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   Nov 21

The Traditional Japanese Herbal Medicine Hachimijiogan Elicits Neurite Outgrowth Effects in PC12 Cells and Improves Cognitive in AD Model Rats via Phosphorylation of CREB

ORIGINAL: https://doi.org/10.3389/fphar.2017.00850

• 1Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

• 2Institute for Aging and Brain Sciences, Fukuoka University, Fukuoka, Japan

• 3Community Medicine Education Unit, Department of Clinical Medicine, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

Hachimijiogan (HJG) is a traditional herbal medicine that improves anxiety disorders in patients with dementia. In this study, we tested the hypothesis that HJG exerts neurotrophic factor-like effects to ameliorate memory impairment in Alzheimer disease (AD) model rats. First, we describe that HJG acts to induce neurite outgrowth in PC12 cells (a rat pheochromocytoma cell line) like nerve growth factor (NGF) in a concentration-dependent manner (3 μg/ml HJG, p < 0.05; 10-500 μg/ml HJG, p < 0.001). While six herbal constituents of HJG, Rehmannia root, Dioscorea rhizome, Rhizoma Alismatis, Poria sclerotium, Moutan bark, and Cinnamon bark, could induce neurite outgrowth effects, the effect was strongest with HJG (500 μg/ml). Second, we demonstrated that HJG-induced neurite outgrowth was blocked by an inhibitor of cAMP response element binding protein (CREB), KG-501 (10 μM, p < 0.001). Moreover, HJG was observed to induce CREB phosphorylation 20-90 min after treatment (20 min, 2.50 ± 0.58-fold) and CRE-mediated transcription in cultured PC12 cells (500 μg/ml, p < 0.01; 1000 μg/ml, p < 0.001). These results suggest a CREB-dependent mechanism underlies the neurotrophic effects of HJG. Finally, we examined improvements of memory impairment following HJG treatment using a Morris water maze in AD model animals (CI + Aβ rats). Repeated oral administration of HJG improved memory impairment (300 mg/kg, p < 0.05; 1000 mg/kg, p < 0.001) and induced CREB phosphorylation within the hippocampus (1000 mg/kg, p < 0.01). Together, our results suggest that HJG possesses neurotrophic effects similar to those of NGF, and can ameliorate cognitive dysfunction in a rat dementia model via CREB activation. Thus, HJG could potentially be a substitute for neurotrophic factors as a treatment for dementia.


Recently, there have been global increases in the number of patients with dementia disorders, such as AD. However, as the pathogenesis of dementia has not yet been fully elucidated, radical curative therapies have not been established. Novel dementia drugs that do not adversely affect ADL are required. In the study of neurodegenerative diseases characterized by neuronal cell death (such as AD), the role of neurotrophic factors has recently drawn attention. Neurotrophic factors are substances that promote neuronal survival, differentiation, and regeneration. Although various trophic factors have been identified, research has indicated particularly important roles for NGF and BDNF (Obara and Nakahata, 2002).

Nerve growth factor is abundant throughout the hippocampus and cerebral cortex, where it stimulates the production, transportation, and secretion of acetylcholine (Ayer-LeLievre et al., 1988; Jonhagen, 2000). Cholinergic neurons, which are NGF susceptible and play an important role in learning and memory, have been shown to be markedly degenerated in the brain of patients with AD. NGF can modulate the metabolism of amyloid precursor protein from amyloidogenic toward non-amyloidogenic processing via binding to the TrkA (Canu et al., 2017). These features guide the design of therapeutic for AD intending to preserve cholinergic function and anti-amyloidogenic activity. However, neurotrophic factors are macromolecular proteins, with an in vivo half-life that is too short to enable delivery to a target tissue or cell. This makes it difficult to directly apply neurotrophic factors as therapeutic agents for AD (Pollack and Harper, 2002; Skaper, 2008). Therefore, attention has turned to developing compounds that show neurotrophic factor-like effects by activating intracellular neurotrophic factor signal transduction pathways and/or promoting biosynthesis of neurotrophic factors. For example, we demonstrated that the Japanese herbal medicine Yokukansan can improve symptoms of dementia (Nogami et al., 2013; Uchida et al., 2013), and its action involves neurotrophic factors such as NGF (Kubota et al., 2013).

In recent years, an association has been reported between neuronal death in AD and decreased activity of CREB (Yamamoto-Sasaki et al., 1999). CREB is a downstream transcriptional regulator of intracellular signaling by neurotrophic factors. CREB is activated through the following three pathways: (1) ERK 1/2; (2) PI3K/Akt; and (3) phospholipase C-γ, all of which lead to CREB phosphorylation (Kaplan and Miller, 2000). In addition to these pathways, AC and the cAMP signaling pathway are also involved in activating CREB and CREB-regulated gene transcription. Activated CREB regulates the transcription of various genes, subsequently inducing spine morphological changes such as neurite outgrowth and branching, and producing long-term potentiation. Additionally, the involvement of CREB in the formation of short-term memories via BDNF induction has been reported (Lonze and Ginty, 2002; Frankland and Bontempi, 2005). Thus, CREB and neurotrophic factor signaling pathways are deeply involved in the formation of both long-term and short-term memory, and are potential targets for drugs to improve cognitive function.

Hachimijiogan (Ba-Wei-Di-Huang-Wan) is an herbal medicine used in China and Japan comprising eight herbs: RR, Cornus fruit, DR, AR, PS, MB, CB, and Aconite root. HJG has traditionally been used to treat diabetes mellitus, hypertension, and nephrotic syndrome. It has also been used to treat dysuria, lumbago, lack of energy, and poor eyesight in older individuals. Furthermore, it has been reported that HJG treatment improved cognitive dysfunction in patients with dementia (Iwasaki et al., 2004). Additionally, several studies, including one of our own, have reported that HJG improves cognitive dysfunction in model animals (Hirokawa et al., 1994, 1996; Moriyama et al., 2017). However, further research is needed to confirm the effects of HJG on cognitive dysfunction. Therefore, to investigate the appropriate clinical application of HJG, we investigated the effects of HJG and its constituent herbs on CREB and neurotrophic factor signaling pathways.

Materials and Methods


Dry powdered extracts of HJG and its constituents (excluding Cornus fruit and Aconite root) supplied by Tsumura & Co. (Tokyo, Japan) were dissolved in distilled water. HJG is a dried extract of the following eight herbs: RR (6.0 g, the root of Rehmannia glutinosa Libosch var. purpurea Makino, Scrophulariaceae), Cornus fruit (3.0 g, the fruit of Cornus officinalis Siebold et Zucc, Cornaceae), DR (3.0 g, the rhizome of Dioscorea batatas Decne, Dioscoreaceae), AR (3.0 g, the rhizome of Alisma orientale Juzepczuk, Alismataceae), PS (3.0 g, the sclerotium of Poria cocos Wolf, Polyporaceae), MB (2.5 g, the root bark of Paeonia suffruticosa Andrews, Paeoniaceae), CB (1.0 g, the bark of Cinnamomum cassia Blume, Lauraceae), and Aconite root (0.5 g, the root of Aconitum carmichaelii Debeaux, Ranunculaceae).

Nerve growth factor 2.5S was obtained from Life Technologies (Carlsbad, CA, United States). Dimethyl sulfoxide and KG-501 (a CREB inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Anti-CREB, anti-phospho-CREB, and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States). Anti-rabbit β-actin antibodies were obtained from Abcam (Cambridge, United Kingdom).

Donepezil hydrochloride was purchased from Tokyo Chemical Industry (Tokyo, Japan). For animal treatments, HJG and DPZ were dissolved in distilled water. Three weeks after CI, HJG, DPZ, and vehicle (distilled water) were orally administered daily for 7 days.

Cell Culture

PC12 (rat pheochromocytoma) cells were cultured in RPMI-1640 with 5% (v/v) fetal bovine serum, 10% (v/v) horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were grown to confluence at 37°C in 5% CO2. The medium was changed two or three times per week. Cells treated with NGF (50 ng/ml) were used as positive controls.

Neurite Outgrowth Assay

PC12 cells (1 × 105 cells/ml) were seeded on collagen-coated six-well plates and cultured for 24 h. Cells were treated with different dilutions of HJG (1-500 μg/ml), or each herb at the concentration based on the original mixing ratio of constituents, and cultured for 72 h. PC12 cells were photographed using an inverted microscope (Olympus, Tokyo, Japan) and phase-contrast objectives. Images of two fields per well were taken with 40-50 cells per field. The length and number of neurites were measured for 20 independent cells per field using Image J 1.44 software (NIH Image Software), and the neurite lengths for each cell were summed. Each experiment was conducted in triplicate.

Western Blotting

Proteins were extracted from cells at appropriate time points after drug treatment using RIPA buffer [50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Protein concentrations were measured using a BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, United States). Proteins were separated on SDS polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and probed with specific antibodies. Immunoreactive polypeptides were detected with chemiluminescence using ImmunoStar LD (Wako, Osaka, Japan).

Luciferase Reporter Assay

PC12 cells (1 × 105 cells/well) were seeded in collagen-coated 12-well plates for 24 h. Cells were co-transfected with the pGL4.29 luciferase reporter vector containing a CRE and pGL4.74 Renilla luciferase vector (Promega) using Lipofectamine 3000 (Life Technologies). Forty-eight hours after transfection, cells were treated with vehicle (0.1% DMSO), HJG, or its constituents for 8 h and harvested using Passive Lysis Buffer (Promega).

Luciferase activities were determined with a Dual-Luciferase Reporter Assay System Kit (Promega) according to the manufacturer’s instructions. We normalized the intensity of luciferase reactions measured in the lysates of transfectants to their Renilla luciferase activity, which was used as an internal control.


Male Wistar rats weighing 250-300 g were obtained from Kyudo Co., Ltd. (Saga, Japan). Rats were housed in groups of 4-5 per cage at 23 ± 2°C with a relative humidity of 60 ± 10%, and maintained under a 12-h light-dark cycle. Food and water were available ad libitum except during the restricted feeding period. We restricted food intake for rats (8-10 g each day), and their body weight was maintained at ∼80% of free-feeding levels during the eight-arm radial maze tasks. We conducted all procedures relating to animal care and use according to regulations dictated by the Experimental Animal Care and Use Committee of Fukuoka University (No. 1405744).

Preparation of Aggregated Aβ and CI + Aβ Rats

Aggregated Aβ was prepared as previously described (Moriyama et al., 2017). Aβ42 peptides were purchased from AnaSpec Inc. (Fremont, CA, United States), dissolved in HEPES-buffered solution to a final concentration of 10 μM, and incubated at 37°C for 7 days.

Cerebral ischemia + Aβ rats were prepared by combining an intracerebroventricular Aβ injection with transient CI as previously described (Moriyama et al., 2017). Rats were anesthetized with sodium pentobarbital. After exposure and threading of their bilateral common carotid arteries, rats were placed in a stereotaxic frame. The bilateral vertebral arteries were electrocauterized with a bipolar coagulator (MICRO-3D; Mizuho Industrial, Tokyo, Japan). For intracerebroventricular infusion, a guide cannula (0.71 ± 0.02 mm o.d., 0.41 ± 0.02 mm i.d., 13-mm length) was implanted bilaterally into the lateral cerebral ventricles at AP = -0.8 mm, L = ±1.3 mm, and H = 3.3 mm from the bregma. Three days after cannulation, the common carotid arteries were compressed by clips and cerebral circulation was interrupted for 10 min to create CI…

Source: Frontiers

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