Introduction

Depressive disorders are among the leading causes of disability and impose a big health burden to the society (Nemeroff 2007). Women are more susceptible to depression due to the fluctuations in estrogen levels in their life, such as perimenopausal and postpartum periods (Oppenheim 1983; Halbreich et al. 1986; Hamilton et al. 1988). Clinical evidence indicates that estrogen decreases the depressive symptoms in perimenopausal women (Ahokas et al. 1999). At present, there are some classical antidepressants in clinical practice, including tricyclic antidepressants (imipramine), selective serotonin reuptake inhibitors (SSRIs) and monoamine oxidase inhibitors (MAOIs). However, their mechanisms of action, particularly on the menopause depression, have not been satisfactorily resolved. Although the estrogen replacement therapy is also effective in the treatment of depressive symptoms in menopausal women, the onset of other side effects after receiving this therapy has been observed (Nelson et al. 2002; Beral et al. 2007). Therefore, the development of safe and effective pharmacotherapy to relieve this symptom in women is in great need.

Flaxseed is a rich source of plant lignans (Thompson et al. 1991). Previous studies suggested the multi-faceted biological activities of flaxseed, such as the anti-oxidant and anti-cancer effects (Dabrosin et al. 2002; Birkved et al. 2011; Tuluce et al. 2011). Although the reports on the effects of flaxseed are really inadequate, many women still prefer taking flaxseed as a source of phytoestrogens. Indeed, an increasing number of postmenopausal women start to take phytoestrogen-rich foods to prevent the risk of breast cancer, osteoporosis and some menopausal symptoms (Duffy and Cyr 2003). Moreover, in the hypercholesterolemic menopausal women, 40 g of flaxseed demonstrated similar efficacy to the hormone replacement therapy in improving the menopausal symptoms (Lemay et al. 2002). All these data suggested the potential antidepressant activities of flaxseed in menopausal women. However, the information regarding to this activity of flaxseed is still lacking.

It has been shown that major depression is associated with elevated peripheral inflammatory biomarkers, particularly pro- and anti-inflammatory cytokines mediating the innate immune response, such as IL-1β, TNF-α and IL-6; patients treated with cytokines are at greater risk of developing major depression (Schiepers et al. 2005; Raison et al. 2006). Fluoxetine-resistant depressed patients express higher levels of IL-6 and TNF-α when compared to the normal controls (O’Brien et al. 2007), which parallel to the decrease in Brain-derived neurotrophic factor (BDNF) expression. Chronic stress induces a reduced BDNF expression in both hippocampus and frontal cortex; while chronic treatment with antidepressants can enhance the BDNF expression in rat hippocampus(Smith et al. 1995; Russo-Neustadt et al. 2000; Karege et al. 2002). These studies implicate the involvement of neuroendocrine-immune network and BDNF in the pathology of depression as well as the action of antidepressants.

In the current study, we assessed the antidepressant-like effect of chronic administration of Secoisolariciresinoldiglycoside (SDG), which is the predominant lignan in flaxseed (Kitts et al. 1999), in the animal model of menopausal depressive-like state in ovariectomized female mice. We first tested the antidepressant-like activity of flaxseed SDG in mouse model of despair tests, forced swim and tail suspension tests (FST and TST) after the chronic stress. Then the serum corticosterone and adrenocorticotropic hormone (ACTH) levels, the cytokines and BDNF expression in the frontal cortex were also measured to further understand the possible mechanism of SDG in the overiectomized (OVX) mice. The tricyclic antidepressant imipramine that has been shown to reduce the immobility time in the ovariectomied mice dose-dependently (Bekku and Yoshimura 2005) was chosen as a positive drug in this study.

Materials and methods

Animals

ICR female mice (18~20 g) were obtained from the Animal Center of Peking University Health Science Center (Beijing, China) and were adapted to the laboratory conditions for 2 weeks before the experiments. On arrival, the animals were housed ten per cage under standard colony conditions, with controlled ambient temperature (25±1°C), humidity (50±10 %) and a 12 h light/12 h dark cycle. The mice have free access to food and water and they are weighed daily for 21 days. All experiment procedures were conducted in accordance to the National Institutes of Health Guide for Animal experimentation and the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Drugs and drug administration

Secoisolariciresinaldiglycoside (SDG) was purchased from Chroma Dex (USA). Imipramine hydrochloride was purchased from Sigma (USA). The anti-BDNF antibody and the secondary antibodies were purchased from Santa Cruz (USA). SDG and imipramine were dissolved in redistilled water and given orally (p.o.) and intraperitoneally (i.p.), respectively. In the present study, SDG (40, 80, 160 mg/kg) and imipramine (10 mg/kg) were administered daily for 21 days. The behavioral testing commenced 60 min after the last drug treatment.

Surgery

Mice were bilaterally ovariectomized under pentobarbital sodium (50 mg/kg, i.p.) anesthesia. The surgery was performed via a ventral approach with a small midline ventral skin incision. The oviducts were then removed and the abdominal wall was closed with sutures. The sham control groups were subject to the same surgical procedure without removing their ovaries. The mice were allowed to recover for 1 week before the experiment. Vaginal smears were obtained daily for 5 days in both sham and OVX groups and were used to confirm the success of ovariectomy.

Chronic stress procedure

The mice were subjected to the chronic stress protocol developed by Molina et al. (1990) and Murua et al. (1991) with slight modifications. Stress was given once per day over a period of 20 days between 8:00 am to 12:00 am. The order of the stressors used was as follows:

Days

1

2

3

4

5

6

7

8

9

10

Stressors

Shaking (high speed, 45 min)

Cold swim (10 °C, 5 min)

Restraint (1.5 h)

Tail pinch "(1 min)

Water deprivation (24 h)

Foot shock (30 min; 1 mA, 1 s duration, average 1 shock/min)

Cold swim (10 °C, 5 min)

Food deprivation (24 h)

Restraint (2 h)

Shaking (high speed, 1 h)

Days

11

12

13

14

15

16

17

18

19

20

Stressors

Tail pinch (1 min)

Water deprivation (24 h)

24 h social isolation (mice were individually placed in 30×15×10 cm cages in another room)

Foot shock (45 min; 1 mA, 1 s duration, average 1 shock/min)

Cold swim (8 °C, 5 min)

Shaking (high speed, 1.5 h)

Restraint (2.5 h)

Tail pinch (2 min)

Food deprivation (24 h)

24 h social isolation (same as day 13)

On day 21 (60 min after the drug or vehicle administration), mice were subjected to different behavioral tests or sacrificed to assess the neuroendocrine changes (including serum corticosterone and ACTH concentrations).

Forced Swimming Test (FST)

The forced swimming test was performed according to the procedure that has been standardized and validated in our laboratory (Xu et al. 2005). Briefly, each mouse was subjected to a pre-test for 15 min in glass containers (height: 25 cm; diameter: 10 cm; containing 19 cm of fresh water at 24±1 °C). After 24 h, mice were placed in the containers again for a 6-min test following the same procedure described above. The duration of immobility was measured during the final 4 min interval of the test.

Tail Suspension Test (TST)

The tail suspension test was carried out based on the previously reported method (Steru et al. 1985). The protocol has been previously validated in our lab (Xu et al. 2005). Mice were individually suspended 50 cm above the floor by using an adhesive tape affixed 1 cm from the tip of the tail. The time during which the mouse was immobile was recorded during the 6 min of the test. The duration of immobility was considered only when the mouse was completely motionless.

Locomotor activity

The assessment of locomotor activity was carried out on mice using a slightly modified method (Xu et al. 2005). Briefly, the locomotor activity of the mice was measured by electronic counters with five activity chambers (JZZ98, Institute of Materia Medica, Chinese Academy of Medical Sciences, China). Each mouse was placed in the chambers 15 min before the evaluation for acclimatization and then locomotion counts were recorded for a period of 10 min.

Measurement of serum corticosterone and adrenocorticotropic hormone (ACTH), and cytokines in the frontal cortex

After unpredictable chronic mild stress procedure, mice were sacrificed and serum samples were collected, aliquoted and frozen at −80 °C until assayed. The levels of corticorsterone was measured by using a competitive enzyme immune assay (EIA) kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) and the serum ACTH was also assayed with an EIA kit (Phoenix Pharmaceuticals Inc, Burlingame, CA, USA). The levels of IL-6 and IL-1β in the frontal cortex were measured using ELISA kits (Enzo Life Sciences, Plymouth Meeting, PA, USA). All the assays were carried out according to the manufacturer’s instruction.

Western blot analysis

Mice were decapitated and the frontal cortex were rapidly removed and stored at −70 °C. Tissue samples were suspended in a solution containing 2 % SDS, 10 % glycerol, 100 mM dithiothreitol, 0.01 % (w/v) bromophenol blue and 60 mM Tris-HCl (pH=6.8) and sonicated for 10s. The protein concentration was measured using Bradford assay. The western blot analysis was subsequently carried out according to our previously established protocol (Xu et al. 2006).

Statistical analysis

All data were expressed as the means± SEM. The significant differences among groups were analyzed using a one-way analysis of variance (ANOVA) followed by a post hoc Student-Newman-Keuls (SNK) test. Effects were considered significant when p <0.05.

Results

The effects of flaxseed SDG on the duration of immobility in FST and TST

Ovariectomy did not induce any behavioral changes in FST and TST. Exposure to different stressors increased the immobility time significantly in the ovariectomized mice compared to the sham-operated mice (p<0.01) in the FST (Fig. 1). Chronic administration with flaxseed SDG (80 and 160 mg/kg) prevented the increase of immobility significantly [F(4, 75)=12.67, p<0.01] (Fig. 1). The similar results were observed in the tail suspension test [F(4, 70)=10.98, p<0.01] (Fig. 2). The efficacy of SDG at 80 mg/kg was more efficient than that of the higher dose (160 mg/kg) in both FST and TST.

Fig. 1
figure 1

The effects of flaxseed SDG on the immobility time in the forced swimming test. The mice were either subjected to an unpredictable chronic mild stress for 20 days (stress groups) or left undisturbed (control groups) following the ovariectomy. The behavior test was performed 24 h after the last treatment with vehicle, SDG (40, 80 and 160 mg/kg, p.o.) or imipramine (10 mg/kg, i.p.). Values are the mean ± S.E.M. with 15 mice in each group. * P < 0.05, ** P < 0.01 and *** P <0.001 vs. the stressed OVX + vehicle group

Full size image
Fig. 2
figure 2

The effects of flaxseed SDG on the immobility time in the tail suspension test. The mice were either subjected to an unpredictable chronic mild stress for 20 days (stress groups) or left undisturbed (control groups) following the ovariectomy. The behavior test was performed 24 h after the last treatment with vehicle, SDG (40, 80 and 160 mg/kg, p.o.) or imipramine (10 mg/kg, i.p.). Values are the mean ± S.E.M. with 15 mice in each group. * P < 0.05, ** P < 0.01 and *** P <0.001 vs. the stressed OVX + vehicle group

Full size image

The effects of flaxseed SDG on the locomotor activity

Figure 3 showed the effects of SDG on locomotor activity in mice. There is no significant difference between the ovariectomized and sham-operated groups in the activity. Moreover, neither SDG (40 to 160 mg/kg) nor imipramine (10 mg/kg) affected the locomotor activity at doses that significantly reduced the immobility response in the FST and TST.

Fig. 3
figure 3

The effects of flaxseed SDG on locomotor activity in mice. The mice were administered with vehicle, SDG (40, 80 and160 mg/kg, p.o.) or imipramine (10 mg/kg, i.p.). The locomotion counts were recorded for 10 min. Values are the mean ± S.E.M. with 15 mice in each group. There were no significant differences compared to control groups

Full size image

The effects of flaxseed SDG on the body weight

The effects of chronic stress and administration with flaxseed SDG on the body weight have been summarized in Table 1. There is no difference in the initial body weight in all the experimental groups (pre-stress). However, the chronic stress significantly decreased the body weight in the ovariectomized group (p<0.01). Treatment with flaxseed SDG (80 mg/kg) significantly prevented the reduction of the body weight (p<0.01), which is similar to the effect of the positive drug imipramine (10 mg/kg). The lower and the higher doses (40 and 160 mg/kg) of SDG seemed less effective, which were consistent with the results in FST and TST.

Table 1 The effect of flaxseed SDG on the body weight (g) of the ovariectomized mice subjected to chronic mild stress (mean ± S.E.M., n=15)
Full size table

The effects of flaxseed SDG on the corticosterone and ACTH levels in the serum, and cytokines in the frontal cortex

As shown in Table 2, chronic stress induced significant increases in the serum corticosterone and ACTH levels; the IL-6 and IL-1β levels in the frontal cortex were also increased in the ovariectomiced mice compared to the non-stressed group. However, the administration of flaxseed SDG (80 and 160 mg/kg) inhibited these changes in serum corticosterone [F(4, 75)=13.60, p<0.001] and ACTH [F(4, 75)=6.41, p<0.05] levels. The increased IL-6 [F(4, 75)=12.97, p<0.001] and IL-1β [F(4, 75)=9.83, p<0.01] levels in the frontal cortex were also prevented by treatment with high doses of SDG. However, the effects of treatment with SDG or imipramine were not able to reduce the corticosterone, ACTH and cytokine levels to the non-stressed baseline levels.

Table 2 Effects of flaxseed SDG on the levels of corticorsterone and ACTH in serum and cytokines in the frontal cortex of ovariectomized mice subjected to chronic mild stress (mean ± S.E.M., n=15)
Full size table

The effects of flaxseed SDG on the BDNF expression

As shown in Fig. 4, western blot analysis demonstrated that BDNF levels were decreased in the frontal cortex of the stressed OVX mice compared to sham-operated non-stressed group (p<0.001). This reduction was reversed by treatment with flaxseed SDG (80 and 160 mg/kg) [F(4, 75)=14.05, p<0.001].

Fig. 4
figure 4

Immuno-blot analysis of BDNF expression in the frontal cortex of mice. The intensity of the protein bands was quantified with a densitometric scanner. Each column represents the group mean ± S.E.M of 15 mice. * P < 0.05 and ** P < 0.01 vs. the stressed OVX + vehicle group. ## P < 0.01 vs. the non-stressed OVX + vehicle group

Full size image

Discussion

Recent research has pointed out that women during the menopausal period are at a growing risk of having depression. Over the years, increasing evidence has demonstrated that there is an association between depressive disorders and the menopause symptoms (Weissman and Olfson 1995; Halbreich and Kahn 2001; Kessler 2003). Among the 460 women aged between 36 and 45 years, the menopausal group has much higher chance to experience significant depressive symptoms than the premenopausal group (Cohen et al. 2006). Moreover, in an 8-year study of 231 women with no history of depression, people found that the high depression score for women in the menopausal transition phase is four times higher than women in the premenopausal phase (Freeman et al. 2006). However, the currently used hormone replacement therapy (HRT) has been shown to link with an increased cancer incidence (Nelson et al. 2002, Beral et al. 2007). Moreover, some of the patients are intolerant to the HRT or HRT in combination with antidepressant medication (Nelson et al. 2002). Therefore, people started to search for other powerful and secure alternatives for the treatment of menopausal depression.

Stress, as an everyday burden, is a precipitating factor in the onset of major depression (Kessler 1997). Unpredictable chronic mild stress, which mimics the environmental stressors people might have in daily life, is an accepted animal model of depression and has been widely used in the antidepressant studies (Garcia 2002). Animals exposed to an unexpected chronic stress have shown various depressive symptoms and learning and memory disorders, which can be reversed by the treatment of various types of antidepressants (Garcia 2002). Bilateral ovariectomy serves as a useful model to study the menopausal depression as well as the promising pharmacotherapy (Bekku et al. 2006). The changes in regional skin temperature of ovariectomized mice are similar to the hot-flush symptom in menopausal women (Okada et al. 1997). Biochemical results suggest that bilateral ovariectomy induces significant decrease in plasma 17β-estradiol level in mice (Davidge et al. 2001). The behavioral despair test for depression shows that the immobility time in the forced swimming test significantly increased 2 weeks after the surgery. Moreover, the prolonged immobility in FST can be reversed by treatment with either estradiol or antidepressants (Bekku and Yoshimura 2005; Bekku et al. 2006). A recent research also pointed out the antidepressant-like effects of fluoxetine and desipramine in forced swimming test of rats after long-term ovariectomy (1, 3 and 12 weeks) (Estrada-Camarena et al. 2011). In this study, we used the unpredictable chronic mild stress paradigm following the ovariectomy in mice as an animal model of menopausal depression to study the efficacy of flaxseed SDG treatment. Flaxseed is the richest source of plant lignans, among which SDG is the dominant one (Thompson et al. 1991). It has been shown that SDG can be metabolized into the mammalian lignansenterolactone (EL) and enterodiol (ED) by bacteria in the colon (Eeckhaut et al. 2008). SDG has been shown to have multiple biological effects, such as antioxidant and anti-cancer effects (Kitts et al. 1999; Saggar et al. 2010), but it is still not clear if SDG has antidepressant-like effect and the related mechanism. Our results indicated that mice subjected to a chronic unpredictable stress regime for 20 consecutive days displayed much greater immobility periods in both FST and TST, which were significantly reversed by treatment with flaxseed SDG (80 and 160 mg/kg). Since changes in the duration of immobility might be due to effects on locomotor activity caused by central nervous system stimulants, the mice were tested in a locomotor activity chamber. The results suggested that SDG, at doses ranging from 40 to 160 mg/kg, did not change locomotor activity significantly. Therefore, SDG appears to have antidepressant-like effects. In addition, we did not observe significant differences for the ovariectomized mice receiving vehicle compared to the sham groups after chronic stress in the behavioral and neurochemical analyses in the preliminary study (data not shown). Moreover, the middle dose of SDG (80 mg/kg) seems highly effective in both FST and TST, with significant decrease in immobility. In agreement with this, the stress-induced loss of body weight was also reversed by treatment with 80 mg/kg SDG, with comparable effect of imipramine (10 mg/kg). This observation is potentially interesting since lower dosage of SDG may be used as an alternative treatment for menopausal depression.

Chronic stress is proved to induce depression and many depressed patients have an impairment of HPA axis feedback inhibition, which is characterized by HPA hyperactivity (Sachar et al. 1973; Carroll et al. 1976). This disruption of the HPA axis activity will result in enhanced serum cortisol (in primates) or corticosterone (in rodents) and ACTH levels, which may in fact be a marker of depression (Amsterdam et al. 1987; Centeno and Volosin 1997). Previous studies have demonstrated that the behavioral deficits and the abnormalities in the neuroendocrine system that are induced by chronic stress, such as aberrations in the HPA system, can be reversed by the treatment of antidepressants (Arborelius et al. 1999; Ibarguen-Vargas et al. 2008). In the present study, we found that treatment with SDG (80 and 160 mg/kg) reversed stress-induced increased levels of serum ACTH and corticorsterone. Therefore, the antidepressant-like effect of SDG should be, at least partly, related to its regulation on the HPA system.

The modern depression hypothesis believes that the dysfunction of neuro-endocrine immune network system is the basis of pathology of depression. Many studies have suggested that inflammatory mediators, including IL-6 and IL-1β, are implicated in the pathophysiology of depression (Schiepers et al. 2005). In the present study, chronic stress increased IL-6 and IL-1β expression in the frontal cortex, which were prevented by treatment with SDG or imipramine. These results consist with the previous study, which suggested that Major depressive patients in the clinic demonstrate higher levels of serum IL-6 and IL-1 (Dowlati et al. 2010; Liu et al. 2012). Interestingly, cytokines may have an impact on BDNF function and affect the BDNF signaling pathway (Murphy et al. 2000; Tong et al. 2008). As the most abundant neurotrophic factor, BDNF plays an important role in the regulation of cell survival and neurotransmission (Kuipers and Bramham 2006). Local infusion of BDNF into the midbrain of the hippocampus produces antidepressant effects in animal models of depression (Shirayama et al. 2002). Moreover, chronic administration of different antidepressants significantly increases the BDNF level (Russo-Neustadt et al. 2000). Moreover, BDNF also plays a central role in brain development and plasticity by opposing neuronal damage and promoting neurogenesis and cell survival (Takahashi et al. 1999; Banasr et al. 2004). In the present study, chronic SDG administration reversed the stress-induced decrease in BDNF levels in the frontal cortex. These results suggest that BDNF expression may prevent the neuronal damage and may be part of a critical link between stress-induced decreases in cell proliferation and the subsequent SDG’s action on depression.

In conclusion, in the present study, we elucidated the antidepressant-like effect of flaxseed SDG on chronic stress-induced depression in ovariectomized mice that mimic the menopausal depressed state. The underlying mechanism may be related to its normalization of neuron-endocrine immune network. The work for the receptors and signal transduction involved in the antidepressant-like effect of SDG during the menopausal period are processing in our laboratory. Due to its long history of use as a dietary supplement, flaxseed SDG should have promising therapeutic application in the treatment of depression. However, efficacy and safety of SDG should be further evaluated.