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Protective effect of liquiritigenin on depressive-like behavior in mice after lipopolysaccharide administration
Psychiatry Research 240 (2016) 131–136
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Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Protective effect of liquiritigenin on depressive-like behavior in mice after lipopolysaccharide administration Qiang Su a,b, Weiwei Tao a,b,n, Huang Huang a, Yan Du a, Xing Chu a, Gang Chen b,nn a
Wuxi Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China Center for Translational Systems Biology and Neuroscience, School of Basic Biomedical Science, Nanjing University of Chinese Medicine, Nanjing 210023, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 January 2016 Received in revised form 27 March 2016 Accepted 1 April 2016 Available online 14 April 2016
Liquiritigenin (Liq), the main active ingredient of traditional Chinese medicine licorice, possesses antiinflammatory and neuroprotective properties. The current investigation was designed to explore whether liquiritigenin could relieve lipopolysaccharide (LPS)-induced depression-like behavior in mice and the underlying mechanism. Liquiritigenin (7.5 mg/kg, 15 mg/kg) and fluoxetine (20 mg/kg) were pretreated intragastrically once daily for 7 consecutive days. LPS (0.5 mg/kg) was injected subcutaneously to establish the depression model 30 min after pretreatment on day 7. Interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels in serum and hippocampus were detected by enzyme-linked immunosorbent assay (ELISA). Behavioral assessment was conduct 24 h post LPS injection. The expressions of p65NF-κB, IκBα, brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) in hippocampus were determined by western blot. The obtained results showed that liquiritigenin effectively reduced the levels of pro-inflammatory cytokines and the expressions of p-p65NF-κB and p-IκBα. Furthermore, liquiritigenin preconditioning could down-regulate the immobility time in tail suspension test (TST), forced swimming test (FST) and up-regulate BDNF and TrkB contents in hippocampus. Thus, it is assumed that the antidepressant activity of liquiritigenin might be attributed to its anti-inflammatory property and BDNF/TrkB signaling pathway. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Liquiritigenin Depression BDNF/TrkB Inflammation
1. Introduction The World Health Organization (WHO) reported that major depressive disorder (MDD) will be the leading cause of disability and have the highest burden of disease by the year 2030. Currently, the focus of researches treating depression was on 5-hydroxytryptamin system, hypothalamicpituitary-adrenal axis (Rohleder, Wolf et al., 2010), neurotrophic system and neurogenesis (Duman and Monteggia, 2006). Numberous clinical studies and animal experiments confirmed that depression led to nerve tissue atrophy and neuronal loss of emotion regulation zone in brain including hippocampus, amygdala, prefrontal cortex and insula. Neurotrophic factors, as such nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) are critical regulators of the formation and plasticity of neural n Corresponding author at: Wuxi Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China nn Correspondence to: Wuxi Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China. E-mail addresses: [email protected] (W. Tao), [email protected] (G. Chen).
http://dx.doi.org/10.1016/j.psychres.2016.04.002 0165-1781/& 2016 Elsevier Ireland Ltd. All rights reserved.
networks (Huang and Reichardt, 2001) and several studies have highlighted the role of neurotrophic factors in depression, as well in actions of antidepressant treatment (Garcia et al., 2009; Trajkovska et al., 2009). Among those neurotrophin, BDNF neurons are widely distributed in the brain, especially in hippocampal dentate gyrus and cortical. Previous literatures supported the theory that pre-incubation of BDNF could significantly suppressed glutamateinduced excitotoxic insult in the hippocampal neurons (Almeida et al., 2005). TrkB (Jiang et al., 2005), which triggers multiple signaling cascades is also essential for cellular survival. Thus, it is speculated that liquiritigenin might exert antidepressant effect through BDNF/TrkB pathway. Furthermore, pro-inflammatory cytokines may also play a vital role in the pathophysiology of depression. Recent research has suggested a central role of inflammatory processes in mediating this effect (Slavich and Irwin, 2014). What's more, episodes of depression can be induced with many inflammatory stimulus (Capuron et al., 2002; Bull et al., 2009) and alleviated with antiinflammatory medication (Köhler et al., 2014). Lipopolysaccharide (LPS) is reported that it can elicit systemic inflammatory responses (Müller et al., 2015), and heightened inflammation commonly is
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Q. Su et al. / Psychiatry Research 240 (2016) 131–136 15 mg/kg respectively for 7 days. Model group were given normal saline for 7 days. Then on the 7th day, except the control group, all groups were injected LPS (0.5 mg/ kg) subcutaneously just 30 min after the Liq, Flu and saline were given. All the control animals were given normal saline in the same volume orally and subcutaneously, respectively. Behavioral tests were measured with 24 h after LPS injection. After behavioral tests, all the mice were sacrificed, the hippocampus were rapidly separated from the brain for biochemical assays and Western blot analysis. 2.4. Behavioral evaluation Fig. 1. Chemical structure of liquiritigenin.
associated with periods of depressive illness (Haapakoski et al., 2015; Strawbridge et al., 2015).So it is reasonable to infer that inflammation-related mediators and proteins are also implicated in the depression-like behavior (Maes, 2008). Liquiritigenin (7,4′-dihydroxyflavanone, Fig. 1), a flavonoid extracted from the radix of Glycyrrhiza, is used clinically for the treatment of liver toxicity in vivo and in vitro (Huang and Reichardt, 2001; Kim et al., 2004; Kimet al., 2006), β-amyloid peptide (Aβ)-induced neurotoxicity in vivo and in vitro (Liu et al., 2009, 2010), ischemia, cancer and inflammation (Kim et al., 2008). However, the antidepressant function of liquiritigenin has not been reported. We extended our study by investigating the effects of liquiritigenin on LPS-induced depression-like behavior in mice and the related mechanism. 2. Materials and methods
2.4.1. Forced swimming test (FST) The forced swimming test was conduct according to the conventional method described previously (Porsolt et al., 1977). Briefly, 24 h post LPS stimulation, every mouse was forced to swim for 6 min in an open cylindrical container (diameter¼14 cm, height¼ 20 cm) containing water up to a height of 12 cm at 257 1 °C. The period of escape-oriented behaviors were considered as immobility time. The total immobility time was recorded during the last 4 min period by two independent observers blinded to the experiment. The water was changed after each group. 2.4.2. Tail suspension test (TST) The tail suspension test was conduct according to the conventional method described previously (Steru et al., 1985). Briefly, 24 h post LPS stimulation, every mouse both acoustically and visually isolated was individually suspended using adhesive tape (approximately 2 cm from the end) for 6 min with 50 cm above the floor. The duration of immobility were measured for the last 4 min by two independent observers blinded to the experiment. 2.5. Cytokine measurement in serum Blood samples were collected from carotid artery and centrifuged at 5000 rpm for 15 min The supernatant were collected for serum cytokines analysis. The levels of IL-6 and TNF-α were detected by ELISA kits according to the manufacturer's instructions.
2.1. Main reagents and kits 2.6. Cytokine measurement in hippocampus Liquiritigenin (purity 98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Fluoxetine hydrochloride, provided by Changzhou Siyao Pharmaceuticals Co., Ltd. (Changzhou, PR China), was dissolved in 0.03% sodium carboxymethyl cellulose (CMC-Na). LPS (Escherichia coli serotype 055: B5, No. L-2880, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile, pyrogenfree physiological saline. TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were produced by Nanjing KeyGEN Biotech. CO., Ltd. (Nanjing, China). All antibodies were purchased from Cell Signaling Technology Inc (Beverly, MA, USA). All other chemicals and reagents used for study were of analytical grade and were purchased from approved organizations. 2.2. Animals 50 male ICR mice were provided by the Experimental Animal Center in Jiangsu Province (Nanjing, China). All the mice were maintained at a constant temperature (22 7 1 °C) under a 12 h light/12 h dark cycle environment and standard food and water were provided ad libitum. The experimental protocol was approved by an Institutional Review Committee for the use of Human or Animal Subjects or that procedures are in compliance with at least the Declaration of Helsinki for human subjects, or the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85–23, revised 1985), the UK Animals Scientific Procedures Act 1986 or the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Mice were sacrificed after behavioral assessment. Hippocampus was harvested, minced and homogenized for proteins and cytokines analysis. The levels of IL-6 and TNF-α in hippocampus were detected by ELISA kits According to the standard protocol. 2.7. Western blot analysis Minced and homogenized hippocampus in lysis buffer were centrifugated at 12,000g for 5 min at 4 °C to remove the debris. BCA protein assay kit (Beyotime, Nanjing, China) was used to measure the total protein concentration in supernatant. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride membrane. The membrane was incubated with primary antibody overnight at 4 °C blocked in skim milk, and then they were incubated with secondary antibody for 1 h at room temperature after washing with TBST three times. The blotted protein bands were developed and fixed by an ECL Advanced kit. The image analysis software was applied to quantify protein intensity. 2.8. Statistical analysis The data were expressed as mean values 7SD. Comparison between groups were analyzed by T-test. All data were processed with Graphpad, while po 0.05 was considered significant.
2.3. Experimental protocol Mice were randomly assigned to five groups (n¼ 10) as follows: control group, model group, LPS þFlu (20 mg/kg) group, LPS þ Liq (7.5 mg/kg) group and LPS þ Liq (15 mg/kg) group. Mice were pretreated with liquiritigenin (7.5 mg/kg, 15 mg/kg) and fluoxetine (20 mg/kg) intragastrically once daily for 7 consecutive days. LPS (0.5 mg/kg) was injected subcutaneously to establish the depression model 30 min after pretreatment on day 7. The control group and model group were given equal volume normal saline. Mice were anesthetized 1.5 h posterior to LPS injection and blood samples were collected from orbit. Behavioral assessment was conduct 24 h after LPS challenge, and then mice were sacrificed to get hippocampus. Mice were randomly assigned to five groups (n¼ 10) as follows: control group, model group, LPS þFlu (20 mg/kg) group, LPS þ Liq (7.5 mg/kg) group and LPS þ Liq (15 mg/kg) group. LPS þ Flu (20 mg/kg) group were treated intragastrically with Flu at the doses of 20 mg/kg for 7 days. LPS þ Liq (7.5 mg/kg) group and LPS þ Liq (15 mg/kg) group were treated intragastrically with Liq at the doses of 7.5 and
3. Result 3.1. Effect of liquiritigenin on behavioral assessments 3.1.1. Effects on immobility time in forced swimming test (FST) The forced swimming test was performed 24 h post-LPS administration. LPS-induced depressive mice showed significant increases in immobility duration compared with the control group. Flu (20 mg/kg) as well as Liq (7.5 mg/kg, 15 mg/kg) notably shortened the immobility period in comparison with the model group (Fig. 2A).
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Fig. 2. Effect of liquiritigenin on behavioral assessments. Values represent the mean 7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group.
3.1.2. Effects on immobility time in tail suspension test (TST) The tail suspension test was also performed 24 h posterior to LPS injection. Mice with depression present remarkable increases in immobility duration versus the control group, while Flu (20 mg/ kg) and Liq (7.5 mg/kg, 15 mg/kg) significantly reduced immobility time compared with the LPS-induced group (Fig. 2B). 3.2. Effect of liquiritigenin on cytokines in serum The anti-inflammation effect of liquiritigenin was confirmed by the measurement of pro-inflammatory cytokines IL-6 and TNF-α. The contents of them dramatically increased compared with those in the control group. As expected, the expressions of IL-6 and TNFα in Flu (20 mg/kg) and Liq (7.5 mg/kg, 15 mg/kg) groups were obviously lower than those in the model group (Fig. 3). 3.3. Effect of liquiritigenin on cytokines in hippocampus Similar to the alterations of cytokines in serum, IL-6 and TNF-α in hippocampus were also up-regulated in model group versus the
control group. However, Flu (20 mg/kg) and Liq (7.5 mg/kg, 15 mg/ kg) pretreatment decreased the levels of IL-6 and TNF-α (Fig. 4). 3.4. Effect of liquiritigenin on inflammation-related proteins in hippocampus To further verify the anti-inflammatory property of liquiritigenin in antidepressant activity, the classical inflammation-related protein was detected (Fig. 5). As shown in Fig. 6, the expressions of p-p65NF-κB and p-IκBα potently increased in the LPS-induced mice, while Flu (20 mg/kg) and Liq (7.5 mg/kg, 15 mg/ kg) preconditioning reversed the situation. 3.5. Effect of liquiritigenin on BDNF/TrkB pathway To further determine the potential mechanism, BDNF/TrkB pathway was studied through the detection of protein contents of BDNF and p-TrkB. The expressions of BDNF and p-TrkB were up-regulated in the model group in contrast with the control group with GAPDH and TrkB expressions as internal controls,
Fig. 3. Effect of liquiritigenin on cytokines in serum. Values represent the mean7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group.
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Fig. 4. Effect of liquiritigenin on cytokines in hippocampus. Values represent the mean 7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group.
Fig. 5. Effect of liquiritigenin on inflammation-related proteins and BDNF/TrkB pathway in hippocampus.
respectively, while different degrees of down-regulation of them were exhibited in pretreatment groups.
4. Discussion The occurrence of depression is a complex process related to physiology, pathology, environment and personal knowledge. Major depressive disorder has long been treated with antidepressants, whose exact mechanism is still unclear. Most antidepressants exert effects through enhancing the concentrations of serotonergic transmitters in the synaptic space (Gulbins et al., 2015). Recently, there are novel investigations focusing on the effects of antidepressants on neurogenesis in the hippocampus and hippocampal neuronal networks, which has revealed the relationship between the occurrence of depression and neuroplasticity. Thus, the current study combined various factors from behavior to molecular level to illustrate the possible mechanism. The tail suspension test (TST) and forced swimming test (FST) are the most extensively used animal models for assessing depression and anxiety, partially due to their high predictive validity
(Peng et al., 2007). In the test, it was observed that liquiritigenin could significantly decline the stationary time in forced swimming test (FST) and tail suspension test (TST) without affecting spontaneous locomotor activity 24 h after LPS stimulation, similar to the positive control fluoxetine, which is in agreement with prior work (Santarelli et al., 2003; Ji et al., 2014). Above results indicated that liquiritigenin could alleviate LPS-induced depressive-like behavior. LPS is a bacterial endotoxin that activates the innate immune response and induces the secretion of pro-inflammatory cytokines in dose-dependent manners (Miller et al., 2005). LPS injected intraperitoneally has been reported to cause significant depressivelike behavior including decreased exploratory behavior (Engeland et al., 2003) and increased anhedonia (Song and Wang, 2011) in mice. The dose of LPS was choosen in the present experiment based on prior researches (Ohgi et al., 2013). Furthermore, previous literatures also showed that the concentrations of IL-6 and TNF-α were higher in LPS-challenged lymphoid mononuclear cells than normal ones, which was related to resistance and severity of depressive symptoms (Suarez et al., 2004). The obtained data suggested that the levels of IL-6 and TNF-α both in serum and
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Fig. 6. A Effect of liquiritigenin on inflammation-related proteins in hippocampus. Values represent the mean 7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group. B Effect of liquiritigenin on BDNF/TrkB pathway. Values represent the mean 7 SD and are representative of two independent experiments. ##p o0.01, significantly different from the values in the control group; **p o0.01, significantly different from the values in the model group.
hippocampus were elevated in different degrees in LPS-induced mice and liquiritigenin, as well as positive control fluoxetine alleviated this situation. Thus, the current study revealed that the anti-depressive activity of liquiritigenin might be attributed to its anti-inflammatory effect. It is well known that NF-κB is a transcription factor protein family which regulates immune responses and inflammation through the control of the expressions of a variety of inflammatory genes, including IL-6 and TNF-α (Ghosh and Karin, 2002). The separation of NF-κB from inhibitory protein IкB leads to NF-κB translocation from cytoplasm into the nucleus and binds to the promoters. Preclinical and clinical findings suggested that the development of depressive disorders was concerned with a persistent activation of the innate immune system and inflammatory processes (Patapoutian and Reichardt, 2001; Mao et al., 2015), which indicated that hippocampus and serum inflammation was related to depressive symptoms. Therefore, we monitored the
changes of IL-6 and TNF-α in serum and hippocampus. The increased levels of the two pro-inflammatory cytokines in the model group and the decreased levels in the liquiritigenin and positive control fluoxetine group were in line with previous reports and our inference. We next explored the signal transduction pathway that could elaborate the molecular link between depressive-like behavior and up-regulated levels of IL-6 and TNF-α in hippocampus. BDNF, a primary member of neurotrophin family of growth factors, promotes the survival and function of adult neurons, differentiation, neurite proliferation and synaptic plasticity through its interactions with the TrkB (Patapoutian and Reichardt, 2001). TrkB, the signaling receptor for BDNF, triggers multiple signaling cascades which are required for maintaining the physiological function of hippocampal neurons (Zhang et al., 2012; Mao et al., 2015). Likewise, results from our present work depicted that pretreatment with liquiritigenin dramatically recovered the contents of BDNF
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and TrkB. Highly significant associations of BDNF-induced TrkB phosphorylation with depressive-like behavior and elevated proinflammatory cytokines were also found. In conclusion, our present investigation disclosed that liquiritigenin decreased IL-6 and TNF-α levels in serum and hippocampus, alleviated LPS-induced depressive-like behavior and increased the expressions of BDNF and p-TrkB. All the obtained results evidenced our hypothesis that the antidepressant activity of liquiritigenin might be associated with BDNF/TrkB signaling pathway and its anti-inflammatory property.
Conflict of interest The authors have declared that there is no conflict of interest.
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Contents lists available at ScienceDirect
Psychiatry Research journal homepage: www.elsevier.com/locate/psychres
Protective effect of liquiritigenin on depressive-like behavior in mice after lipopolysaccharide administration Qiang Su a,b, Weiwei Tao a,b,n, Huang Huang a, Yan Du a, Xing Chu a, Gang Chen b,nn a
Wuxi Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China Center for Translational Systems Biology and Neuroscience, School of Basic Biomedical Science, Nanjing University of Chinese Medicine, Nanjing 210023, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 January 2016 Received in revised form 27 March 2016 Accepted 1 April 2016 Available online 14 April 2016
Liquiritigenin (Liq), the main active ingredient of traditional Chinese medicine licorice, possesses antiinflammatory and neuroprotective properties. The current investigation was designed to explore whether liquiritigenin could relieve lipopolysaccharide (LPS)-induced depression-like behavior in mice and the underlying mechanism. Liquiritigenin (7.5 mg/kg, 15 mg/kg) and fluoxetine (20 mg/kg) were pretreated intragastrically once daily for 7 consecutive days. LPS (0.5 mg/kg) was injected subcutaneously to establish the depression model 30 min after pretreatment on day 7. Interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels in serum and hippocampus were detected by enzyme-linked immunosorbent assay (ELISA). Behavioral assessment was conduct 24 h post LPS injection. The expressions of p65NF-κB, IκBα, brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) in hippocampus were determined by western blot. The obtained results showed that liquiritigenin effectively reduced the levels of pro-inflammatory cytokines and the expressions of p-p65NF-κB and p-IκBα. Furthermore, liquiritigenin preconditioning could down-regulate the immobility time in tail suspension test (TST), forced swimming test (FST) and up-regulate BDNF and TrkB contents in hippocampus. Thus, it is assumed that the antidepressant activity of liquiritigenin might be attributed to its anti-inflammatory property and BDNF/TrkB signaling pathway. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Liquiritigenin Depression BDNF/TrkB Inflammation
1. Introduction The World Health Organization (WHO) reported that major depressive disorder (MDD) will be the leading cause of disability and have the highest burden of disease by the year 2030. Currently, the focus of researches treating depression was on 5-hydroxytryptamin system, hypothalamicpituitary-adrenal axis (Rohleder, Wolf et al., 2010), neurotrophic system and neurogenesis (Duman and Monteggia, 2006). Numberous clinical studies and animal experiments confirmed that depression led to nerve tissue atrophy and neuronal loss of emotion regulation zone in brain including hippocampus, amygdala, prefrontal cortex and insula. Neurotrophic factors, as such nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) are critical regulators of the formation and plasticity of neural n Corresponding author at: Wuxi Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China nn Correspondence to: Wuxi Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China. E-mail addresses: [email protected] (W. Tao), [email protected] (G. Chen).
http://dx.doi.org/10.1016/j.psychres.2016.04.002 0165-1781/& 2016 Elsevier Ireland Ltd. All rights reserved.
networks (Huang and Reichardt, 2001) and several studies have highlighted the role of neurotrophic factors in depression, as well in actions of antidepressant treatment (Garcia et al., 2009; Trajkovska et al., 2009). Among those neurotrophin, BDNF neurons are widely distributed in the brain, especially in hippocampal dentate gyrus and cortical. Previous literatures supported the theory that pre-incubation of BDNF could significantly suppressed glutamateinduced excitotoxic insult in the hippocampal neurons (Almeida et al., 2005). TrkB (Jiang et al., 2005), which triggers multiple signaling cascades is also essential for cellular survival. Thus, it is speculated that liquiritigenin might exert antidepressant effect through BDNF/TrkB pathway. Furthermore, pro-inflammatory cytokines may also play a vital role in the pathophysiology of depression. Recent research has suggested a central role of inflammatory processes in mediating this effect (Slavich and Irwin, 2014). What's more, episodes of depression can be induced with many inflammatory stimulus (Capuron et al., 2002; Bull et al., 2009) and alleviated with antiinflammatory medication (Köhler et al., 2014). Lipopolysaccharide (LPS) is reported that it can elicit systemic inflammatory responses (Müller et al., 2015), and heightened inflammation commonly is
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Q. Su et al. / Psychiatry Research 240 (2016) 131–136 15 mg/kg respectively for 7 days. Model group were given normal saline for 7 days. Then on the 7th day, except the control group, all groups were injected LPS (0.5 mg/ kg) subcutaneously just 30 min after the Liq, Flu and saline were given. All the control animals were given normal saline in the same volume orally and subcutaneously, respectively. Behavioral tests were measured with 24 h after LPS injection. After behavioral tests, all the mice were sacrificed, the hippocampus were rapidly separated from the brain for biochemical assays and Western blot analysis. 2.4. Behavioral evaluation Fig. 1. Chemical structure of liquiritigenin.
associated with periods of depressive illness (Haapakoski et al., 2015; Strawbridge et al., 2015).So it is reasonable to infer that inflammation-related mediators and proteins are also implicated in the depression-like behavior (Maes, 2008). Liquiritigenin (7,4′-dihydroxyflavanone, Fig. 1), a flavonoid extracted from the radix of Glycyrrhiza, is used clinically for the treatment of liver toxicity in vivo and in vitro (Huang and Reichardt, 2001; Kim et al., 2004; Kimet al., 2006), β-amyloid peptide (Aβ)-induced neurotoxicity in vivo and in vitro (Liu et al., 2009, 2010), ischemia, cancer and inflammation (Kim et al., 2008). However, the antidepressant function of liquiritigenin has not been reported. We extended our study by investigating the effects of liquiritigenin on LPS-induced depression-like behavior in mice and the related mechanism. 2. Materials and methods
2.4.1. Forced swimming test (FST) The forced swimming test was conduct according to the conventional method described previously (Porsolt et al., 1977). Briefly, 24 h post LPS stimulation, every mouse was forced to swim for 6 min in an open cylindrical container (diameter¼14 cm, height¼ 20 cm) containing water up to a height of 12 cm at 257 1 °C. The period of escape-oriented behaviors were considered as immobility time. The total immobility time was recorded during the last 4 min period by two independent observers blinded to the experiment. The water was changed after each group. 2.4.2. Tail suspension test (TST) The tail suspension test was conduct according to the conventional method described previously (Steru et al., 1985). Briefly, 24 h post LPS stimulation, every mouse both acoustically and visually isolated was individually suspended using adhesive tape (approximately 2 cm from the end) for 6 min with 50 cm above the floor. The duration of immobility were measured for the last 4 min by two independent observers blinded to the experiment. 2.5. Cytokine measurement in serum Blood samples were collected from carotid artery and centrifuged at 5000 rpm for 15 min The supernatant were collected for serum cytokines analysis. The levels of IL-6 and TNF-α were detected by ELISA kits according to the manufacturer's instructions.
2.1. Main reagents and kits 2.6. Cytokine measurement in hippocampus Liquiritigenin (purity 98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Fluoxetine hydrochloride, provided by Changzhou Siyao Pharmaceuticals Co., Ltd. (Changzhou, PR China), was dissolved in 0.03% sodium carboxymethyl cellulose (CMC-Na). LPS (Escherichia coli serotype 055: B5, No. L-2880, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile, pyrogenfree physiological saline. TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were produced by Nanjing KeyGEN Biotech. CO., Ltd. (Nanjing, China). All antibodies were purchased from Cell Signaling Technology Inc (Beverly, MA, USA). All other chemicals and reagents used for study were of analytical grade and were purchased from approved organizations. 2.2. Animals 50 male ICR mice were provided by the Experimental Animal Center in Jiangsu Province (Nanjing, China). All the mice were maintained at a constant temperature (22 7 1 °C) under a 12 h light/12 h dark cycle environment and standard food and water were provided ad libitum. The experimental protocol was approved by an Institutional Review Committee for the use of Human or Animal Subjects or that procedures are in compliance with at least the Declaration of Helsinki for human subjects, or the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85–23, revised 1985), the UK Animals Scientific Procedures Act 1986 or the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Mice were sacrificed after behavioral assessment. Hippocampus was harvested, minced and homogenized for proteins and cytokines analysis. The levels of IL-6 and TNF-α in hippocampus were detected by ELISA kits According to the standard protocol. 2.7. Western blot analysis Minced and homogenized hippocampus in lysis buffer were centrifugated at 12,000g for 5 min at 4 °C to remove the debris. BCA protein assay kit (Beyotime, Nanjing, China) was used to measure the total protein concentration in supernatant. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride membrane. The membrane was incubated with primary antibody overnight at 4 °C blocked in skim milk, and then they were incubated with secondary antibody for 1 h at room temperature after washing with TBST three times. The blotted protein bands were developed and fixed by an ECL Advanced kit. The image analysis software was applied to quantify protein intensity. 2.8. Statistical analysis The data were expressed as mean values 7SD. Comparison between groups were analyzed by T-test. All data were processed with Graphpad, while po 0.05 was considered significant.
2.3. Experimental protocol Mice were randomly assigned to five groups (n¼ 10) as follows: control group, model group, LPS þFlu (20 mg/kg) group, LPS þ Liq (7.5 mg/kg) group and LPS þ Liq (15 mg/kg) group. Mice were pretreated with liquiritigenin (7.5 mg/kg, 15 mg/kg) and fluoxetine (20 mg/kg) intragastrically once daily for 7 consecutive days. LPS (0.5 mg/kg) was injected subcutaneously to establish the depression model 30 min after pretreatment on day 7. The control group and model group were given equal volume normal saline. Mice were anesthetized 1.5 h posterior to LPS injection and blood samples were collected from orbit. Behavioral assessment was conduct 24 h after LPS challenge, and then mice were sacrificed to get hippocampus. Mice were randomly assigned to five groups (n¼ 10) as follows: control group, model group, LPS þFlu (20 mg/kg) group, LPS þ Liq (7.5 mg/kg) group and LPS þ Liq (15 mg/kg) group. LPS þ Flu (20 mg/kg) group were treated intragastrically with Flu at the doses of 20 mg/kg for 7 days. LPS þ Liq (7.5 mg/kg) group and LPS þ Liq (15 mg/kg) group were treated intragastrically with Liq at the doses of 7.5 and
3. Result 3.1. Effect of liquiritigenin on behavioral assessments 3.1.1. Effects on immobility time in forced swimming test (FST) The forced swimming test was performed 24 h post-LPS administration. LPS-induced depressive mice showed significant increases in immobility duration compared with the control group. Flu (20 mg/kg) as well as Liq (7.5 mg/kg, 15 mg/kg) notably shortened the immobility period in comparison with the model group (Fig. 2A).
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Fig. 2. Effect of liquiritigenin on behavioral assessments. Values represent the mean 7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group.
3.1.2. Effects on immobility time in tail suspension test (TST) The tail suspension test was also performed 24 h posterior to LPS injection. Mice with depression present remarkable increases in immobility duration versus the control group, while Flu (20 mg/ kg) and Liq (7.5 mg/kg, 15 mg/kg) significantly reduced immobility time compared with the LPS-induced group (Fig. 2B). 3.2. Effect of liquiritigenin on cytokines in serum The anti-inflammation effect of liquiritigenin was confirmed by the measurement of pro-inflammatory cytokines IL-6 and TNF-α. The contents of them dramatically increased compared with those in the control group. As expected, the expressions of IL-6 and TNFα in Flu (20 mg/kg) and Liq (7.5 mg/kg, 15 mg/kg) groups were obviously lower than those in the model group (Fig. 3). 3.3. Effect of liquiritigenin on cytokines in hippocampus Similar to the alterations of cytokines in serum, IL-6 and TNF-α in hippocampus were also up-regulated in model group versus the
control group. However, Flu (20 mg/kg) and Liq (7.5 mg/kg, 15 mg/ kg) pretreatment decreased the levels of IL-6 and TNF-α (Fig. 4). 3.4. Effect of liquiritigenin on inflammation-related proteins in hippocampus To further verify the anti-inflammatory property of liquiritigenin in antidepressant activity, the classical inflammation-related protein was detected (Fig. 5). As shown in Fig. 6, the expressions of p-p65NF-κB and p-IκBα potently increased in the LPS-induced mice, while Flu (20 mg/kg) and Liq (7.5 mg/kg, 15 mg/ kg) preconditioning reversed the situation. 3.5. Effect of liquiritigenin on BDNF/TrkB pathway To further determine the potential mechanism, BDNF/TrkB pathway was studied through the detection of protein contents of BDNF and p-TrkB. The expressions of BDNF and p-TrkB were up-regulated in the model group in contrast with the control group with GAPDH and TrkB expressions as internal controls,
Fig. 3. Effect of liquiritigenin on cytokines in serum. Values represent the mean7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group.
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Fig. 4. Effect of liquiritigenin on cytokines in hippocampus. Values represent the mean 7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group.
Fig. 5. Effect of liquiritigenin on inflammation-related proteins and BDNF/TrkB pathway in hippocampus.
respectively, while different degrees of down-regulation of them were exhibited in pretreatment groups.
4. Discussion The occurrence of depression is a complex process related to physiology, pathology, environment and personal knowledge. Major depressive disorder has long been treated with antidepressants, whose exact mechanism is still unclear. Most antidepressants exert effects through enhancing the concentrations of serotonergic transmitters in the synaptic space (Gulbins et al., 2015). Recently, there are novel investigations focusing on the effects of antidepressants on neurogenesis in the hippocampus and hippocampal neuronal networks, which has revealed the relationship between the occurrence of depression and neuroplasticity. Thus, the current study combined various factors from behavior to molecular level to illustrate the possible mechanism. The tail suspension test (TST) and forced swimming test (FST) are the most extensively used animal models for assessing depression and anxiety, partially due to their high predictive validity
(Peng et al., 2007). In the test, it was observed that liquiritigenin could significantly decline the stationary time in forced swimming test (FST) and tail suspension test (TST) without affecting spontaneous locomotor activity 24 h after LPS stimulation, similar to the positive control fluoxetine, which is in agreement with prior work (Santarelli et al., 2003; Ji et al., 2014). Above results indicated that liquiritigenin could alleviate LPS-induced depressive-like behavior. LPS is a bacterial endotoxin that activates the innate immune response and induces the secretion of pro-inflammatory cytokines in dose-dependent manners (Miller et al., 2005). LPS injected intraperitoneally has been reported to cause significant depressivelike behavior including decreased exploratory behavior (Engeland et al., 2003) and increased anhedonia (Song and Wang, 2011) in mice. The dose of LPS was choosen in the present experiment based on prior researches (Ohgi et al., 2013). Furthermore, previous literatures also showed that the concentrations of IL-6 and TNF-α were higher in LPS-challenged lymphoid mononuclear cells than normal ones, which was related to resistance and severity of depressive symptoms (Suarez et al., 2004). The obtained data suggested that the levels of IL-6 and TNF-α both in serum and
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Fig. 6. A Effect of liquiritigenin on inflammation-related proteins in hippocampus. Values represent the mean 7 SD and are representative of two independent experiments. ##p o 0.01, significantly different from the values in the control group; **p o 0.01, significantly different from the values in the model group. B Effect of liquiritigenin on BDNF/TrkB pathway. Values represent the mean 7 SD and are representative of two independent experiments. ##p o0.01, significantly different from the values in the control group; **p o0.01, significantly different from the values in the model group.
hippocampus were elevated in different degrees in LPS-induced mice and liquiritigenin, as well as positive control fluoxetine alleviated this situation. Thus, the current study revealed that the anti-depressive activity of liquiritigenin might be attributed to its anti-inflammatory effect. It is well known that NF-κB is a transcription factor protein family which regulates immune responses and inflammation through the control of the expressions of a variety of inflammatory genes, including IL-6 and TNF-α (Ghosh and Karin, 2002). The separation of NF-κB from inhibitory protein IкB leads to NF-κB translocation from cytoplasm into the nucleus and binds to the promoters. Preclinical and clinical findings suggested that the development of depressive disorders was concerned with a persistent activation of the innate immune system and inflammatory processes (Patapoutian and Reichardt, 2001; Mao et al., 2015), which indicated that hippocampus and serum inflammation was related to depressive symptoms. Therefore, we monitored the
changes of IL-6 and TNF-α in serum and hippocampus. The increased levels of the two pro-inflammatory cytokines in the model group and the decreased levels in the liquiritigenin and positive control fluoxetine group were in line with previous reports and our inference. We next explored the signal transduction pathway that could elaborate the molecular link between depressive-like behavior and up-regulated levels of IL-6 and TNF-α in hippocampus. BDNF, a primary member of neurotrophin family of growth factors, promotes the survival and function of adult neurons, differentiation, neurite proliferation and synaptic plasticity through its interactions with the TrkB (Patapoutian and Reichardt, 2001). TrkB, the signaling receptor for BDNF, triggers multiple signaling cascades which are required for maintaining the physiological function of hippocampal neurons (Zhang et al., 2012; Mao et al., 2015). Likewise, results from our present work depicted that pretreatment with liquiritigenin dramatically recovered the contents of BDNF
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and TrkB. Highly significant associations of BDNF-induced TrkB phosphorylation with depressive-like behavior and elevated proinflammatory cytokines were also found. In conclusion, our present investigation disclosed that liquiritigenin decreased IL-6 and TNF-α levels in serum and hippocampus, alleviated LPS-induced depressive-like behavior and increased the expressions of BDNF and p-TrkB. All the obtained results evidenced our hypothesis that the antidepressant activity of liquiritigenin might be associated with BDNF/TrkB signaling pathway and its anti-inflammatory property.
Conflict of interest The authors have declared that there is no conflict of interest.
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