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Luteolin is a flavonoid contained in many plants and with a variety of known pharmacological properties such as anti-inflammatory, anti-anxiety, and memory-improving effects, suggesting that LUT penetrates into the brain. We investigated the effects of LUT on the behaviors of chronic unpredictable mild stress (CUMS) induced depression model mice and to elucidate the related possible mechanisms. The results showed that CUMS significantly decreased the levels of serum 5-HT and NE, and the activities of superoxide dismutase (SOD) and catalase (CAT), with an increase in lipid peroxidation level as well as a reduction in glutathione (GSH) level and an elevation in the production of malondialdehyde (MDA) in hippocampus. CUMS also reduced open-field activity, sucrose consumption, and increased immobility duration in FST and TST. LUT administration could effectively reverse the alterations in the concentrations of NE and 5-HT, lessen the level of MDA, elevate the activities of SOD and CAT, and the level of GSH, and inhibit lipid peroxidation. Moreover, LUT could effectively reverse alterations in immobility duration, sucrose consumption and open-field activity. In conclusion, LUT administration has exhibited significant antidepressant-like effects in mice with CUMS-induced depression, which might be related to the alteration of monoaminergic response and antioxidant defenses.
Key words: Luteolin, chronic unpredictable mild stress, hippocampus, Monoaminergic neurotransmission, antioxidant
Depression is a common disorder with high morbidity and mortality(Schechter LE et al.,2005). Affective disorder are characterized by a disturbance of mood associated with alteration in behavior, energy, weight, sleep, and appetite. It is a major cause of disability, and raised rates of physical disorders that could lead to suicide. According to the most accepted hypothesis of depression, the monoamine theory, changes in brain monoamine neurotransmitters, specifically norepinephrine (NE) and serotonin (5-HT), reflect patients with major depression. Medications such as tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs), serotonin and norepinephrine reuptake inhibitor (SNRIs), are clinically employed for drug therapy. However, synthetic chemical antidepressants can impose a variety of side-effects including sedation, fatigue, sleep disturbance, apathy, sexual dysfunction, and cognitive impairment, and so forth, because of the the mechanism of depression is quite complex. Hence, there remains a pressing need for more effective antidepressants with lower adverse-effect and more effective. Nature plants, such as Hypericum perforatum, Terminalia bellirica Roxb, Ginkgo biloba, may be an important source of new antidepressant drugs. The safety and effect of nature plant extracts maybe better than that of synthetic antidepressants. Many medicinal plants from natural resources, especially the traditional Chinesemedicine, such as Plantago asiatica, Scrophularia ningpoensis, and Ilex pubescens, were successfully used to treat or prevent the depression-like disorder.
Several studies suggested that stress may contribute to the development of depression, and stress can induce behavioral disturbances and neurochemical changes in animals, some of which are similar to the symptoms and presumed neurochemical changes of depression in humans [17,18]. In our study, we used Chronic unpredictable mild stress (CUMS) model , animals appeared to exhibit behavioral deficits, including anhedonia, behavioral despair and lack of acute activation, which may be relatively as a depression model and is widely used for evaluation of antidepressant activity preclinically[19, 20]. All the behavioral deficits indued by CUMS may be accessed by sucrose preference test, forced swimming and tail suspension test, and open field test, respectively. Some studies indicate a link between the alterations in central monoaminergic systems and increased oxidative load in physiologically adverse conditions. During the process of oxidative stress, free radicals are produced, which, in excess, can cause oxidative damage to lipids. Consequently, proteins and DNA are activated, leading to injury or even apoptosis to neurons. In the present study, we analyzed the hippocampus NE and 5-HT levels. With simultaneous changes in defense systems and lipid peroxidation in the experimental animals, we investigated whether LUT had the activities of regulating monoamine neurotransmitters and scavenging free radicals, and whether these activities are related to its antidepressant-like effect in chronic stress depression animal model.
LUT is a common flavonoid that exists in many types of plants such as Apium graveolens L var. dulce, Petroselium crispum, and Capsicum annuum L. var.'grossum'. It has various pharmacological activities such as antioxidant, memory-improving, and anxiolytic activities, suggesting that LUT could penetrate easily into the brain. In this work, we tried to investigate the antidepressant-like effect of chronic administration of LUT and explore its possible mechanism by using chronic stress depression animal model and biochemical test.
Materials and Methods
Animals and materials
The behavioral experiments were conducted using male adult Kunming mice (18-28 g, 7-9 weeks) maintained at 22-25 Â°C with ad libitum access to water and food, under a 12:12 hour light/dark cycle( lights on at 8:00 a.m.). All manipulations were carried out between 08:00 a.m. and 05:00 p.m. Experiments were carried out in accordance with Experimental Animal Center of Xuzhou Medical College (Grade â…¡, Certificate â„-SUA95021)
Chemicals and administration
LUT were dispersed in normal saline. At the end of one week of adaption, the mice were randomized into four groups (n = 8 each): group 1 served as the control group (NS), free of any stress conditions; group 2 was designated as CUMS group (CUMS); groups 3 and 4 were the treatment groups in which animals were subjected to CUMS for five consecutive weeks, with LUT administered 30 min prior to the stress exposure at the graded oral doses of 50 mg/kg or 100 mg/kg (LUTL, LUTH) once a day from the third week of the experiment.
Behavior despair study
Chronic unpredictable mild stress (CUMS) procedures
The CUMS procedures were performed as described with slight modifications. Stressors were administered once daily for 5 consecutive weeks, i.e., 24-h water deprivation, 24-h food deprivation, 1-min tail pinch with a clothes-pin placed (1 cm distal from the base of tail), 5-min cold swimming (at 4Â°C), damp sawdust 200 ml of water per cage (sufficient to reach the moisture of the sawdust bedding) and 24-h cage tilting, 4-h immobilization and overnight illumination. The same stressor was not applied successively so that mice could not anticipate the occurring of stress. Immediately after the conclusion of each stress session the animals were returned to their home cages and maintained in standard conditions until the next stress session. Normal control animals were housed in group of 4 per cage without disturbing except for necessary procedure such as weighting or cage cleaning. They had free access to food and water except for a 21-h period of food and water deprivation before the sucrose consumption test.
Sucrose preference test
Sucrose preference test was performed at the end of 5-week CUMS exposure, as previously described except for minor modifications. Briefly, 72 h before the test, mice were trained to consume 1% sucrose solution (w/v): two bottles of 1% sucrose solution were placed in each cage, followed by replacement with drinking water in one feeder for next 24-h period. At the end of adaptation, mice were deprived of water and food for 24 h and they were exposed to both 100 ml of the test solution (1% sucrose and tap water) for the next 1-h period, respectively. 3 h later, the consumption volumes of sucrose solution and tap water were recorded and the sucrose preference was calculated by the following formula:
Forced swim test
Forced swim test (FST) was conducted according to the published procedure except for slight modifications. FST was conducted on 2 successive days after the last stress period. Briefly, mice were forced to swim in a vertical glass cylinder (diameter 10 cm, height 25 cm) containing 10cm of water maintained at 25Â±1â-¦C. The total duration of immobility for the last 4 min was measured for a single 6-min test session. During the test session, the immobility time was evaluated by two observers who were blind to the kind of treatment each mouse. A mouse was defined immobile as the absence of active escape directed movements whenever it remained floating passively with its head just above the water. At the conclusion of the swim test, the animal was removed from the cylinder, dried by a towel, and returned to its home cage. The water was changed after each mouse swimming[29, 30].
Tail suspension test
TST was performed based on the method of Steru et al.. Mice were suspended by the tail extremity 50 cm above the floor with adhesive tape placed approximately 1 cm from the tip of the tail. The duration of immobility was recorded during the total 6 min test.
Open field test
The open field test was performed as described by Campos et al.. Briefly, the apparatus consisting of a black square cage with 40Ã-40Ã-40 cm was divided into 25 squares marked with black lines on the floor of the arena. A mouse was placed in the center of the cage, the number of ambulation (crossing sector lines with four paws), the number of rears (mouse standing on its hind paws), and the frequencies of grooming (including grooming face, licking/cleaning and and scratching the various parts of the body) were counted manually for 5 min as described in recent studies[33, 34]. During the interval of tests the apparatus was cleaned with 5% alcohol. The open field test was conducted in a quiet room dimly illuminated.
Tissue and blood sample collection
Twenty-four hours after the behavioral test, the mice were sacrificed by decapitation, and the blood samples were collected from the orbital sinus in tubes with or without heparin for plasma and serum preparation, respectively. The samples were collected and centrifuged at 4000Ã-g for 10 min at 4Â°C and the upper layer of each sample was collected. Hippocampus were rapidly removed from mice brain simultaneously and chilled in dry ice.The serum and tissue samples were stored at -80Â°C until assay.
Assay of serum 5-HT and NE levels
5-HT and NE levels in serum were measured by commercially available ELISA kits (Adlitteram Diagnostic Laboratories, USA).
Biochemical investigation of hippocampus
The hippocampus were thawed and homogenized in a proportion of 1:9 (w/v) ice cold normal saline. The homogenates were centrifuged for 10 min at 4000 rpm at 4Â°C, and then these supernatants were obtained for the determination of the levels of malondialdehyde (MDA) and glutathione (GSH), as well as the activities of catalase (CAT) and superoxide dismutase (SOD).
The malondialdehyde (MDA) was measured according to Okhawa et al. , whereas the reduced glutathione (GSH) was determined according to the method described by Ellman. The activity of catalase (CAT) was evaluated according to Aebi with H2O2 as substrate. The activity of the SOD was assessed by the method of by Sun et al., based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, both of which react with nitro-blue tetrazolium (NTB) to form formazan dye. All the designated chemicals were of analytical grade and were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).
Data were presented as mean Â± SD. Differences among groups were examined using one-way ANOVA, followed by LSD post hoc test (two-tailed), except for the analysis of weight change. Because the experimental design involved two within-subjects factors (time and treatment), a repeated measures ANOVA was performed. All the behavior tests were scored live. All statistical analyses were performed using SPSS software 13.0. A p value of < 0.05 was considered to be statistically significant.
Effect of LUT on the percentages of sucrose consumption
The effect of LUT treatment on the percentages of sucrose consumption of mice was assessed in Fig.1. A 5-week CUMS exposure significantly reduced the percentages of sucrose consumption in the animals (P < 0.01), as compared to the control group. LUT at the doses of 50 mg/kg or 100 mg/kg significantly increased the percentages of sucrose consumption by 20% and 43%, respectively, as compared to the CUMS group (P < 0.05, P < 0.01).
Effect of LUT on the immobility time both in the FST and TST
One-way ANOVA showed significant difference in FST on the mice among experimental groups. Both in the FST and TST, CUMS-induced depressive mice exhibited a significant increase in immobility period as compared to the control group (P < 0.01). LUT at 50 mg/kg (p< 0.05) and 100 mg/kg (p< 0.05) treated mice had significantly decreased immobility time compared with CUMS group mice in the FST (Fig. 2A). Likewise, one-way ANOVA also showed significant difference in TST on mice among experimental groups. LUT at 50 mg/kg (p< 0.05) and 100 mg/kg (p< 0.05) treated mice had significantly decreased immobility time compared with CUMS group mice in the TST (Fig. 2B).
Effects of LUT on locomotor activity in the open field test
Open field test is used to assess locomotor activity, exploration and anxiogenic-like behavior of rats or mice. One-way ANOVA indicated that there were significant differences in rearing ï¼ˆp< 0.05ï¼‰ and grooming ï¼ˆp< 0.05ï¼‰ among experimental groups. Compared to the CUMS-treated mice, LUT-treated mice showed a significant increasment in rearing (p< 0.01) and crossing (p< 0.01) activities (Fig. 3).
Effects of LUT on serum concentrations of NE and 5-HT
CUMS procedure markedly induced decrease in serum NE level compared to mice in the control group (p< 0.01). After the 21-day treatment, LUT at 50 mg/kg (p< 0.05) and 100 mg/kg (p< 0.01) significantly increased the levels of serum NE in CUMS-treated mice. Similarly, CUMS procedure also evoked a significant decrease in mouse's serum 5-HT levels compared with the vehicle control group (p< 0.01). After 21 days of treatment with LUT at 50 mg/kg (p< 0.05) and 100 mg/kg (p< 0.01), the serum levels of 5-HT increased significantly (Fig.4).
Effect of LUT on biochemical assay
The results show that CUMS induced an increase in MDA level in the hippocampus, which was significantly decreased by LUT administration at the doses of 50 mg/kg (P < 0.05) and 100 mg/kg (P < 0.01) ( Fig. 5A). GSH was dramatically decreased by stress exposure and significantly increased by LUT at the doses of 50 mg/kg (P < 0.05) and 100 mg/kg (P < 0.01) (Fig. 5B). As to the activity of antioxidase, the CUMS group markedly decreased the activities of SOD (P < 0.01, Fig. 5C) and CAT (P < 0.01, Fig. 5D) as compared to the control group, while a significant elevation was observed in LUT at the doses of 50 mg/kg (P < 0.05) and 100 mg/kg (P < 0.01).
Flavonoids have a variety of pharmacological properties. It had been reported that LUT has the protective effects against endoplasmic reticulum stress-induced depression and/or cell damage. Our results suggested that LUT not only exerted antidepressant-like effect on CUMS induced depression model mice but also improved the damaged defenses against oxidative stress of mice exposed to stressors. In the present study, we investigated the effects of LUT on behaviors of CUMS induced depression model mice by using tail suspension test, open field and sucrose consumption test as well as determining hippocampus CAT and SOD activities, MDA and GSH levels, and serum NE and5-HT level.
LUT decreased the immobility time in the tail suspension test. A elevation of SOD and CAT activities, and an elevation in GSH level as well as an reduction in MDA level in the hippocampus. Furthermore, the elevation of serum NE and5-HT levels were also observed in the LUT administrated mice. These results suggest that chronic treatment of LUT has an potential antidepressant-like effect in the animal models, which might be related to the alteration of monoaminergic response and antioxidant defenses. Previously, it had been reported that chronic LUT (5 mg/kg, p.o.) treatment increased the immobility time in the forced swim test and showed anxiolytic effect in several behavioral tests. These finding indicate that low doses of LUT show the anxiolytic effect, on the other hand, high doses of LUT show the antidepressant-like effect.
Many studies have indicated changes in behavioral and biochemical characteristics in depressed patients. In the present study we found that CUMS resulted in some analogical changes in an animal model of depression. Exposure to chronic stress, mice appeared to have behavioral deficits including decreased sucrose consumption and open-field activities, and increased immobility time in tail suspension test. Three weeks treatment of LUT from the third week displayed an antidepressant-like effect on depression model mice. LUT administration at doses of 50mg/kg and 100mg/kg increased the sucrose consumption (P < 0.05) and open-field activities, and decreased the immobility time (P < 0.05) significantly. There was a significant dose-effect relationship between LUT and behavioral changes. Above results of behavioral trials showed that LUT was effective in the treatment of some depressive symptoms, such as anhedonia and behavioral despair in CUMS induced depression model.
Monoaminergic neurotransmission in the central nervous system has been shown to be involved in the pathogenesis and the therapeutic targets of depression and other psychiatric disorders[42,43]. Four brain regions were studied: the frontal cortex, the striatum, the hippocampus, and the hypothalamus, which are involved Integrating in important behavioral functions, such as emotion, motivation, and learning and memory[44,45]. Decreases in brain concentrations of 5-HT, NE and DA were reported in patients with stress and depression, suggesting a dysregulation of monoaminergic systems[46,47]. Several studies have shown that chronic stress produces changes in the concentrations of neurotransmitters in CUMS-induced depression mice, mainly expressed as the significant decrease in the levels of 5-HT and NE[48,49]. Our data also supported these studies. In the present work, the CUMS mice exhibited decreased serum 5-HT and NE levels compared to normal control group. Our results show that LUT at doses of 50 mg/kg and 100 mg/kg significantly increased the levels of NE and 5-HT in hippocampus.
LUT showed antidepressant-like activity in CUMS models. The mechanism of anti-depressive-like activity of LUT was mediated by increasing the monoamines level, particularly 5-HT and NE in hippocamupus of mice and antioxidant defenses. From the present study, we conclude that LUT is a worth developing.
The authors are cordially indebted to these financial supports: China Postdoctoral Science Foundation funded project(2012M521125), Jiangsu Planned Projects for Postdoctoral Research Funds, the Natural Science Fund for Universities and Colleges in Jiangsu Province (11KJB350005), "Qing-Lan" Project of Jiangsu Province, the Industrialization of Scientific Research Promotion Projects of Universities and Colleges in Jiangsu Province (2011-16); Laboratory of Biological Therapy for Cancer, Xuzhou Medical College (C0903; C0904; JSBL0803), the Science and Technology Plan Projects of Xuzhou (XF11C037; XF11C062), Superiority Academic Discipline Construction Project of Jiangsu Higher Education Institutions, and Xuzhou Public Service Platform Projects of New Drug Discovery and Research.