig. 4A, P < 0.01). Similarly, Vit C increased the time latency at 1, 24, and 72 hours post-shock (280 ± 13.12, 268 ± 20.53, and 257 ± 24.85 seconds, respectively) (Fig. 4A, P < 0.001). Furthermore, all doses of the N. sativa extract could significantly reduce the number of entries to the dark compartment (P < 0.01 – P < 0.001). Vit C also decreased the number of entries to the dark compartment at 1, 24, and 72 hours after the shock (Fig. 4B, P < 0.001, P < 0.05, P < 0.01). Treatment by 100–400 mg/kg of N. sativa extract or Vit C similarly reduced the amount of spent time in

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The serum thyroxine concentration in PTU treated animals was significantly lower compared to that of control animals (P < 0.001, Fig. 6). In addition, the PTU exposure influenced MDA and thiol concentrations both in hippocampal and cortical tissues. PTU increased hippocampal MDA (Fig. 7A; P < 0.001) and also cortical MDA compared to the control group (Fig. 8A; P < 0.05), while reduced the thiol concentrations in both hippocampal (Fig. 7B; P < 0.01) and cortical tissues compared to the control group (Fig. 8B; P < 0.001). Treatment by all three doses of the N. sativa extract or Vit C significantly attenuated the PTU-induced reduction in serum thyroxine (P < 0.001, Fig. 6). In addition, pretreatment of the animals by 200 and 400 mg/kg of N. sativa extract or 100 mg/kg of Vit C resulted in decreased level of MDA in hippocampal tissues compared to the PTU group (Fig. 7A; P < 0.01 and P < 0.001, respectively). In addition, treatment of the animals by 400 mg/kg N. sativa extract or 100 mg/ kg Vit C significantly reduced the MDA

Figure 6 Serum thyroxine concentrations in offspring. Data are presented as mean ± SEM (n = 10). ***P < 0.001 vs. control group, +++P < 0.001 vs. PTU group. Rats in the control group received tap drinking water, the PTU group 0.005% PTU, PTU-NS 100, PTU-NS 200, and PTU-NS 400 groups 0.005% PTU plus 100, 200, and 400 mg/kg of N. sativa extract, respectively. The animals of PTU-Vit C group received 100 mg/kg of Vit C instead of the extract. The thyroxine level in the PTU group was lower, while in all PTU-NS 100, PTU-NS 200, PTU-NS 400, and PTU-Vit C groups was higher than the PTU group. One-way ANOVA followed by Tukey’s post hoc was used.

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Figure 7 Hippocampal concentrations of MDA and thiol groups. The MDA concentrations (A) and total thiol concentrations (B) in hippocampal tissues of six groups. Data are presented as mean ± SEM (n = 10). Rats in the control group received tap drinking water, the PTU group 0.005% PTU, PTU-NS 100, PTU-NS 200, and PTU-NS 400 groups 0.005% PTU plus 100, 200, and 400 mg/kg of N. sativa extract, respectively. The animals of the PTU-Vit C group received 100 mg/kg of Vit C instead of the extract. The hippocampal MDA in the PTU group was higher, while thiol was lower than that of the control group. The hippocampal MDA in all PTUNS 200, PTU-NS 400, and the PTU-Vit C groups was lower, while thiol was higher than that of the PTU group. One-way ANOVA followed by Tukey’s post hoc was used. **P < 0.01 and ***P < 0.001 vs. control group, ++P < 0.01, +++P < 0.001 vs. PTU group.

concentrations in cortical tissues (Fig. 8A and 8B; P < 0.05). Both 400 mg/kg N. sativa extract and Vit C were able to significantly improve the hippocampal (Fig. 7B; P < 0.01) and cortical levels of total thiol compared to the PTU group (Fig. 8A and 8B; P < 0.01).

Discussion The current study demonstrates that PTU exposure during neonatal and juvenile period results in development of hypothyroidism, and negatively influences on memory and learning abilities in rats. These findings

Figure 8 Cortical concentrations of MDA and thiol groups. The MDA concentrations (A) and total thiol concentrations (B) in cortical tissues of six groups. Data are presented as mean ± SEM (n = 10). Rats in the control group received tap drinking water, the PTU group 0.005% PTU, PTU-NS 100, PTU-NS 200, and PTU-NS 400 groups 0.005% PTU plus 100, 200, and 400 mg/kg of N. sativa extract, respectively. The animals of the PTU-Vit C group received 100 mg/kg of Vit C instead of the extract. The cortical MDA in the PTU group was higher, while thiol was lower than that of the control group. The cortical MDA in all PTU-NS 100, PTU-NS 200, PTU-NS 400, and PTU-Vit C groups was lower, while thiol was higher than that of the PTU group. One-way ANOVA followed by Tukey’s post hoc was used. *P < 0.05 and ***P < 0.001 vs. control group, +P < 0.05, ++P < 0.01, +++P < 0.001 vs. PTU group.

are consistent with our previous study which showed that experimental induction of hypothyroidism (by methimazole) is accompanied with learning and memory deficits during neonatal and juvenile growth in rats.40,50 Previously, it has been shown that neonatal hypothyroidism during developmental periods is associated with cognitive, learning, and memory impairments.51,52 Hypothyroidism disturbs hippocampal-dependent learning, short- and long-term memory, and early and late phases of long-term potentiation (LTP).53,54 The adverse effects of hypothyroidism on crucial periods of brain development are partially through

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alterations in the expression of some proteins which have critical role in neurotransmitter release (e.g. synapsin I, synaptotagmin I, and syntaxin), c-jun and c-fos proteins, and extracellular signal regulated kinases.55 In addition, it has been suggested that thyroid hormone deficiency impacts glutamate release.16,56,57 In particular, neonatal hypothyroidism postpones the myelinogenesis, while reduces the number of myelinated axons.58 In the present study, it was shown that thyroid hormones deficiency during neonatal and juvenile periods was accompanied with learning impairments, which was reflected in animals’ performance in both MWM and PA tests. These findings are consistent with our previous study, which showed an impaired MWM in adult rats with methimazole-induced hypothyroidism.40,59 Also, it has been reported that treatment of thyroidectomized rats with thyroxine improves radial arm water maze tasks and LTP in the CA1 area of the hippocampus.60 Alzoubi et al. 60 suggested that cyclic-AMP response element binding protein (CREB) and mitogen activated protein kinases may contribute to the impairment of hippocampal-dependent learning and memory. Previously, we found that hypothyroidism-induced learning and memory impairments in neonatal and juvenile period were accompanied with a high level of hippocampal nitric oxide (NO) metabolites.50 Findings about NO and its influence on memory are controversial. Some studies have suggested NO as a neurotransmitter that plays an important role in enhancing memory.46,47 In contrast, others have claimed that high levels of NO in hippocampus may result in memory deficits,61 probably through its toxic effects that can lead to neuronal death.62 One of the major mechanisms involved in toxic effect of NO in the central nervous system (CNS) is due to glutamate neurotransmission and the activation of N-methyl-D-aspartate receptors, which remarkably elevates the intracellular calcium, and stimulates neuronal nitric oxide synthase (NOS).63 Previous studies have shown that 3 weeks administration of methimazole to rats enhances NOS activity, reactive oxygen species (ROS) levels, and lipid peroxidation in their amygdala and hippocampus.20 Thus, it has been suggested that the increased NO production in the hippocampus during experimentally induced hypothyroidism may be involved in increased oxidative stress in brain tissue. In the present study, it was shown that hypothyroidism-associated learning and memory impairments during neonatal and juvenile growth were accompanied with oxidative stress in brain tissues. These effects were reflected in higher MDA concentrations in both hippocampal and cortical tissues and lower total thiol groups in hypothyroid rats compared to control ones. In addition, the results of the current study confirmed previous studies which

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demonstrated that hypothyroidism induces oxidative stress in the hippocampus.20 There is a well-known relationship between oxidative stress, neuronal damage, cognitive dysfunction, and learning and memory impairment.41 Furthermore, antioxidants have frequently demonstrated beneficial effects in prevention of memory impairment under different experimental conditions.64 In summary, the findings of current study suggest that the brain tissues’ oxidative damage as one of the possible mechanisms involved in deleterious effects of PTU-induced hypothyroidism on learning and memory during neonatal and juvenile periods. Several studies have showed the increased oxidative stress in hypothyroidism,65,66 while some other studies showed reduced67 or unchanged68 lipid peroxidation in hypothyroidism. In a clinical trial it was found that MDA, a biomarker of lipid peroxidation, was elevated in both hypothyroid and subclinical hypothyroid patients compared to the controls.69 Also, it has been suggested that thyroid hormones may directly play a role in the regulation of expression of some genes involved in antioxidative defense, cell growth, and proliferation.70,71 Previous studies demonstrated that aqueous and methanolic extracts of N. sativa seeds have sedative and depressive effects on CNS and analgesia.72 In addition, the volatile oil extracted from N. sativa plant induces relaxation in different smooth muscles.73 Previous studies have demonstrated beneficial effects of N. sativa components on learning and memory. For instance, N. sativa oil showed to enhance learning and memory abilities of the rats in eight-arm radial arm maze.37 Using MWM also demonstrated that N. sativa may improve learning and memory impairments induced by global cerebrovascular hypoperfusion in rats.35 In addition, N. sativa has shown protective effects against diabetes-induced learning and memory impairment.38 In addition to animal studies, several human studies have also demonstrated the beneficial effects of N. sativa on memory, attention, and cognition.36 The results of the current study confirm previous findings about protective effects of N. sativa on learning and memory. In the present study, administration of different doses of N. sativa extract attenuated hypothyroidism-induced learning and memory impairments during neonatal and juvenile period. To the best of our knowledge, the effects of N. sativa on hypothyroidism-induced memory impairments have not been investigated. However, the beneficial effects of the plant on scopolamine-induced learning and memory impairments have been attributed to its antioxidant activities.74,75 The exact mechanisms involved in beneficial effects on N. sativa in CNS function are not clear yet. Thymoquinone, the main pharmacological ingredient in N. sativa, has shown to be protective against

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lipopolysaccharide-induced hepatic toxicity, increased MDA concentrations, and apoptosis in rats.76 Furthermore, it has been observed that treatment by thymoquinone prevents the experimental autoimmune encephalomyelitis (EAE) in rats probably due to its antioxidant and anti-inflammatory properties.26,77 It has also been suggested that N. sativa displays its antioxidant and immunoregulatory effects via inflammatory cells rather than the host tissues (brain and medulla) in EAE rats.78 The results of the present study also showed that treatment by N. sativa extract prevents the elevation of MDA concentrations, while enhances total thiol concentrations in brain tissues. In another study, N. sativa oil and thymoquinone prevented the hippocampal neurodegeneration after chronic toluene exposure in rats.39 It was also shown that N. sativa and thymoquinone reduced lipid peroxidation in hippocampal tissues of rats in a global cerebral ischemia–reperfusion injury model.33 In addition, N. sativa has showed antiepileptic effect and the ability to prevent excessive ROS formation in pentylenetetrazole-induced seizures.79 Previous studies suggested that neuroprotective effect of N. sativa in an experimental model of spinal cord injury in rats may be through its antioxidant capability.80 It has been shown that N. sativa extract and thymoquinone ameliorate oxidative stress and prevent neuropathy in diabetic rats.81 The results of the current study also suggest that protective effects of N. sativa against brain tissues’ oxidative damage may be involved in its beneficial effects in hypothyroidism-induced memory impairment during neonatal and juvenile period. These results are consistent with the results of previous studies in which the memory enhancing effects of N. sativa was attributed to its antioxidant and antiinflammatory activities.74 Besides thymoquinone, other N. sativa component(s), such as melanin, may also be involved in N. sativa demonstrated effects on learning and memory in this study. Melanin has been shown to be abundant in N. sativa seed coats.82 Melanin has been found to act as an immunomodulator.83 Therefore, this ingredient may also be suggested as a possible N. sativa compound which has a role in the result of the present study. In conclusion, the findings of this study imply that treatment with N. sativa extract may improve deleterious effects of hypothyroidism on learning and memory during neonatal and juvenile growth. Protection against brain tissues’ oxidative damage may be involved in the beneficial effects of N. sativa in brain function in hypothyroidism.

Acknowledgements The results described in this paper were from a MSc student’s thesis. The authors would like to thank the

Vice Presidency of Research of Mashhad University of Medical Sciences for their financial support.

Disclaimer statements Contributors F.B. and S.G.: performing the behavioral experiments; M.H.: supervision of performing the experiments, writing the manuscript, and statistical analysis; F.V.: performing the biochemical measurements; M.N.S.: helping to perform the behavioral experiments; M.S.: adviser for performing biochemical measurements, L.Z.: adviser for the experiments, writing the manuscript, adviser for design of the experiment, adviser for statistical analysis. Funding None. Conflicts of interest None. Ethics approval Animal handling and all related procedures were carried out in accordance with the procedures approved by the Mashhad University of Medical Sciences Ethical Committee.

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The effects of Nigella sativa extract on hypothyroidism-associated learning and memory impairment during neonatal and juvenile growth in rats.

It has been shown that hypothyroidism-induced oxidative damage in brain tissue is involved in its adverse effects on learning and memory. Nigella sati...
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