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7,8-Dihydroxyflavone reverses the depressive symptoms in mouse chronic mild stress
Neuroscience Letters 635 (2016) 33–38
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Research article
7,8-Dihydroxyflavone reverses the depressive symptoms in mouse chronic mild stress Min-Wang Zhang a , She-feng Zhang b , Zhen-Hua Li a , Fang Han a,∗ a b
Department of Encephalopathy (II), Zhengzhou Traditional Chinese Medicine Hospital, Zhengzhou 450007, Henan province, PR China Henan Province Chinese medicine research institute, Zhengzhou 450004, Henan province, PR China
h i g h l i g h t s • 7,8-DHF produced antidepressant-like effects in CMS mice. • 7,8-DHF promoted the expression of synaptic proteins. • K252a pretreatment blocked the effects of 7,8-DHF.
a r t i c l e
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Article history: Received 19 August 2016 Received in revised form 18 October 2016 Accepted 19 October 2016 Available online 20 October 2016 Keywords: 7,8-Dihydroxyflavone (7,8-DHF) Antidepressant-like effects Chronic mild stress (CMS) TrkB PSD95 Synaptophysin
a b s t r a c t 7,8-Dihydroxyflavone (7,8-DHF) is a naturally-occurring flavone which possesses good bioavailability. Due to its ability to cross the blood-brain barrier, previous studies have demonstrated that 7,8-DHF was a potent tropomyosin-related kinase B (TrkB) agonist, and produced antidepressant-like effects in mouse forced swimming test and tail suspension test. However, it has not been evaluated in chronic mild stress (CMS), a classical depression model modulating the processes of major depression in human. In the present study, we not only evaluated the depressive-like behaviors, but also measured the key proteins of TrkB signaling in mice exposed to CMS. Our results firstly found that long term but not single injection of 7,8-DHF restored the depressive-like behaviors in sucrose preference test and novelty suppressed feeding test. In addition, 7,8-DHF not only increased TrkB phosphorylation and brain-derived neurotrophic factor (BDNF) levels, but also activated the expression of TrkB downstream synaptic proteins such as PSD95 and synaptophysin. Furthermore, the TrkB antagonist K252a blocked the antidepressant-like effects of 7,8DHF. In summary, the present results demonstrated that chronic 7,8-DHF treatment exerted significant antidepressant-like effects, which were likely attributed to regulating TrkB signaling and thus promoting synaptic protein expression. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction 7,8-Dihydroxyflavone (7,8-DHF) is a naturally-occurring and orally-bioavailable flavone that could activate a tropomyosinrelated kinase B (TrkB) [1,2]. Due to the character of crossing the blood-brain-barrier, it attracts many focuses in the field of mental disease treatment [3]. Generally, the main substrate brain-derived neurotrophic factor (BDNF) binds with TrkB and then activates TrkB downstream signaling. This signaling activation in turn leads to increasing synaptic protein expression and promoting the growth of these dendrites into the synapse [4]. However, due to the poor brain absorption ability of BDNF, we cannot directly use BDNF to
∗ Corresponding author. E-mail address: [email protected] (F. Han). http://dx.doi.org/10.1016/j.neulet.2016.10.035 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
treat some mental disease related to BDNF deficits. As a result, 7,8DHF theoretically could be used to mimic the function of BDNF, and has been demonstrated to produce therapeutic efficacy in a variety of central nervous system disorders in animals [5]. Recently, a growing number of evidence suggest that BDNF deficits are involved in the pathogenesis of depression [6]. Clinical available antidepressants can significantly increase BDNF expression and thus cure the depressive symptoms [7]. Therefore, 7,8-DHF was initially evaluated in some depression-like animals models [8]. Previous studies have shown that 7,8-DHF shortened the immobility time in both forced swimming test and tail suspension test [1,9], without altering locomotor activity [10], indicating the antidepressant-like effects of 7,8-DHF. However, it has been also found that single 7,8-DHF treatment did not increase synaptic protein in social defeat stress model of stress [9].
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Therefore, in our present study, we tried to assess whether long term 7,8-DHF injection could alleviate the behavioral deficits and stimulate synaptic protein in the prefrontal cortex in chronic mild stress (CMS) model of depression. To confirm that the effects of 7,8-DHF were mediated by TrkB signaling, we used K252a, a TrkB antagonist combined with 7,8-DHF to evaluate. 2. Materials and methods 2.1. Animals Male C57BL/6 mice (8 weeks) were obtained from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All animals were housed in a 12-h dark/light cycle, temperature (20 ± 2 ◦ C) and humidity-controlled environment with unlimited access to water and food. Experimental procedure was approved by the Committee of Animal care of Zhengzhou Traditional Chinese Medicine Hospital. Every effort was taken to reduce both the number of animals and their suffering. 2.2. Chemicals 7,8-DHF was purchased from TCI Co. Ltd (Shanghai, China). Fluoxetine hydrochloride was from Sigma Aldrich Co., Ltd. (St. Louis, USA). K252a was purchased from Alomone Labs (Jerusalem, Israel). Antibody for TrkB was purchased from Boster Biotechnology Co., Ltd (Wuhan, China). Antibody for p-TrkB(515) was purchased from Bioss Biotechnology Co., Ltd (Beijing, China). Antibody for PSD95 was purchased from Cell Signaling Technology Inc (Beverly, USA). Antibodies for BDNF and synaptophysin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). Antibody for -actin was purchased from Zhongshan Golden Bridge, Biotechnology Co., Ltd. (Beijing, China). Ketamine hydrochloride was purchased from Fujian Gutian Yuanhang Medicine Ltd., Co. (Ningde, China).
prior to 7,8-DHF or vehicle 2 injection. The dose of 7,8-DHF and K252a was 10 mg/kg and 25 g/kg, respectively. 2.5. Sucrose preference test Sucrose preference test was performed at the end of week 4 and week 8 according to a previous study with some modifications [12]. Mice were firstly trained to adapt to a 1% sucrose solution (w/v) for 24 h, with two bottles of 1% sucrose solution in each cage. On the second day, a bottle of 1% sucrose solution and a bottle of water were accessible for 24 h. After food and water deprivation for 12 h, the mice were given to both 1% sucrose solution and water for 24 h. Sucrose preference was calculated according to the following formula: sucrose preference (%) = sucrose intake (g)/[sucrose intake (g) + water (g)] × 100%. 2.6. Novelty suppressed feeding test The novelty suppressed feeding test was performed after sucrose preference test according to a previous study with some modifications [13]. Briefly, the mice were deprived of food for 24 h. On the test day, the mice were individually placed in the corner of the arena (80 cm × 80 cm × 40 cm), five pellets were placed on the center of the arena. The animal was placed in a corner of the cage. The latency to get the food was recorded. In addition, the total food intake during the first 5 min was record after the animals were put back into the cage. 2.7. Sample collection One day after the novelty suppressed feeding test, the animals were sacrificed. The prefrontal cortex was collected into liquid nitrogen and stored at −80 ◦ C until protein detection. 2.8. Western blotting
2.3. CMS procedure The mouse CMS procedure was based on the original arrangement [11], with some modifications. Briefly, the following mild stresses were exposed each week in a random order for 8 weeks: food or water deprivation, cold water swimming, switch of light/dark cycle, cage tilting, placement in the dirty cage, overnight illumination, exposure to a foreign object, empty drinking bottle and white noise. All the stresses were randomly organized in order to ensure the unpredictable characteristic of the experiment. 2.4. Drug treatments and experimental design 2.4.1. Experiment 1 After one week adaption, forty mice were exposed to various stresses for consecutive 28 days. Another group of mice (n = 10) was as the control. After the first sucrose preference test, the CMS mice were randomly divided into four experimental subgroups (n = 10): CMS + vehicle group, CMS + 7,8-DHF (10 mg/kg) group, CMS + 7,8DHF (20 mg/kg) group, CMS + fluoxetine (20 mg/kg) group. 7,8-DHF and fluoxetine were administered intraperitoneally (i.p.) to mice in a volume of 10 ml/kg for 28 days. 2.4.2. Experiment 2 After one week adaption, forty mice were exposed to various stresses for consecutive 28 days. After the first sucrose preference test, the CMS mice were randomly divided into four experimental subgroups (n = 10): CMS + vehicle 1 + vehicle 2, CMS + vehicle 1 + 7,8-DHF, CMS + K252a + vehicle 2, CMS + K252a + 7,8-DHF. All agents were administered intraperitoneally (i.p.) to mice in a volume of 10 ml/kg for 28 days. K252a or vehicle 1 was injected 30 min
Proteins were extracted with lysis buffe and then centrifuged at 12000 × g for 10 min at 4 ◦ C. The concentration of total protein was determined by bicinchoninic acid protein assay kit (Nantong, China). Equal amounts of protein were loaded per well on a 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The blots were incubated with the appropriate concentration of specific BDNF (1:500), TrkB (1: 500), p-TrkB (1:500), PSD95 (1:1000), synaptophysin (1:1000) and -actin (1: 2500) antibody overnight at 4 ◦ C blocked in skim milk and then treated with horseradish peroxidase-conjugated second antibody for 1 h at room temperature after washing with TBST for three times. The bands were established and fixed by an advanced ECL kit. 2.9. Statistical analyses All data are expressed as mean ± S.E.M. A one-way or two-way ANOVA followed by post hoc Newman-Keuls multiple comparison test was performed. A value of P < 0.05 was considered statistically significant for analysis. 3. Results 3.1. 7,8-DHF reversed depressive-like behavior induced by CMS in mice The effects of 7,8-DHF on the sucrose preference and the latency to feed are shown in Fig. 1A, B. CMS induced a significant decrease of the sucrose preference [F(1,18) = 12.57, P < 0.01] and an increase of the latency to feed [F(1,18) = 21.75, P < 0.01]. A significant treatment effect was also observed on sucrose preference [F(3,36) = 6.32,
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Fig. 1. Effects of 7,8-DHF on the sucrose preference (A), latency to feed (B) and total food intake (C) during the first 5 min. The results are expressed as the mean ± S.E.M, n = 10 in each group. ## P < 0.01 vs Control group; ** P < 0.01 vs CMS group.
P < 0.01] and latency to feed [F(3,36) = 6.34, P < 0.01] in CMS mice. Post-hoc test indicated that chronic treatment with 7,8-DHF at doses of 10 mg/kg (P < 0.01, P < 0.01, respectively) and 20 mg/kg (P < 0.01, P < 0.01, respectively) reversed these deficits of sucrose preference and latency to feed induced by CMS. The positive drug fluoxetine also increased the sucrose preference (P < 0.01) and decreased the latency to feed (P < 0.01) in mice exposed to CMS. In addition, we did not found any alteration in total food intake during the first 5 min after the novelty suppressed feeding test (Fig. 1C), indicating that the change of latency to feed was not due to the change of food intake. 3.2. 7,8-DHF reversed TrkB phosphorylation reduction induced by CMS in mice CMS resulted in a significant decrease in the prefrontal cortex TrkB phosphorylation [F(1,6) = 11.62, P < 0.05] and BDNF levels [F(1,6) = 19.56, P < 0.01]. A significant treatment effects on TrkB phosphorylation [F(3,12) = 11.82, P < 0.01] and BDNF levels [F(3,12) = 9.38, P < 0.01] were observed in CMS mice. 7,8-DHF (10 and 20 mg/kg) treatment normalized the reduction in TrkB phosphorylation (P < 0.01, P < 0.01, respectively) and BDNF levels (P < 0.01, P < 0.01, respectively) in CMS mice. The results are given in Fig. 2. 3.3. 7,8-DHF reversed the decreased synaptic protein expression induced by CMS in mice The expression of PSD95 and synaptophysin in the prefrontal cortex were significantly decreased in CMS mice, as compared with that of Control mice [F(1,5) = 7.12, P < 0.05; F(1,6) = 8.40, P < 0.05]. A significant treatment effect on PSD95 and synaptophysin levels was observed in CMS mice [F(3,11) = 4.69, P < 0.05; F(3,10) = 30.75, P < 0.01]. As expected, 7,8-DHF (10 and 20 mg/kg) and fluoxetine (20 mg/kg) significantly attenuated CMS-induced decreases in PSD95 (P < 0.05, P < 0.05, P < 0.05, respectively) and synaptophysin (P < 0.01, P < 0.01, P < 0.01, respectively) expression in prefrontal cortex (Fig. 3). 3.4. K252a blocked the antidepressant-like effects of 7,8-DHF in CMS mice According to Fig. 4, K252a significantly blocked the effects of 7,8DHF on sucrose preference and latency to feed. Two-way ANOVA showed that the interaction [F(1, 36) = 6.73, P < 0.05] between treatment and pretreatment was significant, although the treatment effect [F(1, 36) = 2.45, P > 0.05] and pretreatment effect [F(1, 36) = 1.47, P > 0.05] were not significant in the sucrose preference test. Also, two-way ANOVA showed that the interaction [F(1, 36) = 6.00, P < 0.05] and the effect of treatment [F(1, 36) = 4.39, P < 0.05] were significant, although the pretreatment effect [F(1,
Fig. 2. Effects of 7,8-DHF on the prefrontal cortex TrkB phosphorylation and BDNF levels in CMS mice. The results are expressed as the mean ± S.E.M, n = 4 in each group. # P < 0.05 vs Control group; ** P < 0.01 vs CMS group.
36) = 4.10, P > 0.05] was not significant in latency to feed. Post hoc test revealed that sucrose preference (P < 0.05) and latency to feed (P < 0.05) were significantly reversed after treatment with 7,8-DHF, as compared with the CMS group. Additionally, pretreatment with K252a dramatically prevented the 7,8-DHF-induced increase in sucrose preference and decrease in latency to feed in CMS mice. 3.5. K252a prevented the 7,8-DHF-induced increase in the prefrontal cortex TrkB phosphorylation and BDNF levels in CMS mice As shown in Fig. 5, K252a significantly abolished the effects of 7,8-DHF on prefrontal cortex TrkB phosphorylation and BDNF lev-
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Fig. 3. Effects of 7,8-DHF on the prefrontal cortex PSD95 and synaptophysin in CMS mice. The results are expressed as the mean ± S.E.M, n = 3-5 in each group. # P < 0.05 vs Control group; * P < 0.05 and ** P < 0.01 vs CMS group.
els. Two-way ANOVA showed the significant treatment effect [F(1, 16) = 23.54, P < 0.01; F(1, 12) = 16.10, P < 0.01], pretreatment effect [F(1, 16) = 29.23, P < 0.01; F(1, 12) = 11.71, P < 0.01] and their interaction [F(1, 16) = 19.99, P < 0.01; F(1, 12) = 8.01, P < 0.05] in TrkB phosphorylation and BDNF levels. Post hoc test revealed that after treatment with 7,8-DHF, TrkB phosphorylation and BDNF levels (P < 0.01; P < 0.01) were significantly elevated as compared with the CMS group. Additionally, pretreatment with K252a dramatically prevented the 7,8-DHF-induced increase in TrkB phosphorylation and BDNF levels. 3.6. K252a prevented the 7,8-DHF-induced increase in the prefrontal cortex synaptic protein expression in CMS mice As shown in Fig. 6, K252a significantly blocked the effects of 7,8DHF on synaptic protein expression. Two-way ANOVA showed a significant treatment effect [F(1, 8) = 7.57, P < 0.05; F(1, 12) = 19.20,
Fig. 5. K252a blocked the improvement of TrkB phosphorylation and BDNF levels induced by 7,8-DHF in CMS mice. The results are expressed as the mean ± S.E.M, n = 4-5 in each group. ## P < 0.01 vs CMS group; ** P < 0.01 vs 7,8-DHF group.
P < 0.01], a pretreatment effect [F(1, 8) = 11.47, P < 0.01; F(1, 12) = 8.98, P < 0.05] and their interaction [F(1, 8) = 13.30, P < 0.01; F(1, 12) = 9.60, P < 0.01] on PSD95 and synaptophysin expressions. Post hoc test revealed that after treatment with 7,8-DHF, PSD95 (P < 0.01) and synaptophysin (P < 0.01) were significantly increased as compared with the CMS group. Additionally, pretreatment with K252a dramatically prevented the 7,8-DHF-induced increase in PSD95 and synaptophysin in CMS mice.
Fig. 4. K252a blocked the improvement of sucrose preference (A), latency to feed (B) and total food intake (C) induced by 7,8-DHF in CMS mice. The results are expressed as the mean ± S.E.M, n = 10 in each group. # P < 0.05 vs CMS group; * P < 0.05 and ** P < 0.01 vs 7,8-DHF group.
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Fig. 6. K252a blocked the improvement of PSD95 and synaptophysin expression induced by 7,8-DHF in CMS mice. The results are expressed as the mean ± S.E.M, n = 3-4 in each group. ## P < 0.01 vs CMS group; * P < 0.05 and ** P < 0.01 vs 7,8-DHF group.
4. Discussion Our present study, for the first time, demonstrated that 7,8DHF treatment produced antidepressant-like effects in CMS model. Chronic 7,8-DHF treatment restored the CMS-induced downregulation of TrkB phosphorylation and synaptic protein expression in the prefrontal cortex in mice. However, these effects were completely abolished by pretreatment with TrkB antagonist, K252a, which suggested that the TrkB signaling activation was required for 7,8-DHF-induced antidepressant-like effects. Compared to forced swimming test and tail suspension test, CMS models a chronic depressive-like state that develops gradually over time in response to various stresses and is considered to provide more natural induction [14]. More importantly, CMS has been verified to cause long lasting changes of behavioral, neurotrophic and neuroendocrinological variables [15]. Therefore, it is a better model to evaluate the mechanism of antidepressant agents. In the present study, the results showed that CMS reduced sucrose preference and increased latency to feed, which reflected the depressive state of the animals. Four weeks administration with 7,8-DHF significant reversed these behavioral deficits, showing the antidepressant-like effects of 7,8-DHF. Combined with the positive results from other depressive-like models [1,9], it suggests that 7,8-DHF is a valid antidepressant for further development. On the other hand, we also evaluated whether 7, 8-DHF produced rapid antidepressant-like effects in an independent experiment, as 7, 8-DHF can directly activate TrkB phosphorylation and promote mTOR signaling [1,10]. However, according to our supplementary results (Fig.S1), we cannot observe the rapid antidepressant-like effects of 7, 8-DHF in mice exposed to CMS. In fact, it has been reported that acute treatment of traditional antide-
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pressants, such as fluoxetine and citalopram, could also rapidly activate TrkB phosphorylation in the cortex and hippocampus [16]. Therefore, it seems that activating TrkB does not directly result in antidepressant-like effects. It should be noted that our results were inconsistent with the recent studies which indicated that 7, 8-DHF produced fast-acting/rapid antidepressant-like effects in animals induced by chronic mild stress/social defeat stress [9,17]. To be honest, we did not completely agree with their conclusion. First of all, although Chang’s et al. just found that one week injection rather than single injection with 7, 8-DHF increased sucrose consumption, they still described that 7, 8-DHF produced fast onset antidepressant effects[17]. In this way, we don’t think that the “fast onset antidepressant” in their study is equal to the “rapid antidepressant” in experiments using ketamine. In addition, in the other experiment reported by Zhang et al. [9], although there was an increase in sucrose preference after single treatment of 7, 8-DHF in mice exposed to social defeat stress, the sucrose preference of 7, 8DHF treatment was just 55% and was far less than that of ketamine (nearly to 80%). Actually, the difference between 7, 8-DHF and vehicle was attributed to the extremely lower sucrose preference in vehicle group (30%). Moreover, their results indicated that single 7, 8-DHF treatment cannot reverse the decrease of synaptic proteins in brain. Thus, it seems that the results of rapid antidepressant of 7, 8-DHF are controversial. Further study is required to clearly elucidate this issue. Considering that the major function of 7,8-DHF [2], we firstly evaluated whether chronic 7,8-DHF could activate TrkB signaling. Our results clearly demonstrated that four weeks administration with 7,8-DHF increased TrkB phosphorylation, which was parallel to the another vivo study indicating that 7,8-DHF reversed the TrkB phosphorylation in depressive mice exposed to lipopolysaccharide [10]. In addition, our study also indicated that 7,8-DHF increased BDNF levels in the prefrontal cortex. Therefore, it suggested that 7,8-DHF was a potent candidate for BDNF-TrkB signaling impaired diseases in chronic treatment. Generally, once TrkB receptor is activated, neurons tend to experience proliferation and differentiation [18]. During this process, neurons are regulated to increase synaptic protein expression [19]. PSD95, a widely studied synaptic protein is located in the post synaptic density of neurons [20]. In contrast, synaptophysin is another synaptic protein that occurs in presynaptic vesicles of neurons [21]. These two synaptic proteins have been confirmed to play an important role in neurogenesis and synaptogenesis [22]. Clinical study has shown that PSD95 and synaptophysin were significantly decreased in patients with major depressive disorder [23,24]. Besides, a growing number of experimental evidences also indicated that chronic stress reduced the expression of PSD95 and synaptophysin, but antidepressant treatment restored their expression [25,26]. Therefore, it is accepted that PSD95 and synaptophysin are involved in the pathophysiology of depression and treatment of antidepressants. The results from our present study also showed that CMS caused a significant reduction of PSD95 and synaptophysin expression in the prefrontal cortex, which was consistent with previous studies [26,27]. Four weeks treatment with 7,8-DHF reversed this down-regulation, indicating that 7,8-DHF can stimulate the expression of synaptic proteins. Due to 7,8-DHF being an effective activator of TrkB and our present results from experiment 1, it led to the hypothesis that 7,8-DHF may produce antidepressant-like effects by activating TrkB signaling pathways. To verify this hypothesis, K252a, an antagonist of TrkB, was used. K252a was widely used to evaluate the mechanism whether BDNF-TrkB signaling pathway was involved [28,29]. K252a was firstly shown to prevent the neuronal differentiation of PC12 cells by inhibition of TrkB activity [30]. Recently, K252a has been shown to abolish the BDNF-TrkB signaling in animal studies [12,13]. In the present study, pretreatment with K252a blocked the
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effects of 7,8-DHF on behavioral tests, as well as the expression of PSD95 and synaptophysin. In addition, the inhibitory expression of BDNF and inhibitory phosphorylation of TrkB confirmed again the target of 7,8-DHF. These results verified that the antidepressantlike effects of 7,8-DHF was associated with TrkB receptor activation. In summary, the present study clearly demonstrated the antidepressant-like effects of 7,8-DHF in a long term treatment. Notably, our study strongly suggested that TrkB signaling pathway activation in the prefrontal cortex might be involved in the therapeutic action of 7,8-DHF in depression. The present findings are beneficial for understanding the antidepressant-like mechanism of 7,8-DHF. However, due to the limitation of available resources, we cannot perform an immunofluorescence experiment to investigate the effects of 7,8-DHF on the neurogenesis. Because 7,8-DHF acts as an activator of TrkB and promotes the expression of synaptic proteins, we speculate that 7,8-DHF may produce antidepressant-like effects through improved neurogenesis in hippocampus. Future studies are also needed to demonstrate the influence of 7,8-DHF on neurogenesis. In this way, this is one of the limitations of our present study. Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2016.10. 035. References [1] X. Liu, C.B. Chan, S.W. Jang, S. Pradoldej, J. Huang, K. He, L.H. Phun, S. France, G. Xiao, Y. Jia, H.R. Luo, K. Ye, A synthetic 7,8-dihydroxyflavone derivative promotes neurogenesis and exhibits potent antidepressant effect, J. Med. Chem. 53 (2010) 8274–8286. [2] S.W. Jang, X. Liu, M. Yepes, K.R. Shepherd, G.W. Miller, Y. Liu, W.D. Wilson, G. Xiao, B. Blanchi, Y.E. Sun, K. Ye, A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 2687–2692. [3] X. Liu, C.B. Chan, Q. Qi, G. Xiao, H.R. Luo, X. He, K. Ye, Optimization of a small tropomyosin-related kinase B (TrkB) agonist 7,8-dihydroxyflavone active in mouse models of depression, J. Med. Chem. 55 (2012) 8524–8537. [4] H.D. Schmidt, R.S. Duman, The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments and animal models of depressive-like behavior, Behav. Pharmacol. 18 (2007) 391–418. [5] C. Liu, C.B. Chan, K. Ye, 7,8-Dihydroxyflavone, a small molecular TrkB agonist, is useful for treating various BDNF-implicated human disorders, Transl. Neurodegener. 5 (2016) 2. [6] B. Gutierrez, J.A. Bellon, M. Rivera, E. Molina, M. King, L. Marston, F. Torres-Gonzalez, B. Moreno-Kustner, P. Moreno-Peral, E. Motrico, C. Monton-Franco, M.J. GildeGomez-Barragan, M. Sanchez-Celaya, M.A. Diaz-Barreiros, C. Vicens, J. de Dios Luna, I. Nazareth, J. Cervilla, The risk for major depression conferred by childhood maltreatment is multiplied by BDNF and SERT genetic vulnerability: a replication study, J. Psychiatry Neurosci. 40 (2015) 187–196. [7] D. Munno, S. Sterpone, S. Fania, F. Cappellin, G. Mengozzi, M. Saroldi, E. Bechon, G. Zullo, Plasma brain derived neurotrophic factor levels and neuropsychological aspects of depressed patients treated with paroxetine, Panminerva Med. 55 (2013) 377–384. [8] O. Obianyo, K. Ye, Novel small molecule activators of the Trk family of receptor tyrosine kinases, Biochim. Biophys. Acta 1834 (2013) 2213–2218. [9] J.C. Zhang, W. Yao, C. Dong, C. Yang, Q. Ren, M. Ma, M. Han, K. Hashimoto, Comparison of ketamine, 7,8-dihydroxyflavone, and ANA-12 antidepressant effects in the social defeat stress model of depression, Psychopharmacology (Berl.) 232 (2015) 4325–4335.
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Research article
7,8-Dihydroxyflavone reverses the depressive symptoms in mouse chronic mild stress Min-Wang Zhang a , She-feng Zhang b , Zhen-Hua Li a , Fang Han a,∗ a b
Department of Encephalopathy (II), Zhengzhou Traditional Chinese Medicine Hospital, Zhengzhou 450007, Henan province, PR China Henan Province Chinese medicine research institute, Zhengzhou 450004, Henan province, PR China
h i g h l i g h t s • 7,8-DHF produced antidepressant-like effects in CMS mice. • 7,8-DHF promoted the expression of synaptic proteins. • K252a pretreatment blocked the effects of 7,8-DHF.
a r t i c l e
i n f o
Article history: Received 19 August 2016 Received in revised form 18 October 2016 Accepted 19 October 2016 Available online 20 October 2016 Keywords: 7,8-Dihydroxyflavone (7,8-DHF) Antidepressant-like effects Chronic mild stress (CMS) TrkB PSD95 Synaptophysin
a b s t r a c t 7,8-Dihydroxyflavone (7,8-DHF) is a naturally-occurring flavone which possesses good bioavailability. Due to its ability to cross the blood-brain barrier, previous studies have demonstrated that 7,8-DHF was a potent tropomyosin-related kinase B (TrkB) agonist, and produced antidepressant-like effects in mouse forced swimming test and tail suspension test. However, it has not been evaluated in chronic mild stress (CMS), a classical depression model modulating the processes of major depression in human. In the present study, we not only evaluated the depressive-like behaviors, but also measured the key proteins of TrkB signaling in mice exposed to CMS. Our results firstly found that long term but not single injection of 7,8-DHF restored the depressive-like behaviors in sucrose preference test and novelty suppressed feeding test. In addition, 7,8-DHF not only increased TrkB phosphorylation and brain-derived neurotrophic factor (BDNF) levels, but also activated the expression of TrkB downstream synaptic proteins such as PSD95 and synaptophysin. Furthermore, the TrkB antagonist K252a blocked the antidepressant-like effects of 7,8DHF. In summary, the present results demonstrated that chronic 7,8-DHF treatment exerted significant antidepressant-like effects, which were likely attributed to regulating TrkB signaling and thus promoting synaptic protein expression. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction 7,8-Dihydroxyflavone (7,8-DHF) is a naturally-occurring and orally-bioavailable flavone that could activate a tropomyosinrelated kinase B (TrkB) [1,2]. Due to the character of crossing the blood-brain-barrier, it attracts many focuses in the field of mental disease treatment [3]. Generally, the main substrate brain-derived neurotrophic factor (BDNF) binds with TrkB and then activates TrkB downstream signaling. This signaling activation in turn leads to increasing synaptic protein expression and promoting the growth of these dendrites into the synapse [4]. However, due to the poor brain absorption ability of BDNF, we cannot directly use BDNF to
∗ Corresponding author. E-mail address: [email protected] (F. Han). http://dx.doi.org/10.1016/j.neulet.2016.10.035 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
treat some mental disease related to BDNF deficits. As a result, 7,8DHF theoretically could be used to mimic the function of BDNF, and has been demonstrated to produce therapeutic efficacy in a variety of central nervous system disorders in animals [5]. Recently, a growing number of evidence suggest that BDNF deficits are involved in the pathogenesis of depression [6]. Clinical available antidepressants can significantly increase BDNF expression and thus cure the depressive symptoms [7]. Therefore, 7,8-DHF was initially evaluated in some depression-like animals models [8]. Previous studies have shown that 7,8-DHF shortened the immobility time in both forced swimming test and tail suspension test [1,9], without altering locomotor activity [10], indicating the antidepressant-like effects of 7,8-DHF. However, it has been also found that single 7,8-DHF treatment did not increase synaptic protein in social defeat stress model of stress [9].
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Therefore, in our present study, we tried to assess whether long term 7,8-DHF injection could alleviate the behavioral deficits and stimulate synaptic protein in the prefrontal cortex in chronic mild stress (CMS) model of depression. To confirm that the effects of 7,8-DHF were mediated by TrkB signaling, we used K252a, a TrkB antagonist combined with 7,8-DHF to evaluate. 2. Materials and methods 2.1. Animals Male C57BL/6 mice (8 weeks) were obtained from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All animals were housed in a 12-h dark/light cycle, temperature (20 ± 2 ◦ C) and humidity-controlled environment with unlimited access to water and food. Experimental procedure was approved by the Committee of Animal care of Zhengzhou Traditional Chinese Medicine Hospital. Every effort was taken to reduce both the number of animals and their suffering. 2.2. Chemicals 7,8-DHF was purchased from TCI Co. Ltd (Shanghai, China). Fluoxetine hydrochloride was from Sigma Aldrich Co., Ltd. (St. Louis, USA). K252a was purchased from Alomone Labs (Jerusalem, Israel). Antibody for TrkB was purchased from Boster Biotechnology Co., Ltd (Wuhan, China). Antibody for p-TrkB(515) was purchased from Bioss Biotechnology Co., Ltd (Beijing, China). Antibody for PSD95 was purchased from Cell Signaling Technology Inc (Beverly, USA). Antibodies for BDNF and synaptophysin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). Antibody for -actin was purchased from Zhongshan Golden Bridge, Biotechnology Co., Ltd. (Beijing, China). Ketamine hydrochloride was purchased from Fujian Gutian Yuanhang Medicine Ltd., Co. (Ningde, China).
prior to 7,8-DHF or vehicle 2 injection. The dose of 7,8-DHF and K252a was 10 mg/kg and 25 g/kg, respectively. 2.5. Sucrose preference test Sucrose preference test was performed at the end of week 4 and week 8 according to a previous study with some modifications [12]. Mice were firstly trained to adapt to a 1% sucrose solution (w/v) for 24 h, with two bottles of 1% sucrose solution in each cage. On the second day, a bottle of 1% sucrose solution and a bottle of water were accessible for 24 h. After food and water deprivation for 12 h, the mice were given to both 1% sucrose solution and water for 24 h. Sucrose preference was calculated according to the following formula: sucrose preference (%) = sucrose intake (g)/[sucrose intake (g) + water (g)] × 100%. 2.6. Novelty suppressed feeding test The novelty suppressed feeding test was performed after sucrose preference test according to a previous study with some modifications [13]. Briefly, the mice were deprived of food for 24 h. On the test day, the mice were individually placed in the corner of the arena (80 cm × 80 cm × 40 cm), five pellets were placed on the center of the arena. The animal was placed in a corner of the cage. The latency to get the food was recorded. In addition, the total food intake during the first 5 min was record after the animals were put back into the cage. 2.7. Sample collection One day after the novelty suppressed feeding test, the animals were sacrificed. The prefrontal cortex was collected into liquid nitrogen and stored at −80 ◦ C until protein detection. 2.8. Western blotting
2.3. CMS procedure The mouse CMS procedure was based on the original arrangement [11], with some modifications. Briefly, the following mild stresses were exposed each week in a random order for 8 weeks: food or water deprivation, cold water swimming, switch of light/dark cycle, cage tilting, placement in the dirty cage, overnight illumination, exposure to a foreign object, empty drinking bottle and white noise. All the stresses were randomly organized in order to ensure the unpredictable characteristic of the experiment. 2.4. Drug treatments and experimental design 2.4.1. Experiment 1 After one week adaption, forty mice were exposed to various stresses for consecutive 28 days. Another group of mice (n = 10) was as the control. After the first sucrose preference test, the CMS mice were randomly divided into four experimental subgroups (n = 10): CMS + vehicle group, CMS + 7,8-DHF (10 mg/kg) group, CMS + 7,8DHF (20 mg/kg) group, CMS + fluoxetine (20 mg/kg) group. 7,8-DHF and fluoxetine were administered intraperitoneally (i.p.) to mice in a volume of 10 ml/kg for 28 days. 2.4.2. Experiment 2 After one week adaption, forty mice were exposed to various stresses for consecutive 28 days. After the first sucrose preference test, the CMS mice were randomly divided into four experimental subgroups (n = 10): CMS + vehicle 1 + vehicle 2, CMS + vehicle 1 + 7,8-DHF, CMS + K252a + vehicle 2, CMS + K252a + 7,8-DHF. All agents were administered intraperitoneally (i.p.) to mice in a volume of 10 ml/kg for 28 days. K252a or vehicle 1 was injected 30 min
Proteins were extracted with lysis buffe and then centrifuged at 12000 × g for 10 min at 4 ◦ C. The concentration of total protein was determined by bicinchoninic acid protein assay kit (Nantong, China). Equal amounts of protein were loaded per well on a 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The blots were incubated with the appropriate concentration of specific BDNF (1:500), TrkB (1: 500), p-TrkB (1:500), PSD95 (1:1000), synaptophysin (1:1000) and -actin (1: 2500) antibody overnight at 4 ◦ C blocked in skim milk and then treated with horseradish peroxidase-conjugated second antibody for 1 h at room temperature after washing with TBST for three times. The bands were established and fixed by an advanced ECL kit. 2.9. Statistical analyses All data are expressed as mean ± S.E.M. A one-way or two-way ANOVA followed by post hoc Newman-Keuls multiple comparison test was performed. A value of P < 0.05 was considered statistically significant for analysis. 3. Results 3.1. 7,8-DHF reversed depressive-like behavior induced by CMS in mice The effects of 7,8-DHF on the sucrose preference and the latency to feed are shown in Fig. 1A, B. CMS induced a significant decrease of the sucrose preference [F(1,18) = 12.57, P < 0.01] and an increase of the latency to feed [F(1,18) = 21.75, P < 0.01]. A significant treatment effect was also observed on sucrose preference [F(3,36) = 6.32,
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Fig. 1. Effects of 7,8-DHF on the sucrose preference (A), latency to feed (B) and total food intake (C) during the first 5 min. The results are expressed as the mean ± S.E.M, n = 10 in each group. ## P < 0.01 vs Control group; ** P < 0.01 vs CMS group.
P < 0.01] and latency to feed [F(3,36) = 6.34, P < 0.01] in CMS mice. Post-hoc test indicated that chronic treatment with 7,8-DHF at doses of 10 mg/kg (P < 0.01, P < 0.01, respectively) and 20 mg/kg (P < 0.01, P < 0.01, respectively) reversed these deficits of sucrose preference and latency to feed induced by CMS. The positive drug fluoxetine also increased the sucrose preference (P < 0.01) and decreased the latency to feed (P < 0.01) in mice exposed to CMS. In addition, we did not found any alteration in total food intake during the first 5 min after the novelty suppressed feeding test (Fig. 1C), indicating that the change of latency to feed was not due to the change of food intake. 3.2. 7,8-DHF reversed TrkB phosphorylation reduction induced by CMS in mice CMS resulted in a significant decrease in the prefrontal cortex TrkB phosphorylation [F(1,6) = 11.62, P < 0.05] and BDNF levels [F(1,6) = 19.56, P < 0.01]. A significant treatment effects on TrkB phosphorylation [F(3,12) = 11.82, P < 0.01] and BDNF levels [F(3,12) = 9.38, P < 0.01] were observed in CMS mice. 7,8-DHF (10 and 20 mg/kg) treatment normalized the reduction in TrkB phosphorylation (P < 0.01, P < 0.01, respectively) and BDNF levels (P < 0.01, P < 0.01, respectively) in CMS mice. The results are given in Fig. 2. 3.3. 7,8-DHF reversed the decreased synaptic protein expression induced by CMS in mice The expression of PSD95 and synaptophysin in the prefrontal cortex were significantly decreased in CMS mice, as compared with that of Control mice [F(1,5) = 7.12, P < 0.05; F(1,6) = 8.40, P < 0.05]. A significant treatment effect on PSD95 and synaptophysin levels was observed in CMS mice [F(3,11) = 4.69, P < 0.05; F(3,10) = 30.75, P < 0.01]. As expected, 7,8-DHF (10 and 20 mg/kg) and fluoxetine (20 mg/kg) significantly attenuated CMS-induced decreases in PSD95 (P < 0.05, P < 0.05, P < 0.05, respectively) and synaptophysin (P < 0.01, P < 0.01, P < 0.01, respectively) expression in prefrontal cortex (Fig. 3). 3.4. K252a blocked the antidepressant-like effects of 7,8-DHF in CMS mice According to Fig. 4, K252a significantly blocked the effects of 7,8DHF on sucrose preference and latency to feed. Two-way ANOVA showed that the interaction [F(1, 36) = 6.73, P < 0.05] between treatment and pretreatment was significant, although the treatment effect [F(1, 36) = 2.45, P > 0.05] and pretreatment effect [F(1, 36) = 1.47, P > 0.05] were not significant in the sucrose preference test. Also, two-way ANOVA showed that the interaction [F(1, 36) = 6.00, P < 0.05] and the effect of treatment [F(1, 36) = 4.39, P < 0.05] were significant, although the pretreatment effect [F(1,
Fig. 2. Effects of 7,8-DHF on the prefrontal cortex TrkB phosphorylation and BDNF levels in CMS mice. The results are expressed as the mean ± S.E.M, n = 4 in each group. # P < 0.05 vs Control group; ** P < 0.01 vs CMS group.
36) = 4.10, P > 0.05] was not significant in latency to feed. Post hoc test revealed that sucrose preference (P < 0.05) and latency to feed (P < 0.05) were significantly reversed after treatment with 7,8-DHF, as compared with the CMS group. Additionally, pretreatment with K252a dramatically prevented the 7,8-DHF-induced increase in sucrose preference and decrease in latency to feed in CMS mice. 3.5. K252a prevented the 7,8-DHF-induced increase in the prefrontal cortex TrkB phosphorylation and BDNF levels in CMS mice As shown in Fig. 5, K252a significantly abolished the effects of 7,8-DHF on prefrontal cortex TrkB phosphorylation and BDNF lev-
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Fig. 3. Effects of 7,8-DHF on the prefrontal cortex PSD95 and synaptophysin in CMS mice. The results are expressed as the mean ± S.E.M, n = 3-5 in each group. # P < 0.05 vs Control group; * P < 0.05 and ** P < 0.01 vs CMS group.
els. Two-way ANOVA showed the significant treatment effect [F(1, 16) = 23.54, P < 0.01; F(1, 12) = 16.10, P < 0.01], pretreatment effect [F(1, 16) = 29.23, P < 0.01; F(1, 12) = 11.71, P < 0.01] and their interaction [F(1, 16) = 19.99, P < 0.01; F(1, 12) = 8.01, P < 0.05] in TrkB phosphorylation and BDNF levels. Post hoc test revealed that after treatment with 7,8-DHF, TrkB phosphorylation and BDNF levels (P < 0.01; P < 0.01) were significantly elevated as compared with the CMS group. Additionally, pretreatment with K252a dramatically prevented the 7,8-DHF-induced increase in TrkB phosphorylation and BDNF levels. 3.6. K252a prevented the 7,8-DHF-induced increase in the prefrontal cortex synaptic protein expression in CMS mice As shown in Fig. 6, K252a significantly blocked the effects of 7,8DHF on synaptic protein expression. Two-way ANOVA showed a significant treatment effect [F(1, 8) = 7.57, P < 0.05; F(1, 12) = 19.20,
Fig. 5. K252a blocked the improvement of TrkB phosphorylation and BDNF levels induced by 7,8-DHF in CMS mice. The results are expressed as the mean ± S.E.M, n = 4-5 in each group. ## P < 0.01 vs CMS group; ** P < 0.01 vs 7,8-DHF group.
P < 0.01], a pretreatment effect [F(1, 8) = 11.47, P < 0.01; F(1, 12) = 8.98, P < 0.05] and their interaction [F(1, 8) = 13.30, P < 0.01; F(1, 12) = 9.60, P < 0.01] on PSD95 and synaptophysin expressions. Post hoc test revealed that after treatment with 7,8-DHF, PSD95 (P < 0.01) and synaptophysin (P < 0.01) were significantly increased as compared with the CMS group. Additionally, pretreatment with K252a dramatically prevented the 7,8-DHF-induced increase in PSD95 and synaptophysin in CMS mice.
Fig. 4. K252a blocked the improvement of sucrose preference (A), latency to feed (B) and total food intake (C) induced by 7,8-DHF in CMS mice. The results are expressed as the mean ± S.E.M, n = 10 in each group. # P < 0.05 vs CMS group; * P < 0.05 and ** P < 0.01 vs 7,8-DHF group.
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Fig. 6. K252a blocked the improvement of PSD95 and synaptophysin expression induced by 7,8-DHF in CMS mice. The results are expressed as the mean ± S.E.M, n = 3-4 in each group. ## P < 0.01 vs CMS group; * P < 0.05 and ** P < 0.01 vs 7,8-DHF group.
4. Discussion Our present study, for the first time, demonstrated that 7,8DHF treatment produced antidepressant-like effects in CMS model. Chronic 7,8-DHF treatment restored the CMS-induced downregulation of TrkB phosphorylation and synaptic protein expression in the prefrontal cortex in mice. However, these effects were completely abolished by pretreatment with TrkB antagonist, K252a, which suggested that the TrkB signaling activation was required for 7,8-DHF-induced antidepressant-like effects. Compared to forced swimming test and tail suspension test, CMS models a chronic depressive-like state that develops gradually over time in response to various stresses and is considered to provide more natural induction [14]. More importantly, CMS has been verified to cause long lasting changes of behavioral, neurotrophic and neuroendocrinological variables [15]. Therefore, it is a better model to evaluate the mechanism of antidepressant agents. In the present study, the results showed that CMS reduced sucrose preference and increased latency to feed, which reflected the depressive state of the animals. Four weeks administration with 7,8-DHF significant reversed these behavioral deficits, showing the antidepressant-like effects of 7,8-DHF. Combined with the positive results from other depressive-like models [1,9], it suggests that 7,8-DHF is a valid antidepressant for further development. On the other hand, we also evaluated whether 7, 8-DHF produced rapid antidepressant-like effects in an independent experiment, as 7, 8-DHF can directly activate TrkB phosphorylation and promote mTOR signaling [1,10]. However, according to our supplementary results (Fig.S1), we cannot observe the rapid antidepressant-like effects of 7, 8-DHF in mice exposed to CMS. In fact, it has been reported that acute treatment of traditional antide-
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pressants, such as fluoxetine and citalopram, could also rapidly activate TrkB phosphorylation in the cortex and hippocampus [16]. Therefore, it seems that activating TrkB does not directly result in antidepressant-like effects. It should be noted that our results were inconsistent with the recent studies which indicated that 7, 8-DHF produced fast-acting/rapid antidepressant-like effects in animals induced by chronic mild stress/social defeat stress [9,17]. To be honest, we did not completely agree with their conclusion. First of all, although Chang’s et al. just found that one week injection rather than single injection with 7, 8-DHF increased sucrose consumption, they still described that 7, 8-DHF produced fast onset antidepressant effects[17]. In this way, we don’t think that the “fast onset antidepressant” in their study is equal to the “rapid antidepressant” in experiments using ketamine. In addition, in the other experiment reported by Zhang et al. [9], although there was an increase in sucrose preference after single treatment of 7, 8-DHF in mice exposed to social defeat stress, the sucrose preference of 7, 8DHF treatment was just 55% and was far less than that of ketamine (nearly to 80%). Actually, the difference between 7, 8-DHF and vehicle was attributed to the extremely lower sucrose preference in vehicle group (30%). Moreover, their results indicated that single 7, 8-DHF treatment cannot reverse the decrease of synaptic proteins in brain. Thus, it seems that the results of rapid antidepressant of 7, 8-DHF are controversial. Further study is required to clearly elucidate this issue. Considering that the major function of 7,8-DHF [2], we firstly evaluated whether chronic 7,8-DHF could activate TrkB signaling. Our results clearly demonstrated that four weeks administration with 7,8-DHF increased TrkB phosphorylation, which was parallel to the another vivo study indicating that 7,8-DHF reversed the TrkB phosphorylation in depressive mice exposed to lipopolysaccharide [10]. In addition, our study also indicated that 7,8-DHF increased BDNF levels in the prefrontal cortex. Therefore, it suggested that 7,8-DHF was a potent candidate for BDNF-TrkB signaling impaired diseases in chronic treatment. Generally, once TrkB receptor is activated, neurons tend to experience proliferation and differentiation [18]. During this process, neurons are regulated to increase synaptic protein expression [19]. PSD95, a widely studied synaptic protein is located in the post synaptic density of neurons [20]. In contrast, synaptophysin is another synaptic protein that occurs in presynaptic vesicles of neurons [21]. These two synaptic proteins have been confirmed to play an important role in neurogenesis and synaptogenesis [22]. Clinical study has shown that PSD95 and synaptophysin were significantly decreased in patients with major depressive disorder [23,24]. Besides, a growing number of experimental evidences also indicated that chronic stress reduced the expression of PSD95 and synaptophysin, but antidepressant treatment restored their expression [25,26]. Therefore, it is accepted that PSD95 and synaptophysin are involved in the pathophysiology of depression and treatment of antidepressants. The results from our present study also showed that CMS caused a significant reduction of PSD95 and synaptophysin expression in the prefrontal cortex, which was consistent with previous studies [26,27]. Four weeks treatment with 7,8-DHF reversed this down-regulation, indicating that 7,8-DHF can stimulate the expression of synaptic proteins. Due to 7,8-DHF being an effective activator of TrkB and our present results from experiment 1, it led to the hypothesis that 7,8-DHF may produce antidepressant-like effects by activating TrkB signaling pathways. To verify this hypothesis, K252a, an antagonist of TrkB, was used. K252a was widely used to evaluate the mechanism whether BDNF-TrkB signaling pathway was involved [28,29]. K252a was firstly shown to prevent the neuronal differentiation of PC12 cells by inhibition of TrkB activity [30]. Recently, K252a has been shown to abolish the BDNF-TrkB signaling in animal studies [12,13]. In the present study, pretreatment with K252a blocked the
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effects of 7,8-DHF on behavioral tests, as well as the expression of PSD95 and synaptophysin. In addition, the inhibitory expression of BDNF and inhibitory phosphorylation of TrkB confirmed again the target of 7,8-DHF. These results verified that the antidepressantlike effects of 7,8-DHF was associated with TrkB receptor activation. In summary, the present study clearly demonstrated the antidepressant-like effects of 7,8-DHF in a long term treatment. Notably, our study strongly suggested that TrkB signaling pathway activation in the prefrontal cortex might be involved in the therapeutic action of 7,8-DHF in depression. The present findings are beneficial for understanding the antidepressant-like mechanism of 7,8-DHF. However, due to the limitation of available resources, we cannot perform an immunofluorescence experiment to investigate the effects of 7,8-DHF on the neurogenesis. Because 7,8-DHF acts as an activator of TrkB and promotes the expression of synaptic proteins, we speculate that 7,8-DHF may produce antidepressant-like effects through improved neurogenesis in hippocampus. Future studies are also needed to demonstrate the influence of 7,8-DHF on neurogenesis. In this way, this is one of the limitations of our present study. Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2016.10. 035. References [1] X. Liu, C.B. Chan, S.W. Jang, S. Pradoldej, J. Huang, K. He, L.H. Phun, S. France, G. Xiao, Y. Jia, H.R. Luo, K. Ye, A synthetic 7,8-dihydroxyflavone derivative promotes neurogenesis and exhibits potent antidepressant effect, J. Med. Chem. 53 (2010) 8274–8286. [2] S.W. Jang, X. Liu, M. Yepes, K.R. Shepherd, G.W. Miller, Y. Liu, W.D. Wilson, G. Xiao, B. Blanchi, Y.E. Sun, K. Ye, A selective TrkB agonist with potent neurotrophic activities by 7,8-dihydroxyflavone, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 2687–2692. [3] X. Liu, C.B. Chan, Q. Qi, G. Xiao, H.R. Luo, X. He, K. Ye, Optimization of a small tropomyosin-related kinase B (TrkB) agonist 7,8-dihydroxyflavone active in mouse models of depression, J. Med. Chem. 55 (2012) 8524–8537. [4] H.D. Schmidt, R.S. Duman, The role of neurotrophic factors in adult hippocampal neurogenesis, antidepressant treatments and animal models of depressive-like behavior, Behav. Pharmacol. 18 (2007) 391–418. [5] C. Liu, C.B. Chan, K. Ye, 7,8-Dihydroxyflavone, a small molecular TrkB agonist, is useful for treating various BDNF-implicated human disorders, Transl. Neurodegener. 5 (2016) 2. [6] B. Gutierrez, J.A. Bellon, M. Rivera, E. Molina, M. King, L. Marston, F. Torres-Gonzalez, B. Moreno-Kustner, P. Moreno-Peral, E. Motrico, C. Monton-Franco, M.J. GildeGomez-Barragan, M. Sanchez-Celaya, M.A. Diaz-Barreiros, C. Vicens, J. de Dios Luna, I. Nazareth, J. Cervilla, The risk for major depression conferred by childhood maltreatment is multiplied by BDNF and SERT genetic vulnerability: a replication study, J. Psychiatry Neurosci. 40 (2015) 187–196. [7] D. Munno, S. Sterpone, S. Fania, F. Cappellin, G. Mengozzi, M. Saroldi, E. Bechon, G. Zullo, Plasma brain derived neurotrophic factor levels and neuropsychological aspects of depressed patients treated with paroxetine, Panminerva Med. 55 (2013) 377–384. [8] O. Obianyo, K. Ye, Novel small molecule activators of the Trk family of receptor tyrosine kinases, Biochim. Biophys. Acta 1834 (2013) 2213–2218. [9] J.C. Zhang, W. Yao, C. Dong, C. Yang, Q. Ren, M. Ma, M. Han, K. Hashimoto, Comparison of ketamine, 7,8-dihydroxyflavone, and ANA-12 antidepressant effects in the social defeat stress model of depression, Psychopharmacology (Berl.) 232 (2015) 4325–4335.
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