Total Saikosaponins of Bupleurum yinchowense reduces depressive, anxiety-like behavior and increases synaptic proteins expression in chronic corticosterine-treated mice

Bupleurum yinchowense Shan et Y. Li, a widely used Chinese medicine, was first documented in the“Shennong’s Herbal”, which is the oldest Chinese materia medica monographs. It was recorded that Bupleurum yinchowense has action of soothing liver and was used to treat depressive and mood related disorders [1]. Our preliminary experiments indicated that total saikosaponins (TSS) from Bupleurum yinchowense produced antidepressant-like effect in the forced swim test (FST), tail suspension test in normal mice. Meanwhile, TSS exhibited antidepressant-like effect in chronic mild stress model in mice [2]. However, the underlying mechanisms of these effects are not yet clear.

Recently, clinical and basic studies demonstrate that depression is associated with synaptic dysfunction in important regions such as prefrontal cortex and hippocampus [3]. Simultaneously, accumulating evidence has shown that A-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and subsequent mammalian target of rapamycin (mTOR) signaling way is responsible for the induction and maintenance of synaptic plasticity and may contribution to the synaptic alteration underlying depression [4, 5]. Chronic AMPA potentiators and AMPA treatment produce antidepressant-like effect and stimulate mTOR protein transduction signaling way [6]. The mTOR singaling is a downstream intracellular signal pathway which controls the synthesis of synaptic proteins and contributes to synaptogenesis [7]. Ketamine fast induction of mTOR singaling and synaptogenesis may be dependent on AMPAR activation [8]. Sarcosine, an endogenous amino acid, exhibits antidepressant-like effects by activating AMPAR–mTOR signaling pathway [9]. These data suggest that AMPA receptors and subsequent mTOR signaling may be a potential therapeutic target for emerging antidepressant-like agent actions.

Chronic stress-mediated elevated levels of circulating glucocorticoids (corticosterone, CORT, in rodents) are widely believed to be involved in the pathophysiology of depression [10]. Chronic and direct administration of corticosterone to male rodents has been used as a model to induce anxiety/depression-like behavior [11, 12]. In such models, hippocampal dendritic spines and spine synapses were decreased which is accompanied by a loss of key synaptic proteins such as postsynaptic density protein 95 (PSD-95), and the presynaptic protein synapsin-1 [12]. Interestingly, some findings indicated CORT exerted its impact on synaptic plasticity by modulating AMPA receptor [13].

Our recent research showed that TSS induced protective effect in corticosterone-induced damage in PC12 cells [14]. On the basis of above findings, we hypothesize that chronic CORT treatment-induced depressive and anxiety like behaviour of mice is caused by dysregulation of AMPA receptor and subsequent mTOR signaling pathway and treatment with TSS might ameliorate these behavioural and molecular changes.

The present study indicates TSS exerts antidepressant-like and anxiolytic effects that are common to citalopram in chronic corticosterone-treated mice. Moreover, TSS increases synaptic proteins (synapsin-1 and PSD-95) which may be mediated by activation of AMPA receptor and mTOR signaling pathway. Dose-response assessment shows TSS produces antidepressant-like effect in the FST both in 25 and 50 mg/kg CORT-treated mice. Notably, the dose of 25 mg/kg presents maximum anti-immobility effect in the FST. Our previous work has shown that TSS at dose of 25 mg/kg was more efficacious than that of 50 mg/kg at different time point post the last TSS administration [19]. Previous study also indicated saikosaponin at dose of 25 mg/kg produced antidepressant-like effects in the chronic mild stress model [20]. Herein, the dose of 25 mg/kg was selected to further research.

To evaluate behavioral changes, in the present study, OFT, EPM, NSFT and FST were used to assess anxious-depressive like behavior of the chronic corticosterone treatment model in mice. Our study showed that 5 week administration of CORT at dose of 35 μg/ml per day induced depressive like behavior as reflected by the increase in the immobility time in the FST. This model also exerted anxiety behavior as assessed by the decrease in the center area in the OFT and by the decreased percentage of open arm entries and percentage of time spent on the open arms in EPM test, as well as by the increased latency to feed in the NSFT. Thus, in this study, an anxiety-depressive model was successfully modeled by chronic treatment of CORT. These behavior results obtained from our study are consistent with previous findings [11, 21, 22].

Depressive and anxiety disorder commonly occur together in clinic [23]. Clinic research has shown that more than 70% patients with depressive disorder present symptoms of anxiety disorder [24]. Co-morbid depression and anxiety increase suicide risk and contribute to treatment resistance [25]. The selective serotonin reuptake inhibitors (SSRIs) are usually used to treat co-morbid depression with anxiety, although there are drawbacks including sexual and weight gain. In the present study, it is the first time to detect the anxiolytic and antidepressant-like effect of TSS in chronic CORT treatment model by behavior tests such as OFT, EPM, NSFT and FST. Citalopram, a classic prototypical selective serotonin reuptake inhibitor was the positive control. Our results showed that both TSS and citalopram exerted anxiolytic effect, as measured by a significant increase of time spent in the center of the open field and the increased percentage of open arm entries and percentage of time spent on the open arms in the EPM test, as well as by the decreased latency to feed in the NSFT. Furthermore, TSS was effective in reducing the immobility time in the FST, suggesting an antidepressant-like effect. Since locomotor activity may lead to false positive or negative effects in the FST, OFT was used to assess the locomotor activity of animals. Our research has shown that TSS did not produce significant changes in locomotion activity in the OFT. Therefore, the effect of TSS in the FST was not based on the unspecific action such as stimulation of general motor. Whereas, the mice treated with citalopram increased the distance moved and velocity, which is in line with previous study [26]. Taken together, TSS and citalopram exerted anxiolytic and antidepressant-like effects in chronic CORT treatment model. Our preliminary experiment indicated treatment with TSS produced a significant increase of 5-HT level in the brain as compared to chronic mild stress model in mice. Increasing the 5-HT level in the brain may be the common path of antidepressant-like effect of citalopram and TSS. Glucocorticoid receptor (GR) mediates the expression of the serotonin transporter (SERT) and increases the 5-HT level in the brain. We have previous shown that Saikosaponins D, the main active ingredient of TSS, up-regulated the GR protein expression in corticosterone-induced PC12 cells. This result suggests that TSS induced increasing 5-HT level in the brain may be mediated by activating GR [14]. In the subsequent study, we should analyze the alteration of GR in the brain in the chronic corticosterone treatment model and investigate the effect of TSS on the GR expression.

It has been postulated that synaptic plasticity and synaptogenesis play an important role in the pathogenesis of depression and mechanisms of action of current antidepressant drugs [3]. Synapsin-1 is a presynaptic marker and is widely used for measurement of synaptic density [27]. PSD-95 is a post synaptic marker and is involved in glutamatergic synaptogenesis [28]. To detect whether costicosterone decreased the synaptic proteins level, synapsin-1 and PSD-95 levels were assessed in the hippocampus of chronic corticosterone-treated mice. Our present western blotting analysis shows that chronic treatment costicosterone decreases synapsin-1 and PSD-95 level in mice. TSS and citalopram treatment shows an increase in synapsin-1 and PSD-95 levels in the hippocampus of mice exposed to 5 week CORT treatment. To reveal localized changes of synaptic density in the hippocampus, immunohistochemical analysis was used to determine synapsin-1 expression in sub-regions of hippocampus in CORT-treated mice. The present study shows that 5 week CORT treatment induces reduction in synapsin-1level in the CA1 and CA3 of hippocampus. This finding supported previous work which exhibited prolonged corticosterone exposure decreased dendritic spine densities in the hippocampus CA1 and CA3 [29]. In the previous studies, saikosaponin treatment induced molecular changes in the CA3 of hippocampus in the depression models [30, 31]. Consistent with these findings, we found TSS induced a pronounced increase of synapsin-1 expression in the CA3 of hippocampus. Citalopram increased synapsin-1 level both in the CA1 and CA3 region are in agreement with previous study showing that citalopram reversed the decreased synaptic plasticity in the hippocampus in the social isolation model [32]. These results suggest that TSS and citalopram might increase synaptic proteins expression and promote synaptogenesis in the hippcampus. Based on the above mentioned data, the mechanisms by which TSS and citalopram exert their antidepressant-like effects maybe involve both monoamine neurotransmission and synaptogenesis. Further studies should be performed to confirm this hypothesis.

Accumulating evidence shows that AMPA glutamatergic activation triggers the synaptogenic process in important regions such as the prefrontal cortex and hippocammpus [7]. In addition, several research results have shown that phosphorylation of mTOR and kinases ERK and Akt, is important mechanism underlying the activation of AMPA receptors in synaptogenesis [33‐35]. Therefore, AMPA receptor-mTOR signaling pathway may be involved in the synaptic alteration underlying depression. To determine whether the antidepressant and anxiolytic like effects of TSS was accompanied by the activation of AMPA receptor-mTOR signaling pathway, the phosphorylation of GluR1 Ser845 (AMPA receptor subunit), mTOR and mTOR upstream regulator proteins (ERK and Akt) were assessed in chronic corticosterone-treated mice. Our research showed that TSS reversed the decreased level of P- GluR1 (ser845) induced by 5 week CORT administration. Simultaneously, 5 week CORT administration decreased the expression level of p-mTOR, p-ERK and p-Akt in the hippocampus, which was reversed by TSS and citalopram treatment. This finding of citalopram is in agreement with previous study showing that activation of the mTOR signaling was implicated in the antidepressant-like effect of subchronic citalopram treatment in the chronic mild stress model [36]. Our another study showed that pretreatment with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo (f) quinoxaline-7-sulfonamide (NBQX), an AMPA receptor antagonist, blocked the TSS induced antidepressant-like effects of decreased immobility time in the FST (data not shown). These results indicated that AMPA receptor-mTOR signaling pathway may be involved in the antidepressant-like effect of TSS. Further research is needed to examine whether pretreatment with NBQX would affect the activity of the mTOR signaling pathway (p-mTOR, p-ERK and p-Akt) after TSS treatment.

The present study indicates that repeated administration of TSS exerts antidepressant-like and anxiolytic effects in chronic CORT administration mice. Our data provide evidence that TSS improves the depressive and anxiety like behavior via induction of AMPA receptor and subsequent mTOR signaling pathway which are involved in synaptogenesis. Together with our previous research, TSS could be a potential efficacious candidate as a new therapy for depression.