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GC-MS-Based Metabolomic Study on the

Antidepressant-Like Effects of Diterpene
Ginkgolides in Mouse...

Article in Behavioural brain research August 2016

DOI: 10.1016/j.bbr.2016.08.001

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Behavioural Brain Research

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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: [email protected] (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 117

the brain [2]. Despite a vast number of established antidepressant 2. Materials and methods
medication options for MDD, a signicant percentage of patients
fail to respond to these rst-line treatments [4]. In addition, the 2.1. Animals
efcacy and safety of antidepressant medications remain contro-
versial, and an increased risk of suicidal behavior exists in patients This study was approved by the Ethics Committee of Chongqing
treated with some of these medications [5,6]. Owing to the limited Medical University, and all procedures were conducted in accor-
therapy options, treatment of MDD is still a major focus of current dance with the National Institutes of Healths Guide for the Care
biomedical research, with the investigation of alternative treat- and Use of Laboratory Animals. C57BL/6J male mice (age, 8 weeks;
ments urgently needed to reduce the burden of MDD on patients, weight, 1820 g) were obtained from Chongqing Medical Univer-
families, and society. sitys animal center in the Peoples Republic of China. Throughout
The reputed efcacy, low cost, and relative absence of side the study, all mice were maintained in a light, temperature, and
effects of traditional Chinese medicines have led to increasing humidity controlled environment (12-h light/dark cycle, 23 1 C,
interest in the use of them for the treatment of neuropsychiatric 50 5%) with access to food and water ad libitum.
disorders. GBE termed EGb761 is a patented extract from the leaves
of the Ginkgo biloba tree. Since the development of GBE in 1964, 2.2. Reagents
its effects on an extensive range of disorders and diseases have
been investigated [7]. GBE has been suggested to have many ben- DGMI (25 mg/mL, containing ginkgolides A, B, and K at
ecial effects on central nervous system (CNS) function and is 1.70, 3.10, and 0.20 mg/mL, respectively) was provided by
widely used for treating neurologic, psychiatric, functional, and Jiangsu Kanion Pharmaceutical Co., Ltd. Pyridine, methoxyamine
physiologic symptoms [79]. Recent work has indicated that some hydrochloride, l-2-chlorophenylalanine, venlafaxine, and
components of GBE may act as an antidepressant and improve N,O-bis(trimethylsilyl)triuoroacetamide (BSTFA) with 1%
spatial learning and memory [1014]. Meanwhile, GBE is a mix- trimethylchlorosilane (TMCS) were purchased from Sigma-
ture of substances with a wide variety of physical and chemical Aldrich. HPLC-grade methanol and chloroform were purchased
properties and activities. Numerous pharmacological investiga- from the Chongqing Chuandong Chemical (Group) Co. Ultrapure
tions have led to the conclusion that DG mainly composed of water was prepared using a Millipore Milli-Q purication system.
ginkgolide A, B and K in EGb761, are responsible for the main
pharmacological neuroprotective effects [7,12,15,16]. Therefore, 2.3. Treatment
it is reasonable to propose that DG may be effective in treating
depression. After one week of habituation, the mice were randomly divided
Diterpene Ginkgolides Meglumine Injection (DGMI), a novel into the following ve groups: (1) healthy control (CON, 0.9% NaCl
medicinal plant products (manufactured by Jiangsu Kanion Phar- solution, n = 9), (2) low-dose DG (DG Low, DGMI diluted in 0.9%
maceutical Co., Ltd.) has been awarded marketing approval from NaCl solution to a dose of 4.06 mg/kg, n = 10), (3) medium-dose
the China Food and Drug Administration for treating patients with DG (DG Medium, DGMI diluted in 0.9% NaCl solution to a dose
mild to moderate cerebral infarction [17,18]. This injection con- of 12.18 mg/kg, n = 11), (4) high-dose DG (DG High, DGMI diluted
tains the vast majority of DG from GBE (mainly ginkgolide A, B and in 0.9% NaCl solution to a dose of 36.54 mg/kg, n = 11), and (5)
K) [17]. Therefore, DGMI is an ideal t to study the antidepressant venlafaxine (VLX, venlafaxine diluted in 0.9% NaCl solution to a
function of DG. dose of 16 mg/kg, n = 11). Mice were injected intraperitoneally daily
The hippocampus is a brain region that has been extensively between 8:00 AM and 10:00 AM with the same amount of solution
studied in patients with mood disorders and has been selected as for 14 days. Venlafaxine was used as positive control drug to explore
a relevant region for studying MDD molecular pathways [19,20]. the efcacy and mechanism of the DG effects.
Previous studies have shown that hippocampal dendritic or spine
remodeling is dysregulated in stress-induced depression and stress 2.4. Behavioral testing
resilience [21,22]. Furthermore, hippocampal plasticity is not only
altered in patients with MDD and in stressed animals but also After 14 days of drug treatment, mice were weighed and sub-
affected by antidepressant treatments. jected to the SPT, as a means to measure hedonic behavior, and
Recently, metabolomics has been widely used to discover then the TST. Mice were habituated to the testing room for 30 min
the molecular biomarkers for diagnosing or prognosing disease, before behavioral analyses began.
and to prole potential pathophysiological mechanisms and to Mice were habituated to 1% sucrose solution for 72 h before the
assess therapeutic effects of antidepressants [2,2325]. Compared SPT [26]. After 24 h of water and food deprivation, a two-bottle pref-
with methods such as nuclear magnetic resonance or liquid erence test was used in which all mice had access to both water and
chromatographymass spectrometry, gas chromatographymass a 1% sucrose solution for 24 h during a no-stress period. Sucrose
spectrometry (GCMS) has been widely applied because of its high preference was scored using the following formula: sucrose pref-
sensitivity, peak resolution, and reproducibility [25]. erence (%) = (sucrose intake/total intake) 100.
In the present study, we investigated the antidepressant- The tail suspension test was performed as previously described
like effects of DG in C57BL/6J mice using the measures of [27]. Briey, mice were suspended by a small metal hook xed
SPT and TST. We then applied a non-targeted metabolomics with adhesive tape wrapped around the tail. After 120 s of adapta-
approach based on GCMS technology to characterize the global tion, the duration of immobility was recorded with a video-tracking
metabolic prole in mouse hippocampus following the sys- system (SMART, Panlab SL, Barcelona, Spain) for 4 min. Mice were
temic administration of DG and compared it with the prole returned to their home cages after every animal was tested.
obtained following venlafaxine administration. Our primary aims
were to determine whether DG induced an antidepressant-like 2.5. Tissue collection and sample preparation for GCMS analysis
effect, to identify probable biological mechanisms underly-
ing this effect of DG, and to search for potential therapeutic Twenty-four hours after the administration of the nal drug or
targets for MDD, providing new directions for future stud- NaCl, mice in the CON, DG (DG Medium), and VLX groups were
ies. 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- to be statistically signicant. The SPSS software package (SPSS pro-
tem. The GC capillary column was an Agilent J&W HP-5 ms UI gram, version 21.0) was used for all statistical tests. The graphs
(30 m 0.25 mm 0.25 m). The temperatures of the injector, the were generated using Prism 5.0 (GraphPad Software, Inc. USA).
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
2.9. Bioinformatics analysis
was applied for metabolite separation. The column temperature
was initially kept at 70 C for 2 min and increased to 240 C at a
Ingenuity Pathways Analysis (IPA, Ingenuity Systems, www.
rate of 10 C min1 , then raised to 300 C and maintained at that
ingenuity.com) was used to analyze the metabolomic data from
temperature for 6 min. MS spectra were acquired m/z 50600.
DG and CON groups and to obtain information about relationships,
biological mechanisms, functions, and pathways [30]. An Excel le
2.7. Identication of the metabolites and metabolomic data
containing metabolite identiers and corresponding mean fold-
analysis
change values was uploaded into IPA. Those molecules previously
identied as being statistically signicant were overlaid onto net-
Low molecular weight metabolites were represented as chro-
works developed from information contained in the Ingenuity
matographic peaks in total ion current chromatograms. A total of
Pathways Knowledge Base. Networks of these metabolites were
229 individual peaks with intensities threefold higher than the
algorithmically generated based on their connectivity. The activ-
signal-to-noise ratio were recorded and integrated. In this study,
ity of molecules highly connected to these networks was assessed
compound annotation was performed by comparing the accurate
with the IPA molecule activity predictor.
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 3. Results
TagFinder [29]. The resulting three-dimensional matrix contain-
ing peak indexes (RT-m/z pairs), sample names (observations), and 3.1. Behavioral testing
normalized peak intensities were introduced into SIMCA-P 11.5
software (Umetrics AB, Umea, Sweden). After 14 days of the different drug treatments, no differences
Partial least squares discriminant analysis (PLSDA) with UV- were observed in body weight (data not shown). The SPT has been
scaled spectral data was performed to visually discriminate mice used extensively to assess stress-induced anhedonia [31]. After
in the CON, DG, and VLX groups. R2 Y and Q2 were used to quantify drug treatments, the sucrose preference signicantly increased for
the goodness of t and predictability of the model, respectively. mice in the DG Medium, DG High, and VLX groups (all P < 0.05 com-
Parameter values approaching 1.0 indicated a stable model with pared with the CON group; Fig. 1). No signicant effect on sucrose
predictive reliability. The discriminating metabolites were identi- preference was observed for mice in the DG Low.
ed using a statistically signicant threshold of variable inuence Fig. 2 shows the results of the immobility time in the TST.
on projection (VIP) values obtained from the PLSDA model and a The duration of immobility was signicantly reduced in the DG
two-tailed Students t-test. The heat map of differential metabo- Medium group (P < 0.05 compared with the CON group), suggesting
lites was obtained using MetaboAnalyst 3.0 (www.metaboanalyst. an antidepressant-like effect of DG. Although there was also a trend
ca) for each metabolite. for a decrease in the duration of immobility in the VLX group, this
decrease was not statistically signicant.
2.8. Statistical analysis for behavioral tests As hypothesized, DG induced antidepressant-like effects in the
SPT and TST. Moreover, the medium-dose group showed a better
The data from the sucrose preference test and tail suspension antidepressant-like effect, suggesting that the DG antidepressant-
test are expressed as means SEM. All data were analyzed using like effect was dose related. Thus, we used this medium dose
one-way analyses of variance (ANOVA) followed by the post hoc to investigate the biological mechanisms underlying diterpene
least signicant difference test. Values of P < 0.05 were considered ginkgolides exposure.
 Z. Liang et al. / Behavioural Brain Research 314 (2016) 116124 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-
Fig. 2. The immobility time in the tail suspension test. Only the DG Medium group
aspartic acid, inosine, d-glycerol-1-phosphate, and pyrophosphate.
shows a signicantly reduced duration of immobility in the test. *P < 0.05, compared
with the control (CON) group. 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,
3.2. Metabolomic analysis -aminobutyric acid, N-acetyl-beta-alanine, and pyrophosphate.
These results indicated that DG and VLX might function as antide-
The total ion current chromatograms of all hippocampal sam- pressants through a similar pathway (Fig. 4).
ples displayed strong signals for analysis as well as large peak
capacity and good reproducibility of retention time. After exclud- 3.3. Metabolites bioinformatics analysis
ing those from the internal standards, 229 individual peaks were
detected in each of the CON, DG, and VLX groups. These peaks were The initial IPA results showed that several of the observed
used in the subsequent multivariate analysis. metabolites were involved in two key networks, the tumor necro-
The pairwise PLSDA score plots demonstrated statistical dif- sis factor (TNF) and extracellular signal-regulated kinase (ERK) 1/2
ferences between DG and CON groups (R2 X = 0.508, R2 Y = 0.786, pathways (Fig. 5A, B). The results from the IPA database predicted
Q2 = 0.568; Fig. 3A), the VLX and CON groups (R2 X = 0.769, that the inhibition of TNF and the activation of ERK1/2 and the
R2 Y = 0.989, Q2 = 0.94; Fig. 3B), and the DG and VLX groups NMDA receptor were critical for the therapeutic mechanisms of
(R2 X = 0.643, R2 Y = 0.846, Q2 = 0.618; Fig. 3C). To further validate the DG in mouse hippocampus.

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.
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

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

evidence that the glutamatergic system has been an effective tar-
The most signicant alterations were attributed to changes in get for treating major depressive disorder [38,39]. The increase of
neurotransmitter metabolism. These neurotransmitters can be cat- glutamate biosynthesis in our study suggested that DG might exert
egorized into three classes: glutamate biosynthesis (phosphate, its antidepressant effect by reversing the alterations of glutamate
pyrophosphate, and l-glutamine), aspartate metabolism (alanine, biosynthesis in the hippocampus.
N-acetyl-beta-alanine, asparagine, and N-acetyl-l-aspartic acid) A signicant decrease in aspartate metabolism was also
and others (-aminobutyric acid, glycine, and N-oleoyldopamine). responsible for discriminating the metabolic proles between the
The biosynthesis of hippocampal glutamate, an important exci- DG-treated mice and the controls. Alanine, N-acetyl-beta-alanine,
tatory neurotransmitter in the mammalian CNS, was increased in and asparagine are synthetic materials for N-acetyl-l-aspartic acid
DG-treated mice, as demonstrated by the upregulation of phos- (NAA). NAA, which is considered a marker for neuronal density and
phate, pyrophosphate, l-glutamine. Previous studies by others and synaptic proliferation [40,41], is altered in patients with depres-
our research group have revealed that glutamate metabolism is 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 123

determining the antidepressant metabolic changes incident to DG [17] Y.J. Li, Simultaneous determination of ginkgolides A, B and K in human plasma
treatment in mice, this study also provided directions for future by UPLCMS/MS and its application to the pharmacokinetic study of Ginkgo
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cell survival in adult hippocampal neurogenesis without altering
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[20] J.E. by Malberg, R.S. Duman, Cell proliferation in adult hippocampus is
decreased by inescapable stress: reversal by uoxetine treatment,
Author contributions Neuropsychopharmacology (2010).
[21] X. Han, W. Shao, Z. Liu, S. Fan, J. Yu, J. Chen, et al., iTRAQ-based quantitative
analysis of hippocampal postsynaptic density-associated proteins in a rat
Z.L., S.B. and P.X. conceived the study. The experiments were chronic mild stress model of depression, Neuroscience 298 (2015) 220292.
performed by P.S., X.W. and Q.H. The data analysis was performed [22] J. Zhou, Z. Liu, J. Yu, X. Han, S. Fan, W. Shao, et al., Quantitative proteomic
analysis reveals molecular adaptations in the hippocampal synaptic active
by M.D., W.W., J.L., K.C. and H.W. The manuscript was revised by
zone of chronic mild stress-unsusceptible rats, Int. J. Neuropsychopharmacol.
D.Z. and Y.H. (2016) 19.
[23] P. Zheng, Y. Wang, L. Chen, D. Yang, H. Meng, D. Zhou, et al., Identication and
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Program of China (973 Program, Grant no. 2009CB918300),
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the National Natural Science Foundation of China (Grant No. dysfunction revealed in the prefrontal cortex of a rat model of depression,
31300917), the Natural Science Foundation of Inner Mongolia Behav. Brain Res. 278 (2015) 286292.
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