Japan Brain Science Society

Journal of Brain Science, December 30, 2014,Vol.44

Reseach and Report

Yokukansan increases serum Brain-derived neurotrophic factor

(BDNF) levels in Gunn rat

Motohide Furuya1,2*, Tsuyoshi Miyaoka1, Sadayuki Hashioka1, Rei Wake1, Keiko

Tsuchie1, Jun Horiguchi1

1 Department of Psychiatry, Shimane (BDNF) is expressed at high levels in the

University Faculty of Medicine, 89-1 hippocampal dentate gyrus (DG), and

Enya-cho, Izumo 693-8501, Japan decreased levels of BDNF have been

2 Department of Developmental Biology, implicated in the pathophysiology of

Shimane University Faculty of Medicine, schizophrenia (SCZ). We have previously

89-1 Enya-cho, Izumo 693-8501, Japan reported that yokukansan (YKS), which is

* Corresponding to: Motohide Furuya, a traditional Japanese medicine, is

M.D., Ph.D. effective for SCZ and promotes

Department of Psychiatry, Shimane neurogenesis in the DG of Gunn rats, an

University Faculty of Medicine, animal model of SCZ. In this study, we

Assistant Professor investigated the effect of YKS on serum

89-1 Enya-cho, Izumo 693-8501, Japan BDNF levels in Gunn rats. The results

Phone: 853-20-2263, Fax: 853-20-2260 showed that YKS increased serum BDNF

e-mail: pancho@med.shimane-u.ac.jp in this model, which may suggest that

Running title: Yokukansan increases BDNF expression in the DG leads to

serum BDNF levels in Gunn rat increased neurogenesis. Our findings

Received: 2014-11-26 may help to explain the efficacy of YKS in

Accepted: 2014-12-6 treating SCZ.

Abstract Key words: brain-derived neurotrophic

Brain-derived neurotrophic factor factor; yokukansan; schizophrenia;

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unconjugated bilirubin; rat disease [6].

Introduction We previously reported that

Schizophrenia (SCZ) is a yokukansan (YKS), one of the traditional

heterogeneous group of mental illnesses Japanese medicines known as “Kampo”

with a pathogenesis resulting from medicines in Japan, is effective as an

multiple factors, including genetic, adjunctive therapy for treatment-resistant

[7]
biological and environmental ones. From SCZ . We also revealed that YKS

the standpoint of the heterogeneity of promotes neurogenesis in the

SCZ, previous studies have indicated a hippocampal dentate gyrus (DG) of Gunn

close association between unconjugated rats [3].

bilirubin (UCB) and SCZ [1, 2]. On the basis BDNF is expressed at high levels in the

of these findings, we suggested that DG [8]. The ability of BDNF to freely cross
[9]
elevated levels of UCB play an important the blood-brain barrier suggests that

role in SCZ etiology, and that Gunn rats, serum BDNF levels may reflect the BDNF

which exhibit a high concentration of UCB, levels in the DG. Therefore, in this study,

are useful as an animal model of SCZ [3]. we investigated whether YKS affects the

Brain-derived neurotrophic factor serum BDNF levels in Gunn rats and

(BDNF), the most abundant neurotrophin normal rats.

in the brain, regulates neuronal survival, Methods

differentiation and growth during Animals

[4]
development . BDNF is also active Seven-week-old male homozygous (j/j)

during a critical developmental period Gunn rats and male Wistar rats (Japan

and likely to influence the neuroplasticity SLC Inc., Shizuoka, Japan) were used in
[5]
of SCZ . Therefore, researchers have this study. The rats were housed in plastic

indicated that BDNF is a key factor for the cages (39 × 27 × 18 cm) under standard

pathogenesis and treatment of this conditions (temperature, 23 ± 2oC;

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humidity, 55 ± 5%; 12 h light/dark cycle technique was used to produce dried

[light phase from 0700 to 1900h]) and extract powder. The yield of the extract

were given free access to food and water. was about 15.9%. The rats were divided

One week before the experiment, the rats into 4 groups: a Wistar-control (WC)

underwent a handling procedure once group, Wistar-YKS (WY) group,

daily to reduce stress during the Gunn-control (GC) group, and Gunn-YKS

experiment. All procedures were (GY) group. The rats in the control groups

performed with the approval of the (WC and GC) were given drug-free water

Shimane University Animal Ethics ad libitum for 6 weeks, whereas those in

Committee, under the guidelines of the the YKS-treated groups (WY and GY)

National Health and Medical Research were given water containing 0.6% YKS

Council of Japan. (corresponding to a dosage of 1 g/kg of

Drugs body weight) for the same period.

YKS (Tsumura & Co., Tokyo, Japan) is Blood sampling

composed of 7 dried medical herbs Twenty-four hours after the last

(Table 1). Each plant material was administration, blood samples were

authenticated by identifying the external collected into sampling tubes under deep

morphology and marker compounds of intraperitoneal anesthesia with sodium

plant specimens according to the pentobarbital (80 mg/kg body weight).

methods of the Japanese The blood samples were centrifuged at

Pharmacopoeia and the company’s 2000 g for 20 min. Serum was stored at –

standard. The 7 medical herbs were 80°C until analysis. After the blood

mixed and extracted with purified water at samples were collected, the rats were

95°C for 1 h, and the extraction solution perfused transcardially with 500 ml of

was filtered and concentrated under physiological saline, followed by 500 ml

reduced pressure. The spray-drying of 4% paraformaldehyde in 0.1 M

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phosphate buffer (PB; pH 7.3). The serum BDNF levels in the WC, WY,

Serum BDNF determination by GC, and GY groups were 2.65 ± 0.15,

enzyme-linked immunosorbent assay 3.27 ± 0.40, 2.60 ± 0.16, and 4.07 ± 0.53

(ELISA) ng/ml, respectively (Figure 1). No

A commercial sandwich ELISA kit significant difference was observed

(Abnova, Taipei, Taiwan) was used to among the WC, WY, and GC groups.

quantify the serum BDNF level according However, the serum concentration of

to the manufacturer’s instructions. The BDNF in the GY group was significantly

plate was read in an increased compared with that in the GC

ELISA-spectrophotometer reader with an group (P = 0.039) .

absorbance wavelength of 405 nm. Discussion

Standard curves were obtained from In the present study, we found that

values generated from known chronic YKS treatment increased the

concentrations of BDNF in provided kits. serum BDNF levels in Gunn rats. This

All assays were performed in triplicate. finding could support our previous result

Statistical analyses that YKS promotes hippocampal

Results were analyzed by one-way neurogenesis in association with its

analysis of variance (ANOVA) and post anti-inflammatory action [3].

hoc Bonferroni test to determine Several studies have indicated that

differences among groups. Values are BDNF has a crucial role for neurogenesis

expressed as the mean ± SEM. Analyses in the DG. For example, infusion of

were performed using the Statistical endogenous BDNF into the DG leads to

Package for the Social Sciences (SPSS) increased neurogenesis [10]. Other studies

software. In the analyses, P values < 0.05 have shown that BNDF is critically

were considered statistically significant. required for the increased neurogenesis

[11]
Results following dietary restriction ,
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[12]
antidepressant treatment and treatment-resistant SCZ, while we

environmental enrichment [13]. reported that YKS is effective as an

On the other hand, an extensive review adjunctive therapy for treatment-resistant

[7]
has pointed out that BDNF has a close SCZ . Thus, there may be a common

association between the nervous and pharmacological characteristic between

immune systems and plays an important CLZ and YKS. Further work will be
[14]
role in brain-related disorders . In needed to investigate this possibility.

addition, serum BDNF levels are In conclusion, this study was the first to

negatively correlated with inflammatory report that YKS increased the serum

marker IL-6 and TNF-α in psychosis [15]. BDNF levels in an animal model of SCZ.

A recent meta-analysis study has These results may provide a clue to the

shown that serum BDNF levels are not effects of YKS in SCZ, and could

significantly different between naive SCZ contribute to the identification of

patients and SCZ patients medicated with candidate biomarkers and a better
[16]
antipsychotics , while other studies understanding of this disease.

have reported that serum BDNF levels Acknowledgment

are positively correlated with clozapine We are grateful to Tsumura & Co. for
[17, 18]
(CLZ) treatment . CLZ is the only generously supplying YKS.

effective antipsychotic for

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Crude drug name Composition (g)

Atractylodes lancea rhizome 4.0

Poria sclerotium 4.0

Cnidium rhizome 3.0

Uncaria hook 3.0

Japanese angelica root 3.0

Bupleurum root 2.0

Glycyrrhiza 1.5
Table 1. Crude Drug Composition of YKS

Figure 1. YKS increased serum BDNF levels in Gunn rats

Data are presented as the mean ± SEM (n = 4, respectively).*P <0.05, ANOVA

followed by post hoc Bonferroni test.

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