Improvement of Depression-Like Behavior and Memory Impairment with the Ethanol Extract of Pleurotus eryngii in Ovariectomized Rats

Abstract

Ethanol extract of Pleurotus eryngii (DC.) QUÉL has estrogen-like activities that protect against bone loss caused by estrogen deficiency. In the present study, we investigated the effect of P. eryngii on depression-like behavior and memory impairment in ovariectomized (OVX) rats. Immobility time during a forced swimming test was significantly longer for OVX rats than for sham-operated rats. The depression-like behavior in OVX rats was improved by long-term administration of the ethanol extract of P. eryngii (500 mg/kg body weight (b.w.)/d). Spatial memory impairment in OVX rats assessed by the Morris water maze test was also improved by P. eryngii extract without any effect on motility. These results suggested that P. eryngii extract has estrogen-like improvement activity against depression-like behavior and memory impairment in OVX rats. Additionally, increase in the amount of synaptosomal zinc after ovariectomy was inhibited by P. eryngii extract. Since zinc in synaptic vesicles is important for memory function and is linked to the pathophysiology of depression, normalization of zinc signaling would be involved in the beneficial effect of P. eryngii extract on neurological disorders after ovariectomy.

Women who are in a state of estrogen deficiency are at risk for neurological symptoms such as depression and memory impairment. The risk for depression is twice higher in women than men and increases with menopause.¹⁾ Estrogen can be used as an adjunct therapy for patients with depression who do not respond to conventional antidepressant treatment.²⁾ Also, episodic memory after surgical menopause is improved by prompt estrogen administration, although natural menopause is not associated with substantial cognitive change.³⁾

Estrogen or estrogen-progestin replacement therapy, however, increase the risk of breast and endometrial cancers with long-term usage.⁴⁾ A clinical trial, HABITS: hormonal replacement therapy after breast cancer—is it safe?, was terminated because the risk of breast cancer in women who had breast cancer previously reached an unacceptably high level with hormone replacement therapy (HRT).⁵⁾ In the 2002 Women’s Health Initiative, health risks exceeded benefits from HRT in healthy postmenopausal women, resulting in termination of the trial.⁶⁾ Considering the negative aspect of HRT, it would be helpful to find alternative and naturally occurring substances that minimize postmenopausal syndrome.

Recently, mushrooms are gaining importance as functional food and therapeutics in maintaining brain health.⁷⁾ Among the mushrooms, Pleurotus giganteus was reported to have neurite outgrowth effects.⁸⁾ Pleurotus eryngii (DC.) QUÉL, also known as king oyster mushroom, is gaining popularity as a culinary mushroom and is commercially grown in several countries including Japan. P. eryngii produced several biological active compounds such as β-1,3-glucans, lovastatin, pleureryn, eryngin, ribonuclease, 17β-estradiol, eryngeolysin, ergothioneine and protein xb68AB.⁹⁾ These compounds from P. eryngii have been reported to show various medicinal effects including immune modulation, anti-tumor, anti-infection and anti-osteoporosis effects.⁹⁾ Shimizu et al. reported that the ethanol extract of P. eryngii contains estrogen-like compounds that protect against bone loss caused by estrogen deficiency.¹⁰⁾ In the present study, we investigated the effect of estrogen-like compounds contained in P. eryngii on depression-like behavior and memory impairment in ovariectomized (OVX) rats.

Blood zinc level is affected by ovarian steroid deprivation and menopausal hormone therapy.^11,12) In the brain, approximately 20% of total zinc exists in presynaptic vesicles. Synaptic zinc is an important modulator of neurotransmission, synaptic plasticity and recognition memory and is involved in the pathogenesis of depression, Alzheimer’s disease and excitotoxic neuron injury.^13–17) Synaptosomal zinc level is affected by both endogenous and exogenous estradiol.¹⁸⁾ Therefore, we also investigated the effect of P. eryngii extract on the change in synaptosomal zinc level after ovariectomy.

MATERIALS AND METHODS

Ethanol Extractions of P. eryngii

Freeze-dried of P. eryngii (1.4 kg, Hokuto PLE No. 2, Hokuto, Nagano, Japan) was milled and extracted with ethanol (8 L, Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 40°C for 24 h. After filtration through a filter paper (No. 2, Advantec, Tokyo, Japan), the residue was extracted with ethanol again in the same manner. The extracts were concentrated with an evaporator and then freeze-dried. The freeze-dried extract was dissolved in water (50 mg/mL) and stored at −20°C.

Experimental Animals

Female Wistar rats were purchased from Japan SLC (Hamamatsu, Japan). Rats had ad libitum access to standard chow and tap water and were kept under a controlled 12-h light/dark cycle in a temperature- and humidity-controlled room (23±1°C, humidity of 55±5%). All experiments were performed in accordance with the Japanese Pharmacological Society guide for the care and use of laboratory animals, and the protocols were pre-approved by the Animal Ethical Committee of the University of Shizuoka.

Ovariectomy and Administration

Bilateral ovariectomies were carried out under chloral hydrate anesthesia (400 mg/kg body weight (b.w.), intraperitoneally (i.p.)) in 10-week-old rats. Similar operations were carried out for the sham-operated rats except for removal of ovaries. After 2 d, OVX rats were administered P. eryngii extract (500 mg/kg b.w./d, per os (p.o.)) or tap water (10 mL/kg b.w./d, p.o., OVX), and sham-operated rats were administered tap water (10 mL/kg/d, p.o., Sham) every day for 82 d. Body weights were measured every week.

Forced Swim Test

The forced swim test was performed in an acrylic cylinder (20 cm in diameter, 50 cm in height) filled to a height of 22 cm with water maintained at (22±2°C). Rats were individually placed in the cylinder for 6 min. Behavior during the test was recorded with a video camera to measure immobility time accurately. A rat was judged to be immobile when it remained floating passively in the water.

Morris Water Maze Test

A pool of 130 cm in diameter was filled with water (22±2°C), and a transparent platform was submerged in the pool. Rats were released into the water and trained to find the platform within a maximum time of 40 s. The trials were repeated 8 times in one day for 3 d. Releasing location was changed randomly in each trial. The last 8 trials were analyzed as the test session.

Motility Measurement

The pool was divided into four quadrants virtually, and total entry times into each quadrant zone were measured during the first 4 trials of the Morris water maze test. Motility was assessed as entry times/latency time (s).

Synaptosome Preparation

The right hemisphere in 5 mL of 0.32 M sucrose was homogenized with 12 strokes of a Potter–Elvehjem tissue grinder at 800 rpm. After centrifugation at 1000×g for 10 min, the supernatant was centrifuged again. The supernatant was layered onto 4 mL of 1.2 M sucrose and centrifuged at 160000×g for 15 min (himac CP65 β, Hitachi Koki, Tokyo, Japan). The synaptosome-containing band at the interface between the 1.2 M and 0.32 M sucrose layers was collected, diluted with 4 mL of 0.32 M sucrose, and then layered onto 4 mL of 0.8 M sucrose. After centrifugation at 160000×g for 15 min, the pellet was collected as synaptosomes.

Zinc Measurement

The synaptosomes were diluted with 100 µL of 1% nitric acid and then centrifuged at 16000×g for 3 min. Zinc concentration in the supernatant was measured using the colorimetric method with 5-Br-PAPS (zinc assay kit LS-MPR, Metallogenics, Chiba, Japan) and a microplate reader (Infinite M200, Tecan, Männedorf, Switzerland) with absorbance at 560 nm.

Statistical Analysis

Student’s t-test was used for comparison of the means of unpaired data. For multiple comparisons, ANOVA followed by Newman–Keuls multiple comparison test was performed.

RESULTS

At 16 d after ovariectomies, the body weight of OVX rats (187.1±3.0 g) was significantly greater than that of sham-operated rats (177.0±1.6 g, p<0.01, Student’s t-test). At 79 d after ovariectomies, OVX rats continued to show significantly greater body weight than that of sham-operated rats. On the other hand, long-term administration of the ethanol extract of P. eryngii did not affect the body weight of OVX rats (Fig. 1).

Fig. 1. Effect of P. eryngii Extract on Body Weight of OVX Rats

A, Time table for the experiment. Ovariectomies were carried out at day 0 in 10-week-old rats. P. eryngii extract was administered from day 2 for 82 d. B, Changes in body weights of sham-operated (Sham), OVX and P. eryngii extract-administered OVX (OVX+P. ery.) rats are shown. C, Body weights at day 79 are shown. Each value is the mean±S.E.M. (Sham, n=10; OVX, n=10; OVX+P. ery., n=8). Asterisks indicate significant differences (* p<0.05, ANOVA followed by Newman–Keuls multiple comparison test).

The effect of P. eryngii extract on depression-like behavior in OVX rats was investigated by the forced swim test. Immobility time was significantly longer for OVX rats than for sham-operated rats at 79 d after surgery. When the OVX rats were administrated P. eryngii extract for 77 d, immobility time of the OVX rats was significantly shortened to the levels of sham-operated rats (Fig. 2).

Fig. 2. Improvement of Depression-Like Behavior in OVX Rats by Long-Term Administration of P. eryngii Extract

A, Accumulated immobility time during the forced-swim test was measured in sham-operated, OVX and P. eryngii extract-administered OVX rats at day 79 after ovariectomy. B, Immobility time for 6 min is shown. Each value is the mean±S.E.M. (Sham, n=10; OVX, n=10; OVX+P. ery., n=8). Asterisks indicate significant differences (* p<0.05, ANOVA followed by Newman–Keuls multiple comparison test).

We also investigated the effect of P. eryngii extract on memory impairment in OVX rats by the Morris water maze test. Latency time until arriving at the hidden platform was significantly increased for OVX rats compared to that for sham-operated rats at 65–67 d after surgery. This memory impairment in OVX rats was improved by the administration of P. eryngii extract (Fig. 3A). Motility during the training session did not show a significant difference within each group (Fig. 3B).

Fig. 3. Improvement of Memory Impairment in OVX Rats with P. eryngii Extract

A, Ability of hippocampal-dependent spatial memory in sham-operated (n=9), OVX (n=10) and P. eryngii extract-administered OVX (n=8) rats was assessed by the Morris water maze test at days 65–67 after ovariectomy. Latency time until arriving at the platform is shown. Each value is the mean±S.E.M. Asterisks indicate significant differences (* p<0.05, ANOVA followed by Newman–Keuls multiple comparison test). B, Motility was assessed during the training session of the Morris water maze test. Total entry times into each virtual quadrant zone were counted and the motility was assessed as entry times/latency time (s).

Since estrogen influences zinc transporter expression and zinc level in synaptic vesicles, we studied the effect of P. eryngii extract on synaptosomal zinc level of OVX rats. Zinc level in synaptosomal fractions prepared from OVX rats was significantly higher than that in fractions from sham-operated rats. The increased synaptosomal zinc level in OVX rats was significantly decreased by administration of P. eryngii extract (Fig. 4).

Fig. 4. Effect of P. eryngii Extract on Synaptosomal Zinc Level of OVX Rats

A, Synaptosomal zinc concentrations in the right hemisphere of sham-operated, OVX and P. eryngii extract-administered OVX rats are shown. Each bar and line represent the mean±S.E.M. (Sham, n=6; OVX, n=7; OVX+P. ery., n=6). Asterisks indicate significant differences (* p<0.05, ANOVA followed by Newman–Keuls multiple comparison test).

DISCUSSION

Estrogen deficiency increases the risk for metabolic syndrome and osteoporosis as well as depression and memory impairment.^1,3,19,20) The importance of estrogen levels for leading to these symptoms is supported by the OVX rodent, which is in a state of estrogen deprivation. OVX rodents show body weight gain, excessive feeding^21–24) and osteoporosis²⁵⁾ following ovariectomy, which are attenuated by administration of estradiol. OVX rats also show depression-like behavior as assessed by the forced swim test^21,26,27) or the tail suspension test.²⁸⁾ The depression-like behavior of OVX rats is improved by administration of estradiol^29–32) or phytoestrogen,³³⁾ the effect of which is probably mediated by monoaminergic systems.^34–38) Additionally, OVX rats display impairment of spatial memory and decrease of spine density in the hippocampus and prefrontal cortex.^39–41) Administration of estradiol improves spatial memory deficits in OVX rats by increase in spine density and alteration of monoaminergic activity.^42,43)

Our data indicate that body weight of OVX rats was significantly increased following bilateral ovariectomy. Additionally, immobility time during the forced swim test and latency time to find the hidden platform during the Morris water maze were significantly increased in OVX rats compared to those in sham-operated rats. These results indicate that OVX rats at 9–12 weeks after ovariectomy are useful as a model for postmenopausal depression and memory impairment.

Two different types of estrogen receptor (ER), ERα and ERβ, have been identified. The beneficial effect of estradiol on excessive feeding and body weight gain in OVX mice is not shown in ERα knockout mice.²³⁾ ERα is necessary for normal food intake and body weight, while ERβ is not. On the other hand, The depression-like behavior of OVX rats was improved by the ERβ-selective agonist diarylpropionitrile,^44,45) but it was not improved by estradiol in ERβ knockout mice⁴⁶⁾ and it was aggravated by infusion of antisense oligodeoxynucleotides for ERβ in OVX rats.⁴⁷⁾ Recognition memory assessed by object recognition and placement memory tasks is enhanced by administration of estradiol or an ERβ-selective agonist, but not by administration of an ERα-selective agonist, in OVX rats.^43,48) Therefore, the beneficial effect of estradiol on depression-like behavior and spatial memory deficits after ovariectomy is mediated by activation of ERβ rather than by activation of ERα.

We examined the effect of the ethanol extract of P. eryngii on postmenomausal depression and memory impairment. The depression-like behavior and memory impairment in OVX rats were improved by long-term administration of P. eryngii extract without any effect on motility. It is thought that estrogen-like substances contained in P. eryngii have a beneficial effect on postmenopausal depression and memory impairment. Since the body weight of OVX rats was not influenced by P. eryngii extract, estrogen-like substances in P. eryngii extract would be more selective for ERβ than for ERα.

The zinc level in a mammalian body is influenced by estrogen. After ovariectomy, zinc level in hippocampal synaptosomes is significantly increased¹⁸⁾ but that in serum is lowered.⁴⁹⁾ Also in our study, the amount of synaptosomal zinc was significantly increased by estrogen deficiency in OVX rats. Synaptic vesicles abundantly contain zinc, especially in hippocampal mossy fiber terminals. Vesicular zinc is an important modulator of neurotransmission and is linked to the pathophysiology of depression.^14,15,50) It has been reported that a transient increase in zinc in hippocampal CA1 pyramidal neurons causes reversible memory deficit.⁵¹⁾ Therefore, appropriate zinc level is important for maintenance of normal neural functions.

The zinc level in synaptic vesicles is controlled by zinc transporter 3 (ZnT3), which is involved in cognitive function.⁵²⁾ Since the expression level of ZnT3 is lowered by estrogen, the high concentration of synaptosomal zinc in the OVX mouse is lowered by administration of estrogen.¹⁸⁾ We examined the effect of P. eryngii extract on the synaptosomal zinc level in OVX rats, and we found that the increment of synaptosomal zinc level in OVX rats was decreased to the level in sham-operated rats by administration of P. eryngii extract. This result suggested that P. eryngii extract enables the abnormal vesicular zinc level in OVX rats to recover.

In conclusion, the ethanol extract of P. eryngii has an estrogen-like beneficial effect on depression and memory impairment in OVX rats. Normalization of zinc signaling is thought to be involved in this effect of P. eryngii extract on neurological disorders after ovariectomy.

Acknowledgment

This research was supported in part by the JMS Bayer Schering Pharma Grant from the Japan Menopause Society to H.M., and the Scientific Research Grant from Amano Foundation of Industrial Technology to A.M.

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