| Background and ObjectiveDepression is considered to be the most common mental illnesss, and is a seriously disabling public health problem of very high prevalence, which affecting 20% of the population people worldwide. The World Health Organization has estimated that depression will become the second cause of disability worldwide with heavy economic burden for the human by 2020. However, the pathogenesis of depression has not been perfect interpreted. It has been considered that the most relevant pathophysiological mechanism of depression includes changes in brain monoaminergic transmission, the dysregulation of hypothalamic-pituitary-adrenal (HPA) axis, reduced neurotrophic factors, abnormality of inflammatory cytokines, and increased oxidative stress. The treatment of depression with conventional antidepressants, based on the monoamine-deficiency hypothesis, is not perfect and only provides a complete remission just for less than 50% of the individuals. It was important to emphasize the need to discover novel and effective antidepressants.Over the last two decades, new developments in depression research have led to the cytokine hypothesis that inflammatory processes and brain-immune interactions are involved in depression. Some cytokines are believed to be important contributors to the pathogenesis of depression. Research has shown immune activation and the production of inflammatory cytokines in patients with depression; conversely inflammatory cytokines induced the changes of mood and behaviors in individual. The activities of pro-inflammatory cytokines and anti-inflammatory cytokines coexist in depression. The pro-inflammatory cytokines, such as interleukin-1 (IL-1)、IL-6、 IL-17 and tumor necrosis factor-α (TNF-α), involved in immune activation and inflammatory processes, while anti-inflammatory cytokines, such as IL-10 and IL-13, resist immune response through inhibiting of cell activation and inflammatory modulators production.Recent evidence suggests that oxidative stress processes might play a relevant role in the pathogenic mechanisms underlying depression, which is related to increased oxidative stress and to lowered concentrations of several endogenous antioxidant compounds. Activation of immune and inflammatory response favors production of reactive oxygen species (ROS), which caused oxidative damage of body. ROS has been shown to modulate the levels and activities of norepinephrine,5-HT, dopamine and glutamate, the principal neurotransmitters involved in the pathogenesis of depression. Depression is characterized by significantly lower plasma concentrations and antioxidant enzyme activity, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px).There is considerable evidence that HPA axis involved in depression. Patients with depression may have impaired regulation of the HPA axis, elevated cortisol levels in plasma, and increased corticotropin-releasing hormone (CRH) levels in cerebrospinal fluid. Long term administration of exogenous glucocorticoid increased depressive behavior of rats. Mice with the knockout of the glucocorticoid receptor have increased activity of the HPA axis and depressive behavior.Brain-derived neurotrophic factor (BDNF), a neurotrophic peptide, is critical for axonal growth, neuronal survival, and synaptic plasticity. BDNF is believed to involve in the pathogenesis of depression. Animal and clinical studies showed BDNF levels were decreased in individuals with depression, and antidepressant management could restore the decreased BDNF levels. Studies also showed levels of BDNF are positively correlated with 5-HT. Moreover, central administration of BDNF produced antidepressant-like activity in a rat model of depression, which directly supported the protective role of BDNF in depression. Besides, evidence showed that inflammatory cytokines could decrease systemic BDNF levels in humans, which integrates the roles of inflammation and BDNF in depression.Ginkgo biloba extract EGb761 has showed anti-inflammatory, anti-oxidative, anti-arteriosclerosis and neuroprotective activities. There is also study that pretreatment with EGb761 significantly decreased the immobility time in the forced swimming test in BALB/c mice, and improved the symptoms of depression effect. The animal models are important tool for the study of the pathogenesis, drugs screening and treatment strategies of depression. However, it is unknown whether EGb761 has antidepressant-like effects in lipopolysaccharides (LPS)-induced depression, which is a well established animal model of inflammation associated depression.Thus, acute and chronic depressive-like behavior mice model were prepared with the single and repeated intermittent intraperitoneal injection of LPS respectively. The changes of depressive behaviors, the levels of inflammatory cytokines, BDNF, oxidative stress and corticosterone in mice model were observed after pretreatment and treatment of EGb761. To clarify the antidepressant effect of EGb761 and further explore its possible mechanisms, which provide a theoretical basis for clinical application of EGb761 for treatment of depression.Methods1. Establishment and evaluation of animal model(1) Preparation of mice with acute depressive-like behaviorsC57BL/6J male mice were intraperitoneally administrated LPS (dose 830 μg/kg). Forced swim test (FST), tail suspension test (TST), sucrose preference test and open field test were performed to evaluate the depressive-like behaviors of the mice.(2) Preparation of mice with chronic depressive-like behaviorsC57BL/6J female mice were intraperitoneally administrated LPS for 5 days every month in four months. The LPS dose was increased every day for the first three days and was then gradually decreased on day 4 and 5, i.e. day 1:750 μg/kg, day 2: 1000 μg/kg, day 3:1250 μg/kg, day 4:1000 μg/kg and day 5:750 μg/kg. Forced swim test (FST), tail suspension test (TST) and sucrose preference test were performed to evaluate the depressive-like behaviors of the mice.2. The group and process of experiment(1) Effects and mechanisms of EGb761 pretreatment on acute depressive-like behaviors of miceForty male mice were randomly divided into control group, model group, low dose group, middle dose group and high dose group with 8 mice in each group. Mice in low, middle and high dose group were orally treated with different doses of EGb761 (respectively 50,100 or 150 mg/kg/day, dissolved in saline) from day 1 to day 12, and mice in the control group and model group were treated with the same volume of vehicle saline instead. After 10 days pretreatment, the mice in model group, the low, middle and high dose groups were intraperitoneally administrated LPS (0.83 mg/kg). Twenty four hours after the LPS administration, spontaneous locomotor activity of the mice was assessed by open field test. We carried out FST, TST and sucrose preference test, which can evaluate the effect of EGb761 to depressive-like behaviors of mice.The mice were sacrificed under deep anesthesia after behavior evaluation. The hippocampus was immediately removed from the brain on the ice and was used to made tissue homogenate using a homogenizer. After centrifugation (4℃,5000 rpm, 15 min), the supernatant was sub-packaged and stored at minus 20 ℃ for further measurements. Levels of TNF-α, IL-1β, IL-6, IL-17A, IL-10 and BDNF in the supernatant of the hippocampus were measure using specific ELISA kits.(2) Effects and mechanisms of EGb761 treatment on chronic depressive-like behavior of miceForty female mice were randomly divided into control group, model group, low dose group, middle dose group and high dose group with 8 mice in each group. Mice in low, middle and high dose group were orally treated with different doses of EGb761 (respectively 50,100 or 150 mg/kg/day, dissolved in saline) for 1 month after 4 months LPS intraperitoneal administration, and mice in the control group and model group were treated with the same volume of vehicle saline instead. We carried out FST, TST and sucrose preference test to evaluate the effect of EGb761 to depressive-like behaviors of mice.The mice were sacrificed under deep anesthesia after behavior evaluation. Blood was collected and centrifuged at 3000rpm for 15 min at 4℃; the supernatant was removed and stored at minus 20 ℃ until assay. The hippocampus was immediately removed from the brain on the ice and was used to made tissue homogenate using a homogenizer. After centrifugation (4℃,5000 rpm,15 min), the supernatant was sub-packaged and stored at minus 20℃ for further measurements.Levels of TNF-α、IL-6 and IL-10 in serum and hippocampus were measure using specific ELISA kits. Biochemistry methods were used to determine the content of malonaldehyde (MDA) and activities of SOD, CAT and GSH-Px in both of serum and hippocampus. Fluorescence method was used for determination of the corticosterone (CORT) concentration in serum of mice.Results1. Acute and chronic inflammation induced by LPS caused depressive-like behaviors in miceThe model group showed markedly longer immobility time than the control group in FST and TST (P<0.05); LPS injection significantly decreased the sucrose preference in the model group compared to the control group (P<0.05). Both single and repeated, intermittent LPS administration induced significant depressive-like behaviors in mice. There were less rearings and crossings in the model group than the control group (both P<0.05), which indicated that single LPS injection reduced the locomotor activity of the mice.2. Effects of EGb761 pretreatment on mice of acute depressive-like behaviors(1) Improving the acute depressive-like behaviorThe middle dose group and the high dose group showed shorter immobility time than the model group in FST and TST (both P<0.05). Moreover, the high dose group’s immobility time was much shorter than that of the low and the middle dose group (both P<0.05).Sucrose preferences of the middle and the high dose group were much higher than the model group (both P<0.05). Moreover, sucrose preference of the high dose group was higher than that of the low dose group (P<0.05). The results suggested that EGb761 pretreatment had antidepressant-like effect in mice with single LPS injection, and the effect was dose-related.There were no significant differences in numbers rearing and crossings of open field test among the model group, low, middle and high dose group in acute depressive-like mice (all P>0.05), suggesting that EGb761 pretreatment had no marked effects on the locomotor activity of the mice.(2) Regulating production of hippocampus inflammatory cytokinesThe model group showed much higher levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-17A, but lower level of anti-inflammatory cytokine IL-10 in the hippocampus tissue homogenate than the control group (all P<0.05), indicating that single LPS injection induced inflammation in hippocampus. Yet, EGb761 pretreatment significantly inhibited the elevation of hippocampal TNF-α, IL-1β, IL-6 and IL-17A and increased the level of IL-10 in the middle dose group and high dose group if compared to the model group (all P<0.05). Moreover, the high dose was more potent than the low dose and middle dose (all P<0.05).(3) Increasing the level of hippocampus BDNFThe model group showed much lower level of BDNF in the hippocampus tissue homogenate than the control group (P<0.05). Yet, EGb761 pretreatment significantly increased the level of BDNF in the middle dose group and high dose group if compared to the model group (all P<0.05). Moreover, the high dose was more potent than the low dose and middle dose (P<0.05).3. Effects of EGb761 treatment on mice of chronic depressive-like behaviors(1) Improving the chronic depressive-like behaviorThe middle and the high dose group showed shorter immobility time than the model group in FST and TST (both P<0.05). Moreover, the high dose group’s immobility time was much shorter than that of the low and the middle dose group (both P<0.05).Sucrose preferences of the middle and the high dose group were much higher than the model group (both P<0.05). Moreover, sucrose preference of the high dose group was higher than that of the low dose group (P<0.05). The results suggested that EGb761 treatment had antidepressant-like effect in mice with repeated and intermittent LPS injection, and the effect was dose-related.(2) Regulating production of inflammatory cytokines in hippocampus and serumThe model group showed much higher levels of TNF-a and IL-6, but lower level of IL-10 in the hippocampus tissue than the control group (all P<0.05). EGb761 administration for 1 month significantly inhibited the elevation of hippocampal TNF-a and IL-6, and increased the level of IL-10 in the middle dose group and high dose group if compared to the model group (all P<0.05). Moreover, the high dose was more potent than the low dose and middle dose (both P<0.05).There were no significant differences in levels of TNF-a and IL-6 of mice serum among the control group, model group, low, middle and high dose group (all P>0.05). The model group showed lower level of IL-10 in the serum than the control group (P<0.05). EGb761 administration for 1 month significantly improve level of serum IL-10 in the middle dose group and high dose group if compared to the model group (all P<0.05).(3) Reducing the level of oxidative stress in hippocampus and serumThe repeated and intermittent LPS administration produced a marked increase in the content of MDA and the decreased activities of SOD, CAT, GSH-Px in the hippocampus and serum of mice. EGb761 administration for 1 month significantly reduced the content of MDA in the low, middle and high dose group if compared to the model group (all P<0.05), and improved the activities of SOD, CAT, GSH-Px in the hippocampus and serum of mice (all P<0.05). Moreover, the high dose was more potent than the low dose and middle dose (both P<0.05).(4) Reducing the concentration of CORT in the serumThe repeated and intermittent LPS administration produced a marked increase in the levels of CORT in the serum of mice. EGb761 administration for 1 month significantly inhibited the elevation of serum CORT in the middle dose group and high dose group if compared to the model group (both P<0.05). Moreover, the high dose was more potent than the low dose and middle dose (both P<0.05).Conclusion1. Both single and repeated, intermittent LPS administration induced significant depressive-like behaviors in C57BL/6J mice. The chronic administration of LPS may induce a long-lasting depressive effect.2. Pretreatment with EGb761 significantly attenuated acute depressive-like behaviors, and the antidepressant-like effect was dose-related. Meanwhile, EGb761 pretreatment inhibited the production of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-17A, as well as restored the levels of BDNF and anti-inflammatory cytokine IL-10 in hippocampus of mice with single LPS administration, which should contribute to the antidepressant-like activities of EGb761.3. EGb761 treatment significantly attenuated chronic depressive-like behaviors, and the antidepressant-like effect was dose-related. Meanwhile, EGb761 treatment inhibited the production of TNF-α, IL-6 in the hippocampus and MDA in the hippocampus and serum, and restored the level of IL-10 and the activities of SOD, CAT, GSH-Px in the hippocampus and serum, as well as inhibited the rise of CORT level in serum.4. The antidepressant-like effect of EGb761 involves multiple signaling pathways, including inhibition of inflammation, increased expression of BDNF, regulation of oxidative stress and restored the function of HPA axis. |
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