Depression, a psychiatric disorder characterized by a low self-esteem, altered mood, hopelessness, reduced interest/pleasure in daily activities and persistent thoughts of death or suicide, has become a significant global health issue and economic burden [1
]. The lifetime prevalence of depression is approaching 20% of the population and is expected to be the second leading cause of incapacity worldwide by the year 2020 based on data from the World Health Organization [2
]. The etiopathology as well as the clear mechanisms underlying depressive disorders are still far from understood, since depression is a highly complicated psychiatric illness. Stress, which can induce neuroinflammation, mitochondrial damage, neuroplastic deficits and intracellular signaling pathways, has been implicated to act as a major determinant for the onset of depression, and may provide a novel target for preventing neurodegeneration [3
]. The animal models and clinical studies on the link between stress and depressive disorders suggest that antioxidant agents can reduce oxidative stress through scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS), which further protect against neuronal damage induced by stress [5
]. In addition, stress-induced depression has been shown to alter the levels of monoamine neurotransmitters such as serotonin (5-HT), along with behavioral changes in animal models [10
]. Numerous studies reported that normalizing the disturbed monoaminergic neurotransmitters is associated with treating depressive disorders [12
]. Furthermore, a growing body of evidence has demonstrated that stress negatively regulates the level of brain-derived neurotrophic factor (BDNF), which may contribute to the impairment of the dendritic plasticity and hippocampal neurogenesis and be responsible for neuron damage and onset of depression [15
]. Although there are many pharmacotherapies available nowadays, over 30% of depressed patients do not achieve a clinically appreciable improvement with current treatments. The significant limitations of conventional antidepressants include the slow onset for therapeutic actions (weeks to months) and undesirable side-effects such as nausea, diarrhea, migraine headache, sleep disturbance and sexual problems [18
]. In the view of the impact on depressors, especially for those suicide-risk patients, research focused on the discovery and development of agents with promising efficacy and fewer side effects is urgent.
(HE), Houtou mushroom in Chinese, has been used as food and folk medicine in several East-Asia countries for centuries [20
]. HE has been documented to display a wide range of beneficial properties, including anticancer, antimicrobial, antihyperglycemic, antioxidant and hypolipidemic activities, and immune modulation [21
]. A group of diterpenoids isolated from the cultured mycelia of HE, namely erinacines, were demonstrated to be potential enhancers of nerve growth factor (NGF) biosynthesis in cultured astrocytes [25
]. The increased production of NGF is correlated with proper neural growth and maintenance [28
]. Importantly, in particular, erinacine A has been reported to exhibit the protective effect against ischemic injury, Parkinson’s and Alzheimer’s diseases in vivo [30
]. Therefore, erinacine A-enriched HE is attracting attention and may serve as a promising agent having neurotrophic activity with potential application in ameliorating neurodegenerative disorders.
Restraint stress (RS) has been extensively applied to induce a depression-like state for screening the effectiveness of antidepressant activities [33
]. However, there is no quantitative data regarding the antidepressant-like activities of HE in a repeated RS-induced mouse model of depression. The aim of this present study, thus, was to study the effects of erinacine A-enriched HE mycelium and reveal the possible mechanisms using an RS mouse model. In relation to that, the behavioral alterations and the contents of monoamines, proinflammatory cytokines, and depression-related protein expressions were assessed.
The chromatograms generated by high-performance liquid chromatogram (HPLC) and liquid-chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) with positive and negative ionization modes of the ethanolic extract from mycelia of H. erinaceus
are displayed in Figure 1
A. Peak 2 was verified to be erinacine A (2
) and the other three peaks were tentatively identified comparing to the prepared standards (kindly provided by Dr. CC Chen, HungKuang University, Taichung, Taiwan) as previously reported [32
]. The chemical structures and mass spectral characteristics of four major peaks are illustrated and described in Figure 1
B and Table 1
, respectively. The contents of those peaks were quantified from the established calibration curve as erinacine A (2
) with the highest amount of 5.0 mg/g dry weight (Table 1
To examine the antidepressant-like effect of HE treatment, behavioral responses of the immobility time in the mouse tail suspension test (TST) and forced swimming test (FST) were carried out and are shown in Figure 2
A,C, respectively. The results indicated a significant anti-immobility effect elicited by the treatment of HE at the doses of 200 and 400 mg/kg in the TST (p
< 0.01) and FST (p
< 0.01) as compared to the vehicle-treated stressed mice (RS group). In addition, HE at the doses of 100, 200 and 400 mg/kg increased the swimming time in the FST (p
< 0.01, p
< 0.001 and p
< 0.001, respectively) as compared to the RS group (Figure 2
The ability of HE to modulate emotional reactivity in stressed mice was examined and Table 2
reveals the results of HE on the assayed parameters in the elevated plus maze over the 5-min test. The data showed that there was a significant increase of the number of entries in open arm (POAE) in stressed mice treated with medium and high doses (200 and 400 mg/kg) of HE (p
< 0.01) as compared to the RS group. The percentage increases in the POAE were 21.1% and 24.1%, respectively. Furthermore, stressed mice treated with HE at 200 and 400 mg/kg significantly increased the time spent in the open arm (PTOA) by 22.4% and 22.1%, respectively, as compared to the RS group. No significant difference in the number of closed-arm entries (CAE) was observed in all groups.
To exclude the changes in behavior observed in TST and FST that were attributed to the false-positive effect, the responses of HE treatment on locomotor activities in mice were tested. Table 3
depicts the mean locomotor responses of testing mice. The administration of vehicle or various doses of HE to repeated restraint-stressed animals did not give rise to any obvious changes in number of crossing and rearing. On the other hand, the RS-alone group showed higher numbers of defecation by ~82% (p
< 0.01), and middle and high doses of HE reduced defecation significantly by ~27% as compared to the RS group (p
The concentrations of norepinephrine (NE), dopamine (DA) and 5-HT were drastically reduced after repeated restraint stress in the vehicle-treated group (RS group) compared with the control group (p
< 0.001) (Figure 3
). Although significant elevation of NE level was found only in high dose of HE treatment (p
< 0.05), HE (100, 200 and 400 mg/kg) produced profound increases in DA levels in the hippocampal region (p
< 0.001) as compared to the RS group (Figure 3
A,B). Supplementation of medium and high doses of HE helped to revert the stress-induced 5-HT depletion (by raising about 81.6% and 92.5%, respectively) (Figure 3
Effect of HE on the concentrations of plasma cytokines is illustrated in Figure 4
. The levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were markedly elevated in repeated restraint stress-treated mice compared with the control group (p
< 0.001). Supplementation with HE at 200 and 400 mg/kg significantly inhibited stress-induced increases in IL-6 levels (p
< 0.05 and p
< 0.01, respectively) and treatment with HE at all doses drastically suppressed plasma TNF-α contents (p
< 0.05, p
< 0.01 and p
< 0.01, respectively) as compared to the RS group.
To understand the molecular mechanism underlying the antidepressant-like effect of HE, the expressions of BDNF, TrkB and PI3K signaling pathway proteins with β-actin as control in the hippocampus of mice were examined (Figure 5
). Repeated restraint stress decreased the expression levels of BDNF, TrkB and PI3K in the mice brain tissue compared to the control group. HE at tested concentrations was effective to reverse the stress-induced downregulation. Western blotting data revealed that Akt and GSK-3β expressions did not change in all groups. However, repeated restraint stress significantly downregulated Akt-p and GSK-3β-p expressions and the stress-induced decreases in both proteins were prevented by the treatment with HE in the hippocampus of mice.
As a typical signal transduction pathway of pro-inflammatory cytokines, NF-κB and IκB expressions were examined in stressed mice with HE treatment. As shown in Figure 6
, significantly lower expressions of NF-κB and IκB in the cytosol fraction of the hippocampus were observed in stressed mice, indicating that the nuclear factor was translocated into nucleus, and enhanced the production of inflammatory mediators. An increasing tendency of both protein expressions could be detected with the treatment of HE, demonstrating that HE could block the NF-κB-induced inflammation, and this was in line with plasma cytokine studies.
Accumulating data suggest that stress plays an important role in the development and manifestation of depression [3
], and restraint stress (RS) has been applied as a major promoter of depression-like condition to verify the effectiveness of antidepressant activities [33
]. The present study investigated the antidepressant-like effects of erinacine A-enriched HE mycelium in the RS mouse model. For the first time, based on the evidence that supplementation of HE decreased immobility times in the mouse TST and FST without affecting the locomotor activity in the mouse open field test (OFT), we have demonstrated that HE exerted remarkable antidepressant-like effects in the RS-induced depressive mice.
Both TST and FST are the most common tools used for evaluating antidepressant potential. In line with other results, mice treated by RS displayed significant immobility in the TST and FST [36
]. These behaviors were reversed by the treatment of HE (at 200 or 400 mg/kg), indicating an antidepressant-like effect. The antidepressant activity of oral HE treatment was further confirmed by an increase in swimming time analyzed by the FST. Meanwhile, in the OFT, the numbers of crossings and rearings were not altered among groups, indicating that the anti-immobility effects of HE observed in the TST and FST were not attributable to changes in locomotor activities.
Modulating monoamine neurotransmitters, including NE, DA and 5-HT, has been recognized as a major target for elucidating the mechanisms underlying the antidepressant-like effect. The present study demonstrated a significant decrease in the levels of neurotransmitter contents in the hippocampus following 14 days of restraint stress. Our results are in keeping with other studies showing RS induced significantly decreased levels of biogenic amines [38
]. Interestingly, HE was effective in restoring these changes in the hippocampus induced by RS following treatment. In the current study, we found that the antidepressant-like effects of HE might stem from increasing the levels of hippocampal NE, DA and 5-HT, which is consistent with previous studies that showed that some botanical extracts mediated the antidepressant-like effect by virtue of an increase of brain monoamines [39
]. Thus, this result supported the finding that HE administration may lead to antidepressant-like effect by reducing TST and FST immobility time through noradrenergic, dopaminergic and serotonergic modulation in the RS mice. Therefore, we speculated that a possible mechanism underlying the activity is that erinacine A (enriched erinacine in HE constituents) might act as a monoamine neurotransmitter receptor agonist or monoamine neurotransmitter reuptake inhibitor. This possibility needs to be further verified in the future investigation.
There is evidence to suggest that pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, contribute to the onset and progression of depressive disorders [41
]. Studies have pointed out inflammation could activate some signals which can trigger the transition from inflammation to depression [42
]. In fact, increased circulating levels of pro-inflammatory cytokines have been reported with stressed and depressed patients [43
]. The present study substantiated the enhancement of pro-inflammatory cytokines in the RS depressive mice model. The levels of IL-6 and TNF-α were markedly elevated. Our data are consistent with other reports, which revealed that stressful life events and depressive symptoms are associated with the increase of circulating cytokines in clinical and stress-treated animals [45
]. Recent studies showed that erinacine A protected from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity as a result of oxidative stress signaling and the JNK/p38/NF-κB pathways in mice [30
], and had a protective effect on ischemic myocardial injury via the inhibition of iNOS/p38 mitogen-activated protein kinase (MAPK) and nitrotyrosine in rats [31
]. Based on the close link between inflammation and depression, it is reasonable to expect a favorable effect of anti-inflammatory response of erinacine A-enriched HE on depression-like behavior. In fact, our results signified that supplementation with HE drastically inhibited the stress-induced rise of plasma IL-6 and TNF-α contents. Furthermore, HE exhibited antidepressant effects on RS-induced depressive behaviors. Thus, these findings can support the possibility that HE might have an antidepressant effect via regulating the inflammatory response. Although HE shows antidepressant effects via suppressing inflammation, the underlying precise molecular mechanisms remain to be determined. A recent study showed that benzyl alcohol derivatives from H. erinaceum
attenuate the lipopolysaccharide (LPS)-stimulated inflammatory response through the regulation of NF-κB and AP-1 activity in macrophage cells [47
]. The present findings also demonstrated that repeated restraint-stressed animals accompanied by depression-like behavior reduced the expression levels of NF-κB and IκB in the cytosol fraction of hippocampal tissue, and HE-treated mice normalized these levels. It has been believed that NF-κB is a pivotal transcription factor, and it translocates into the nucleus and initiates the transcription of an array of relevant genes (such as pro-inflammatory cytokines) and inducible enzymes (such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 following activation. Accordingly, targeting of the NF-kB pathway is an interesting tactic in the treatment of depression because inflammation plays a critical role in the progression of the disorder [39
]. In this way, the normalization of NF-κB level could be, at least in part, responsible for the pharmacological effects of HE after repeated restraint stimulation.
It is generally accepted that synaptic plasticity could be influenced by stress condition and the weakening in neuroplasticity might be a key factor in the process of depression [48
]. Accordingly, neuroplasticity turns out to be the therapeutic target of antidepressant agents. In this study, the expression of BDNF, the pivotal marker of synaptic plasticity, was examined to reveal the molecular mechanism by which HE drives to normalize depression-like behavior. BDNF is a member of the neurotrophic factor known to participate in the life of neurons during development and to modulate hippocampal-dependent learning and memory [49
]. Accumulating evidence supports that BDNF is indispensable for exerting antidepressant effects because it can modulate synaptic efficacy by changing transmitter release and sensitivity [50
]. There is also evidence that the lack of BDNF is linked to the pathophysiology of mood disorders [51
]. Recently, Wittstein et al. suggested that corallocin C isolated from Hericium coralloids
is able to induce the mRNA levels of NGF and BDNF for neurite outgrowth of PC12 cells, and the mechanism, at least in part, is connected to act on an upstream target [52
]. This is in line with our present study, which found reduced expressions of BDNF and TrkB in the hippocampal region of mice after RS, and that the treatments with HE were effective in restoring the BDNF levels in the brain region. Since the BDNF content was greatly influenced by monoamine transmission [53
], the restoration of BDNF content may be an effect of the normalized monoamine content (NE, DA and 5-HT).
Glycogen synthase kinase-3β (GSK-3β) is an enzyme that phosphorylates glycogen synthase, which in turn inhibits glycogen biosynthesis. Moreover, GSK-3β is now believed to play an important role in the pathophysiology of depression and is implicated to be a drug target for the treatment of depression. Furthermore, GSK-3β inhibitors such as thiadiazolidinone NP031115 and AR-A014418 have been reported to be associated with antidepressant effects, as proven by reduced immobility in the forced swimming test [54
]. A large amount of evidence has implicated that the pathology of depression might be associated with neuronal inflammation [42
]. Literature data indicate that phosphatidylinositol 3-kinase (PI3K) and serine/threonine protein kinase AKT seem to activate immune cells by modulation of the key inflammatory cytokines [55
]. In addition, the PI3K/Akt pathway has been reported to play the role as an upstream mechanism of GSK-3β activity regulation, in which Akt might directly phosphorylate GSK-3β, resulting in GSK-3β inactivation. [56
]. Irregularities in the PI3K/Akt/GSK-3β pathway are linked in patients with psychiatric illnesses. Therefore, regulation of AKT and GSK-3β may form an important signaling center for depressive therapy. In the present study, we demonstrated that HE was able to increase phosphorylation of Akt and GSK-3β. Altogether, the results presented herein firstly reveal that the antidepressant-like effect of HE involves the activated pathway of PI3K/Akt and inhibition of GSK-3β that converge to increase BDNF.