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GC-MS-Based Metabolomic Study on the

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Article in Behavioural brain research August 2016

DOI: 10.1016/j.bbr.2016.08.001

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Research report

GCMS-based metabolomic study on the antidepressant-like effects

of diterpene ginkgolides in mouse hippocampus
Zihong Liang a,b,c,d,1 , Shunjie Bai b,c,e,1 , Peng Shen a,b,c,1 , Qingchuan Hu b,c,e,1 ,
Xingfa Wang b,c,f , Meixue Dong a,b,c , Wei Wang b,c,f , Juan Li a,b,c , Ke Cheng a,b,c ,
Shuxiao Zhang b,c,e , Dezhi Zou a,b,c , Yu Han a,b,c , Haiyang Wang b,c , Peng Xie a,b,c,f,
a
Department of Neurology, The First Afliated Hospital of Chongqing Medical University, Chongqing, China
b
Chongqing Key Laboratory of Neurobiology, Chongqing, China
c
Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing, China
d
Department of Neurology, The Inner Mongolia Autonomous Region peoples Hospital, Hohhot, Inner Mongolia, China
e
Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China
f
Department of Neurology, Yongchuan Hospital, Chongqing Medical University, Chongqing, China

h i g h l i g h t s

Diterpene ginkgolides (DG) show antidepressant-like effects in behavioral tests.

DG treatment also causes 18 differentially expressed mouse hippocampal metabolites.
They are related to neurotransmitter, glutathione, lipid, and energy metabolism.
The DG- and venlafaxine-induced alterations in seven metabolites are similar.

a r t i c l e i n f o a b s t r a c t

Article history: Ginkgo biloba extract (GBE), including EGb-761, have been suggested to have antidepressant activity
Received 14 July 2016 based on previous behavioral and biochemical analyses. However, because GBE contain many con-
Received in revised form 28 July 2016 stituents, the mechanisms underlying this suggested antidepressant activity are unclear. Here, we
Accepted 1 August 2016
investigated the antidepressant-like effects of diterpene ginkgolides (DG), an important class of con-
Available online 3 August 2016
stituents in GBE, and studied their effects in the mouse hippocampus using a GCMS-based metabolomics
approach. Mice were randomly divided into ve groups and injected daily until testing with 0.9% NaCl
Keywords:
solution, one of three doses of DG (4.06, 12.18, and 36.54 mg/kg), or venlafaxine. Sucrose preference
Ginkgo biloba extract
Antidepressant
(SPT) and tail suspension (TST) tests were then performed to evaluate depressive-like behaviors in
Diterpene ginkgolides mice. DG (12.18 and 36.54 mg/kg) and venlafaxine (VLX) administration signicantly increased hedo-
Metabolomics nic behavior in mice in the SPT. DG (12.18 mg/kg) treatment also shortened immobility time in the
Hippocampus TST, suggestive of antidepressant-like effects. Signicant differences in the metabolic prole in the DG
(12.18 mg/kg) compared with the control or VLX group indicative of an antidepressant-like effect were
observed using multivariate analysis. Eighteen differential hippocampal metabolites were identied that
discriminated the DG (12.18 mg/kg) and control groups. These biochemical changes involved neurotrans-
mitter metabolism, oxidative stress, glutathione metabolism, lipid metabolism, energy metabolism, and
kynurenic acid, providing clues to the therapeutic mechanisms of DG. Thus, this study showed that DG
has antidepressant-like activities in mice and shed light on the biological mechanisms underlying the
effects of diterpene ginkgolides on behavior, providing an important drug candidate for the treatment of
depression.
2016 Elsevier B.V. All rights reserved.

1. Introduction

Major depressive disorder (MDD) is a one of the most frequently

Corresponding author at: Department of Neurology, Yongchuan Hospital,
encountered forms of mental illness and a leading cause of disabil-
Chongqing Medical University, Chongqing 402460, China.
E-mail address: xiepeng@cqmu.edu.cn (P. Xie). ity worldwide [13]. Most antidepressants available today inhibit
1
These authors contributed equally to the manuscript. the reuptake or breakdown of serotonin, noradrenaline, or both in

http://dx.doi.org/10.1016/j.bbr.2016.08.001
0166-4328/ 2016 Elsevier B.V. All rights reserved.
 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124
the brain [2]. Despite a vast number of established antidepressant
medication options for MDD, a signicant percentage of patients
fail to respond to these rst-line treatments [4]. In addition, the
efcacy and safety of antidepressant medications remain contro-
versial, and an increased risk of suicidal behavior exists in patients
treated with some of these medications [5,6]. Owing to the limited
therapy options, treatment of MDD is still a major focus of current
biomedical research, with the investigation of alternative treat-
ments urgently needed to reduce the burden of MDD on patients,
families, and society.
The reputed efcacy, low cost, and relative absence of side
effects of traditional Chinese medicines have led to increasing
interest in the use of them for the treatment of neuropsychiatric
disorders. GBE termed EGb761 is a patented extract from the leaves
of the Ginkgo biloba tree. Since the development of GBE in 1964,
its effects on an extensive range of disorders and diseases have
been investigated [7]. GBE has been suggested to have many ben-
ecial effects on central nervous system (CNS) function and is
widely used for treating neurologic, psychiatric, functional, and
physiologic symptoms [79]. Recent work has indicated that some
components of GBE may act as an antidepressant and improve
spatial learning and memory [1014]. Meanwhile, GBE is a mix-
ture of substances with a wide variety of physical and chemical
properties and activities. Numerous pharmacological investiga-
tions have led to the conclusion that DG mainly composed of
ginkgolide A, B and K in EGb761, are responsible for the main
pharmacological neuroprotective effects [7,12,15,16]. Therefore,
it is reasonable to propose that DG may be effective in treating
depression.
Diterpene Ginkgolides Meglumine Injection (DGMI), a novel
medicinal plant products (manufactured by Jiangsu Kanion Phar-
maceutical Co., Ltd.) has been awarded marketing approval from
the China Food and Drug Administration for treating patients with
mild to moderate cerebral infarction [17,18]. This injection con-
tains the vast majority of DG from GBE (mainly ginkgolide A, B and
K) [17]. Therefore, DGMI is an ideal t to study the antidepressant
function of DG.
The hippocampus is a brain region that has been extensively
studied in patients with mood disorders and has been selected as
a relevant region for studying MDD molecular pathways [19,20].
Previous studies have shown that hippocampal dendritic or spine
remodeling is dysregulated in stress-induced depression and stress
resilience [21,22]. Furthermore, hippocampal plasticity is not only
altered in patients with MDD and in stressed animals but also
affected by antidepressant treatments.
Recently, metabolomics has been widely used to discover
the molecular biomarkers for diagnosing or prognosing disease,
and to prole potential pathophysiological mechanisms and to
assess therapeutic effects of antidepressants [2,2325]. Compared
with methods such as nuclear magnetic resonance or liquid
chromatographymass spectrometry, gas chromatographymass
spectrometry (GCMS) has been widely applied because of its high
sensitivity, peak resolution, and reproducibility [25].
In the present study, we investigated the antidepressant-
like effects of DG in C57BL/6J mice using the measures of
SPT and TST. We then applied a non-targeted metabolomics
approach based on GCMS technology to characterize the global
metabolic prole in mouse hippocampus following the sys-
temic administration of DG and compared it with the prole
obtained following venlafaxine administration. Our primary aims
were to determine whether DG induced an antidepressant-like
effect, to identify probable biological mechanisms underly-
ing this effect of DG, and to search for potential therapeutic
targets for MDD, providing new directions for future stud-
ies.
117
2. Materials and methods
2.1. Animals
This study was approved by the Ethics Committee of Chongqing
Medical University, and all procedures were conducted in accor-
dance with the National Institutes of Healths Guide for the Care
and Use of Laboratory Animals. C57BL/6J male mice (age, 8 weeks;
weight, 1820 g) were obtained from Chongqing Medical Univer-
sitys animal center in the Peoples Republic of China. Throughout
the study, all mice were maintained in a light, temperature, and
humidity controlled environment (12-h light/dark cycle, 23 1 C,
50 5%) with access to food and water ad libitum.
2.2. Reagents
DGMI (25 mg/mL, containing ginkgolides A, B, and K at
1.70, 3.10, and 0.20 mg/mL, respectively) was provided by
Jiangsu Kanion Pharmaceutical Co., Ltd. Pyridine, methoxyamine
hydrochloride, l-2-chlorophenylalanine, venlafaxine, and
N,O-bis(trimethylsilyl)triuoroacetamide (BSTFA) with 1%
trimethylchlorosilane (TMCS) were purchased from Sigma-
Aldrich. HPLC-grade methanol and chloroform were purchased
from the Chongqing Chuandong Chemical (Group) Co. Ultrapure
water was prepared using a Millipore Milli-Q purication system.
2.3. Treatment
After one week of habituation, the mice were randomly divided
into the following ve groups: (1) healthy control (CON, 0.9% NaCl
solution, n = 9), (2) low-dose DG (DG Low, DGMI diluted in 0.9%
NaCl solution to a dose of 4.06 mg/kg, n = 10), (3) medium-dose
DG (DG Medium, DGMI diluted in 0.9% NaCl solution to a dose
of 12.18 mg/kg, n = 11), (4) high-dose DG (DG High, DGMI diluted
in 0.9% NaCl solution to a dose of 36.54 mg/kg, n = 11), and (5)
venlafaxine (VLX, venlafaxine diluted in 0.9% NaCl solution to a
dose of 16 mg/kg, n = 11). Mice were injected intraperitoneally daily
between 8:00 AM and 10:00 AM with the same amount of solution
for 14 days. Venlafaxine was used as positive control drug to explore
the efcacy and mechanism of the DG effects.
2.4. Behavioral testing
After 14 days of drug treatment, mice were weighed and sub-
jected to the SPT, as a means to measure hedonic behavior, and
then the TST. Mice were habituated to the testing room for 30 min
before behavioral analyses began.
Mice were habituated to 1% sucrose solution for 72 h before the
SPT [26]. After 24 h of water and food deprivation, a two-bottle pref-
erence test was used in which all mice had access to both water and
a 1% sucrose solution for 24 h during a no-stress period. Sucrose
preference was scored using the following formula: sucrose pref-
erence (%) = (sucrose intake/total intake) 100.
The tail suspension test was performed as previously described
[27]. Briey, mice were suspended by a small metal hook xed
with adhesive tape wrapped around the tail. After 120 s of adapta-
tion, the duration of immobility was recorded with a video-tracking
system (SMART, Panlab SL, Barcelona, Spain) for 4 min. Mice were
returned to their home cages after every animal was tested.
2.5. Tissue collection and sample preparation for GCMS analysis
Twenty-four hours after the administration of the nal drug or
NaCl, mice in the CON, DG (DG Medium), and VLX groups were
anesthetized with chloral hydrate and decapitated, and their whole
brains were removed. The hippocampi were dissected, weighed,
118 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124

rapidly frozen in liquid nitrogen, and stored at 80 C until used in

GCMS analyses.
The procedure was performed according to our previous study
[28]. Prior to analysis, each frozen mouse hippocampus was added
to 500 L of a solution containing methanol:water:chloroform
(5:2:2, v/v/v) and l-2-chlorophenylalanine (0.03 mg/mL), which
was used as an internal standard. The sample was ground using
a tissue homogenizer (multi-sample TissueLyser, Jingxin Tech-
nology). The mixture was then sonicated and centrifuged at
14,000 rpm for 10 min. The supernatant (300 L) was collected and
freeze-dried. The dried extract was derivatized into its methoxime
derivatives through a reaction with 80 L of methoxyamine
hydrochloride solution and pyridine (15 mg/mL) at room tempera-
ture for 90 min with continuous shaking. Subsequently, the solution
was derivatized with 80 L of BSTFA (1% TMCS) and 20 L of n- Fig. 1. Sucrose preference at baseline and after 14 days treatment. CON, control
group; DG Low, mice treated with diterpene ginkgolides meglumine injection
hexane at 70 C for 60 min, and then placed at room temperature (DGMI) diluted in 0.9% NaCl solution to a dose of 4.06 mg/kg; DG Medium, mice
for 30 min before GCMS analysis. treated with DGMI at a dose of 12.18 mg/kg; DG High, mice treated with DGMI at a
dose of 36.54 mg/kg; VLX, mice treated with venlafaxine.
2.6. GCMS analysis

GCMS analysis was performed using the Agilent GC system

7890A equipped with a 5975C mass selective detector sys-
tem. The GC capillary column was an Agilent J&W HP-5 ms UI
(30 m 0.25 mm 0.25 m). The temperatures of the injector, the
EI iron source, and quadrupole rods were adjusted to 280 C, 230 C,
and 150 C, respectively. High-purity helium carrier gas owed at
a constant rate of 1 mL min1 . A total of 0.5 L for each sample
was applied for metabolite separation. The column temperature
was initially kept at 70 C for 2 min and increased to 240 C at a
rate of 10 C min1 , then raised to 300 C and maintained at that
temperature for 6 min. MS spectra were acquired m/z 50600.
2.7. Identication of the metabolites and metabolomic data
analysis
Low molecular weight metabolites were represented as chro-
matographic peaks in total ion current chromatograms. A total of
229 individual peaks with intensities threefold higher than the
signal-to-noise ratio were recorded and integrated. In this study,
compound annotation was performed by comparing the accurate
mass (m/z) and retention time (RT) and the accurate mass of com-
pounds obtained from web-based resources (National Institute of
Standards and Technology online databases). GCMS metabolic
proles were processed after conversion into a NetCdf format using
TagFinder [29]. The resulting three-dimensional matrix contain-
ing peak indexes (RT-m/z pairs), sample names (observations), and
normalized peak intensities were introduced into SIMCA-P 11.5
software (Umetrics AB, Umea, Sweden).
Partial least squares discriminant analysis (PLSDA) with UV-
scaled spectral data was performed to visually discriminate mice
in the CON, DG, and VLX groups. R2 Y and Q2 were used to quantify
the goodness of t and predictability of the model, respectively.
Parameter values approaching 1.0 indicated a stable model with
predictive reliability. The discriminating metabolites were identi-
ed using a statistically signicant threshold of variable inuence
on projection (VIP) values obtained from the PLSDA model and a
two-tailed Students t-test. The heat map of differential metabo-
lites was obtained using MetaboAnalyst 3.0 (www.metaboanalyst.
ca) for each metabolite.
2.8. Statistical analysis for behavioral tests
The data from the sucrose preference test and tail suspension
test are expressed as means SEM. All data were analyzed using
one-way analyses of variance (ANOVA) followed by the post hoc
least signicant difference test. Values of P < 0.05 were considered
to be statistically signicant. The SPSS software package (SPSS pro-
gram, version 21.0) was used for all statistical tests. The graphs
were generated using Prism 5.0 (GraphPad Software, Inc. USA).
2.9. Bioinformatics analysis
Ingenuity Pathways Analysis (IPA, Ingenuity Systems, www.
ingenuity.com) was used to analyze the metabolomic data from
DG and CON groups and to obtain information about relationships,
biological mechanisms, functions, and pathways [30]. An Excel le
containing metabolite identiers and corresponding mean fold-
change values was uploaded into IPA. Those molecules previously
identied as being statistically signicant were overlaid onto net-
works developed from information contained in the Ingenuity
Pathways Knowledge Base. Networks of these metabolites were
algorithmically generated based on their connectivity. The activ-
ity of molecules highly connected to these networks was assessed
with the IPA molecule activity predictor.
3. Results
3.1. Behavioral testing
After 14 days of the different drug treatments, no differences
were observed in body weight (data not shown). The SPT has been
used extensively to assess stress-induced anhedonia [31]. After
drug treatments, the sucrose preference signicantly increased for
mice in the DG Medium, DG High, and VLX groups (all P < 0.05 com-
pared with the CON group; Fig. 1). No signicant effect on sucrose
preference was observed for mice in the DG Low.
Fig. 2 shows the results of the immobility time in the TST.
The duration of immobility was signicantly reduced in the DG
Medium group (P < 0.05 compared with the CON group), suggesting
an antidepressant-like effect of DG. Although there was also a trend
for a decrease in the duration of immobility in the VLX group, this
decrease was not statistically signicant.
As hypothesized, DG induced antidepressant-like effects in the
SPT and TST. Moreover, the medium-dose group showed a better
antidepressant-like effect, suggesting that the DG antidepressant-
like effect was dose related. Thus, we used this medium dose
to investigate the biological mechanisms underlying diterpene
ginkgolides exposure.
 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124
Fig. 2. The immobility time in the tail suspension test. Only the DG Medium group
shows a signicantly reduced duration of immobility in the test. *P < 0.05, compared
with the control (CON) group.
3.2. Metabolomic analysis
The total ion current chromatograms of all hippocampal sam-
ples displayed strong signals for analysis as well as large peak
capacity and good reproducibility of retention time. After exclud-
ing those from the internal standards, 229 individual peaks were
detected in each of the CON, DG, and VLX groups. These peaks were
used in the subsequent multivariate analysis.
The pairwise PLSDA score plots demonstrated statistical dif-
ferences between DG and CON groups (R2 X = 0.508, R2 Y = 0.786,
Q2 = 0.568; Fig. 3A), the VLX and CON groups (R2 X = 0.769,
R2 Y = 0.989, Q2 = 0.94; Fig. 3B), and the DG and VLX groups
(R2 X = 0.643, R2 Y = 0.846, Q2 = 0.618; Fig. 3C). To further validate the
Fig. 3. Metabolomic analysis of hippocampal samples from DG-treated (DG medium), VLX-treated, and saline-treated (CON) mice. PLSDA score plots for pairwise compar-
isons between DG and CON (A), VLX and CON (B), and DG and VLX (C). Statistical validation of the PLSDA model by permutation testing between DG and CON (D), VLX and
CON (E), and DG and VLX (F). Permutation tests (D, E, F) showed the original R2 and Q2 values (top right) were signicantly higher than the corresponding permuted values
(bottom left), demonstrating the PLS-DA models robustness.
119
quality of the PLSDA models, permutation tests were conducted
that consisted of a randomly permuting class membership and 200
iterations. The results shown in Fig. 3D, E, and F conrmed that the
constructed PLSDA models were robust and not overt.
Metabolites with VIP values >1.0 and P values <0.05 in every
group were deemed to be statistically signicant. A total of 18 dif-
ferential metabolites were differently expressed in the DG and CON
groups (Tables 1 and 2). Compared with the CON group, 10 metabo-
lites were upregulated in the DG group, including phosphate,
kynurenic acid, inosine, -aminobutyric acid, n-oleoyldopamine, l-
glutamine, l-cysteine, glycine, ethanolamine, and pyrophosphate.
By contrast, 8 metabolites were downregulated in the DG group
relative to the CON group, including lactic acid, pyroglutamic
acid, alanine, asparagine, O-phosphoethanolamine, N-acetyl-l-
aspartic acid, d-glycerol-1-phosphate, and N-acetyl-beta-alanine.
Among these 18 metabolites, 6 showed lower levels in the VLX
than in the CON group, including alanine, N-acetyl-beta-alanine,
asparagine, l-glutamine, glycine, and O-phosphoethanolamine,
whereas 6 metabolites were increased in the VLX relative to the
CON group, namely, phosphate, -aminobutyric acid, N-acetyl-l-
aspartic acid, inosine, d-glycerol-1-phosphate, and pyrophosphate.
Relative to the CON group, the changes in 7 metabolites in the
DG group were consistent with those in the VLX group, including
phosphate, alanine, asparagine, inosine, O-phosphoethanolamine,
-aminobutyric acid, N-acetyl-beta-alanine, and pyrophosphate.
These results indicated that DG and VLX might function as antide-
pressants through a similar pathway (Fig. 4).
3.3. Metabolites bioinformatics analysis
The initial IPA results showed that several of the observed
metabolites were involved in two key networks, the tumor necro-
sis factor (TNF) and extracellular signal-regulated kinase (ERK) 1/2
pathways (Fig. 5A, B). The results from the IPA database predicted
that the inhibition of TNF and the activation of ERK1/2 and the
NMDA receptor were critical for the therapeutic mechanisms of
DG in mouse hippocampus.
120 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124

Table 1
Key differential metabolites among DG, VLX and CON comparison groups.

Metabolites r.t(min) Mass DG vs. CON VLX vs. CON DG vs. VLX

Fold changea VIPb score t-test (p) Fold change VIP score t-test (p) Fold change VIP score t-test (p)

Phosphate 9.56 299 1.02 8.82 0.003 1.03 7.94 0.028

Lactic acid 6.39 117 0.87 5.99 0.009 0.89 5.65 0.035
Pyroglutamic acid 13.15 73 0.92 3.58 0.030
Alanine 6.79 116 0.82 2.82 0.008 0.81 2.61 0.004
Asparagine 7.72 147 0.31 2.69 0.041 0.34 2.51 0.049 1.14 1.44 0.001
Kynurenic acid 15.85 73 1.32 2.45 0.041 1.13 2.72 0.015
Inosine 24.2 73 1.25 2.25 0.004 1.19 1.27 0.002 1.17 1.98 0.010
O-phosphoethanolamine 14.94 299 0.74 2.04 <0.001 0.44 2.84 <0.001 1.7 3.3 <0.001
-aminobutyric acid 9.33 142 1.52 1.95 0.034 2.36 3 <0.001 0.64 5.13 <0.001
N-oleoyldopamine 9.11 93 2.63 1.7 0.000 1.8 1.65 0.001
N-acetyl-l-aspartic acid 14.91 116 0.94 1.68 0.004 1.09 2.41 0.049
L-glutamine 12.06 73 1.22 1.67 0.005 0.39 1.26 0.010 3.13 1.84 <0.001
D-glycerol-1-phosphate 14.51 299 0.89 1.41 0.022 1.16 1.47 0.048 0.81 2.79 0.013
l-cysteine 12.18 73 1.2 1.41 0.046 1.37 2.1 <0.001
Glycine 7.3 147 1.1 1.33 0.044 0.58 1.56 0.000 1.55 1.97 <0.001
Ethanolamine 8.3 133 1.22 1.25 0.026 1.48 1.06 <0.001
N-acetyl-beta-alanine 8.12 129 0.67 1.18 0.010 0.67 1.24 0.022
Pyrophosphate 14.31 73 1.44 1.13 0.005 2.29 1.98 <0.001 0.54 2.78 0.001
a
Values greater than 1 indicate higher levels in DG group relative to CON group; values less than 1 indicate higher levels in CON group relative to DG group, this comparison
method was also applicable in other groups.
b
Variable importance in the projection (VIP) was obtained from PLS-DA with a threshold of 1.0. The short dash line () indicates no signicant variation.

Table 2
A list of signicant metabolites accountable for class discrimination.

Metabolic pathways Metabolites DG vs. CON VLX vs. CON DG vs. VLX

Neurotransmitter metabolism, glutamate biosynthesis Phosphate

Neurotransmitter metabolism, glutamate biosynthesis Pyrophosphate
Neurotransmitter metabolism, glutamate biosynthesis l-glutamine
Neurotransmitter metabolism, aspartate metabolism Alanine
Neurotransmitter metabolism, aspartate metabolism N-acetyl-beta-alanine
Neurotransmitter metabolism, aspartate metabolism Asparagine
Neurotransmitter metabolism, aspartate metabolism N-acetyl-l-aspartic acid
Neurotransmitter metabolism -aminobutyric acid
Neurotransmitter metabolism Glycine
Neurotransmitter metabolism N-oleoyldopamine
Oxidative stress, glutathione metabolism Glycine
Oxidative stress, glutathione metabolism l-cysteine
Oxidative stress, glutathione metabolism Phosphate
Oxidative stress, glutathione metabolism Pyroglutamic acid
Lipid metabolism Ethanolamine
Lipid metabolism d-glycerol-1-phosphate
Lipid metabolism O-phosphoethanolamine
Energy metabolism Lactic acid
Energy metabolism Inosine
Other Kynurenic acid

The short dash line () indicates no signicant variation. These metabolites are veried by reference compounds available. The arrow denotes the VIP value greater than 1.0
and the up- (or down-) regulation of the arrow represents the relative increased (or decreased) concentration.

4. Discussion This study demonstrated that DG had antidepressant-like

Depression has been reported to be associated with neurotrans-
mitters and oxidative stress and can result in neurotransmitter
disorders, oxidation, and an immune response. The mechanisms
of antidepressants are also thought to be related to these mecha-
nisms [3234]. Earlier research ndings have shown that GBE has
antidepressant and antistress activities and is useful for reducing
depressive-like behavior [10,13,35]. However, GBE contains many
different ingredients. Although the diterpene ginkgolides are the
major component in these extracts, whether they have an antide-
pressant effect is still uncertain. Therefore, the present study used
two preclinical models widely used for assessing antidepressant-
like behavior (SPT and TST) to determine whether DG had an
antidepressant-like effect. A GCMS-based metabolomic approach
coupled with PLSDA statistical analysis was applied to investigate
the biological mechanisms of DG.
effects in the SPT and TST and revealed a set of 18 differen-
tial metabolites that clearly distinguished mice in the DG-treated
group from the controls, including 8 metabolites showing
changes consistent with those for mice in the venlafaxine-treated
group. To develop further insight into the underlying molec-
ular mechanisms, these differential metabolites were assigned
to the following three dominating categories according to
their biochemical functions: (i) neurotransmitter metabolism
(phosphate, pyrophosphate, l-glutamine, alanine, N-acetyl-beta-
alanine, asparagine, N-acetyl-l-aspartic acid, -aminobutyric acid,
glycine, N-oleoyldopamine); (ii) oxidative stress and glutathione
metabolism (glycine, l-cysteine, phosphate, pyroglutamic acid);
(iii) lipid metabolism (ethanolamine, d-glycerol-1-phosphate, O-
phosphoethanolamine), energy metabolism (lactic acid, inosine),
and kynurenic acid.
 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124 121

Fig. 4. Heat map of the differential metabolites in CON, DG Medium, and VLX groups.

Fig. 5. Networks associated with DG treatment identied by Ingenuity Pathway Analysis. The activities of molecules highly connected to these networks were assessed with
the IPA molecule activity predictor. The IPA database predicts the downregulation of TNF and upregulation of ERK1/2 and NMDA receptors.

4.1. Neurotransmitter metabolism signicantly decreased in depression [28,36,37]. There is extant

The most signicant alterations were attributed to changes in
neurotransmitter metabolism. These neurotransmitters can be cat-
egorized into three classes: glutamate biosynthesis (phosphate,
pyrophosphate, and l-glutamine), aspartate metabolism (alanine,
N-acetyl-beta-alanine, asparagine, and N-acetyl-l-aspartic acid)
and others (-aminobutyric acid, glycine, and N-oleoyldopamine).
The biosynthesis of hippocampal glutamate, an important exci-
tatory neurotransmitter in the mammalian CNS, was increased in
DG-treated mice, as demonstrated by the upregulation of phos-
phate, pyrophosphate, l-glutamine. Previous studies by others and
our research group have revealed that glutamate metabolism is
evidence that the glutamatergic system has been an effective tar-
get for treating major depressive disorder [38,39]. The increase of
glutamate biosynthesis in our study suggested that DG might exert
its antidepressant effect by reversing the alterations of glutamate
biosynthesis in the hippocampus.
A signicant decrease in aspartate metabolism was also
responsible for discriminating the metabolic proles between the
DG-treated mice and the controls. Alanine, N-acetyl-beta-alanine,
and asparagine are synthetic materials for N-acetyl-l-aspartic acid
(NAA). NAA, which is considered a marker for neuronal density and
synaptic proliferation [40,41], is altered in patients with depres-
sion and animal models of depression [25,37,42,43]. This study as
122 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124

well as others and our own previous metabolomic research studies gesting that DG may relieve the dysregulated immune responses
have shown that aspartate metabolism is upregulated in the brains observed in depression.
of rats subjected to chronic unpredictable mild stress, a rat model
of depression [25,37], and down-regulated by antidepressants [2].
4.3. Lipid metabolism, energy metabolism and other metabolite
Additionally, we found in the present study that the changes in
aspartate metabolism in the VLX-treated group were mostly con-
Our data showed a decrease in the levels of two lipid
sistent with those in the DG-treated group.
metabolism-related molecules, namely, d-glycerol-1-phosphate
NAA acts as a precursor for the synthesis of N-
and o-phosphoethanolamine, and an increase in a lipid catabolite
acetylaspartylglutamate (NAAG), the most abundant peptide
(ethanolamine) in DG-treated mice relative to those in controls.
transmitter in the mammalian nervous system [42]. It is an agonist
As previously described, several studies have determined that
at presynaptic metabotropic glutamate receptors (mGluR3), acting
lipid levels can be inuenced by depression and antidepres-
to inhibit glutamate release [25,42]. This effect may explain our
sants [2,48,58,59]. Based on our data, total lipid levels should be
current ndings for an increase in glutamate biosynthesis but a
decreased, indicating that DG may exert an antidepressant effect
decrease in aspartate metabolism.
by reducing lipid metabolism.
The inhibitory neurotransmitter -aminobutyric acid (GABA)
Additionally, energy metabolism-related molecules (lactic acid
and GABAergic neurons are sensitive to glutamate elevation [44].
and inosine) were also affected by DG. Although lactic acid can
Thus, an elevation of glutamate may stimulate GABAergic neu-
serve as an energy substrate for neurons, it is also a toxic metabo-
rons to release GABA. Since 1999, when Gerard Sanacora showed
lite of anaerobic respiration. The downregulation of lactic acid
that -aminobutyric acid levels are reduced in depressed patients
might mean that DG reduced the metabolic waste of anaerobic
[45], increasing evidence has indicated that MDD is associated with
metabolism. Another important metabolite, inosine, has been used
altered functions of the major excitatory and inhibitory neurotrans-
widely to protect various kinds of cells in many diseases, including
mitters, glutamate and GABA, respectively [46,47]. The elevated
those of the CNS [60]. Inosine preserves the viability of glial cells and
hippocampal GABA levels we observed here in the DG- and VLX-
neuronal cells during hypoxia and stimulates axonal regrowth after
treated mice suggested that DG might exert an antidepressant
injury [61]. Recent studies have revealed that inosine has a potential
effect by upregulating GABA. Glycine, another inhibitory amino acid
antidepressant effect via adenosine receptors [62,63], this suggests
neurotransmitter, has been shown in our previous metabolomics
that DG may exert its antidepressant effect by upregulating the
ndings to be signicantly decreased in patients with MDD and
level of inosine.
rats subjected to chronic unpredictable mild stress [28,48]. Inter-
Kynurenic acid, which is produced mainly by astrocytes, is pro-
estingly, in this study, glycine levels could be reversed by DG.
tective against the excitotoxic action of quinolinic acid [64,65].
N-oleoyldopamine is in the family of lipid-soluble dopamine (DA)
The apoptosis of astrocytes could thus mean lower neuroprotec-
compounds. N-oleoyldopamine may enhance the normally limited
tive action against the neurodegenerative quinolinic acid, leading
DA transport into the brain, which would be of therapeutic poten-
to a loss of neurons [65]. The plasma kynurenic acid concentration
tial in the pathological states characterized by a deranged brain
in depressed patients has been shown to be lower than that in con-
neurotransmitter prole [49].
trol participants, suggesting that the upregulated level of kynurenic
Based on the results of the IPA, elevated levels of glutamate
acid caused by DG might help reverse depression in these patients.
biosynthesis, GABA, and glycine could activate ERK1/2 and NMDA
However, the results and conclusions of our study should be
receptors, both of which have been reported as highly correlated
cautiously interpreted because of some study limitations. First, the
with the pathogenesis of depression.
sample size in this study was relatively small, so future studies with
larger cohorts should be performed to validate our ndings. Second,
the current data were obtained in nave mice; additional studies
4.2. Oxidative stress and neuroimmune response
should investigate using an animal model of depression. Third, the
antidepressant components of the diterpene ginkgolides need to be
Levels of four oxidative stress-related metabolites (glycine, l-
investigated by comparing the effects of ginkgolides A, B, C, and K in
cysteine, phosphate, and pyroglutamic acid) were signicantly
an animal model of depression. Finally, owing to the diverse physic-
affected in DG-treated mice compared with those in CON mice
ochemical properties and wide concentration range of metabolites,
in the present study. Upregulation of three metabolites (glycine,
additional metabolomic methods or multiple metabolomic plat-
l-cysteine, and phosphate) has been suggested to increase
forms (i.e., stable isotope-resolved metabolomic analysis, nuclear
glutathione (GSH) biosynthesis, with the downregulation of pyrog-
magnetic resonance, liquid chromatographymass spectrometry,
lutamic acid reecting a lower degradation of GSH. GSH serves
and targeted metabolomics) should be employed in future studies
as an antioxidant, preventing excessive oxidation of sensitive
[6669].
cellular components, and is a substrate for various antioxidant
enzymes [50]. Loss of glutathione and oxidative damage could
constitute early, possibly signaling events, in apoptotic cell death 5. Conclusions
[51]. Evidence has shown that depressed patients and animals sub-
jected to stress had a decreased antioxidant status [52,53]. Gingko In summary, the administration of diterpene ginkgolides led
biloba extract and antidepressants both have antioxidant proper- to antidepressant-like effects in mice subjected to the SPT and
ties [54,55]. Elevated glutathione metabolism may suggest that the TST. Comparative metabolomic proling revealed signicant alter-
antidepressant effect of DG may be mediated through the antioxi- ations in levels of neurotransmitter metabolism, oxidative stress,
dant system. glutathione metabolism, lipid metabolism, energy metabolism, and
It is generally acknowledged that oxidative stress is accom- kynurenic acid in the hippocampus of DG-treated mice compared
panied by inammatory/immune responses. Previous studies by with saline-treated mice. Thus, DGs may exert their antidepressant
others and our research group have determined that neuroinam- effect through reversing the alterations in glutamate biosynthesis,
mation plays a causal role in the development of MDD and the aspartate metabolism, oxidative stress, neuroinammation, and
antidepressant response [56,57]. Our IPA molecule activity pre- lipid and energy metabolism in the hippocampus. Moreover, DG
dictor analysis found that TNF was inhibited in DG-treated mice, administration enhanced GABA, inosine, and kynurenic acid levels,
indicating an inhibition of the inammatory response and sug- which may have contributed to the antidepressant effect. Through
 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124
determining the antidepressant metabolic changes incident to DG
treatment in mice, this study also provided directions for future
exploration on the effects of DG exposure at the intracellular level
and in humans.
Conict of interest
All authors declare no conicts of interest.
Author contributions
Z.L., S.B. and P.X. conceived the study. The experiments were
performed by P.S., X.W. and Q.H. The data analysis was performed
by M.D., W.W., J.L., K.C. and H.W. The manuscript was revised by
D.Z. and Y.H.
Acknowledgements
This work was supported by the National Basic Research
Program of China (973 Program, Grant no. 2009CB918300),
the National Natural Science Foundation of China (Grant No.
31300917), the Natural Science Foundation of Inner Mongolia
Autonomous Region of China (Grant No. 2016MS0884) and the
Foundation Project of the Inner Mongolia Autonomous Region Peo-
ples Hospital (Grant No. 201551).
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