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2,3,5,4′-Tetrahydroxystilbene-2-O-β-d -glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression
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2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression
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Xi-Xi Li, a Yun Yu, a Xiu-Yuan Lang, a Cheng-Yong Jiang, a Rongfeng Lan b* and Xiao-Yan Qin a*
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b
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Center on Translational Neuroscience, College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China
Department of Cell Biology & Medical Genetics, School of Basic Medical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, China
Abstract—Depression is a long term inhibitory mood that heavily disabled human beings. We have previously demonstrated anti-depression effect of 2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside (THSG) in chronicrestraint stress (CRS) induced depressive-like mice by restoring the oxidative pathway and neuroinflammation. In this study, we examine the conditions of neurotrophins in CRS-induced depressive-like mice and whether THSG could be an antidepressant by ameliorating the neurotrophins and their associated signaling axis. CRS produced downregulation of antioxidants, the decline of brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and associated signaling regulators in the hippocampus and prefrontal cortex, corresponding to the behavioral inability and anhedonia. Administration of THSG restored the expression of antioxidants and neurotrophins BDNF, FGF2. Besides, THSG recovered the Akt signaling pathway and antagonistically restored the expression of Bcl-2 and cleaved-caspase-3 to inhibit apoptosis. Consistently, behavioral performances were recovered from CRS-induced motor inability and anhedonia. In summary, THSG is effective to attenuate stressinduced depression by ameliorating the biochemistry of neurotrophins and their related signaling pathways. These results may provide an avenue to take BDNF as a target to explore folk medicine for anti-depression. Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: BDNF, FGF2, THSG, forced swim test, depression.
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INTRODUCTION
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Depression is a debilitating mood disorder that often includes symptoms of anhedonia, feelings of dejection and hopelessness, lack of motivation, fatigue, sleep disorders and reckless behaviors such as suicide. More than just feeling not good in a short time, depression may severely change the way how you think, feel and the daily behaviors. Psycho-social factors such as loneliness, stressful life and economic strain are considered to make people vulnerable to depression. Genetic conditions and biochemical changes in the brain may also contribute to the onset of depression. Elucidation of the mechanism of depression induced
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neurophysiological changes will contribute to the exploration of therapy strategy. Although many hypotheses have been proposed to explain the pathogenesis of depression, it is well established that restoring the biochemistry of neurotransmitters and attenuating neuroinflammation may contribute to the therapy of depression (Martinowich et al., 2007). In the neurotrophin hypothesis of depression, reduced expression of brain-derived neurotrophic factor (BDNF) may lead to neural atrophy and cell loss in the hippocampus and prefrontal cortex, whereas antidepressants could exert their therapeutic effects by increasing the expression of BDNF and restoring its mediated downstream signaling (Duman and Monteggia, 2006; Martinowich et al., 2007). Although it may not be the sole mediator of depression, BDNF and its receptor TrkB acts in antidepressant medication. Antidepressant medication serves as a simple and quick way of relief objects from suffering the pain of severe depression. In an experimental study, chronic-restraint stress (CRS) induced depressive-like behaviors of rodent models are well established to simulate depression and is used for drug development (Duman and Monteggia, 2006; Nestler and Hyman, 2010). Chronic stress induced anxiety, body weight loss, along with dysfunctions of
*Corresponding authors. Addresses: A7-456, Xili Campus, Shenzhen University Health Science Center, China (R. F. Lan). Room 1301, College of Life and Environmental Sciences, Minzu University of China (X.- Y. Qin). E-mail addresses: [email protected] (R. Lan), [email protected]. cn (X.-Y. Qin). Abbreviations: BDNF, brain-derived neurotrophic factor; CRS, chronicrestraint stress; FGF2, fibroblast growth factor 2; FH, fluoxetine hydrochloride; MDA, malondialdehyde; SOD, superoxide dismutase; SPT, sucrose preference test; T-AOC, total antioxidant capacity; TGSH, total glutathione; THSG, 2,3,5,40 -tetrahydroxystilbene-2-O-b-Dglucoside. https://doi.org/10.1016/j.neuroscience.2020.01.025 0306-4522/Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved. 1 0
Please cite this article in press as: Li X-X et al. 2,3,5,4 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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inflammatory factors, neurotrophins and their receptors are consistently reported (Chiba et al., 2012; Jeong et al., 2013; Jiang et al., 2018). It is well recognized that 2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside (THSG) is the major bioactive ingredients of Polygonum multiflorum, which is a traditional Chinese herb well known as He shou wu that has been used for hair-blacking, tonifying live and kidney, and longevity (Lin et al., 2015; Chen et al., 2016). Pharmacologic studies have verified the effects of THSG in antioxidant, anti-aging, antiinflammation and neuroprotection (Zhang et al., 2007; Lin et al., 2015; Chen et al., 2016; Ling and Xu, 2016; Park et al., 2016). We also previously reported that THSG attenuates staurosporine-induced cytotoxicity in cultured neurons (Yang et al., 2014) or MPP+/MPTP induced neurotoxicity in vitro and in vivo (Yu et al., 2019), and exerts anti-depression effects by restoring inflammatory response and oxidative stress in CRS-induced depressive-like mice (Jiang et al., 2018). THSG medication significantly attenuated the elevation of inflammatory cytokines TNF-a, IL-1 and IL-6 in the hippocampus and prefrontal cortex, and promotes the reactivation of Akt signaling pathway and astrocyte proliferation, so as to alleviate depression. In addition, THSG restores the hippocampal BDNF signaling and neurogenesis to produce antidepressant effects (Wang et al., 2017). However, it is interesting to explore the status of antioxidants, neurotrophins and associated signaling pathways in CRS-induced depressive-like mice and the effects of THSG as antidepressants (Martinowich et al., 2007). In this study, we examined the expression of neurotrophin in CRS-induced depressive-like mice and unravel the mechanistic avenue of THSG-mediated antidepressant medication.
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EXPERIMENTAL PROCEDURES
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Animals
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C57BL/6J mice (6–8 weeks old, n = 40) were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. Mice were housed at a controlled temperature (21 ± 2 °C) and humidity (30–70%) room with a light/dark cycle of 12 h and a free accessible diet and water for acclimation of 7 days. CRS-induced depressive-like mice were established as described previously with minor modifications after 7 days adaption (Nestler and Hyman, 2010; Chu et al., 2016; Jiang et al., 2018). Briefly, mice (n = 32) were restrained in a cylindrical plastic tube (3 cm 10 cm in diameter length) and maintained for 7 h from 9:00 a.m. to 4 p.m. every day for 28 days in light, 21 °C and humidity of 50%. The ventilators (0.5 cm in diameter) are located in the head along the sidewall of the tube to allow air flowing. During the restraint, animals can move their heads and forelimbs, but the bodies and hindquarters cannot move or turn around. Besides, no access to food and water was allowed. Once the restraint was completed, animals were immediately put back in their cages and free to get food and water. The control group (n = 8) (without restraint) remained in the cages
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until the behavioral experiments started. The animals were weighed and behavioral assessed to determine the successful establishment of the model. After 28 days of modeling, the open field test (OFT) (lasting 6 h) and tail suspension test (TST) (after 6 h, lasting 3 h) was conducted on the first day. Then, the mice were subjected to forced swim test (FST) on the second day (after 24 h, lasting 6 h). Finally, the mice were sacrificed for biochemical experiments. All animal experiments were carried out in accordance with the Regulation of the Beijing Municipality for Administration of Laboratory Animals and approved by Animal Care and Use Ethics Committee of Minzu University of China. OFT was performed in a cubic box (50 cm 50 cm 45 cm), which was divided into 25 (10 cm 10 cm) area on the white bottom, whereas all side surfaces were painted black as described (Seibenhener and Wooten, 2015). However, the top of the box is left uncovered. Mouse is placed separately in the middle of the bottom surface to begin free exploration; it was free to explore 6 min in light conditions. Meanwhile, movements are recorded by computer tracking programs to analyze movements (including horizontal movement, vertical movement and self-grooming, etc.) of the animal over time. TST is realized by suspending the animal by the tail to make its body hangs in the air, facing down (Cryan et al., 2005). The animal will actively try to escape, however, if escape is impossible, the animal will eventually stop trying (‘‘give up”). When the animal stops struggling and hangs immobile, it is considered to have ‘‘given up”. Longer periods of immobility are characteristic of a depressive-like state. The test sustained for 6 min, and the immobility time in the last 5 min is recorded. FST is performed to monitor depressive-like behavior in mice (Yankelevitch-Yahav et al., 2015). The test lasted for 6 min consisting of a session of pretest (the first 1 min) and test (the last 5 min). Mouse was placed to a cylinder (40 cm 15 cm in height width) with 2 L water (25 °C) so that it cannot touch the bottom of the container with hind legs and tail. Mouse will attempt to escape by actively swimming. When the animal stops swimming and starts to float on the surface of the water, it is considered to have ‘‘immobility” or ‘‘given up”. An animal that gives up relatively quickly is considered displaying characteristics similar to human depression. After the pretest (1 min), the immobility time of mice was recorded in test session (5 min). Sucrose preference test (SPT) is performed as described previously (Liu et al., 2018). At the end of the behavioral test, a bottle of 1% (mass/volume) sucrose water (200 mL) and a bottle of tap water (200 mL) were placed in each cage; animals were free to drink sucrose or tap waters for pre-test acclimation. The next day, a three-day experiment was conducted. During the experiment, the positions of the two bottles were changed every day to reduce any confound produced by a side bias. Once the test ended, sucrose preference is represented by a percentage of the volume of sucrose consumed over
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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the total volume of fluid intake. The preference toward the sweetened drink is typical, and if not, it indicates anhedonia or depression.
Drugs THSG (2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside) (#B21757, CAS 82373-94-2, HPLC 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Fluoxetine hydrochloride (FH, #F132) (Wong et al., 2005), a selective serotonin reuptake inhibitor used for treating depression was supplied by Sigma-Aldrich LLC. THSG and FH were dissolved in 0.9% NaCl and subcutaneous injected into mice in the nape. Two concentrations of THSG (20 mgkg1 as medium and 40 mgkg1 as high concentration) were used as described previously (Jiang et al., 2018), whereas FH is 10 mgkg1. Mice received the drug treatment one time per day and lasted for 15 days. Injection of 0.9% NaCl (vehicle) served as control.
Determination of antioxidants in the hippocampus and prefrontal cortex Total antioxidant capacity (T-AOC) (#A015-1-2), total GSH activity (#A061-1-1), total superoxide dismutase (T-SOD) (#A001-1-2), and nitric oxide (#A012-1-2) were determined by assay kits provided by Nanjing Jiancheng Bioengineering Institute. Catalase activity assay kit (#S0051) was provided by Beyotime Biotechnology. As a biomarker of lipid peroxidation reflects the effect of diet on the oxidative status in vivo, malondialdehyde (MDA) is measured by MDA assay kit (TBA method) (#A003-1-2). Total protein extracted from the hippocampus and prefrontal cortex was used for assays.
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Western blotting
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Hippocampus and prefrontal cortex were isolated from the mouse brain and quickly frozen in liquid nitrogen, and stored at 80 °C until use. Tissue was dissolved in lysis buffer (#T8200, Beyotime) and subjected to homogenization. Then extracted protein was harvested after centrifugation, and quantified by the BCA protein assay kit (#P0010, Beyotime). Equal total protein was loaded on SDS–PAGE gel and separated, followed by a transblotting, blocking, and antibody probing. Target protein was photographed in the chemiluminescent imager Tanon 4200. Goat anti-mouse IgG-HRP (#sc2005), goat anti-rabbit IgG-HRP (#sc-2004) secondary antibodies were purchased from Santa Cruz Biotechnology. Anti p-Akt (Ser473) (#4060S), total Akt (t-Akt, #9272S), Bcl-2 (#3498S), phospho-TrkB (#4619T), GFAP (#3670S), cleaved-caspase-3 (#9662) and b-actin (#4970) antibodies were acquired from Cell Signaling Technology, Inc. Antibodies anti BDNF (#ab108319) was purchased from Abcam, anti fibroblast growth factor 2 (FGF2) (#sc-136255) and anti HSP70 (#sc-32239) were provided by Santa Cruz Biotechnology, Inc.
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Statistical analysis
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Data were presented as mean ± SD, ns, non-significant, * (#), p < 0.05, ** (##), p < 0.01, *** (###), p < 0.001 represented the significance variance of the difference between the groups that were analyzed by one-way ANOVA (analysis of variance) and followed by Bonferroni’s multiple comparisons test, or determined by Mann-Whitney test for non-parametric significance analysis as indicated. Figures were plotted using GraphPad Prism 7.0 (GraphPad Software, USA).
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RESULTS
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THSG effectively improves the behavioral performances of CRS-induced depressive-like mice
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Mice were subjected to chronic restraint stress as described in Material and methods to simulate the episode of developing depression (Fig. 1A). Accordingly, CRS-induced mice showed a significant loss in body weight compared with their control counterparts (Fig. 1B) (p < 0.001). CRS-induced behavioral inability was examined by motor tests. Firstly, open field test, including horizontal movement, vertical movement and self-grooming confirmed the establishment of CRSinduced depressive-like behaviors in mice and the antidepression activity of THSG (Fig. 1C). In addition, forced swim test and the tail suspension test further confirmed the CRS-induced depressive-like behaviors, indicating by extended immobility time (Fig. 1D, CRS group vs. control group). However, medication of THSG recovered the loss of body weight and reduced the immobility time in FST and TST to normal (Fig. 1B, D, CRS + THSG group vs. control). The effects of THSG were similar to that of FH (CRS + FH group), which is a clinical antidepressant. Moreover, THSG recovered the level of experiencing pleasure of CRS-induced mice suggested by SPT (Fig. 1E), which is a reward-based test to measure the level of anhedonia. Anhedonia is one of the core symptoms of depression. From the results aforementioned, we concluded that THSG is effective in restoring the behavioral performances of CRS-induced depressive-like mice.
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THSG restores the biochemistry of antioxidant factors to attenuate CRS-induced depression
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We have previously shown that the expression of neuroinflammatory factors TNF-a, IL-6 and IL-b in the brain is significantly increased, suggesting the elevation of inflammation upon stress (Jiang et al., 2018). To investigate the effect of THSG on restoring the redox status of CRS-induced depressive-like mice, the redox factors were determined in the lysis of the hippocampus and prefrontal cortex by kits as described in Materials and methods. Consistently, the metabolic capacity of oxidative stress in the brain of CRS-mice was dramatically decreased, as indicated by the decline of T-AOC, total glutathione (T-GSH), superoxide dismutase (SOD), catalase activity and NO (Fig. 2A–E). In contrast, the content of MDA was notably elevated in the hippocampus and prefrontal cortex (Fig. 2F). Luckily, THSG medication res-
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Fig. 1. THSG effectively ameliorates the behavioral performance of CRS-induced depressive-like mice. (A) Timeline for establishment of CRSinduced depressive-like mice models along with behavioral tests and drug administration. (B) Chronic-restrained stress (CRS) induced a significant loss of mice body weight. THSG and fluoxetine hydrochloride (FH) rescued the body weight of CRS-induced depressive-like mice. THSG (M), medium concentration, 20 mgkg1; THSG (H), high concentration, 40 mgkg1, FH, 10 mgkg1. (C) THSG reduced the immobility time to normal in FST (Forced swim test) and TST (Tail suspension test) experiments. (D) THSG improved the performances of CRS-induced depressive-like mice examined by open field test, including horizontal movement, vertical movement and self-grooming. (E) THSG recovered the level of experiencing pleasure of CRS-induced mice to normal in sucrose preference test. Statistical significance between groups was detected by one-way ANOVA followed by Bonferroni’s multiple comparisons test, except the data of self-grooming that was determined by Mann-Whitney test. *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; ns, non-significant; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 8.
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cued the decline of antioxidant capacity and restored the content of MDA to normal (Fig. 2). Besides, THSG restored the redox system to normal with a similar effect as that of FH (CRS + FH group), an antidepressant which inhibiting selective reuptake of serotonin. Therefore, we confirmed that there is a significant body weight loss in CRS-mice, corresponding to a reduction in cellular antioxidant capacity. However, THSG is effective in restoring the biochemistry of redox systems to attenuate depression. THSG restores the expression of BDNF, FGF2 in CRSinduced depressive-like mice In addition, CRS-induced impairments of glial cells in the hippocampus and prefrontal cortex were indicated by
reduced expression of GFAP (glial fibrillary acidic protein), which is a commonly used protein marker of astrocytes (Fig. 3A). In spite of this, the administration of THSG ameliorated the expression of GFAP that suggests the recovery of the neuroglia. Consistently, additional examination of the expression of FGF2 and BDNF mimicked the situation of GFAP. CRS induced downregulation of FGF2 and BDNF as well as BDNFTrkB signaling pathway (Fig. 3B). These results suggested that neurotrophic functions are significantly inhibited in CRS induced depressive-like mice. Luckily, the administration of THSG restored the expression of FGF2 and BDNF, reactivated BDNF-TrkB signaling pathway (Fig. 3) and finally exerted antidepressant effects.
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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Fig. 2. THSG effectively restores the antioxidant ability in the hippocampus and prefrontal cortex of CRS-induced depressive-like mice. (A–E) CRSinduced depressive-like mice showed a significant decrease in T-AOC, T-GSH, SOD, catalase activity and NO. THSG recovered the above redox associated indicators to normal. (F) MDA was significantly increased in CRS-induced depressive-like mice, while THSG ameliorated the conditions and restored the MDA content to normal. In the aforementioned assays, the treatment of fluoxetine hydrochloride (FH) was used as a positive control. Significance of variance between groups was examined by one-way ANOVA and followed by Bonferroni’s multiple comparisons test. ns, non-significant; *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 8.
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THSG ameliorates the Akt signaling pathway and inhibits apoptosis to exert antidepressant functions As aforementioned, we confirmed that BDNF and FGF2 could be restored by THSG. We detected the downstream cellular signaling pathway in subsequent experiments. Moderate activity of the Akt signaling pathway is essential for neuron survival, neurite outgrowth and branching, as well as anti-inflammation and inhibition of apoptosis. In the hippocampus and prefrontal cortex of CRS-induced depressive-like mice,
both Akt activation and the expression of HSP70 were severely inhibited (Fig. 4A); these results are consistent with our previous work (Jiang et al., 2018). Administration of THSG effectively reactivated the Akt signaling pathway and restored the expression of HSP70. In addition, upon CRS-induced depressive stress, the onset of neuron apoptosis was observed as indicated by the down regulation of the anti-apoptotic protein Bcl-2 and the significant elevation of apoptosis promoting enzyme cleavedcaspase-3. However, the administration of THSG
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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Fig. 3. THSG effectively ameliorates the conditions of CRS-induced depressive-like mice through restoring the expression of BDNF and FGF2. (A) Representative western blot showed significant downregulation of astrocyte marker GFAP in CRS-induced depressive-like mice, while THSG restored the expression of GFAP to normal with similar effects compared to the treatment of fluoxetine hydrochloride (FH). The relative expression level of GFAP was quantified according to the density of western blot bands and normalized to b-actin. (B) THSG restored the expression of FGF2 and BDNF-TrkB signaling pathway to attenuate depression. The relative protein expression level of FGF2, BDNF and its receptor TrkB was measured by western blotting and normalized to b-actin. The significance of variance between groups was examined by one-way analysis of variance (ANOVA) and followed by Bonferroni’s multiple comparisons test to correct the p value. *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 3.
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restored the expression of Bcl-2 and cleaved-caspase-3 to maintain the integrity of neurons for anti-depression (Fig. 4B). These results suggest that in CRS-mice, the downregulation of neurotrophins are accompanied by damage to glial cells, and further lead to apoptosis of nerve cells. THSG effectively ameliorated the secretion of neurotrophins BDNF and FGF2, activated the downstream signaling pathways and inhibited neuron apoptosis. Thus, our work establishes the antidepressant effects of THSG, which may provide a mechanism avenue for the development of novel antidepressants by restoring the expression of neurotrophins (see Fig. 5).
DISCUSSION
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Depression severely disables an object with low mood and anhedonia as the main characteristics. Both genetic and socio-economic factors contribute to the onset of depression, although the duration and time of episodes over a lifetime are variable (Malhi and Mann, 2018). In addition to cognitive therapy, antidepressant medication is usually the first line of treatment for depression, although there is no absolute effective prescription to treat this complex mental disease (Anthes, 2014). Neurotrophin hypothesis of depression argues that BDNF
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Fig. 4. THSG restores the Akt signaling pathway and the expression of Bcl-2/caspase-3/HSP70 to inhibit apoptosis. (A) THSG attenuated CRSinduced downregulation of p-Akt and HSP70 in the hippocampus and prefrontal cortex. The relative protein expression levels of p-Akt and HSP70 were normalized to total Akt (t-Akt) or b-actin, respectively. (B) THSG antagonistically ameliorated the expression of the anti-apoptotic protein Bcl-2 and apoptosis promoting enzyme cleaved-caspase-3 (cleaved-Csp-3) to alleviate depression in CRS-induced mice. The relative protein expression level of Bcl-2 and cleaved-caspase-3 was normalized to b-actin. Analysis of variance among groups was calculated by one-way ANOVA and followed by Bonferroni’s multiple comparisons test. *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 3.
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and its mediated signaling axis could be a target of antidepressant medication (Martinowich et al., 2007). It is conceived that decreased expression of BDNF and other neurotrophic factors could result in atrophy of the hippocampus and prefrontal cortex in depression (Gronli et al., 2006; Chiba et al., 2012; Wang et al., 2017), whereas restoring the expression of BDNF and its signaling axis can reverse the decay of neuron systems to produce antidepressant effects. Among antidepressants, traditional Chinese medicine could be a rich source. Poly-
gonum multiflorum has long been empirically used as a tonic and anti-aging medicine (Lin et al., 2015). Preclinical studies have characterized its main active component THSG and established its effects in antioxidant, antidepression and anti-aging, for THSG was reported to promote the expression of longevity gene klotho and reduce senescent cells in related tissues and organs (Ling et al., 2016). We previously established CRS-induced mouse depression model in which the dysfunction of Akt and
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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regulatory role of neurotrophins in depressive-like model mice. CRS seriously inhibits the expression of neurotrophic factors such as BDNF and FGF2 in the hippocampus and prefrontal cortex of the brain, which suggests the decreased activity of glial cells, indicating that chronic depression stress profoundly affect the functional state of the brain and cause severe nerve damage. Moreover, the activation of a moderate level of the Akt signaling pathway required for normal neural activity is also significantly inhibited under the stress of depression. On the contrast, inflammatory factors were obviously elevated. However, in CRS-mice treated with THSG, the expression of BDNF and FGF2 was rescued consistently with the attenuation of astrocyte apoptosis and recovery of neurotrophic function. Therefore, we concluded that in CRS-induced depressive-like mouse model, neurotrophins and their related signaling pathway could be a target for antidepressant medication. THSG Fig. 5. A schematic model suggests THSG as an antidepressant in CRS-induced depression-like is a promise component of Polymodels. CRS impaired the expression of BDNF and FGF2 in the hippocampus and prefrontal cortex, inactivated Akt signaling axis and caused neuronal death. In contrast, THSG restored the expression gonum multiflorum that could be of BDNF and FGF2, reactivated Akt signaling axis and antagonistically restored the expression of antiused as an antidepressant by apoptotic protein Bcl-2 and apoptosis promoting enzyme cleaved-caspase-3 to attenuate CRSrestoring the biochemistry of the induced depression. nervous system and promoting neural plasticity and functions. In CRS-induced depressive-like Erk signaling accompanying an elevation of inflammation mice, the expression of BDNF and FGF2 along with assoand decreased capacity of antioxidant (Jiang et al., 2018). ciated signaling pathways is strongly impaired. The major In view of this, we carefully tested the total proteins colbioactive ingredients of Polygonum multiflorum THSG are lected in the hippocampus and prefrontal cortex, and effective to ameliorate the biochemistry of neurons in the detected the changes in BDNF, FGF2 and associated sigbrain for anti-depression. naling effectors. We found that CRS significantly reduced BDNF and FGF2 in the hippocampus and prefrontal cortex and significantly decreased the activity of glial cells, ACKNOWLEDGMENTS indicting by the diminished expression of glial marker We are grateful to the grants supported by the National GFAP. GFAP is an important biological marker indicating Natural Science Foundation of China the activity of astrocyte, for astrocyte atrophy is closely (8187308821778038), and Shenzhen science and related to the injury of the central nervous or degenerative technology innovation committee program diseases (Volterra and Meldolesi, 2005). Meanwhile, in (JCYJ20180305163349116) and the MUC 111 project addition to the significantly increased expression of (URTP2017110030). inflammatory cytokines TNF-a, IL-1 and IL-6 that we previously verified (Jiang et al., 2018), the antioxidant capacity in the brain of CRS-mice was significantly declined, REFERENCES confirmed by the decline of T-AOC, catalyse activity, TAnthes E (2014) Depression: a change of mind. Nature 515:185–187. GSH, NO, and SOD and elevation of MDA (Fig. 2). FortuChen T, Yang YJ, Li YK, Liu J, Wu PF, Wang F, Chen JG, Long LH nately, medication of THSG significantly restored the (2016) Chronic administration tetrahydroxystilbene glucoside expression of BDNF and FGF2 in the hippocampus and promotes hippocampal memory and synaptic plasticity and cortex and reactivated the downstream Akt signaling activates ERKs, CaMKII and SIRT1/miR-134 in vivo. J pathway. Meanwhile, apoptosis of nerve cells was preEthnopharmacol 190:74–82. vented, which was indicated by the recovered expression Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H (2012) Chronic restraint stress causes anxiety- and of anti-apoptotic protein Bcl-2 and the inhibition of depression-like behaviors, downregulates glucocorticoid receptor caspase-3. In this work, we demonstrated the important expression, and attenuates glutamate release induced by brain-
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(Received 2 July 2019, Accepted 16 January 2020) (Available online xxxx)
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2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression
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Xi-Xi Li, a Yun Yu, a Xiu-Yuan Lang, a Cheng-Yong Jiang, a Rongfeng Lan b* and Xiao-Yan Qin a*
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a
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Center on Translational Neuroscience, College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China
Department of Cell Biology & Medical Genetics, School of Basic Medical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, China
Abstract—Depression is a long term inhibitory mood that heavily disabled human beings. We have previously demonstrated anti-depression effect of 2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside (THSG) in chronicrestraint stress (CRS) induced depressive-like mice by restoring the oxidative pathway and neuroinflammation. In this study, we examine the conditions of neurotrophins in CRS-induced depressive-like mice and whether THSG could be an antidepressant by ameliorating the neurotrophins and their associated signaling axis. CRS produced downregulation of antioxidants, the decline of brain-derived neurotrophic factor (BDNF), fibroblast growth factor 2 (FGF2) and associated signaling regulators in the hippocampus and prefrontal cortex, corresponding to the behavioral inability and anhedonia. Administration of THSG restored the expression of antioxidants and neurotrophins BDNF, FGF2. Besides, THSG recovered the Akt signaling pathway and antagonistically restored the expression of Bcl-2 and cleaved-caspase-3 to inhibit apoptosis. Consistently, behavioral performances were recovered from CRS-induced motor inability and anhedonia. In summary, THSG is effective to attenuate stressinduced depression by ameliorating the biochemistry of neurotrophins and their related signaling pathways. These results may provide an avenue to take BDNF as a target to explore folk medicine for anti-depression. Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: BDNF, FGF2, THSG, forced swim test, depression.
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INTRODUCTION
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Depression is a debilitating mood disorder that often includes symptoms of anhedonia, feelings of dejection and hopelessness, lack of motivation, fatigue, sleep disorders and reckless behaviors such as suicide. More than just feeling not good in a short time, depression may severely change the way how you think, feel and the daily behaviors. Psycho-social factors such as loneliness, stressful life and economic strain are considered to make people vulnerable to depression. Genetic conditions and biochemical changes in the brain may also contribute to the onset of depression. Elucidation of the mechanism of depression induced
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neurophysiological changes will contribute to the exploration of therapy strategy. Although many hypotheses have been proposed to explain the pathogenesis of depression, it is well established that restoring the biochemistry of neurotransmitters and attenuating neuroinflammation may contribute to the therapy of depression (Martinowich et al., 2007). In the neurotrophin hypothesis of depression, reduced expression of brain-derived neurotrophic factor (BDNF) may lead to neural atrophy and cell loss in the hippocampus and prefrontal cortex, whereas antidepressants could exert their therapeutic effects by increasing the expression of BDNF and restoring its mediated downstream signaling (Duman and Monteggia, 2006; Martinowich et al., 2007). Although it may not be the sole mediator of depression, BDNF and its receptor TrkB acts in antidepressant medication. Antidepressant medication serves as a simple and quick way of relief objects from suffering the pain of severe depression. In an experimental study, chronic-restraint stress (CRS) induced depressive-like behaviors of rodent models are well established to simulate depression and is used for drug development (Duman and Monteggia, 2006; Nestler and Hyman, 2010). Chronic stress induced anxiety, body weight loss, along with dysfunctions of
*Corresponding authors. Addresses: A7-456, Xili Campus, Shenzhen University Health Science Center, China (R. F. Lan). Room 1301, College of Life and Environmental Sciences, Minzu University of China (X.- Y. Qin). E-mail addresses: [email protected] (R. Lan), [email protected]. cn (X.-Y. Qin). Abbreviations: BDNF, brain-derived neurotrophic factor; CRS, chronicrestraint stress; FGF2, fibroblast growth factor 2; FH, fluoxetine hydrochloride; MDA, malondialdehyde; SOD, superoxide dismutase; SPT, sucrose preference test; T-AOC, total antioxidant capacity; TGSH, total glutathione; THSG, 2,3,5,40 -tetrahydroxystilbene-2-O-b-Dglucoside. https://doi.org/10.1016/j.neuroscience.2020.01.025 0306-4522/Ó 2020 IBRO. Published by Elsevier Ltd. All rights reserved. 1 0
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inflammatory factors, neurotrophins and their receptors are consistently reported (Chiba et al., 2012; Jeong et al., 2013; Jiang et al., 2018). It is well recognized that 2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside (THSG) is the major bioactive ingredients of Polygonum multiflorum, which is a traditional Chinese herb well known as He shou wu that has been used for hair-blacking, tonifying live and kidney, and longevity (Lin et al., 2015; Chen et al., 2016). Pharmacologic studies have verified the effects of THSG in antioxidant, anti-aging, antiinflammation and neuroprotection (Zhang et al., 2007; Lin et al., 2015; Chen et al., 2016; Ling and Xu, 2016; Park et al., 2016). We also previously reported that THSG attenuates staurosporine-induced cytotoxicity in cultured neurons (Yang et al., 2014) or MPP+/MPTP induced neurotoxicity in vitro and in vivo (Yu et al., 2019), and exerts anti-depression effects by restoring inflammatory response and oxidative stress in CRS-induced depressive-like mice (Jiang et al., 2018). THSG medication significantly attenuated the elevation of inflammatory cytokines TNF-a, IL-1 and IL-6 in the hippocampus and prefrontal cortex, and promotes the reactivation of Akt signaling pathway and astrocyte proliferation, so as to alleviate depression. In addition, THSG restores the hippocampal BDNF signaling and neurogenesis to produce antidepressant effects (Wang et al., 2017). However, it is interesting to explore the status of antioxidants, neurotrophins and associated signaling pathways in CRS-induced depressive-like mice and the effects of THSG as antidepressants (Martinowich et al., 2007). In this study, we examined the expression of neurotrophin in CRS-induced depressive-like mice and unravel the mechanistic avenue of THSG-mediated antidepressant medication.
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EXPERIMENTAL PROCEDURES
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Animals
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C57BL/6J mice (6–8 weeks old, n = 40) were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. Mice were housed at a controlled temperature (21 ± 2 °C) and humidity (30–70%) room with a light/dark cycle of 12 h and a free accessible diet and water for acclimation of 7 days. CRS-induced depressive-like mice were established as described previously with minor modifications after 7 days adaption (Nestler and Hyman, 2010; Chu et al., 2016; Jiang et al., 2018). Briefly, mice (n = 32) were restrained in a cylindrical plastic tube (3 cm 10 cm in diameter length) and maintained for 7 h from 9:00 a.m. to 4 p.m. every day for 28 days in light, 21 °C and humidity of 50%. The ventilators (0.5 cm in diameter) are located in the head along the sidewall of the tube to allow air flowing. During the restraint, animals can move their heads and forelimbs, but the bodies and hindquarters cannot move or turn around. Besides, no access to food and water was allowed. Once the restraint was completed, animals were immediately put back in their cages and free to get food and water. The control group (n = 8) (without restraint) remained in the cages
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until the behavioral experiments started. The animals were weighed and behavioral assessed to determine the successful establishment of the model. After 28 days of modeling, the open field test (OFT) (lasting 6 h) and tail suspension test (TST) (after 6 h, lasting 3 h) was conducted on the first day. Then, the mice were subjected to forced swim test (FST) on the second day (after 24 h, lasting 6 h). Finally, the mice were sacrificed for biochemical experiments. All animal experiments were carried out in accordance with the Regulation of the Beijing Municipality for Administration of Laboratory Animals and approved by Animal Care and Use Ethics Committee of Minzu University of China. OFT was performed in a cubic box (50 cm 50 cm 45 cm), which was divided into 25 (10 cm 10 cm) area on the white bottom, whereas all side surfaces were painted black as described (Seibenhener and Wooten, 2015). However, the top of the box is left uncovered. Mouse is placed separately in the middle of the bottom surface to begin free exploration; it was free to explore 6 min in light conditions. Meanwhile, movements are recorded by computer tracking programs to analyze movements (including horizontal movement, vertical movement and self-grooming, etc.) of the animal over time. TST is realized by suspending the animal by the tail to make its body hangs in the air, facing down (Cryan et al., 2005). The animal will actively try to escape, however, if escape is impossible, the animal will eventually stop trying (‘‘give up”). When the animal stops struggling and hangs immobile, it is considered to have ‘‘given up”. Longer periods of immobility are characteristic of a depressive-like state. The test sustained for 6 min, and the immobility time in the last 5 min is recorded. FST is performed to monitor depressive-like behavior in mice (Yankelevitch-Yahav et al., 2015). The test lasted for 6 min consisting of a session of pretest (the first 1 min) and test (the last 5 min). Mouse was placed to a cylinder (40 cm 15 cm in height width) with 2 L water (25 °C) so that it cannot touch the bottom of the container with hind legs and tail. Mouse will attempt to escape by actively swimming. When the animal stops swimming and starts to float on the surface of the water, it is considered to have ‘‘immobility” or ‘‘given up”. An animal that gives up relatively quickly is considered displaying characteristics similar to human depression. After the pretest (1 min), the immobility time of mice was recorded in test session (5 min). Sucrose preference test (SPT) is performed as described previously (Liu et al., 2018). At the end of the behavioral test, a bottle of 1% (mass/volume) sucrose water (200 mL) and a bottle of tap water (200 mL) were placed in each cage; animals were free to drink sucrose or tap waters for pre-test acclimation. The next day, a three-day experiment was conducted. During the experiment, the positions of the two bottles were changed every day to reduce any confound produced by a side bias. Once the test ended, sucrose preference is represented by a percentage of the volume of sucrose consumed over
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the total volume of fluid intake. The preference toward the sweetened drink is typical, and if not, it indicates anhedonia or depression.
Drugs THSG (2,3,5,40 -tetrahydroxystilbene-2-O-b-D-glucoside) (#B21757, CAS 82373-94-2, HPLC 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Fluoxetine hydrochloride (FH, #F132) (Wong et al., 2005), a selective serotonin reuptake inhibitor used for treating depression was supplied by Sigma-Aldrich LLC. THSG and FH were dissolved in 0.9% NaCl and subcutaneous injected into mice in the nape. Two concentrations of THSG (20 mgkg1 as medium and 40 mgkg1 as high concentration) were used as described previously (Jiang et al., 2018), whereas FH is 10 mgkg1. Mice received the drug treatment one time per day and lasted for 15 days. Injection of 0.9% NaCl (vehicle) served as control.
Determination of antioxidants in the hippocampus and prefrontal cortex Total antioxidant capacity (T-AOC) (#A015-1-2), total GSH activity (#A061-1-1), total superoxide dismutase (T-SOD) (#A001-1-2), and nitric oxide (#A012-1-2) were determined by assay kits provided by Nanjing Jiancheng Bioengineering Institute. Catalase activity assay kit (#S0051) was provided by Beyotime Biotechnology. As a biomarker of lipid peroxidation reflects the effect of diet on the oxidative status in vivo, malondialdehyde (MDA) is measured by MDA assay kit (TBA method) (#A003-1-2). Total protein extracted from the hippocampus and prefrontal cortex was used for assays.
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Western blotting
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Hippocampus and prefrontal cortex were isolated from the mouse brain and quickly frozen in liquid nitrogen, and stored at 80 °C until use. Tissue was dissolved in lysis buffer (#T8200, Beyotime) and subjected to homogenization. Then extracted protein was harvested after centrifugation, and quantified by the BCA protein assay kit (#P0010, Beyotime). Equal total protein was loaded on SDS–PAGE gel and separated, followed by a transblotting, blocking, and antibody probing. Target protein was photographed in the chemiluminescent imager Tanon 4200. Goat anti-mouse IgG-HRP (#sc2005), goat anti-rabbit IgG-HRP (#sc-2004) secondary antibodies were purchased from Santa Cruz Biotechnology. Anti p-Akt (Ser473) (#4060S), total Akt (t-Akt, #9272S), Bcl-2 (#3498S), phospho-TrkB (#4619T), GFAP (#3670S), cleaved-caspase-3 (#9662) and b-actin (#4970) antibodies were acquired from Cell Signaling Technology, Inc. Antibodies anti BDNF (#ab108319) was purchased from Abcam, anti fibroblast growth factor 2 (FGF2) (#sc-136255) and anti HSP70 (#sc-32239) were provided by Santa Cruz Biotechnology, Inc.
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Statistical analysis
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Data were presented as mean ± SD, ns, non-significant, * (#), p < 0.05, ** (##), p < 0.01, *** (###), p < 0.001 represented the significance variance of the difference between the groups that were analyzed by one-way ANOVA (analysis of variance) and followed by Bonferroni’s multiple comparisons test, or determined by Mann-Whitney test for non-parametric significance analysis as indicated. Figures were plotted using GraphPad Prism 7.0 (GraphPad Software, USA).
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RESULTS
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THSG effectively improves the behavioral performances of CRS-induced depressive-like mice
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Mice were subjected to chronic restraint stress as described in Material and methods to simulate the episode of developing depression (Fig. 1A). Accordingly, CRS-induced mice showed a significant loss in body weight compared with their control counterparts (Fig. 1B) (p < 0.001). CRS-induced behavioral inability was examined by motor tests. Firstly, open field test, including horizontal movement, vertical movement and self-grooming confirmed the establishment of CRSinduced depressive-like behaviors in mice and the antidepression activity of THSG (Fig. 1C). In addition, forced swim test and the tail suspension test further confirmed the CRS-induced depressive-like behaviors, indicating by extended immobility time (Fig. 1D, CRS group vs. control group). However, medication of THSG recovered the loss of body weight and reduced the immobility time in FST and TST to normal (Fig. 1B, D, CRS + THSG group vs. control). The effects of THSG were similar to that of FH (CRS + FH group), which is a clinical antidepressant. Moreover, THSG recovered the level of experiencing pleasure of CRS-induced mice suggested by SPT (Fig. 1E), which is a reward-based test to measure the level of anhedonia. Anhedonia is one of the core symptoms of depression. From the results aforementioned, we concluded that THSG is effective in restoring the behavioral performances of CRS-induced depressive-like mice.
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THSG restores the biochemistry of antioxidant factors to attenuate CRS-induced depression
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We have previously shown that the expression of neuroinflammatory factors TNF-a, IL-6 and IL-b in the brain is significantly increased, suggesting the elevation of inflammation upon stress (Jiang et al., 2018). To investigate the effect of THSG on restoring the redox status of CRS-induced depressive-like mice, the redox factors were determined in the lysis of the hippocampus and prefrontal cortex by kits as described in Materials and methods. Consistently, the metabolic capacity of oxidative stress in the brain of CRS-mice was dramatically decreased, as indicated by the decline of T-AOC, total glutathione (T-GSH), superoxide dismutase (SOD), catalase activity and NO (Fig. 2A–E). In contrast, the content of MDA was notably elevated in the hippocampus and prefrontal cortex (Fig. 2F). Luckily, THSG medication res-
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Fig. 1. THSG effectively ameliorates the behavioral performance of CRS-induced depressive-like mice. (A) Timeline for establishment of CRSinduced depressive-like mice models along with behavioral tests and drug administration. (B) Chronic-restrained stress (CRS) induced a significant loss of mice body weight. THSG and fluoxetine hydrochloride (FH) rescued the body weight of CRS-induced depressive-like mice. THSG (M), medium concentration, 20 mgkg1; THSG (H), high concentration, 40 mgkg1, FH, 10 mgkg1. (C) THSG reduced the immobility time to normal in FST (Forced swim test) and TST (Tail suspension test) experiments. (D) THSG improved the performances of CRS-induced depressive-like mice examined by open field test, including horizontal movement, vertical movement and self-grooming. (E) THSG recovered the level of experiencing pleasure of CRS-induced mice to normal in sucrose preference test. Statistical significance between groups was detected by one-way ANOVA followed by Bonferroni’s multiple comparisons test, except the data of self-grooming that was determined by Mann-Whitney test. *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; ns, non-significant; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 8.
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cued the decline of antioxidant capacity and restored the content of MDA to normal (Fig. 2). Besides, THSG restored the redox system to normal with a similar effect as that of FH (CRS + FH group), an antidepressant which inhibiting selective reuptake of serotonin. Therefore, we confirmed that there is a significant body weight loss in CRS-mice, corresponding to a reduction in cellular antioxidant capacity. However, THSG is effective in restoring the biochemistry of redox systems to attenuate depression. THSG restores the expression of BDNF, FGF2 in CRSinduced depressive-like mice In addition, CRS-induced impairments of glial cells in the hippocampus and prefrontal cortex were indicated by
reduced expression of GFAP (glial fibrillary acidic protein), which is a commonly used protein marker of astrocytes (Fig. 3A). In spite of this, the administration of THSG ameliorated the expression of GFAP that suggests the recovery of the neuroglia. Consistently, additional examination of the expression of FGF2 and BDNF mimicked the situation of GFAP. CRS induced downregulation of FGF2 and BDNF as well as BDNFTrkB signaling pathway (Fig. 3B). These results suggested that neurotrophic functions are significantly inhibited in CRS induced depressive-like mice. Luckily, the administration of THSG restored the expression of FGF2 and BDNF, reactivated BDNF-TrkB signaling pathway (Fig. 3) and finally exerted antidepressant effects.
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Fig. 2. THSG effectively restores the antioxidant ability in the hippocampus and prefrontal cortex of CRS-induced depressive-like mice. (A–E) CRSinduced depressive-like mice showed a significant decrease in T-AOC, T-GSH, SOD, catalase activity and NO. THSG recovered the above redox associated indicators to normal. (F) MDA was significantly increased in CRS-induced depressive-like mice, while THSG ameliorated the conditions and restored the MDA content to normal. In the aforementioned assays, the treatment of fluoxetine hydrochloride (FH) was used as a positive control. Significance of variance between groups was examined by one-way ANOVA and followed by Bonferroni’s multiple comparisons test. ns, non-significant; *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 8.
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THSG ameliorates the Akt signaling pathway and inhibits apoptosis to exert antidepressant functions As aforementioned, we confirmed that BDNF and FGF2 could be restored by THSG. We detected the downstream cellular signaling pathway in subsequent experiments. Moderate activity of the Akt signaling pathway is essential for neuron survival, neurite outgrowth and branching, as well as anti-inflammation and inhibition of apoptosis. In the hippocampus and prefrontal cortex of CRS-induced depressive-like mice,
both Akt activation and the expression of HSP70 were severely inhibited (Fig. 4A); these results are consistent with our previous work (Jiang et al., 2018). Administration of THSG effectively reactivated the Akt signaling pathway and restored the expression of HSP70. In addition, upon CRS-induced depressive stress, the onset of neuron apoptosis was observed as indicated by the down regulation of the anti-apoptotic protein Bcl-2 and the significant elevation of apoptosis promoting enzyme cleavedcaspase-3. However, the administration of THSG
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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Fig. 3. THSG effectively ameliorates the conditions of CRS-induced depressive-like mice through restoring the expression of BDNF and FGF2. (A) Representative western blot showed significant downregulation of astrocyte marker GFAP in CRS-induced depressive-like mice, while THSG restored the expression of GFAP to normal with similar effects compared to the treatment of fluoxetine hydrochloride (FH). The relative expression level of GFAP was quantified according to the density of western blot bands and normalized to b-actin. (B) THSG restored the expression of FGF2 and BDNF-TrkB signaling pathway to attenuate depression. The relative protein expression level of FGF2, BDNF and its receptor TrkB was measured by western blotting and normalized to b-actin. The significance of variance between groups was examined by one-way analysis of variance (ANOVA) and followed by Bonferroni’s multiple comparisons test to correct the p value. *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 3.
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restored the expression of Bcl-2 and cleaved-caspase-3 to maintain the integrity of neurons for anti-depression (Fig. 4B). These results suggest that in CRS-mice, the downregulation of neurotrophins are accompanied by damage to glial cells, and further lead to apoptosis of nerve cells. THSG effectively ameliorated the secretion of neurotrophins BDNF and FGF2, activated the downstream signaling pathways and inhibited neuron apoptosis. Thus, our work establishes the antidepressant effects of THSG, which may provide a mechanism avenue for the development of novel antidepressants by restoring the expression of neurotrophins (see Fig. 5).
DISCUSSION
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Depression severely disables an object with low mood and anhedonia as the main characteristics. Both genetic and socio-economic factors contribute to the onset of depression, although the duration and time of episodes over a lifetime are variable (Malhi and Mann, 2018). In addition to cognitive therapy, antidepressant medication is usually the first line of treatment for depression, although there is no absolute effective prescription to treat this complex mental disease (Anthes, 2014). Neurotrophin hypothesis of depression argues that BDNF
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Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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Fig. 4. THSG restores the Akt signaling pathway and the expression of Bcl-2/caspase-3/HSP70 to inhibit apoptosis. (A) THSG attenuated CRSinduced downregulation of p-Akt and HSP70 in the hippocampus and prefrontal cortex. The relative protein expression levels of p-Akt and HSP70 were normalized to total Akt (t-Akt) or b-actin, respectively. (B) THSG antagonistically ameliorated the expression of the anti-apoptotic protein Bcl-2 and apoptosis promoting enzyme cleaved-caspase-3 (cleaved-Csp-3) to alleviate depression in CRS-induced mice. The relative protein expression level of Bcl-2 and cleaved-caspase-3 was normalized to b-actin. Analysis of variance among groups was calculated by one-way ANOVA and followed by Bonferroni’s multiple comparisons test. *, Control vs. CRS; #, CRS vs. CRS + THSG/FH; * (#) p < 0.05, ** (##) p < 0.01 or *** (###) p < 0.001, n = 3.
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and its mediated signaling axis could be a target of antidepressant medication (Martinowich et al., 2007). It is conceived that decreased expression of BDNF and other neurotrophic factors could result in atrophy of the hippocampus and prefrontal cortex in depression (Gronli et al., 2006; Chiba et al., 2012; Wang et al., 2017), whereas restoring the expression of BDNF and its signaling axis can reverse the decay of neuron systems to produce antidepressant effects. Among antidepressants, traditional Chinese medicine could be a rich source. Poly-
gonum multiflorum has long been empirically used as a tonic and anti-aging medicine (Lin et al., 2015). Preclinical studies have characterized its main active component THSG and established its effects in antioxidant, antidepression and anti-aging, for THSG was reported to promote the expression of longevity gene klotho and reduce senescent cells in related tissues and organs (Ling et al., 2016). We previously established CRS-induced mouse depression model in which the dysfunction of Akt and
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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regulatory role of neurotrophins in depressive-like model mice. CRS seriously inhibits the expression of neurotrophic factors such as BDNF and FGF2 in the hippocampus and prefrontal cortex of the brain, which suggests the decreased activity of glial cells, indicating that chronic depression stress profoundly affect the functional state of the brain and cause severe nerve damage. Moreover, the activation of a moderate level of the Akt signaling pathway required for normal neural activity is also significantly inhibited under the stress of depression. On the contrast, inflammatory factors were obviously elevated. However, in CRS-mice treated with THSG, the expression of BDNF and FGF2 was rescued consistently with the attenuation of astrocyte apoptosis and recovery of neurotrophic function. Therefore, we concluded that in CRS-induced depressive-like mouse model, neurotrophins and their related signaling pathway could be a target for antidepressant medication. THSG Fig. 5. A schematic model suggests THSG as an antidepressant in CRS-induced depression-like is a promise component of Polymodels. CRS impaired the expression of BDNF and FGF2 in the hippocampus and prefrontal cortex, inactivated Akt signaling axis and caused neuronal death. In contrast, THSG restored the expression gonum multiflorum that could be of BDNF and FGF2, reactivated Akt signaling axis and antagonistically restored the expression of antiused as an antidepressant by apoptotic protein Bcl-2 and apoptosis promoting enzyme cleaved-caspase-3 to attenuate CRSrestoring the biochemistry of the induced depression. nervous system and promoting neural plasticity and functions. In CRS-induced depressive-like Erk signaling accompanying an elevation of inflammation mice, the expression of BDNF and FGF2 along with assoand decreased capacity of antioxidant (Jiang et al., 2018). ciated signaling pathways is strongly impaired. The major In view of this, we carefully tested the total proteins colbioactive ingredients of Polygonum multiflorum THSG are lected in the hippocampus and prefrontal cortex, and effective to ameliorate the biochemistry of neurons in the detected the changes in BDNF, FGF2 and associated sigbrain for anti-depression. naling effectors. We found that CRS significantly reduced BDNF and FGF2 in the hippocampus and prefrontal cortex and significantly decreased the activity of glial cells, ACKNOWLEDGMENTS indicting by the diminished expression of glial marker We are grateful to the grants supported by the National GFAP. GFAP is an important biological marker indicating Natural Science Foundation of China the activity of astrocyte, for astrocyte atrophy is closely (8187308821778038), and Shenzhen science and related to the injury of the central nervous or degenerative technology innovation committee program diseases (Volterra and Meldolesi, 2005). Meanwhile, in (JCYJ20180305163349116) and the MUC 111 project addition to the significantly increased expression of (URTP2017110030). inflammatory cytokines TNF-a, IL-1 and IL-6 that we previously verified (Jiang et al., 2018), the antioxidant capacity in the brain of CRS-mice was significantly declined, REFERENCES confirmed by the decline of T-AOC, catalyse activity, TAnthes E (2014) Depression: a change of mind. Nature 515:185–187. GSH, NO, and SOD and elevation of MDA (Fig. 2). FortuChen T, Yang YJ, Li YK, Liu J, Wu PF, Wang F, Chen JG, Long LH nately, medication of THSG significantly restored the (2016) Chronic administration tetrahydroxystilbene glucoside expression of BDNF and FGF2 in the hippocampus and promotes hippocampal memory and synaptic plasticity and cortex and reactivated the downstream Akt signaling activates ERKs, CaMKII and SIRT1/miR-134 in vivo. J pathway. Meanwhile, apoptosis of nerve cells was preEthnopharmacol 190:74–82. vented, which was indicated by the recovered expression Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H (2012) Chronic restraint stress causes anxiety- and of anti-apoptotic protein Bcl-2 and the inhibition of depression-like behaviors, downregulates glucocorticoid receptor caspase-3. In this work, we demonstrated the important expression, and attenuates glutamate release induced by brain-
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(Received 2 July 2019, Accepted 16 January 2020) (Available online xxxx)
Please cite this article in press as: Li X-X et al. 2,3,5,40 -Tetrahydroxystilbene-2-O-b-D-glucoside Restores BDNF-TrkB and FGF2-Akt Signaling Axis to Attenuate Stress-induced Depression. Neuroscience (2020), https:// doi.org/10.1016/j.neuroscience.2020.01.025
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