ORIGINAL RESEARCH

Bilobalide Induces Neuronal Differentiation of P19 EmbryonicCarcinoma Cells via Activating Wnt/b-Catenin Pathway

Mei Liu • Jingjing Guo • Juan Wang •

Luyong Zhang • Tao Pang • Hong Liao

Received: 31 March 2014 / Accepted: 29 April 2014

� Springer Science+Business Media New York 2014

Abstract Bilobalide, a natural product extracted from

Ginkgo biloba leaf, is known to exhibit a number of

pharmacological activities. So far, whether it could affect

embryonic stem cell differentiation is still unknown. The

main aim of this study was to investigate the effect of

bilobalide on P19 embryonic carcinoma cells differentia-

tion and the underlying mechanisms. Our results showed

that bilobalide induced P19 cells differentiation into neu-

rons in a concentration- and time-dependent manner. We

also found that bilobalide promoted neuronal differentia-

tion through activation of Wnt/b-catenin signaling path-

way. Exposure to bilobalide increased inactive GSK-3bphosphorylation, further induced the nuclear accumulation

of b-catenin, and also up-regulated the expression of Wnt

ligands Wnt1 and Wnt7a. Neuronal differentiation induced

by bilobalide was totally abolished by XAV939, an

inhibitor of Wnt/b-catenin pathway. These results revealed

a novel role of bilobalide in neuronal differentiation from

P19 embryonic cells acting through Wnt/b-catenin signal-

ing pathway, which would provide a better insight into the

beneficial effects of bilobalide in brain diseases.

Keywords Bilobalide � Neuronal differentiation � P19

embryonic carcinoma cells � Wnt/b-catenin signaling

Abbreviations

Ab Amyloid b-peptide

ADDL Amyloid-b derived diffusible ligand

APC Adenomatous polyposis coli

bHLH protein Basic helix-loop-helix protein

b-NGF b-Nerve growth factor

BSA Bovine serum albumin

CNPase 20,30-Cyclic-nucleotide 30-phosphodiesterase

CREB Cyclic-AMP response element binding

protein

DMSO Dimethyl sulfoxide

ESCs Embryonic stem cells

GFAP Glial fibrillary acidic protein

Gli Glioma-associated oncogene family zinc

finger

GSK-3b Glycogen synthase kinase-3 beta

Ngn1 Neurogenin1

P19 EC cells P19 embryonic carcinoma cells

RA Retinoid acid

TCF/LEF T-cell factor/lymphoid enhancer binding

factor

Introduction

Embryonic stem cells (ESCs) derived from the inner cell

mass of pre-implantation blastocyst cells have the capacity

to differentiate into all three cell lineages (i.e., endoderm,

mesoderm, and ectoderm) (Thomson et al. 1998). Since

there is a loss of neurons within the brain in stroke, trau-

matic brain injury, and Alzheimer’s and Parkinson’s

M. Liu � J. Guo � J. Wang � L. Zhang � T. Pang (&) �H. Liao (&)

Neurobiology Laboratory, National Center for Drug Screening,

China Pharmaceutical University, #24 Tongjiaxiang Street,

Nanjing 210009, People’s Republic of China

e-mail: tpang@cpu.edu.cn

H. Liao

e-mail: liaohong56@hotmail.com

L. Zhang � T. Pang � H. Liao

Key Laboratory of Drug Quality Control and Pharmacovigilance

(China Pharmaceutical University), Ministry of Education,

Nanjing 210009, People’s Republic of China

123

Cell Mol Neurobiol

DOI 10.1007/s10571-014-0072-7

disease (Duchen 2012), the in vitro generation of neurons

from ESCs has enormous potential to derive specific cell

populations to replace the degenerated cells and provides

an unlimited source for cell-based regenerative therapy.

During ESC neurogenesis, a variety of extrinsic and

intrinsic factors that control developmental stages have

been shown to be crucial for the generation of new neurons.

For instance, when ESCs are cultured with retinoic acid

(RA), they can differentiate into neuron-like cells (Bain

et al. 1996). b nerve growth factor (b-NGF) produced an

effect similar to RA (Schuldiner et al. 2001). Similarly,

basic helix-loop-helix (bHLH) protein neurogenin1 (Ngn1)

was able to facilitate a neuronal differentiation in the

absence of RA (Kim et al. 2004).

Many signaling pathway involved in neural lineage

commitment by ESCs, and one of important candidate

pathways is the Wnt/b-catenin pathway. In canonical Wnt

signaling pathways, binding of Wnts to their receptors can

activate Disheveled (Dsh), then inhibit glycogen synthase

kinase-3beta (GSK-3b), a component of the destruction

complex (consisting of Axin, adenomatous polyposis coli

(APC), and GSK-3b) and lead to b-catenin accumulation.

Nuclear translocation of b-catenin in a concentration-

dependent manner allows it to bind to T-cell factor/lym-

phoid enhancer binding factor (TCF/LEF) transcription

factors, which activate transcription of Wnt target genes

(Wodarz and Nusse 1998; MacDonald et al. 2009). A

number of studies indicate that Wnt signaling pathway has

been played a primary role in neurogenesis. Wnt ligands

such as Wnt1 (Tang et al. 2002) and Wnt3a (Davidson

et al. 2012) could lead to significantly fewer undifferenti-

ated ESCs and promote neuronal differentiation. Wnt7a

promoted neuronal differentiation of neural progenitor

cells in the developing mouse neocortex (Hirabayashi et al.

2004). Except for Wnt ligands, activating Wnt signaling by

expressing stabilized b-catenin, GSK-3 mutant, or by

adding the GSK-3 inhibitor lithium also led to increasing

neuronal marker expression and promoting progenitor

proliferation and neurogenesis in culture and in vivo

(Wexler et al. 2008; Lie et al. 2005). Additionally, several

reports point to a role of Wnt signaling in neurite out-

growth, axon guidance, synapse formation, and plasticity

(Budnik et al. 2011; Ille and Sommer 2005). Thus, small

molecules that target specific regulatory processes and

control cell fate choice would be valuable for the produc-

tion of neurons from ESCs.

Bilobalide is one of important constituents of Ginkgo

biloba leaf extract which is known for its protective actions

on improvement of blood flow, anti-free radicals, and anti-

inflammatory effects (DeKosky et al. 2008; Shi et al. 2009;

Kotakadi et al. 2008). Recently, bilobalide has been drawn

more attention due to its beneficial effects in the central

nervous system. An increasing number of studies have

shown the potential role of bilobalide in the treatment of

symptoms associated with mild-to-moderate dementia,

impairment of other cognitive functions associated with

aging, and cerebrovascular disorders (Defeudis 2002). In

addition, bilobalide has also been proposed to exert trophic

effects in neurons, such as protecting neurons against

damage induced by glutamate, serum deprivation, and

amyloid b-peptide (Ab)1-42 (Ahlemeyer and Krieglstein

2003; Shi et al. 2010). Bilobalide was reported to dose-

dependently enhance neurogenesis and synaptogenesis in

the hippocampal neurons via elevation of the levels of

phosphorylated cyclic-AMP Response Element Binding

Protein (p-CREB) and brain-derived neurotrophic factor

(Tchantchou et al. 2009). Thus, bilobalide might have the

potential role in neuronal differentiation of ESCs. In this

study, P19 embryonic carcinoma (EC) cells which resem-

ble mouse ESCs were used to investigate the neuronal

differentiation effect of bilobalide. Our results showed that

bilobalide could induce P19 EC cells differentiate into

neurons via activation of Wnt/b-catenin signaling pathway.

Materials and Methods

Materials

Traditional Chinese Medicine compound bilobalide

(MUST, purity [98 %, ChengDu, China), all-transRA

(Sigma, USA), and XAV939 (inhibitor of Wnt/b-catenin

pathway, Tocris Bioscience, UK) were dissolved in dime-

thyl sulfoxide (DMSO) as a stock solution at 10 mmol/L.

For all the experiments, these compounds were prepared by

diluting the stock with culture medium (final concentration

of DMSO was below 0.1 %).

Cell Culture, Differentiation and Drug Treatment

P19 EC cells were obtained from the American Type

Culture Collection (ATCC, USA). Undifferentiated P19

cells were grown in a-MEM (Gibco, USA) complete

medium with 7.5 % calf serum (Gibco, USA), 2.5 % fetal

bovine serum (Gibco, USA), 100 units/mL of penicillin,

and 100 lg/mL of streptomycin in humidified incubator

supplemented with 5 % CO2. To induce differentiation,

P19 cells were seeded at 1 9 106 cells/mL in 100-mm

nonadhesive bacteriological-grade dishes (day 0) and

grown as aggregates in complete medium containing

1 lmol/L of RA (positive control) or different concentra-

tions of bilobalide for 4 days. For XAV939 effect, P19

cells were pretreated with XAV939 for half an hour and

then cultured as before. On day 4 of differentiation, cell

aggregates were trypsinized and transferred into tissue

culture dishes containing drug-free neurobasal medium

Cell Mol Neurobiol

123

(Gibco, USA) supplemented with B27TM and 0.5-mmol/L

L-Glutamine (Gibco, USA). The replated cells were cul-

tured for additional 5 days, and the culture medium was

replenished every 2 days.

Western Blot Analysis

P19 cells obtained from different treatments were washed

with cold PBS and suspended in ice-cold lysis buffer (50-

mmol/L Tris–HCl, 2-mmol/L MgCl2, 1 % NP-40, 10 %

glycerol, 100-mmol/L NaCl) with 1 % protease inhibitor

cocktail (Roche Molecular Biochemicals, USA). The mix-

tures were collected as the whole cell extracts. To obtain

nuclear extracts, we used Nuclear and Cytoplasmic Protein

Extraction Kit (Beyotime, China) according to the manufac-

turer’s instructions. Cell extracts were separated by 10 %

SDS-PAGE, transferred onto nitrocellulose membranes, and

probed with specific primary antibody against bIII-tubulin

(1:1000; StemCell), GSK-3b (1:1000; Cell Signaling Tech-

nology, USA), phospho-GSK-3b-Ser9 (1:500; Epitomics,

USA), LaminB1 (1:500; Bioworld, USA), b-catenin (1:1000;

Cell Signaling Technology, USA), and b-actin (1:1000;

Sigma, USA). The blots were developed with secondary

antibodies anti-rabbit IgG (1:4000; Amersham, USA) or anti-

sheep IgG (1:7000; Amersham, USA), then imaged on Gel-

Doc XR system (Bio-Rad, USA).

RNA Isolation and RT-PCR

The total RNA was extracted using Trizol reagent (Invit-

rogen, USA). Reverse transcription was performed with

1 lg of total RNA using PrimeScript 1st strand cDNA

synthesis kit (Takara). For PCR analysis, 0.5 lg of cDNA

was used as a template and amplified using specific primers

as follows: Ngn1 (50-cctttggagacctgcatctc-30 and 50-gatgtag

ttgtaggcgaagc-30), Mash1 (50-caagttggtcaacctgggtt-30 and

50-gctcttgttcctctgggcta-30), Wnt1 (50-cgaacctgttgacggattccaa

g-30 and 50-ggaggtgattgcgaagatgaacg-30), Wnt3a (50-gaactgc

accaccgtcagcaac-30 and 50-tctccctggcatcggcaaac-30), Wnt7a

(50-gaggctgccttcacctatgcga-30 and 50-agaccttggcgaagccgatgc-30),and b-actin (50-ctggacctggctggccgg-30 and 50-gctcttctccagggagg

aaga-30). Each PCR mix contained 2 lg of cDNA, 1 lL of

each of primer (10 mmol/L), 8.5 lL of ddH2O, and 12.5-lL

SYBR Green Premix (Takara, Japan). The relative quantitative

expression was measured in triplicates on a Real-Time PCR

Detection System (PTC-200, BioRad) where the mouse b-actin

served as a control. The specificity of amplification was con-

firmed by a melting curve.

Immunofluorescence Staining

P19 cells after drug treatment were seeded on coverslips

precoated with poly-L-lysine (10 lg/mL, Sigma). For

immunofluorecence, the cells were fixed with 4 % para-

formaldehyde in PBS for 30 min, permeabilized in PBS

containing 0.1 % Triton X-100 for 10 min and blocked

with 5 % bovine serum albumin. Then, coverslips were

incubated overnight at 4 �C with primary antibodies, anti-

bIII-tubulin (1:300; StemCell), anti-glial fibrillary acidic

protein (GFAP, 1:500; DakoCytomation), anti-20,30-cyclic-

nucleotide 30-phosphodiesterase (CNPase, 1:500; Chem-

icon), and anti-b-catenin (1:1000; Cell Signaling Tech-

nology). Nuclei were labeled with Hoechst 33342 (10 lg/

mL, Molecular Probes). After washing three times with

PBS, cell were incubated with fluorescence-conjugated

secondary antibody (Cy2 or Cy3, 1:200; Amersham) for

60 min at about 20 �C. The fluorescent was then examined

by a fluorescence microscopy (Olympus).

Statistical Analysis

All present data represent the results of three independent

experiments. Statistical analysis was performed using

unpaired Student’s t test. All data are presented as

mean ± SD. P \ 0.05 was considered statistically

significant.

Results

Bilobalide-Induced Neuronal Differentiation of P19 EC

Cells

To investigate the role of bilobalide in the differentiation of

P19 EC cells, the effect of bilobalide at different concen-

trations was first determined. Neuronal and glial differen-

tiations were detected by immunostaining after P19 EC

cells treated with bilobalide (0.2, 1, 5 lmol/L) for 4 days

using neuron-specific proteins bIII-tubulin, astrocyte mar-

ker GFAP, and oligodendrocyte marker CNPase. The

immunofluorescent analysis revealed that bilobalide

exhibited a significant neuronal induction in a concentra-

tion-dependent manner (Fig. 1a), while there was no dif-

ference in glial differentiation, including astrocytes and

oligodendrocytes (data not shown). Moreover, as shown in

Fig. 1b, the Western blot results indicated that bilobalide at

1 lmol/L induced the most significant increase of bIII-

tubulin.

Also, different treatment duration of bilobalide was

assessed. Bilobalide-induced P19 differentiation in a time-

dependent manner to be found in both immunocytochem-

istry and Western blot assays. After 4 days treatment, bi-

lobalide (1 lmol/L) significantly increased the population

of bIII-tubulin positive (bIII-tubulin?) neurons (Fig. 2a),

but not GFAP? or CNPase? cells (data not shown). The

expression of bIII-tubulin significantly increased in

Cell Mol Neurobiol

123

Fig. 1 The effect of bilobalide

on P19 cells differentiation in a

concentration-dependent

manner. a Immunofluorescence

staining was performed for

detecting P19 EC cells

differentiation. bIII-tubulin

(1:300) was used as neuron

marker. Scale bar 50 lm. b P19

cells differentiation was

determined by Western blot. b-

actin was used as endogenous

control. Quantitative assessment

of Western blot was shown

below. Values were reported as

mean ± SD. *P \ 0.05,

**P \ 0.01 versus DMSO

group

Cell Mol Neurobiol

123

Fig. 2 The effect of bilobalide

on P19 cells differentiation in a

time-dependent manner.

a Immunofluorescent staining of

bIII-tubulin (Red) after P19

cells differentiation for 2, 4, or

6 days. Scale bar 50 lm. b Cell

lysates from P19 cells were

subjected to immunoblotting

with anti-bIII-tubulin (1:1000)

in Western blot assay. b-Actin

was used as endogenous control.

Quantitative assessment of

Western blot was shown below.

Values were reported as

mean ± SD. *P \ 0.05,

**P \ 0.01 versus DMSO

group (Color figure online)

Cell Mol Neurobiol

123

bilobalide- and RA-treated cultures at 4 days (Fig. 2b).

Based on the results, bilobalide can significantly promote

neuronal differentiation from undifferentiated P19 EC cells

compared with control. The concentration of 1 lmol/L and

the period of 4-day treatment were selected as the optimal

differential conditions for subsequent experiments.

Activation of Canonical Wnt/b-catenin Signaling

Pathway in Neuronal Differentiation Induced

by Bilobalide

To explore the possible molecular mechanisms underlying

the inductive effect of bilobalide, the canonical Wnt/b-

catenin signaling was investigated. It is well known that

Mash1 and Ngn1 were expressed in differentiating neurons

and closely associated with the determination of neuronal

fate (Lo et al. 1998). Moreover, Mash1 and Ngn1 are Wnt-

signaling target genes (Kageyama et al. 1997). Results

showed that the expression level of Mash1 and Ngn1

mRNA in the group of bilobalide was significantly higher

than that of DMSO control (Fig. 3a). To gain further

insights into mechanism of action of bilobalide, the major

components involved in this signaling pathway, including

the GSK-3b and b-catenin, were tested. P19 cells were

treated with bilobalide (1 lmol/L) for 4 days, and cell

lysates were analyzed by Western blot. As shown in

Fig. 3b, bilobalide induced the phosphorylation of GSK-3bat Ser9 (inactive form) by 3.65 ± 0.15-fold (P \ 0.01)

Fig. 3 Bilobalide activated canonical Wnt/b-catenin signaling path-

way during the neuronal differentiation of P19 cells. P19 cells were

cultured in the absence or presence of 1 lmol/L of bilobalide for

4 days. a Action of bilobalide on target genes of Wnt/b-catenin

signaling. After total RNA was isolated, quantitative RT-PCR

analysis was performed to measure mRNA expression of downstream

target genes Ngn1 and Mash1. b Effect of bilobalide on GSK-3bphosphorylation and the stabilization of b-catenin signals in P19 cells.

Cell lysates were collected for Western blot analysis. c Bilobalide

induces b-catenin accumulation in the nucleus during neuronal

differentiation. Nuclear protein was isolated, then immunoblotted

with b-catenin and LaminB1 antibodies (nuclear envelope marker).

Quantitative assessment of Western blot was shown below. d Quan-

titative RT-PCR analysis of Wnt ligands by Wnt1, Wnt3a, and Wnt7a

gene expression. b-actin is used as an internal control for equal cDNA

amount. All values were reported as mean ± SD. **P \ 0.01 versus

DMSO group

Fig. 4 Inhibitory effect of XAV939 on bilobalide-induced neuronal

differentiation of P19 cells. Cells were pretreated with or without

1 lmol/L XAV939 for 30 min before the addition of bilobalide

(1 lmol/L). a, b Western blotting confirmed that b-catenin and bIII-tubulin levels were effectively down-regulated in XAV939-treated

P19 cells. c Immunofluorescence staining with b-catenin (Red) was

used to assess changes in nuclear and cytoplasmic b-catenin. Scale

bar 25 lm. d The examination of expression of bIII-tubulin using

immunofluorescence staining. Scale bar 50 lm. All values were

reported as mean ± SD. **P \ 0.01 versus DMSO group (Color

figure online)

c

Cell Mol Neurobiol

123

Cell Mol Neurobiol

123

compared to untreated controls, increased intracellular b-

catenin levels, and induced b-catenin accumulation in the

nucleus (Fig. 3c). Moreover, we also assessed the effect of

bilobalide on mRNA expression levels of Wnt ligands

Wnt1, Wnt3a, and Wnt7a. As shown in Fig. 3d, bilobalide

treatment significantly increased the mRNA expression of

Wnt1 and Wnt7a, but has no effect on the expression of

Wnt3a. These findings suggest that Wnt/b-catenin signal-

ing pathway is activated during neuronal differentiation

from P19 cells induced by bilobalide.

Blockade of Wnt/b-catenin Signaling Pathway

Abrogates Neuronal Differentiation Induced

by Bilobalide

The above results showed that Wnt/b-catenin signaling

pathway is activated by bilobalide during neuronal differ-

entiation of P19 cells. To investigate whether this pathway

activation is specifically involved in bilobalide-induced

neuronal differentiation, the Wnt/b-catenin pathway

inhibitor XAV939 was used. XAV939, a tankyrase1/2

inhibitor, stimulates b-catenin degradation by stabilizing

Axin, considered as a specific inhibitor of Wnt/b-catenin

signaling pathway (Huang et al. 2009). Undifferentiated

P19 cells incubated with 1 lmol/L of XAV939 half an hour

before treatment with bilobalide decreased the level of

intracellular b-catenin expression (Fig. 4a), and the level of

b-catenin translocation into the nucleus (Fig. 4c).

To further verify whether inhibitor XAV939 blocked the

effect of bilobalide on neuronal differentiation, immuno-

staining and Western blot analysis were performed. Our data

showed that there was no difference between XAV939 group

and DMSO group, but XAV939 treatment remarkably

blocked bIII-tubulin level which was promoted by bilobalide

(Fig. 4b, d). These results suggested that the critical role of

canonical Wnt/b-catenin signaling pathway in neuronal

differentiation of P19 EC cells induced by bilobalide.

Discussion

Recently, bilobalide has been drawn more attention due to

its neurotrophic effects on the central nervous system,

especially its memory enhancing effects (DeKosky et al.

2008; Snitz et al. 2009). The mechanisms of bilobalide in

neuroprotection have been understudied compared with its

terpenoid ginkgolide partners (Vitolo et al. 2009; Chatter-

jee et al. 2003). However, its ability to affect ES cell

behaviors has not been fully understood yet. In the present

study, for the first time, we reported that bilobalide can

induce the neuronal differentiation of P19 EC cells through

activation of Wnt/b-catenin signaling pathway.

Fig. 4 continued

Cell Mol Neurobiol

123

P19 EC cells share the general properties of pluripotent

stem cells in that they exhibit unlimited self-renewal and can

give rise to derivatives of all three-embryonic germ layers

(McBurney and Rogers 1982; Jones-Villeneuve et al. 1982).

Neurogenesis from P19 EC cells is similar to mouse ESCs

(Wichterle et al. 2002; Lang et al. 2004). According to the

treatment of cells, neuronal differentiation of P19 cells can

be divided into two sequential stages, induction and differ-

entiation (McBurney et al. 1982). During the first stage, P19

cells are allowed to aggregate in the bacterial dishes and

incubated with bilobalide for 4 days. In the second stage, the

induced P19 cells are replated into cell culture dishes as

single cell suspension to differentiate into mature neurons.

Using this differentiation protocol, we evaluated the effect of

bilobalide on inducing neuronal differentiation. Bilobalide

at 1 lM exhibited maximum induction effects on bIII-

tubulin expression as indicated by immunostaining and

immunoblots, but bIII-tubulin expression in immunoblots is

not as significant as that in immunostaining, which may be

due to different type of anti-tubulin antibody used in dif-

ferent experiment conditions. Our results show that after

4-day bilobalide treatment, P19 cells differentiated into

neurons, but not glial cells.

The possible mechanism of bilobalide involved in neuronal

differentiation was determined. The molecular signals involved

in neural induction are not yet totally elucidated, but it is known

that canonical Wnt signaling pathway involves in this crucial

event. Results showed that Wnt-signaling target genes Mash1

and Ngn1 are up-regulated by bilobalide during the differenti-

ation stage. Both Mash1 and Ngn1 are activator-type bHLH

genes which have been shown, in addition to their neural

determination function, to play important roles in the neuronal

versus glial fate decision (Kageyama et al. 1997). Ngn1 could

promote neurogenesis by functioning as a transcriptional acti-

vator, while inhibiting astrocyte differentiation (Sun et al.

2001). Mash1 defines cells with long-term neurogenic potential

in subgranular and subventricular zones in adult-mouse brain

(Kim et al. 2011). Besides, GSK-3b antagonizes the canonical

Wnt pathway, facilitating phosphorylation of b-catenin. b-

catenin is another key signal that can translocate into the

nucleus in a concentration-dependent manner (Kuhl and Kuhl

2013). The levels of phosphorylated GSK-3b (inactive form)

and b-catenin protein were significantly increased by biloba-

lide, indicating that bilobalide inhibited GSK-3b activity and

promoted the nuclear translocation of b-catenin.

There are two possible mechanisms on how the

canonical Wnt/b-catenin signaling is induced during bi-

lobalide-induced neuronal differentiation from ES cells

(illustrated in Fig. 5): first, bilobalide may interact with

GSK-3b as a potential GSK-3b inhibitor to activate Wnt/

b-catenin signaling pathway; second, bilobalide might

activate the Wnt signaling through increasing Wnt

ligands. Although it is well known that Wnt signaling is

considered as a critical regulator of neurogenesis (Val-

vezan and Klein 2012), the relationship between biloba-

lide and Wnt/b-catenin activation has not been identified.

Our results indicated that bilobalide could trigger the

canonical Wnt/b-catenin signaling cascade in promoting

neuronal differentiation from ESCs.

Stroke, traumatic brain injury, and many neurodegenera-

tive diseases, such as Alzheimer’s and Parkinson’s disease,

characterized by a loss of neurons within the brain (Duchen

Fig. 5 Scheme for the proposed

mechanisms involved in

bilobalide-induced neuronal

differentiation from P19 EC

cells. Exposure to bilobalide

results in a significant increase

in the inactive GSK-3b (Ser9

phosphorylation), further

induces the nuclear

translocation of b-catenin, also

up-regulated the expression of

Wnt ligands Wnt1 and Wnt7a,

which promote the P19 EC cells

into neurons

Cell Mol Neurobiol

123

2012), has seriously reduced the quality of human life, and

eventually lead to devastating cognitive and motor deficits.

However, currently clinic treatments unmet medical needs.

The generation of neurons from ESCs is a promising approach

for neural tissue repair and cell-based replacement therapies of

the nervous system. Neurogenesis induced by bilobalide may

be one explanation of its memory enhancing effect. The effect

of bilobalide inducing neuronal differentiation of ESCs

in vitro might be reached in vivo, and the related study is

ongoing. It has been demonstrated that bilobalide can promote

the dendritic process and significantly prevent synaptic loss

induced by amyloid-b derived diffusible ligand (ADDL) in

hippocampal neurons (Tchantchou et al. 2009). ESCs-derived

neurons by bilobalide may have the potential role to replace

the lost neurons to show some degree of functional recovery

after transplantation in the brain of patients, which would

provide a translational value in regenerative therapy.

Another important extract of Ginkgo biboba leaves EGb

761 which contains 24 % flavonol glycosides, 6 % ginkgo-

lide, and bilobalide has been ongoing for many years (DeF-

eudis and Drieu 2000). Basic and clinical studies, conducted

both in vitro and in vivo, support these beneficial neuropro-

tective effects of EGb 761, in which bilobalide increases the

respiratory control ratio of mitochondria by protecting against

uncoupling of oxidative phosphorylation (IhI 2012).

In conclusion, our results have demonstrated a novel

role of bilobalide on enhancing P19 EC cells to differen-

tiate into neurons via canonical Wnt/b-catenin signaling

pathway, which may provide drug discovery clues and a

potential combination strategy for more effective therapy

in both basic and clinical settings, although the develop-

ment of a cell-based drug to treat neurological conditions is

still at a relatively early stage for clinic use.

Acknowledgments This work was supported by National Natural

Science Foundation of China (81271338, 81070967), the Specialized

Research Fund for the Doctoral Program of Higher Education of

China (20130096110011), Natural Science Foundation of Jiangsu

Province (BK20130653), the Fundamental Research Funds for the

Central Universities (JKZD2013006), and 2011’ Program for Excel-

lent Scientific and Technological Innovation Team of Jiangsu Higher

Education.

Conflict of interest The authors declare no conflicts of interest.

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