The effection of Traditional Chinese Herbal Bu-Wang-San on Neuronic Protection in Ovariectomized Rats

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Author: Hui Li, Shu-Ling Li, Zhi-Hong Wu ,Li Gong, Jiu-Ling Wang, Yu-Zhu Li

The effection of Traditional Chinese Herbal Bu-Wang-San on Neuronic Protection in Ovariectomized Rats

Hui Li, Shu-Ling Li, Zhi-Hong Wu ,Li Gong, Jiu-Ling Wang, Yu-Zhu Li Department of Traditional Chinese Medicine, Qilu Hospital Affiliated to Shandong University, Jinan 250012, Shandong, PR China

Abstract

Bu-Wang-San (BWS) is a traditional Chinese herbal medicine for the treatment of learning and memory impairment. The effect of BWS on neuroprotection and how BWS increases spine synapse number in menopaused women was investigated in the model of ovariectomized (OVX) rats. Sixteen OVX rats were divided into two groups, the OVX group and OVX+BWS group. After three months, Morris water maze was used to assess spatial acquisition and spatial retention. Swim time, swim distance, swim speed, quadrant time and platform crossing were recorded. The ultrastructure of spine synapse number in hippocampus were examined by transmission electron microscopy (TEM). In the spatial acquisition and spatial retention phase of testing, BWS group functioned significantly better than control group. Ultrastructural observation of the hippocampus of OVX group showed significantly less spine synapse number, however, BWS significantly increased spine synapse number. In addition, BWS significantly increased synaptophysin P38 (P38) mRNA expression in hippocampus. These results suggested that BWS could improve cognitive ability of menopause-induced learning and memory impairment. The positive effect of BWS on mouse learning and memory was associated with increase of spinal synapse number and increased the expression of synaptophysin P38 (P38) mRNA in hippocampus from menopause-induced injury. Keywords: herbal medicine; Bu-Wang-San; learning; memory

1. Introduction

Menopause marks the end of reproductive capacity of women and results from the permanent cessation of ovarian function. Natural or surgical menopause is confirmed by absence of menstrual periods for 12 consecutive months, excluding other obvious pathologic or physiologic causes(Notelovitz, 1989).Some symptoms such as hot flashes, tiredness, irritability, insomnia, palpitations, memory or concentration difficulties, and mood swings or depression begin in the peri-menopause and increase as women progress through the menopause (Hardy and Kuh, 2002). Many studies have shown that learning and memory may be negatively affected altered by the loss of estrogen after menopause (Sherwin, 1988). These changes can be ameliorated by estrogen replacement therapy (ERT)( Ditkoff et al., 1991; Sherwin, 1994; Kimura, 1995). As a neuroprotective and neurotrophic factor, Estrogen( E2 ) helps maintain memory and cognition(Wise et al., 2001 and Sughrue and Merchenthaler, 2000),decreases the risk and delays the onset of neurological disorders, e.g. Alzheimer's disease(AD). Indeed, estrogen has been shown to increase cerebral blood flow, to act as an anti-inflammatory agent and enhance neural synapse activity(Toran-Allerand et al., 1999 and Roof and Hall, 2000). Numerous studies indicate that estrogen is essential for optimal brain function (Wise et al., 2001; Sughrue and Merchenthaler, 2000; Toran-Allerand et al., 1999; Roof and Hall, 2000; Wise et al., 2000). However, the above-mentioned health benefits of ERT was often overshadowed by the serious side-effects of estrogen use in menopaused women. Specifically, long term use of estrogen in postmenopausal women may lead to the increased risk of endometrial and breast cancer (Hammond, 1994;Grady et al., 1995; Anonymous, 1997). Accordingly, there has been a growing interest in alternative therapies. Chinese herbal medicine has been used for thousands of years in China and other Asian countries. In clinical practice of traditional Chinese medicine (TCM), it is common to modify original formula by adding or substituting herbs in accordance with a patient's condition to enhance the efficacy of the original formula. Most of the regular use of traditional Chinese medicine is not associated with serious side effects. Specific formula of traditional Chinese herbal medicines has been reported to be effective against cognitive disorder (Junping et al., 2005; Young-Ju et al., 2007; Hai-Fa et al., 2006). However, there was little information available in literature about whether herbal medicines with neuroprotective effect could affect postmenopausal congnitive disorder. Bu-Wang-San( 不忘散，BWS), transliterally meaning the decoction for ' enhancing the memory', is a traditional Chinese formula. It has been used to treat the postmenopausal cognitive disorder for years in clinic. The mechanism as to how BWS protects learning and memory has not been studied. We have used ovariectomized rat as the estrogen-depleted, postmenopausal model(Kalu, 1991) to examine the effect of BWS on postmenopausal congnitive disorder. In addition, we have studied the effect of BWS, for the first time, on the change of synapse number in hippocampus with electron microscope and synaptophysin P38(P38) mRNA expression in hippocampus.

2. Materials and methods

2.1．Herbal materials

BWS consists of four medicinal compositions as shown in Table 1. All fresh roots of these herbs were harvested from Jilin, Sichuan, Yunnan and Hebei province, respectively, in China. The herbal farms in these areas have been shown to produce the best quality herbs. All plant materials were collected in September or October after six years (Panax ginseng C.A. Mey) or two years (Acorus gramineus Soland, Poria cocos(Schw.) Wolf and Polygala tenuifolia Willd) of cultivation. The plants were carefully authenticated by a botanist during field collection, and the roots and sclerote were ground into powder and stored in a desiccated condition prior to delivery to the laboratory. Voucher specimens (numbers were listed in Table 1) were deposited at the Herbarium of Shandong University (Jinan, China).

2.2． Herbal extraction

After drying, these herbs were mixed in proportion. Forty eight grams of the mixed material was mixed with 300 ml of distilled water and boiled for 1h at 100℃. The extract was filtered, and the residual medicine was boiled in water following the same procedure once more. Finally, the pool of the extracts from two boiling and filtering was lyophilized to form a dried powder. The yield of BWS extract was 25% (w/w) of the original herbs. The resulting lyophilized powder, stored at 4℃, was diluted to the appropriate concentrations with distilled water and filtered before use.

2.3. Analysis of BWS by HPLC

BWS (0.5g) was extracted with 20 mL of methanol with ultrasonic for 30 min followed by centrifugation. The analysis of the supernatant solution by high-performance liquid chromatography ( HPLC) equipped with LC-10 AD pumps, an PAD photodiode-array detector and a CTO-10A column over (Shimadzu, Kyoto, Japan) was performed using a Kromosil C18 column (5μm, 200 ×4.6 mm, Alltech Associates Inc, Columbia, Maryland) with a mobile phase composed of acetonitrile and H3PO4 acidifield water. The flow rate and the column temperature were 1.0 mL/min and 25 °C, respectively. The UV data of the effluent from the column ranging from 200 to 450 nm were collected, and the peak analysis and assignment were performed using the system analysis software, CLASS-LC10 (Shimadzu, Kyoto, Japan).

2.4. Animals and treatment

Ten-week-old virgin female Wistar rats weighing 260-300 grams were purchased from Shandong University Laboratory Animal Shelter (Shandong, China). Rats were housed in individual cages under controlled environmental conditions (22±2℃ relative humidity 40-60%, 12-hour dark/light cycles, food and water ad libitum). All rats were treated parallelly in terms of daily manipulation. The rats were divided randomly into 3 groups; the first group was given a sham operation (Sham group), the others were ovariectomized(OVX ). They were either bilaterally ovariectomized or sham-operated through dorsal incision under anaesthesia with sodium pentobarbital (150mg/kg,i.p.). The next day, ALL post-op rats were injected with penicillin ( 22,000 u.i./kg ) for three days, and the vaginal smear was taken from each rats for four days. Rats of the Sham group with a classic estrous cycle were selected (n=8). Success of the ovariectomization was confirmed by demonstration of predominantly leukocytes with few epithelial cells in vaginal smears over at least 4 days. The successful OVX rats were further randomly divided into two groups (n=8 equally): OVX group, OVX+ BWS group. The next day, the Sham group and the OVX group received distilled water; and the OVX+BWS group received Bu-Wang-San(1.2g/kg/day) by the oral gavage daily for three months. The dose of BWS was determined by conversion of regular dose for human to that for rat and also by a pilot experiment with different doses of BWS for rat. All surgical procedures and protocols used were in accordance with the Guidelines for Ethical Care of Experimental Animals, which was approved by the Shandong University Animal Care and Use Committee.

2.5. Morris water maze

All rats were put into the Morris water maze to assess learning and memory performance on a spatial orientation task(Morris, 1981). A circular 180cm diameter swimming pool made of black polyethylene was filled 32cm deep with 25±2℃ water. The water was made opaque by the addition of pure milk powder ( Inner Mongolia Yili Industrial Group Co., Ltd., China ). A platform, which consisted of a round transparent lucite platform(10cm diameter, 30cm high) invisible to the rat, was hidden below the surface of water in one of the four quadrants of the pool. Conspicuous visual cues outside the pool were provided for orientation. A video camera suspended above the pool was connected to a video tracking system (MI-200, Chengdu Taimeng Technology & Market Co.,Ltd., China) that recorded that recorded the swimming pattern including the length of the swim path on each trial.

2.6. Behavioral test Spatial acquisition.

Testing was conducted between 8:30 to 11:30am and 1:30 to 4:30pm, rats were trained throughout four days, with eight trials per day, and there was a 2-min break between each trial. The first day, rats were initially placed on the platform and allowed to stay there for 30s. They were then placed in the pool at the edge of the platform with their front paws touching it and were allowed to climb out of the water onto the platform and stand for 30s. This was repeated three additional times. Finally, they were placed at the edge of the pool and allowed to swim to the cued platform and climb onto it. Animals that failed to locate the platform within 120 s were manually guided to it. From the second day to the fourth day, the test was carried out in following way: rats were placed at the edge of the pool. The point of placing rats was each alternative midpoint of the four quadrants. Rats were allowed to swim to the submerged platform and climb onto it and stayed there for 30s. Rats that failed to locate the platform within 120s were manually guided to it. We recorded the swim time(s), swim distance (cm) and swim speed (cm/s) on each trial. Spatial retention. Spatial probe trial was conducted 24h after the spatial acquisition phase to determine long-term memory. Each rat was allowed to swim for 60s. During this trial, the platform was removed from the pool. We measured the parameters including quadrant time (percentage of time spent in the quadrant in which the platform was located in the spatial acquisition phase) and platform crossings (the number of times the rat crossed the exact location of the platform).

2.7. Ultrastructural observation and spine synapse number

Rats were deeply anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused through the ascending aorta sequentially with 100 ml of physiological saline (0.9% NaCl ) and 60ml of 3% glutaraldehyde. The region of the forebrain containing the hippocampal formation was removed and cut into blocks. Blocks were then retrimmed into 1mm3 and further fixed in 3% glutaraldehyde overnight. The next day, the blocks were washed three times with 0.2 mol/l phosphate buffer and were fixed with 1% osmium tetraoxide, washed with 0.2 mol/l phosphate buffer again, and dehydrated by different concentrations of ethanol. The sections were immersed in fresh Spon812 resin/acetone (1:1) for 30min, and embedded and convergenced overnight at 70℃. Semi-thin sections were obtained and stained with toluidine blue for light microscopic examination to locate the pyramidal cell. Thin sections (50nm) were made with an ultramicrotome and stained with 2%uranyl acetate and lead citrate. The synaptic density was observed using H-7000FA transmission electron microscopy (TEM) (Hitachi Co.Ltd., Tokyo, Japan). The photos were taken with magnifying times of 5,000- 10,000.

2.8. RNA isolation and real-time PCR assay

Frozen specimens about 1cm were homogenized and the total RNA was sequentially extracted using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA, USA)according to the manufacturer's instruction. The RNA was treated with DNase (DNA-freeTM, Ambion Inc., Austin, TX, USA) in order to remove contaminating genomic DNA, followed by phenol, chloroform extraction and ethanol precipitation. The quality of RNA was checked using a spectrophotometer (DU®800, Beckman, Palo Alto, CA, USA). Total RNA was performed in a two-step procedure as described by SYBR RT-PCR Kit (Perfect Real Time) (TaKaRa Corp., Kyoto, Japan). Briefly, in the first step, cDNA was prepared from 500ng RNA by reverse transcription in a final volume of 20μl in a thermal cycler (Tgradient 96, Whatman Biometra, Niedersachsen, Germany). The samples were incubated at 37 ℃ for 60 min and 95 ℃ for 5 min. The cDNA were stored at −80 ◦C. The rat-specific primers for the genes of synaptophysin P38 and β-actin were designed using Primer Premier 5 (Premier Biosoft Ltd., Palo Alto, CA, USA). The primer sequences are shown in Table 2. Primers were synthesized by BioAsia Corp. (Shanghai, China). In the second step, quantitative real-time PCR was performed on the LightCycler apparatus (Roche Diagnostics, Mannheim, Germany) using SYBR Green I in SYBR RT-PCR Kit (Perfect Real Time). The reaction was conducted with an initial denaturing at 95 ℃ for 10 s, and then involved 50 cycles of 57 ℃ 5s, 72◦C 10 s, and terminated by a cooling step 30 s at 65 ℃. A melting-curve analysis was performed to confirm the absence of primer dimmers in specific PCR products. A quantitative standard curve was created between 1 and 40 ng of cDNA with a Roche Light Cycler by monitoring increasing fluorescence of PCR products during amplification. Quantification of the unknowns was performed in duplicate and then determined with the Light Cycler 4.0 (Roche Diagnostics) data analysis software and adjusted to the quantitative expression of β-actin from the corresponding unknows.

2.9. Statistical analysis

All statistical analysis was performed using SPSS software (Version 10.0, SPSS Inc., Chicago, IL, USA). Data was expressed as mean±SEM. Statistical comparisons were performed by one-way analysis of variance (ANOVA). When one or more of the groups were found to follow a non-normal distribution, nonparametric statistics were used for analysis. The level of significance was accepted at p< 0.05.

3. Result

3.1. Spatial acquisition In the spatial acquisition phase of testing,

OVX groups had significantly longer swim time than Sham and BWS groups, which was confirmed by conducting a separated repeated measure ANOVA for each group. Animals in Sham and BWS groups did achieve the shorter swim time at the end of the whole trial. Comparison of the three groups at the end of the experiment showed that untreated OVX rats had significantly longer swim time than did both two other groups, whereas OVX rats treated with BWS demonstrated reduced swim time, which was close to swim time of Sham rats. Moreover, there was a significantly decreased trend for swim time both in Sham group and BWS group (P<0.01), but but no significantly decreased trend in OVX group (P>0.05) (Fig.1A). As was done for swim time, BWS again showed a dramatic positive effect when swim distance is used for analysis. OVX rats had significantly longer swim distances than did both two other groups. These animals in BWS group required significantly less swim distance during the trial. Swim distance of BWS group is close to that of Sham group. And there was a decreased trend for swim distance both in Sham and BWS group (P0.05) (Fig.1B). There was no effect of block or treatment on swim speed, and no treatment by block interaction (Fig.1C).

3.2. Spatial retention

In the spatial retention trial, both quadrant time and platform crossings were significantly affected by BWS (Table 3). BWS treatment protected against the OVX-induced decrease in the retention for the target quadrant during the trial. In spatial retention trial, quandrant time in OVX rats (18.40±4.71) are significantly reduced in comparison to that in Sham rats(31.70±4.58), but BWS treatment prevented this decline in OVX rats. Rats in BWS group had more significantly platform crossings than those in OVX group.

3.3. Spinal synapse number

Ultrastructural observation in the hippocampus of the OVX group showed that the number of spine synapse was apparently decreased. The number was significantly increased by BWS treatment. The BWS group had no apparent difference from the Sham group. (Fig. 2)

3.4. Expression of P38 mRNA in hippocampus

Our data showed that P38 gene expression in the hippocampus of OVX group(7.41±1.98 fold) was down-regulated compared with Sham rats(21.72±2.36 fold), and in BWS group( 18.56±2.16 fold), a significant increase of P38 expression was observed compared with OVX group (Fig. 3).

4. Discussion

The present study clearly showed a neuroprotective role of BWS in ovariectomized rat in association with an increase of spinal synapse number and the expression of synaptophysin P38 in hippocampus. It has been recognized that OVX for six days in young animals results in synaptic loss between noradrenergic terminals and gonadotropin hormone releasing hormone (GnRH) neurons, Long-term OVX, hypothesized to protect against neuroendocrine aging, failed to guard against any age-related changes (Miller et al., 1998). Interactions of the estrogen receptor system with various growth factors were important for the neurite growth and differentiation (Singh et al., 1999). Some factors can ameliorate the memory disorder of ovariectomized rat by increasing synaptic sprouting, increasing cholinergic activity in the hippocampal formation, protecting neurons against amyloid-induced toxicity or other excitotoxic events (Stone et al., 1998; Gibbs, 1994; Morrison and Hof, 1997).

The Morris water maze is a well-established paradigm for evaluating deficits in hippocampal-dependent memory. In particular, learning and memory deficit is demonstrated by the extended time in acquisition and retention. The Morris water maze was used to test spatial memory in many studies, e.g. a study of exogenous estrogen replacement found that estrogen given to ovariectomized female rats can improve spatial memory (Markham et al., 2002). Others have found that chronic exogenous estrogen can impair spatial memory in the water maze in both rats and mice (Holmes et al., 2002 and Fugger et al., 1998). In this study, the fact that spatial memory continued to improve significantly in BWS group during the three training days, but not in the control OVX group, suggests that BWS ameliorated disordered learning of OVX mice.

The data of spatial probe trial demonstrated that BWS protects against the OVX-induced decrease of the spatial retention. P38 is a calcium protein with molecular weight of 380 000, synthesized in neuronal body and transported to axonal terminals, specifically expressed on the presynaptic vesicle membrane (Franke WW et al.). The P38 mRNA expressing position and quantity can correctly reflect the distribution and density of synapses, as well as the numerical change of synapse.

Estrogen-induced increases in hippocampal plasticity leads to enhanced memory function. Estrogen may prevent impairment of transport systems that maintain ion homeostasis and energy metabolism, and thereby forestall excitotoxic synaptic degeneration and neuronal loss in disorders such as AD and ischemic stroke (Keller et al., 1997). The ability of estrogen to preserve mitochondrial function, suppress oxidative stress, and counteract the pro-apoptotic actions of mutant presenilin-1(PS-1) suggests a generalized neuroprotective action of estrogens in both sporadic and inherited forms of AD.

In this study, BWS increased spine synapse number and the expression of synaptophysin P38 in hippocampus. In light of the behavioral findings, BWS-treated rats learned better than OVX. The result of ultrastructural observation and real time PCR in the hippocampus suggested that BWS is capable of protecting neurons from ovariectomized injury and that the protective effect is associated with protection of mitochondrial function. More work will be needed to further elucidate the effect of BWS on memory in OVX rats, including non-water maze spatial memory tasks and other memory tasks (e.g. inhibitory avoidance).

In summary, our present study suggested that BWS could improve cognitive ability and memory in a model of neuronal impairment induced by estrogen depletion. The mechanisms were possibly associated with increase of spinal synapse number and the expression of synaptophysin P38 in hippocampus from estrogen-induced injury. BWS may be a beneficial agent for patients with postmenopausal memory disorder.

Acknowledgment

This work was supported by the Health Department of Shandong Province, Jinan, China.

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