Aerobic training with consumption of Nigella sativa extract ameliorate myelin core protein, axonal regeneration, and cognitive function in a mouse model of multiple sclerosis

doi:10.21203/rs.3.rs-3218529/v1

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Research Article

Aerobic training with consumption of Nigella sativa extract ameliorate myelin core protein, axonal regeneration, and cognitive function in a mouse model of multiple sclerosis

https://doi.org/10.21203/rs.3.rs-3218529/v1

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Purpose

Multiple sclerosis (MS) is a progressive disease of the central nervous system (CNS) that leads to demyelination of the CNS and memory impairment in animals suffering from this disease. It seems that the release of neurotrophic factors after training is one of the possible mechanisms for increasing axonal protection and brain health of these patients.

methods

In order to induce MS, cuprizone was added to rodent food powder at a weight ratio of 0.2%. The state of balance in the studied groups was checked by repeated measurement statistical test, the level of expression of brain derived neurotrophic factor (BDNF) and myelin basic protein (MBP) markers by immunohistochemistry, the amount of tumor necrosis factor-alpha (TNF-α) by ELISA method and the evaluation of memory and spatial learning by Morris water maze test.

Results

The amount of MBP in the corpus callosum in the cuprizone (CUP) group was significantly decreased compared to the sham group and significantly increased in the groups treated with training and oral Nigella sativa compared to the CUP group. A significant increase in BDNF and a significant decrease in TNF-α were observed in the treated groups compared to the CUP group.

Conclusion

probably due to the reduction of inflammation and the improvement of myelin, the balance and motor activity of the animals have also improved. These structural changes in the brain anatomy of mice, which show histological changes and in line with its behavioral function, can have a new path for faster diagnosis and prevention of debilitating symptoms.

Aerobic training

Nigella sativa

MBP

BDNF

Multiple sclerosis

Multiple sclerosis (MS) is an autoimmune, inflammatory, chronic, and progressive disease in which the immune system attacks neurons in the brain and spinal cord, causing damage to the myelin sheath and axons of these cells(Mancuso et al., 2015). The major myelin protein, myelin basic protein (MBP), expressed by oligodendrocytes, plays a major role in the multilayer structure of the myelin sheath. This protein is effective in maturation and compression of the myelin sheath. Due to the close relationship between myelin and axon transport, any myelin dysfunction can lead to different types of axonal pathology(Robinson et al., 2014). Therefore, any abnormality in the myelin sheath is involved in neuronal dysfunction. Neuronal myelin changes are also determined by measuring MBP levels. Inflammatory central nervous system (CNS) infiltration destroys MBP and causes demyelination, axonal damage, and subsequent progressive paralysis of the hind limb(Robinson et al., 2014).

Common motor symptoms of MS include loss of balance, decreased isokinetic strength, isometric, decreased flexibility, decreased endurance and walking speed, fatigue, muscle stiffness, uncontrollable tremors, and factors such as boredom, depression, and fear of falling(Wagner et al., 2015). Due to the fact that there is currently no definitive treatment for this disease, it is possible to alleviate the symptoms of the disease and reduce its progression by formulating and following a special treatment program and using new drugs and rehabilitation techniques(Moradbeygi et al., 2021). One of the pathological symptoms of MS is the infiltration of immune cells into the central nervous system and the destruction of myelin and axonal damage that during the process of tissue repair, immune cells produce brain derived neurotrophic factor (BDNF) (Jørgensen et al., 2019). BDNF is a member of the neurotrophin family, which plays an important role in synaptic flexibility, neural survival, and differentiation, and has beneficial effects on CNS function in animal models(Briken et al., 2016). BDNF secretion improves the regeneration of various tissues. Various studies have shown that in people with MS, the level of this protein decreases. One of the most important factors that increase the level of BDNF is training(Jørgensen et al., 2019). The secretion of neurotrophic factors following training seems to be one of the possible mechanisms to increase axonal protection and brain health of patients.

The neuroprotective and inhibitory potential of training by inducing neurogenesis in the hippocampus, an important brain structure for learning and memory, has been demonstrated in several animal studies. In addition, training is known to improve neurocognitive function in the elderly (Cotman et al., 2007). Physical activity is widely accepted as a behavioral strategy to enhance general health including mental function. Training requires purposeful work of the nervous system, which sends important messages to the muscles and improves the nervous system and better coordination of muscles and nerves. Training also affects the voluntary nerves directly and the involuntary nerves indirectly (Beheshti et al., 2018; Gilhotra & Dhingra, 2011). On the other hand, in recent years, extensive studies have been conducted on the effect of physical activity on MS(Beheshti et al., 2018; Gilhotra & Dhingra, 2011).

It seems that the effect of an environmental stimulus on the immune system leads to demyelination and disruption of CNS processes in genetically predisposed individuals(Sena et al., 2012). The result of CNS demyelination is associated with impaired neural conduction and reduced motor and cognitive abilities (Walton et al., 2020). Such an immune-mediated process includes disruption of the blood-brain barrier as well as a lack of neural support for myelin repair (Golden & Voskuhl, 2017). Various studies indicate the anti-inflammatory, antioxidant and neuroprotective properties of physical activity. Therefore, it has been widely used in the treatment of neurodegenerative diseases such as Parkinson's and Alzheimer's due to its potential antioxidant effects (Hadi et al., 2018).

In Iran, medicinal plants are widely used in traditional medicine to treat diseases. One of these plants is Nigella sativa, which is native to Southwest Asia and belongs to the Ranunculaceae family(Javidi et al., 2016). This plant has long been used in Iran and in traditional medicine to treat various diseases and its healing properties are mainly due to the presence of thymoquinone (Hadi et al., 2018). This plant has anti-inflammatory and antioxidant properties, and studies have examined the effect of thymoquinone (the active ingredient in Nigella sativa) on neurological diseases such as epilepsy, Parkinson's, Alzheimer's, anxiety and memory disorders. In fact, thymoquinone protects brain cells from all kinds of damage due to its antioxidant, anti-inflammatory and anti-apoptotic effects(Beheshti et al., 2018; Gilhotra & Dhingra, 2011).

According to studies, the anti-inflammatory and antioxidant effects of thymoquinone occur through the control of proinflammatory cytokines. On the other hand, in recent years, extensive studies have been conducted on the impact of physical activities on MS disease( Beheshti, Norouzi, Abareshi, Anaeigoudari, & Hosseini, 2018; Gilhotra & Dhingra, 2011). Therefore, this study was designed to investigate the effect of aerobic training with consumption of Nigella sativa extract ameliorate myelin core protein, axonal regeneration, and cognitive function in a mouse model of multiple sclerosis.

2-1 Animals

The statistical population of this study consisted of 64 adult male mice (8 weeks) of C57BL / 6 weights in the weight range of 20-26 g. At the time of the study, these animals had unrestricted access to drinking water and standard food, and for the same period of time (12 hours) in the dark (night) and light (day) and at a temperature of 21 to 23 degrees. Were exposed to Celsius. To avoid any stress and to accustom the animals to the new environment, the study was started after 10 days. These animals were kept in a light and dark period of 12 hours in optimal and standard conditions and were randomly divided into 8 groups of 8 Which included: groups 1-healthy (sham) 2- Multiple sclerosis (CUP) model 3- Healthy with aerobic training (EX) 4- Multiple sclerosis model with aerobic training (CUP+EX) 5- Healthy recipient of Nigella sativa (NS) oral extract 6- Multiple Sclerosis Model Receiving Oral Extract of Nigella sativa (CUP+NS) 7- Healthy Receiving Oral Extract of Nigella sativa with Aerobic Training (EX+NS) 8- Model Multiple Sclerosis Receiving Oral Extract of Nigella sativa along with aerobic training (CUP+NS+EX).

2-2 Create a demyelination model

Feeding mice with cuprizone causes reversible demyelination and the destruction of adult oligodendrocytes, especially in the corpus callosum, while other cells in the central nervous system are not affected. Demyelination occurs during feeding with cuprizone and after discontinuation, remylation begins spontaneously in the brain (Shirazi et al., 2016). 4 groups of samples (CUP+NS, CUP+EX, CUP+NS+EX and CUP) were fed with 0.2% cuprizone-impregnated feed for 7 weeks. Sufficient weight containers were used to prevent food from overturning. We placed the animal cages under the hood to prevent food powder from spreading into the space. Food containing 0.2% cuprizone was given to the animals for 7 weeks without any restrictions to induce the model. To ensure demyelination, a tissue sample from the brain was taken three weeks after induction of multiple sclerosis and the corpus callosum was examined.

2-3 Plant collection, preparation of extract and amount of consumption

Nigella sativa was prepared commercially. The Nigella sativa was thoroughly ground with an industrial crusher. Alcoholic extract was prepared from Nigella sativa powder by Soxhlet method. Extraction was performed three consecutive days from morning to night to ensure that all alcohol-soluble extracts were extracted from the powder. At the end of the alcoholic extract, the solvent was removed. This was done using a rotary device. According to studies, the lethal dose of Nigella sativa in mice was reported to be 870.9 mg/kg body weight. In previous studies, one tenth of the lethal dose was used for oral gavage (Efendi, 2015; Huang et al., 2017), so in this study, the amount for oral gavage was 87 mg per kg body weight.

2-4 Treadmill workout

After assigning the animals to 8 groups, the training groups were trained on a treadmill for one week. The training program included forced running on a treadmill at a speed of 15 to 20 meters per minute for 8 weeks, 6 days a week and twice a day, each session lasting 15 minutes with a minimum interval of 2 hours (Landers et al., 2014). It started at 15 meters per minute in the first week and gradually increased to 20 meters per minute in the fifth week and continued unchanged until the end of the eighth week. We used a sponge as a tactile stimulus to keep the mice at speed while minimizing external stressors. This training program is modeled on a study by Landers et al., Which has been modified according to the study conditions.

2-5 Weight assessment of mice

Weight of all tested mice was obtained before and after receiving cuprizone and once a week and was analyzed for growth changes with increasing age and drug use.

2-6 Clinical assessment

Clinical trials of each animal were evaluated at 7 weeks of study. This test consisted of 0-6 points, which was based on the weakness of the limbs and the way the scoring movement took place. Modified Neurological Severity Scores (MNSS), memory and spatial learning assessment of Morris water maze test (Nichols et al., 2013) and open field behavioral responses (Ghotbeddin et al., 2020) were used to evaluate the Beam balance test, sensory-motor analysis.

BBT is a movement coordination test. It can be used to check the state of equilibrium. This behavioral test is used to evaluate motor defects caused by lesions of the central nervous system in rodents (Luong et al., 2011). Morris Water Maze is a circular metal tank with a black wall, 200-120 cm in diameter and 50-60 cm high, which is filled with water. The optimum water temperature is 25 2 2 ° C. A dark metal platform 10 or 11 cm in diameter was placed 1 to 5 cm below the water surface, invisible to the animal, in the center of one of the four quarters northeast, southeast, northwest, or southwest. The movement and behavior of the animal was tracked and controlled by an infrared television camera located at a height of two meters above the central area of the tank. The digital TV signal enters the computer system with which it is connected and the animal's movements are stored and analyzed with the help of software installed for this purpose, and variables such as: How long it took for the animal to find the Plexiglas platform, length The total distance of the mouse swimming path at each training, the percentage of time that the animal spent in each quarter of the tank, and the speed of movement of the mouse were measured (Mohammadi-Rad et al., 2019; Robinson et al., 2014).

The OFT test used an open box whose bottom was divided into several equal squares by lines. Each animal was placed in a box for 5 minutes and the number of cut lines, the number of times standing on two legs (rearing, urination and defecation in 5 minutes) were counted by two researchers.

2-7 Tissue samples

At the end of the eighth week and 48 hours after the last intervention, the experimental groups were anesthetized by intraperitoneal injection of ketamine and xylazine in a ratio of one to ten and then with normal saline and paraformaldehyde solution of 4% perfusion through fluid transfer in the left atrium. Then a hole was made in the right ventricle. Then the animal's head was removed and two parietal bones were separated from the sagittal area and slowly and without damage to the brain were removed. Fixative was placed. Finally, brain samples were subjected to tissue passage for histopathological examination(Struck et al., 2011).

2-8 Histopathological examinations

Brain samples were subjected to tissue passage for histopathological examination. The steps include: Dehydration, clearing, impregnation, embedding and sectioning. After trimming the prepared blocks, serial sections with a thickness of 8 am were cut using a rotary type microtome. The slices were glued to a blade pre-prepared with 0.1% gelatin.

2-9 Fast Blue Luxury Staining (LFB)

Luxol fast blue specific myelin tissue staining is used to evaluate axon repair and regeneration and the amount of remaining areas is determined by software using differential staining (eosin) (Sanadgol et al., 2018). First, Lux Fast and Acetic Acid were mixed and smoothed with 95% alcohol. After clarification and watering, the slices were placed in the prepared paint solution for 16 hours, and then washed with alcohol and water. Differentiation of tissues was performed with 0.05% lithium carbonate (for a few seconds) and 70% alcohol (20-30 seconds). The slices were washed in distilled water for 2 minutes and then examined under a light microscope. At this stage, the gray tissue of the brain did not stain and the white matter turned blue. The tissue color differentiation step was repeated and finally differential staining was performed with eosin for 20 minutes. In the next step, the tissues were placed in the environment to dry and then mounted with entanel adhesive. A light microscope with a magnification of 50 10 10 am was used to examine the texture.

2-10 Immunohistochemical study

Immunohistochemistry was used to evaluate the expression of MBP marker in corpus callosum and BDNF in hippocampus. MBP immunohistochemistry was performed by paraffin method. In order to identify, first the watering steps were performed on tissue sections and then the samples were exposed to the initial antibody of Bio orbyte company in England and were placed in a humid environment at a temperature of 4 degrees overnight. After washing with PBS (saline buffer solution) then with secondary antibody conjugated to Alexa red at 1: 200, it was placed at room temperature for 2 hours. In the next step, washing again with PBS (in the dark) was stained using DAPI cores. The samples were then dewatered again and fixed on the slide using mount adhesive(Hamano et al., 1996).

2-11 Evaluation of tumor necrosis factor-alpha (TNF-α) by ELISA

At the end of the study, after anesthesia, blood samples were taken from the mice using insulin syringes and the serum was separated by centrifugation at 3000 rpm for 5 minutes. To evaluate the serum levels of TNF-α, Zellbio kit made in France by ELISA method with ELISA reader made by EON company according to the instructions of its manufacturer was used. At the end of the test, ‌ The obtained wavelength, which indicates the concentration of TNF-α, was read and recorded by ELISA reader.

2-12 Statistical analysis

Data were analyzed using SPSS software. The results were reported as Mean±SEM. In order to evaluate the percentage of myelination between the control group and MS in the third week, independent t-test, statistical difference between the studied groups, one-way ANOVA test and Tukey test were used to determine the groups that differed from each other. Repeated measurement test was used to evaluate the balance situation in the zero, third and sixth weeks in the studied groups. In the water maze test, the animal's movement and behavior were tracked and controlled by an infrared TV camera located at a height of two meters above the central area of the tank. The digital TV signal enters the computer system that was connected to it and several variables such as: how long it takes the animal to find the Plexiglas platform, the length of the total distance of the mouse swimming path per training, the percentage of time the animal is in Each quarter passes through the tank and the movement speed of the mouse was measured and stored and analyzed with the help of software that was installed for this purpose. The level of significance in this study was considered P <0.05.

3-1 Weighing results in 8 weeks

The mean weight of mice at 8 weeks of treatment, after induction with cuprizone, was assessed. The mean weight of mice in different experimental groups showed that in sham group (healthy mice) the weight of mice increased with age and this increase was significant in the last three weeks compared to the first week (P <. 05). In the negative control group (cup) that received cuprizone but did not receive treatment, weight loss (from the first to the 8th week) was observed, which was not significant compared to the first week. In the case of healthy groups receiving Nigella sativa (NS), training test (EX) and their combination (NS+EX), the comparison of the two weights of the first and last week shows an increase that is statistically significant. The examination of the treatment groups showed that in the Cup+NS group, weight gain was observed from the first to the eighth week, which is statistically significant (P<.005). No weight change was observed in the Cup+EX group between the first and last two weeks. Also, in the Cup+NS+EX group, there was an increase in weight from the first week after the treatment to the eighth week, which is not significantly different from the Cup+NS group, but it is significantly different from the Cup+EX group (fig.1a).

3-2 Balance beam test results

The results of the Balance Beam Test show that the Balance Beam Test rating was zero in the group and the three groups of the NS, EX and NS+ EX, and no balance and motor disorder was observed in these animals. If the Copicizon group increased the score from the first to eighth week after the induction of dilation, and this increase was significant. In three therapies (CUP+NS, CUP+EX and CUP+NS+Ex) from the first to eighth week, Score reduction was observed, which is significant compared to the first week. Score reduction in three treatment groups (CUP+NS, CUP+EX and CUP+NS+Ex) showed a significant difference from the CUP group (P<0.001). Comparison between therapeutic groups (CUP+NS, CUP+EX and CUP+NS+EX) showed in two groups of CUP+NS and CUP+NS+EX compared to the CUP+EX group (P <.05) (Fig. 1b).

3-3 The effect of Nigella sativa and training test on mouse movement (Open-Field Test)

In experimental studies, the Open Field Test (OFT) is used to assess behavioral responses such as motor activity, hyperactivity, and inquisitive behavior, as well as to measure anxiety (Ghotbeddin et al., 2020). In order to check the amount and intensity of movement of mice in different groups and to check the effect of Nigella sativa and training test on it, different test groups were placed in the environment and the findings of the device were analyzed In the examination of the distance traveled by mice, the findings showed that comparing the sham group with the groups receiving Nigella sativa (NS), training test (EX) and combined (EX+NS), in distance Movement (a) and velocity (b) there is no significant difference. In demyelination groups receiving Nigella sativa (Cup+NS), training test (Cup+EX) and combined (Cup+NS+EX) there is a significant difference in distance Movement (Fig. 2a) and velocity (Fig. 2b) with the sham group )P<0.05). Comparison of the treatment groups showed that there is a significant difference between the Cup+EX group and the Cup+NS and Cup+NS+EX groups (P<0.05). Therefore, it seems that consumption of Nigella sativa extract and aerobic training alone and in combination, possibly due to the reduction of inflammation in the brain tissue, improves the motor symptoms of animals with MS.

3-4 Results from Morris water maze

The Morris water maze is one of the most common tests in neuroscience, developed by Morris et al. In 1982 to assess memory and spatial learning in rodents (Truski et al., 2020). This test demonstrates the effects of improving memory or forgetfulness of drugs, as well as the effects of genetic manipulations related to hippocampal function. In this test, the animal is placed in a pool of water and he must remember the location of the platform that is hidden just below the surface of the water, using the signs and symptoms that are located in the outer space. Due to the analysis of time data, the average time to reach the platform between all groups of Sham, EX, NS and NS+EX as well as therapeutic groups (CUP+NS, CUP+EX And CUP+NS+EX) from day 2 to the sixth day shows a significant decrease. There was no significant difference between the Sham, EX, NS and NS+EX groups. While the time elapsed from day one to sixth day, showed a significant decline. CUP+NS, CUP+EX and CUP+NS+EX also have less time than the CUP group, and this difference is significant (p≤0.01). The comparison of therapeutic groups also showed that the CUP+NS and CUP+NS+EX groups have spent less time than the CUP+EX group (p≤0.05) (Fig. 3a).

To examine the spatial memory of animals, the total distance traveled to reach the platform in the water maze test is measured. Then, the mean of the treated groups and the control group are compared and analyzed by statistical methods (Moradbeygi et al., 2021). According to the analysis of the distance data, the average distance traveled to reach the platform between the sham groups and the three groups receiving NS, EX and NS+ EX from the first day to the sixth day shows a significant decrease (P≤0.01). Also, a comparison between the groups receiving Cuprizon and Cup+NS, Cup+EX and Cup+NS+EX receiving a decrease in the average distance traveled from the first to the sixth day was observed and the results indicate a significant difference between the Cup and groups. NS and Cup+NS+EX are with the Cup+EX group, while there was no difference between the two groups Cup+NS and Cup+NS+EX. It can be concluded that the motility of the animals has probably improved due to the reduction of inflammation as well as the improvement of myelination status(Fig. 3b).

In the recall test, the time taken for the mouse to cross the platform for the first time and the swimming speed are measured. Then, the mean of each of the mentioned parameters is compared between the treated groups and the control group and is analyzed by statistical methods for conclusion (Hadi et al., 2018). According to the data analysis, the movement speed spent to reach the platform between the sham groups and the three groups receiving NS, EX and NS+ EX from the first day to the sixth day does not show a significant change (P≤0.01). Also, the comparison between Cup+NS, Cup+EX and Cup+NS+EX treatment groups shows an increase in the average distance traveled compared to the untreated Cuprizone group, which is a significant difference, but the comparison between groups Cup+NS, Cup+NS+EX and Cup+EX No differences were found. It can be concluded that the motility of the animals has probably improved due to the reduction of inflammation as well as the improvement of myelination status(Fig. 3c).

3-7 The effect of training and Nigella sativa test on MBP in corpus callosum

Fluorescent immunohistochemistry (IHC-Fr) is used to evaluate the level of MBP in the corpus callosum of brain tissue (Moradbeygi et al., 2021). Fig 4 shows the effect of training and Nigella sativa test separately and in combination on the MBP of corpus callosum in different treated groups(fig. 4a). The results were reviewed in three sections. First, statistical analysis using one-way ANOVA showed a significant difference between the studied groups (P<0.0001) then Tukey's post hoc test to investigate the statistical difference between the treatment groups and MS and control groups and also for Statistical differences were used to evaluate the three treatment regimens. The results showed a significant difference between all three treatment regimens and the Cup group (P<0.001). As the fig 4 below shows, the percentage of MBP in the Cup+EX, Cup+NS and Cup+NS+EX groups increased compared to the Cup group at the end of the study. Comparing the treatment groups with the sham group, a statistically significant difference was observed between the Nigella sativa group (P<0.01) and therapeutic group (P<0.01) with the sham group. Finally, in comparing all three treatments, there was a statistically significant difference between Cup+EX and Cup+NS (P<0.05) and Cup+NS+EX (P<0.01) It was seen to indicate the greater effectiveness of the combined diet on MBP (Fig. 4d). Considering the damage of MBP in MS, it can be said that the use of a combination diet with protection of MBP against inflammatory processes has a role in controlling demyelination due to MS.

3-8 The effect of training and Nigella sativa test on BDNF level in The hippocampus

Fluorescent immunohistochemistry (IHC-Fr) is used to evaluate the level of BDNF in the The hippocampus of the brain tissue (Moradbeygi et al., 2021). Fig 4 shows the effect of training and Nigella sativa test separately and in combination on the amount of hippocampus BDNF in different treated groups(fig.4b). As this graph shows, there is a significant difference between the groups with one-way ANOVA analysis (P<0.001). In Tukey's post hoc, a significant difference was observed between the treatment groups and the Cup group (P<0.001). After 8 weeks, BDNF levels increased in the training test group (37.69±2.04), the Nigella sativa group (34.96±1.84) and the combined group (43.17±3.2). In comparison with the treated groups, there is a significant difference between the Nigella sativa group and the other two groups (P<0.05) (Fig. 4c). Considering the role of BDNF in the process of regeneration and reduction of symptoms, it can be concluded that aerobic training and Nigella sativa alone and in combination are effective in improving the demyelination lesions caused by MS by increasing the level of BDNF.

3-9 Investigation of demineralization in corpus callosum using Luxol Fast Blue

Luxol fast blue specific myelin tissue staining is used to evaluate axon repair and regeneration and the amount of remaining areas is determined by software using differential staining (eosin) (Sanadgol et al., 2018). Lux fast Blue stains the areas with the most axonal damage red and the myelin areas blue, which is easily recognizable. In this way, the affected areas and in addition the remaining tissue after treatment was measured with image J software and the results are as follows.

Microscopic examination of tissue sections in the experimental groups with fast blue luxury staining showed that the greatest amount of damage was concentrated in the white matter, especially in the corpus callosum(Fig. 5a). In the Cuprizone group, the most damage was done so that the affected areas were more than the whole tissue and this difference was significant compared to other groups (P<0.05). In the four groups, Sham, NS, EX and NS+EX, the least amount of damage was observed, but there was no significant difference between these four groups, but there was a significant difference between these groups and the Cuprizone group. In the study of treatment groups, Cup+EX showed the highest amount of damage and this difference with the two treatment groups Cup+NS and Cup+NS+EX was significant. Also, this damage is less compared to cuprizone and this difference is significant. Among the treatment groups, the lowest rate of injury is related to the Cup+NS+EX group, which is a significant difference between the other two treatment groups and with Cuprizone (Fig. 5b). This finding confirms that Nigella sativa and training testing repair damaged areas.

3-10 the effect of training and Nigella sativa test on TNF-α level In the blood serum

The results showed a significant decrease in TNF-α between the treatment groups and the Cup group. The diagram shows the effect of training and Nigella sativa test separately and in combination on serum TNF-α levels in different treated groups. As this chart shows, there is a significant difference between the studied groups with one-way ANOVA analysis (P<0.001). In Tukey's post hoc, a significant difference was observed between the treatment groups and the Cup group (P<0.001). After 8 weeks, TNF-α level decreased in the training test group (329.25±9.57), the Nigella sativa group (325.87±9.55) and the combined group (313.62±6.67). In the comparison of treated groups, there is no significant difference between the Cup+NS group and the Cup+EX group (P<0.194), but there is a significant difference between these two groups and the Cup+NS+EX group (P<0.037). Therefore, it seems that Nigella sativa extract in the combined group helps to reduce TNF-α by reducing oxidative stress caused by aerobic training.

In this study, the effectiveness of aerobic training along with the consumption of Nigella sativa extract on myelination, histological changes and the amount of motor activity in the corpus callosum and hippocampus for an animal model of demyelination using CUP administration was investigated. The results of examining the animal's motor activity from the OFT behavioral test, memory and spatial learning from the Morris water maze and evaluating the balance with the BBT test confirmed our tissue observations in this research and showed that aerobic exercise significantly improves motor deficits in animal models of MS. These results are in line with the study (Mandolesi et al., 2019) which showed that training can improve clinical scores in mice suffering from demyelination. Moradbeigi et al. (2021), in a study, showed that the induction of the cuprizone model reduces the balance and motor activity of animals compared to the control group, and after starting the treatment regimen with Nigella sativa extract, the results of OFT and BBT tests were different. They showed a statistically significant difference between the treated groups and the healthy group, which indicates the effect of this treatment regimen on the amount of movement activity and the balance status of the animals and is consistent with the results of the present study (Moradbeygi et al., 2021). Studies have shown that training is associated with significant changes in brain anatomy in terms of size and weight, hippocampal neurogenesis and synaptogenesis and synaptic plasticity (Patten et al., 2013) in rodents. According to the findings of previous studies, the presence of defects in the balance of animals indicates the early stages of EAE (Luong et al., 2011), which provides valuable information about tail function and control of the descending motor system in animals under the EAE model. Therefore, it can be concluded that aerobic training alone and together with Nigella sativa extract probably improves the balance and motor activity level of animals due to the reduction of inflammation and the improvement of the remyelination process. These structural changes in the brain anatomy of C57BL/6 male mice, which show histological changes and in line with its behavioral function, can have a new path for faster diagnosis and prevention of debilitating symptoms. Regarding the exact mechanisms of these changes, more experimental studies and complementary behavioral tests are needed.

Regarding the effect of Nigella sativa on the severity of the symptoms of animals with MS, the study by Fahmy et al. in 2014 showed that the use of oral Nigella sativa 2 weeks before the induction of the EAE model increases the animal's motor activity and reduces anxiety(Noor et al., 2015). Considering that past studies have shown the inflammatory effects of various compounds of Nigella sativa (aqueous extract, oil extract, etc.), it can be concluded that probably due to the reduction of inflammation in the brain tissue, the motor symptoms of animals with MS also It improves. The results of the present study are in line with the results of previous studies, and the increase in the quality of motor activity of the groups under study can be considered to be in line with the decrease in inflammation. In the microscopic examination of the tissue sections in the tested groups with Luxal Fast Blue staining, the highest amount of damage was concentrated in the white matter, especially in the corpus callosum. The highest amount of damage was seen in the Cup group, in such a way that the damaged areas were more than the whole tissue and this difference was significant compared to other groups.

Myelin cells or so-called oligodendrocytes are the main reason for the onset of the disease. The result of CNS demyelination is associated with impaired nerve conduction and reduced motor and cognitive abilities (Mokhtarzade et al., 2017). In MS, plaques form a coating on the myelin of nerve fibers. When myelin is destroyed as a result of plaque formation, nerve fiber conduction is reduced or lost. This phenomenon, i.e. demyelination, causes the interruption of nerve messages sent from the brain (Chiaravalloti & DeLuca, 2008). Regarding the role of immune system attacks against myelin axons of the CNS and as a result of demyelination and degeneration caused by it, it can be briefly said that following the attack of the immune system, the infiltration of inflammatory cells occurs in the CNS, which results in the death of oligodendrocytes, activation of microglia and astrocytes. And finally axonal degeneration will happen. Astrocytes play a role in multiple and complex actions, including the production of pro- and anti-inflammatory cytokines (Noor et al., 2015). Therefore, it is considered that the damage of oligodendrocytes and demyelination occurs as a result of CNS inflammation (Landers et al., 2014). The process of demyelination and remyelination through different stages with several complex mechanisms of differentiation of oligodendrocyte progenitor cells to mature myelinating oligodendrocytes are dependent (Bercury and Macklin, 2015). In the CNS, the activation of microglia and astrocytosis, which are important components of the lesion environment, play a role in affecting demyelination/remyelination processes during the disease (Gudi et al., 2014a). Among the treatment groups, the lowest amount of damage is related to the Cup + NS + EX group, which is significant both with the other two treatment groups and with the Cup group. Overall, studies show that some oligodendrocyte progenitor cells are lost between peak disease and chronic disease, and remyelination of axons is reduced. In addition, training can increase the proliferation, migration and differentiation of oligodendrocyte progenitor cells (Ehninger et al., 2011). In a study, Watanabe et al. showed that in demyelinated models with remyelination, there is a relationship between the decrease in the number of oligodendrocyte progenitor cells and myelin repair (Watanabe et al., 2002). In line with the results of the present study, Jensen et al., in the study of the effect of training on oligodendrogenesis in central myelination, showed an increase in the number of oligodendrocytes with training and stated that the regeneration of oligodendrocytes as a result of training leads to an increase in remyelination (Jensen et al., 2018). Also, Mandolesi et al., in a study by investigating the effect of training in the cuprizone model, concluded that training causes oligodendrocyte regeneration in the cuprizone demyelinating model (Mandolesi et al., 2019), which is consistent with the results of the present study. The result of a study showed that the hydroalcoholic extract of Nigella sativa has a significant ability to prevent hippocampus nerve damage and prevent learning and memory disorders in mice )Javidi et al., 2016(. This finding confirms that Nigella sativa and training restore the damaged areas. From the obtained results, it can be concluded that Nigella sativa oral extract and aerobic training can be effective in reducing the inflammation and severity of multiple sclerosis symptoms by increasing the number of oligodendrocytes and axonal remyelination.

In examining the effectiveness of aerobic training along with the consumption of Nigella sativa extract on BDNF, a significant difference was observed between the treatment groups and the Cup group. In a study titled "Effect of Training on BDNF Serum Level in Patients with Progressive Multiple Sclerosis", Sonbreken et al showed that during acute training, even in disabled patients with progressive MS, BDNF is highly expressed(Wootla et al., 2016). BDNF is believed to play a role in the neuroprotective effects of training in MS. BDNF plays an important role in synaptic flexibility, differentiation and survival of neurons and has beneficial effects on the functioning of the central nervous system in animal models (Jørgensen et al., 2019). Several animal studies have shown that the neuroprotective and inhibitory potential of training is associated with the induction of neurogenesis in the hippocampus, an important brain structure for learning and memory. Studies have shown that training leads to the positive regulation of BDNF and this can help the health of the animal's brain. It has also been shown that serum BDNF levels increased immediately after a session of light aerobic training in people with MS (Gold et al., 2003). BDNF is widely expressed through the CNS and plays a role in the maintenance and repair of the CNS and regulates and controls the expression of inflammatory cytokines in the CNS (Gold et al., 2003).

Mohammadi Rad et al reported in a study that there are interactions between neurotrophic and inflammatory factors in the brain that play an important role in the process of MS. Also, the cause of inflammatory symptoms in MS can be the existence of an inverse relationship between the amount of TNF-α and BDNF in the brain tissue and serum of cuprizone model mice, which is in line with the results of the present study (Mohammadi-Rad et al., 2019). The results of this study show a significant decrease in TNF-α between treatment groups and the Cup group. TNF-α is one of the most important pro-inflammatory cytokines that can activate immune-mediated inflammation in the central nervous system, and as a pro-inflammatory cytokine, it is the main mediator in the defense, development and maintenance of the immune system. And its level increases in all types of CNS disorders such as MS by secretion from autoreactive T cells, microglia and astrocytes, which can show both neuroprotective and neurodegenerative properties and significantly change BDNF levels (VonDran et al., 2011). The secretion of BDNF improves the regeneration of different tissues and it has been shown that the level of this protein may decrease in people with MS. It has been reported that BDNF can regulate the release of pro-inflammatory cytokines such as TNF-α, modulate the expression of cytokines from pro-inflammatory to anti-inflammatory and block the harmful effects of TNF-α on CNS cells during inflammation. To suppress the formation of new lesions, play a protective role (VonDran et al., 2011).

The increase of TNF-α during demyelination causes oligodendrocyte and myelin damage, as well as the removal of damaged myelin sheaths to restart myelination (Rowhani-Rad & Taherianfard, 2019). TNF-α acts as an immunomodulatory cytokine through its two receptors on astrocytes. TNFR1 mainly mediates inflammation, pathological processes and pro-apoptotic functions that are important for MS initiation, and TNFR2 induces cell survival, resolution of inflammation, immunity and proliferation of oligodendrocyte progenitor cells and remyelination (Domingues et al., 2016). Studies have shown that the key components in the inflammatory process are inflammatory cytokines and mediators (Germano et al., 2008). Several studies showed that Nigella sativa oil contains potent and non-toxic compounds that suppress pro-inflammatory cytokines (Noor et al., 2015). Thymoquinone (TQ) is the active substance of Nigella sativa and many studies have investigated its effect on neurological diseases such as Parkinson's, anxiety and memory disorders. It has been experimentally proven that TQ, having antioxidant, anti-inflammatory and anti-apoptotic properties, protects brain cells against all kinds of injuries (Beheshti et al., 2018; Gilhotra & Dhingra, 2011). It has also been reported that TQ inhibits the production of TNF-α. According to the evidence presented and the results of the present study, the beneficial effects of Nigella sativa plant extract on MS can be related to its anti-inflammatory properties (Anaeigoudari et al., 2018).

In the present study, remyelination was observed in the groups treated with Nigella sativa in the histological examination of the corpus callosum. Therefore, the protective and therapeutic effects of Nigella sativa administration on CUP-induced mice in the current study can be due to the anti-inflammatory role of its components, by suppressing pro-inflammatory cytokines (Noor et al., 2015). Studies have shown that one of the main obstacles that prevent axon regeneration after CNS damage is glial scar formation. The proliferation of astrocytes around the lesion site leads to astrocytic hypertrophy and the expression of inhibitory molecules that prevent axon regeneration (Silver et al., 2004). In addition, the absorption of peripheral macrophages from the bloodstream and the activation of microglia play a role in the formation of glial scars and are considered as an important obstacle for the regeneration of axons (Busch et al., 2010).

In the pathological examination of the present research, it was observed that the consumption of Nigella sativa leads to the restoration of myelin in CUP model mice. In confirmation of these observations, Banu et al reported that N.sativa is useful as a potential treatment for neurodegeneration after chronic exposure to toluene in mice. Therefore, it can be suggested that the remyelination power of N.sativa in CUP-induced mice may be due to its inhibitory effect on the expression of astrocytes and microglial cells. As a result, Nigella sativa seeds can be a promising effective agent in the protection and treatment of EAE.

Oxidative stress has been shown in several studies to be a major component in the pathogenesis of MS (Zheng et al., 2010). Oxidative stress is produced by excessive production of ROS by activated microglia/macrophages and astrocytes and leads to tissue damage (Noor et al., 2015). The results of this research showed a decrease in damage in the CUP + EX + NS group compared to the CUP + EX and CUP + NS groups. Therefore, it is possible that N.sativa, having anti-inflammatory properties and suppressing pro-inflammatory cytokines, can reduce the oxidative stress caused by training and help the demyelination of axons.

The study of the effect of aerobic training along with the consumption of N.sativa extract on MBP, BDNF and TNF-a in the cuprizone model of MS in C57BL/6 male mice and the behavioral analysis of the effect of demyelination on brain structures showed that the induction of MS causes changes Brain tissue is located in the hippocampus and corpus callosum areas. These changes are accompanied by cell death, destruction of axons and myelin, induction of gliosis. Behavioral findings show that tissue and morphometric disorders lead to cognitive and memory disorders as well as movement and balance disorders. Aerobic training and Nigella sativa extract alone and in combination can be effective in treating and preventing the exacerbation of symptoms in people suffering from this disease. In the three treated groups, the obtained results showed an improvement in motor activity and also the balance status of the treated groups compared to the CUP group, which according to the results of immunohistochemistry, Luxol Fast Blue and clinical assessment, it can be concluded that it is probably due to a decrease The level of inflammation as well as the improvement of myelination status, balance and the level of movement activity of the animals have also improved. These structural changes in the brain of C57BL/6 male mice, which show histological changes and in line with its behavioral function, can have a new path for faster diagnosis and prevention of debilitating MS symptoms.

CNS: Central Nervous system

BDNF: Brain derived neurotrophic factor

EAE: Experimental autoimmune encephalomyelitis

LFB: Luxol fast blue

MBP: myelin basic protein

CUP: cuprizone

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There is no conflict of interest, and all authors support the submission to this journal.

Consent for publication:

All authors support the publication of the manuscript in this journal.

Availability of data and materials:

Data and materials are made available to individuals upon request from the corresponding author.

Ethical approval:

All experiments were performed under, Lorestan University Animal Ethics Committee (under the code LU.ACRA.2021058) and according to the NIH Guidelines for the Care and Use of Laboratory Animals.

competing interest

The authors have no affiliation or involvement with any organization or institution with any financial or non-financial interest in the topic or content discussed in this manuscript.

Funding:

No organization or institution has provided financial support for this research project.

Author contributions

Aziz zinvand lorestani and Rahim Mirnasouri designed this study; Aziz zinvand lorestani, Rahim Mirnasouri, Masoud Rahmati and Marzieh Darvishi performed research and wrote the paper. Aziz zinvand lorestani, Rahim Mirnasouri and Masoud Rahmati, Marzieh Darvishianalyzed the data. Final approval of the version to be published: all authors.

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Aerobic training with consumption of Nigella sativa extract ameliorate myelin core protein, axonal regeneration, and cognitive function in a mouse model of multiple sclerosis

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