Saffron (Crocus Sativus L.), Combined with Endurance Exercise, Synergistically Enhances BDNF, Serotonin, and NT-3 in Wistar Rats

Background: Evidence indicates that combined approaches based on exercise and nutrition benefit neural development. We aimed to determine the effect of saffron and endurance training on hippocampus neurogenic factors, neurotrophin-3 gene expression in soleus muscle, and short-term memory in Wistar rats. Methods: The study analyzed four groups of ten rats each: control, exercise, saffron, and saffron plus exercise. The rats in the exercise groups were trained on a rodent motor-driven treadmill. All rats were gavage daily with either saffron extract (40 mg/kg) or water. After eight weeks of intervention all rats were evaluated using the novel object recognition (NOR) test. Blood and tissue samples were collected to measure proteins and neurotrophin-3 gene expression. Results: Rats that received saffron treatment combined with exercise had significantly greater brain-derived neurotrophic factor (BDNF) and serotonin in hippocampus compared to the control and saffron-only-treated rats (p< 0.05). Neurotrophin-3 mRNA in soleus muscle was higher in the saffron plus exercise group than rats in the other three groups (p< 0.05). Hippocampus 5-hydroxyindolacetic acid and short-term memory were significantly greater in all the intervention groups than in the control group (p< 0.05). Conclusions: Saffron, combined with endurance exercise, synergistically increased hippocampus BDNF, serotonin, and muscular neurotrophin-3 mRNA in Wistar rats.


Introduction
Studies have revealed that exercise improves neurogenesis and cognitive function (1). Neurogenesis has been linked to exercise through up-regulation of neurotrophins to a great extent in the hippocampal denate gyrus. The hippocampus is a vital region for memory consolidation and learning (2). Therefore, new-born neurons integrate into the hippocampal circuitry and consequently benefit hippocampal-dependent learning and memory tasks (3).
One important neurotrophin is brain-derived neurotrophic factor (BDNF), which appears to be regulated by physical activity. This factor is a member of the nerve growth factors family with about 70-80% of circulating BDNF secreted from the brain (4). Research has shown aerobic exercise elevates BDNF gene (BDNF) expression in the hippocampus, which results in dendritic complexity, synaptic plasticity, and enhanced memory and learning (5).
Serotonin (5-hydroxytryptamine; 5-HT) has also been shown to play a critical regulatory role in hippocampal neurogenesis (6). Accumulating evidence indicates that serotonin production and metabolism during or after exercise increase in various regions of the brain including hippocampus, amygdala, basal ganglia, and cortex, which in turn ameliorates depression (7,8). Further studies have shown that the denate gyrus is rich in serotonergic fibers which, in response to exercise, modulate neurogenesis (6). Insulin-like growth factor-1 (IGF-1) is another neurotrophic factor that has been shown to regulate adult brain neurogenesis (9). Exercise increases the availability of this growth factor, which enhances neurogenesis and improves brain function (10). BDNF, serotonin, and IGF-1 have been shown to activate similar signaling cascades that may act synergistically in neuro-homeostasis and neurogenesis (11).
Moreover, evidence indicates that combined approaches based on exercise and nutritional management can benefit neural development. Brain function undoubtedly requires adequate nutrition. Thus, variations in the composition and quantity of nutrient consumption likely influence cognitive function. Nutricuetical factors provide the essential material for the brain to create and maintain connections critical for neural plasticity and brain function (12). Studies show growing interest in the potential beneficial effects of polyphenols on brain health. Polyphenols, which have strong antioxidant properties, are micronutrients in plant-originated food. Food and beverages including cocoa, coffee, brightlycolored fruits and vegetables, and tea are the major dietary sources of polyphenols. It has been suggested that their neuro-protective effects occur via reducing neuro-inflammation and they enhance cognitive function and promote memory and learning (13).
Crocus sativus L., commonly known as saffron, is a member of the Iridaceae family, which is rich in carotenoids including crocin, crocetin, safranal, and picrocrocin, and flavonoids including quercetin, kaempferol, and naringenin (14,15). In traditional medicine, saffron has been used to treat fever and asthma, and as an analgesic, expectorant, sedative, and mood stabilizer (16). Recent pharmacological studies have shown that saffron increased related brain neurotransmitters in animal models of depression (17) and the possible mechanism has been shown to occur via BDNF and VGF neuropeptide upregulation in the hippocampus (18). However, due to the limitations in human trials, it is not yet known whether saffron can directly induce neurodevelopmental biomarkers to enhance memory. Thus, by relying on the positive effects of exercise on neurogenesis and memory we aimed to determine the effect of saffron and endurance training individually and combined on hippocampus neurogenic factors and short-term memory in Wistar rats.
We also aimed to evaluate the effect of saffron combined with exercise on neurotrophin-3 (NT-3) mRNA level in soleus muscle. NT-3 is an important autocrine neurotrophic factor that stimulates differentiation and neuron outgrowth in central and peripheral nerves in target tissues such as skeletal muscle (19). Studies have shown that voluntary exercise in normal rate increases NT-3 mRNA expression in soleus muscle (20); however, the role of saffron in inducing this neurotrophic factor alone or synergistically with exercise has not yet been studied.

Saffron extract preparation
The saffron used in this study was provided by Saharkhiz Co. (Mashhad, Iran). Its herbarium code is 829-537-06. A hydroalcoholic form was prepared by percolation. Briefly, in an ultrasonic bath each gram of the air-dried stigma was extracted with 33 ml of an 80% ethanol solution. The extract was filtered, isolated on a percolation rotary evaporator, and stored at -20 ºC until use. The dosage of saffron extract was 40 mg/kg which was dissolved in 1.5 ml water according to a previous study (21).

Animals
Forty male Wistar rats with a mean weight of 198 ± 8.20 g at the beginning of the study were provided by the animal house of Iran University of Medical Sciences and 10 rats were randomly allocated to each of four groups: Exercise, Saffron, Exercise plus Saffron, and Control. The animals were maintained in stainless cages with four animals per cage and kept in a standard atmosphere. The room temperature was 22 ± 2 ºC, humidity was 50 ± 5% with a twelve-hour light-dark cycle (8:00-20:00). Food (pellet) and water (specified water bottles) were available at all times. The rats were acclimated to the location for one week prior to intervention. All animals were gavaged with either water as control or saffron extract (40 mg/kg) with the same volume for eight weeks (21). The animals were weighed weekly to maintain the intervention dosage during the trial. All animal experimental approaches were proved and applied by the Animal Care and Use Committee (ACUC) at IUMS, which was in accordance with the NIH guidelines (22). The ethical code registered for this study was IR.IUMS.FMD.REC1396.9223475203.

Endurance training
Endurance training was performed on a treadmill specified for rats. The Exercise and Exercise + Saffron groups were acclimatized for three days on the treadmill for 10 minutes at a speed of 8 m/min. The exercise was performed five days per week (Saturday through Wednesday) at 11:00-13:00 for eight weeks. During the eight weeks the exercise time and intensity were increased progressively. Sixty percent of VO2Max was considered as the initial speed of training. This speed was calculated by a gradually increasing ramp protocol. Accordingly, the starting running speed was 8 m/min, then the speed was increased every two min by 2m/min. Finally, VO2Max was considered at the speed the rats were unable to maintain even by mild taping on their tails for one min. The mean VO2Max was computed at approximately 20 m/min. According to the VO2Max, the training regimen was designed in week: 20 m/min, 45 min. The rats were warmed up for five min at 8m/min before each exercise session. A hard sponge was placed at the end part of the treadmill with no shocker bars connected. In addition, to limit stress on the rats, which would adversely affect the results, the rats' tails were gently tapped.

Blood and tissue sample collection
To prevent the metabolic effects of the final run, 48 h after the last exercise session all animals were anaesthetized with xylazine (10 mg/kg) and ketamine (120 mg/kg). Next, blood was collected via cardiac puncture, plasma was extracted, and samples were stored at -80 ºC. Then, all the animals were euthanized through spinal dislocation to limit suffering immediately after blood collection. Finally, in less than four min, the soleus muscles from both legs and hippocampus from both sides of the brain were dissected, frozen in liquid nitrogen, and stored at -80 ºC for further protein and genetic analysis.
The mRNA expression was evaluated by PCR Master Mix (Takara, Japan). Expression for all samples was measured twice. Each sample included 0.5 µL of each primer, 1 µL of the DNA, 7.5 µL of SYBR Green, 0.3 µL of Rox, and 5.2 µL of RNasefree water. An ABI PRISM (7700 Sequence Detection System, Applied Biosystems) was used for the Real-time PCR through the following procedure: 2 min of heating at 94 ºC followed by 45 cycles of denaturation at 94º C for 30 sec and annealing and extension at 61º C for 30 sec.

Novel object recognition test
The hippocampus is an important brain region for spatial and recognition memory (23). Therefore, we evaluated the effect of saffron and endurance training either individually or combined on the rats' short-term memories by the novel object recognition (NOR) test. This test is based on the rat's natural exploratory behavior. On day one, the rats were habituated to the testing box for one h with no objects. After 24 hours, they were exposed for three minutes to two identical objects set in opposite corners of the testing box. One h later, they were exposed to one familiar object as before and one new object placed in the corners of the box as before for three minutes (Fig. 2A). In the current study, "exploration" was defined as the movement of the snout towards the object, or touching or sniffing with the snout. Sitting on the object, climbing on, or running around it, were not considered exploration. The novel and familiar objects differed in color, size, and shape, which contributed to recognizing the novel object. After each testing session the box and objects where cleaned with 10% ethanol to remove odor cues. To analyze the test, we used the Discrimination Index (DI), which is used to differentiate the exploration time between the familiar and novel objects (24).

Statistics
The data was analyzed using SPSS 24 (IBM, Chicago, IL, USA). The Shapiro-Wilk test was used to evaluate the normal distribution of the data. ANOVA and Post-Hoc LSD test were used to analyze normally-distributed data. The Mann-Whitney U-test was used to analyze the genomic factor NT-3. The threshold cycles (Cts) attained for NT-3 and β-actin were used for the genomic analysis. Quantitative gene expression was obtained by measuring 2 -ΔΔCt for each sample (ΔCt = Ct NT3 -Ct β Actin). In this study P values less than 0.05 were considered statistically significant.

Saffron analysis
The total hydroalcoholic extract of saffron was 56%. The flavonoid content of the extract considered as equivalent to rutin was 65.41 ± 0.89 mg/g, and the phenol content considered as equivalent to gallic acid was 60.07 ± 1.90 mg/g.

BDNF,
IGF-1, Serotonin, 5HIAA, 5HIAA/Serotonin Table 1 shows the brain biomarker concentrations after the various interventions. Plasma BDNF concentration of Exercise + Saffron group was significantly greater than those of the other groups. BDNF in hippocampus was significantly higher in the Exercise + Saffron group compared to Control and Saffron treated rats. No significant difference in IGF-1 concentration was detected between the groups. The serotonin concentration was significantly greater in the Exercise + Saffron group than in the Control and Saffron treated groups. 5HIAA was significantly greater in the Exercise, Saffron, and Exercise + Saffron groups than in the Control group. The 5HIAA/serotonin ratio was significantly greater in the Exercise and Saffron groups than in the Control group; however, no significant difference in this ratio was seen between the Exercise + Saffron and the other groups. Data represented as Mean ± SD *p< 0.05 representative of a significant level by comparing between-group differences using ANOVA. Significant p value by comparing within-group differences using Post-hoc LSD test presented as a: compared to the Control group; b: compared to the Exercised group c: compared to the saffron-treated group.

Real-time q-PCR
The Kruskal-Wallis test indicated a significant difference in NT-3 mRNA expression between four groups (p< 0.001). According to the Mann-Whitney U test both the Exercise and Saffron groups expressed significantly more NT-3 mRNA than the Control group, while the Exercise + Saffron group expressed significantly more NT-3 mRNA than all the other groups (p< 0.01) (Fig. 1).

Novel object recognition test
All rats were habituated to the atmosphere of the novel object box for one h one day before the main test. In the main test two rats from the Control group, one from the Exercise group, two from the Saffron group, and one from the Exercise + Saffron group were excluded due to no exploring movements. The rats in the three treatment groups spent more time than the Control group exploring the novel vs. the familiar object (Fig. 2B). The discrimination index shows this difference was statistically significant (Fig. 2C).

Fig. 2.
Novel object recognition test: (A) Schematic illustration of the object recognition test. Rats were first exposed to an identical pair of objects. After 1 h, they were exposed to the familiar object (FO) and a novel object (NO). (B) Time (s) spent exploring the familiar and novel objects in the second part of the test. (C) The Exercise, Saffron, and Exercise + Saffron groups all spent significantly more time on the novel object than the familiar object compared with the Control group, as determined by the discrimination index. Bars represent means (SEM). * p< 0.05.

Discussion
In this study saffron combined with endurance training increased BDNF protein concentration in hippocampus and plasma. Previous studies mainly studied saffron anti-depressant effect, emphasizing its role in BDNF elevation (18). Because BDNF plays an important role in augmenting synaptic plasticity and consequently improving memory (25), we further assessed the effect of the interventions on rat short-term memory using the NOR test. We found that Saffron, Exercise, and Exercise + Saffron all improved performance on the test. Previous studies showed that aerobic training increases BDNF and consequently improves spatial memory (26)(27)(28). However, the effect of saffron on spatial memory has been less studied. Crocin has been reported to improve cognition and memory in Alzheimer's disease-induced mice (29) and also improve rat memory and learning (30). The cellular and molecular action of BDNF on neurogenesis and consequently on memory is initiated by the tropomyosin-related kinase B (TrKB) receptor, a member of the tyrosine kinase receptor family. The interface between BDNF and the receptor results in tyrosine phosphorylation in the tyrosine kinase domain, and subsequent adaptor protein connection. Finally, these adaptors trigger the ras/mitogen-activated protein kinase (Ras-MAPK) pathway, resulting in neuronal proliferation and differentiation (32).
In our study, hippocampal IGF-1 was greater in the Exercise + Saffron group than in the other groups, but the difference was not significant. Similarly, Gaeini et al. showed that saffron combined with resistance training for six weeks had no effect on IGF-1 serum levels (33); however, evidence is lacking on the effect of saffron on hippocampal IGF-1 levels. A strong association has been shown between musculoskeletal mass and IGF-1; therefore, muscle enlargement during resistance training could increase IGF-1 levels (33); in contrast, in our study aerobic training was employed.
In our study, serotonin concentration in the hippocampus was greater in the Exercise + Saffron group compared to Control and saffron treated rats. During physical exercise serotonin is released from projections in the hippocampus, which affects serotonin receptors and triggers the onset of brain neurogenesis (6). Further, serotonin is a neurotransmitter involved in maintaining memory (34). However, in the Exercise and saffron-only-treated groups, serotonin showed no significant increase even though the rats improved significantly in the NOR test; this could be due to the high metabolism of serotonin. In addition, serotonin metabolism (5HIAA/serotonin) increased in the exercisetrained and saffron-treated groups. Because serotonin is an unstable neurotransmitter catabolized by monoamine oxidases (MAOs) to its metabolite, it is interpreted that saffron and exercise may have increased the main pool of serotonin individually, but its metabolism prevented its maintenance. However, in the Exercise + Saffron group, the main pool of serotonin was significantly greater, while serotonin metabolism was less. This could be due to the potency of saffron to inhibit serotonin reuptake and maintain it at high levels in the brain (35). In light of the positive effects of saffron and exercise on memory in this study, it seems that one possible mechanism could be mediated by BDNF and serotonin-stimulating neurogenic effects.
Our study is the first to evaluate the effect of saffron alone or combined with exercise on NT-3 in soleus muscle. We found that while both saffron and training increased NT-3 mRNA expression individually, when combined, this effect was even greater. NT-3 is produced by neurons, which play an essential role in the plasticity of the central nervous system and regeneration in spinal cord and muscle (19). Exercise could increase expression of NT-3 and its receptor, tyrosine kinase C (Trk C) in soleus muscle (20). Sharma et al. reported that moderateintensity aerobic training induced NT-3 in skeletal muscle and also reduced muscle pain (36). However, no study has yet evaluated the effect of saffron on NT-3. The antioxidant and antiinflammatory properties of saffron may have prevented muscle damage due to exercise (37) and provided an appropriate medium for the regeneration of the neuromuscular region.
This study showed that saffron extract combined with endurance exercise increases hippocampus and plasma BDNF, hippocampus serotonin, and muscular NT-3 gene expression. All three interventions increased hippocampus 5HIAA and improved NOR test results compared to control. However, no significant effect was observed on hippocampal IGF-1 in this study.
Our study has limitations as follows: this was an experimental study accomplished in rats; results may differ in humans, and limited neurotrophins were evaluated in the tissues, which could be expanded in future studies.