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Folic acid exerts antidepressant effects by upregulating brain-derived

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DOI: 10.1097/WNR.0000000000000887

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1078 Cellular, molecular and developmental neuroscience
Folic acid exerts antidepressant effects by upregulating
brain-derived neurotrophic factor and glutamate receptor 1
expression in brain
Liangcai Gaoa,*, Xinnan Liua,*, Li Yua, Junlin Wua, Mingchu Xua,b and Yusi Liua
Folic acid is a vitamin with a variety of pharmacological
effects. The present study aims to explore the beneficial
effects of folic acid on chronic unpredictable mild stress
(CUMS)-induced depression-like behaviors and its possible
mechanisms. The behavioral tests including open-field test,
tail suspension test, and forced swimming test were used to
evaluate the antidepressant effects of folic acid. Then the
changes of brain 5-hydroxytryptamine (5-HT) concentration,
brain-derived neurotrophic factor (BDNF), glutamate
receptor 1 (GluR1) expression levels, and synaptic
organization were assessed to explore the antidepressant
mechanisms of folic acid. Our results showed that CUMS
caused significant depression-like behaviors,
neuropathological changes, and decreased brain 5-HT
concentration, BDNF, and GluR1 expression in the
hippocampus and association cortex. In conclusion, the
results showed that folic acid significantly improved
depression-like behaviors in CUMS-induced rats, and its
Introduction
Depression is a type of mental disorder involving emotional,
cognitive, and physical symptoms with considerable mor-
bidity and mortality [1,2]. WHO forecasts that depression
will be the second highest disease to threaten human’s
health. Depression can be induced by diverse factors,
including psychological, social, environmental, genetic, and
metabolic factors. Clinical depression is characterized by low
mood, anhedonia, reduced cognition, low or impaired psy-
chomotor activity, and sleep disturbance [3].
Currently, the hypotheses on the pathological mechan-
ism of depression are mainly based on the monoamine
deficiency [4], neurotrophin deficiency [5], and
hypothalamic–pituitary–adrenal axis dysfunction [6].
Several studies have shown that long-term exposure to
chronic unpredictable mild stress (CUMS) can cause
decreasing level of serotonin [5-hydroxytryptamine
(5-HT)] in the hippocampus, thus leading to memory
deficits and inducing anxiety-like behaviors [7–9].
Selective serotonin reuptake inhibitors such as fluox-
etine, the most widely prescribed classes of anti-
depressants, have been a first-line pharmacological
therapy for depression [10,11]. In addition, preclinical
research revealed similar trends in that high levels of
stress hormones are known to increase depression-like
behaviors in rodents [12]. In addition, glucocorticoids
have deleterious effects on neurochemistry and
0959-4965 Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.
Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved.
antidepressant effects might be related to the increase of
brain 5-HT concentration, BDNF and GluR1 expression, and
repair of synaptic organization in the brain. NeuroReport
28:1078–1084 Copyright © 2017 Wolters Kluwer Health, Inc.
All rights reserved.
NeuroReport 2017, 28:1078–1084
Keywords: antidepressant, brain-derived neurotrophic factor, folic acid,
glutamate receptor 1
a
Department of Biomedical Sciences, School of Life Science, East China Normal
University, Shanghai, China and bDepartment of Molecular and Human Genetics,
Baylor College of Medicine, Houston, Texas, USA
Correspondence to Liangcai Gao, PhD, School of Life Science, East China
Normal University, Shanghai 200241, China
Tel: + 86 215 434 4130; fax: + 86 215 434 1006; e-mail: lcgao@bio.ecnu.edu.cn
*Liangcai Gao and Xinnan Liu contributed equally to the writing of this article.
Received 7 July 2017 accepted 9 August 2017
neuroanatomy, and many of the behavioral and neurobiolo-
gical changes produced by repeated glucocorticoid adminis-
tration can be reversed by antidepressant treatments [13–15].
Brain-derived neurotrophic factor (BDNF), a member of
the ‘neurotrophin’ family of growth factors, is distributed
throughout the central nervous system [16]. Indeed,
several studies have shown that the BDNF level in brain
areas is reduced in patients with depression, whereas
treatment with antidepressants could elevate BDNF
levels in the brain [17,18]. Recently, a number of studies
have shown that the glutamatergic system may be a novel
target for the treatment of major depressive disorder.
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor is likely to be involved in actions of anti-
depressants [19].
Folic acid (folate) is a water-soluble vitamin B and its
biologically active form is tetrahydrofolic acid. Tetra-
hydrofolic acid participates in the transfer of 1-carbon
units (such as methyl, methylene, and formyl groups) to
the essential substrates involved in the synthesis of
DNA, RNA, and proteins [20]. Several clinical studies
have revealed the correlation between depressive dis-
order and low folic acid levels [21]. However, it remains
unknown whether folic acid possesses antidepression
effects.
DOI: 10.1097/WNR.0000000000000887

In this study, we used the CUMS rat model to explore
the antidepressant-like effect of folic acid and the
underlying mechanisms. The effects of folic acid on
5-HT, corticosterone, BDNF, and glutamate receptor 1
(GluR1) were studied in rats exposed to CUMS.
Materials and methods
Animals
A total of 50 male Sprague-Dawley rats weighing
180–220 g were purchased from the Department of
Experimental Animals (Fudan University, Shanghai,
China). The animals were housed at 22 ± 2°C with free
access to food and water, under a 12 : 12 h light/dark cycle
(lights on at 08:00 h). All experimental methods were
approved by the Institutional Review Boards of East
China Normal University, and they were performed in
accordance with relative guidelines and regulations.
Drugs and administration
Folic acid (50, 75 mg/kg; Sinopharm Group Co. Ltd,
Shanghai, China) was dispersed in 0.5% sodium carbox-
ymethyl cellulose (CMC-Na solution). All drugs and
vehicles (0.5% CMC-Na) were administered through the
oral route at a volume of 10 ml/kg of body weight. Before
the CUMS procedures, all rats were subjected to the
open-field tests. Rats were randomly divided into four
groups (n = 12) on the basis of their test scores: three test
groups, which received folic acid (50 and 75 mg/kg) and
CUMS (0.5% CMC-Na), and a control group (0.5%
CMC-Na). All drug treatment groups were treated once a
day 1 h after the end of the CUMS procedures.
Chronic unpredictable mild stress procedure
The CUMS procedure was performed as described by
Zhao et al. [22] with minor modifications. All groups
except for the control group were exposed to CUMS.
The rats were subjected to a variety of mild stressors:
fasting for 24 h; water deprivation for 24 h; tail pinching
with a clothes-pin placed 1 cm distal from the base of the
tail for 1 min; shaking for 5 min (160 rpm rocking bed);
damp sawdust (200 ml of water in 100 g of sawdust) for
12 h; swimming in 4°C cold water for 5 min; and altera-
tion of light and dark cycles. One of these stressors (in
random order) was given every day for 4 weeks.
Behavioral test
Sucrose preference test
The procedure was performed as described previously
[23], with minor modifications. Sucrose preference test
(SPT) was carried out after the drug administration. All
rats were trained to adapt to the sucrose solution: 24-h
exposure to two bottles of sucrose solution and an addi-
tional 24-h exposure to one bottle of sucrose solution and
one bottle of water. Before the tests, the rats were
deprived of food and water for 12 h. SPT was conducted
for 24 h, during which period the rats were housed in
individual cages and could freely access two bottles, one
Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved.
Folic acid exerts antidepressant effects Gao et al. 1079
being 100 ml of water and the other 100 ml of 1% (w/v)
sucrose solution. The sucrose preference was calculated
using the following formula: the sucrose preference
(%) = (sucrose consumption)/(sucrose consumption +
water consumption) × 100%.
Forced swimming test
The forced swim test (FST) was performed as described
[24] with minor modifications. In brief, individual rats
were forced to swim for 5 min in a transparent plastic
vessel (diameter 26 cm, height 50 cm) filled with 30 cm of
water (25 ± 1°C). The immobility time was counted
during a test period of 6 min (prior 1 min for rats to adapt
and the last 5 min were recorded) using a chronograph.
The immobility time was defined as the duration a rat
floating in the water without struggling and making only
small movements to keep its head above the water.
Tail suspecting test
The tail suspecting test (TST) was performed based on
the previous method [25]: the rat was hung 25 cm above
the floor by the tip of the tail (1 cm) tied up to the level.
The immobility time was counted during a test period of
6 min (prior 1 min for rats to adapt and the last 5 min were
recorded) using a chronograph. In addition, only when
the rat hung passively and completely motionless, it
could be regarded as immobile. Rats that climbed their
tails during the trials were excluded from data analysis.
Blood sampling and tissue extraction
After the final TST, all animals were left without any
treatment until the following morning. The rats were
decapitated, and blood samples were collected into
heparinized tubes and centrifuged at 3000g for 15 min at
4°C. Serum samples were stored at − 20°C. The hippo-
campus and association cortex were quickly removed
from the brains, weighed, frozen in liquid nitrogen, and
transported to − 80°C until assays were performed.
Quantitative reverse transcription-PCR of brain-derived
neurotrophic factor and glutamate receptor 1
Total RNA was extracted from the hippocampus using a
Trizol reagent kit (Invitrogen, Carlsbad, California,
USA), according to the manufacturer’s instructions.
cDNA was synthesized by OligodT primer (Takara
Biotechnology Co. Ltd, Dalian, China). The real-time
primers for the BDNF (forward: 5′-TCATACTTC
GGTTGCATGAAGG-3′; reverse: 5′-AGACCTCTCGA
ACCTGCCC-3′) and the real-time primers for the
GluR1 (forward: 5′-GTTCTGGCAACATCGCTT-3′;
reverse: 5′-CTCACTTCTCCTTTCCGTATG-3′) were
designed by Primer 3 software (ABI, Shanghai, China).
Real-time quantitative PCR was performed with an
CFX96Touch Real-Time PCR Detection System
(ABI, Shanghai, China) using the SYBR Green PCR
Master Mix (ABI, Shanghai, China). Samples were
compared using relative CT method. The fold increase
1080 NeuroReport 2017, Vol 28 No 16
or decrease was determined relative to a vehicle-treated
control after normalizing to a housekeeping gene using
the 2 DDCt method.
Enzyme-linked immunosorbent assay
After the final open-field test, all animals were left without
any treatment until the following morning. The rats were
decapitated, and blood samples were collected into hepar-
inized tubes and centrifuged at 3000g for 15 min at 4°C.
Serum samples were stored at − 20°C. The 5-HT of the
rats’ hippocampus were measured with a commercially
available ELISA kit (Enzyme-linked Biotechnology
Company, Shanghai, China). Serum corticosterone levels
were measured using an ELISA kit (Enzyme-linked
Biotechnology Company, Shanghai, China).
Electron microscopic study
The brains were removed from the skull and placed in 2.5%
glutaraldehyde overnight. The hemispheric tissue blocks
containing hippocampi were cut into 400-µm-thick coronal
slices. Slices were washed in 0.1 MPB and kept in 2.5%
glutaraldehyde in 0.1 MPB until processing. When proces-
sing, the slices were washed in 0.1 MPB, postfixed in 1%
osmium tetroxide in 0.1 MPB for 2 h, and again washed in
0.1 MPB. The hippocampus was identified with a light
microscope Leica MMAF (Hitachi High-Technologies
Corporation, Beijing, China), cut out from the coronal slices,
dehydrated in a graded series of ethanol and acetone, and
embedded in araldite. Blocks were trimmed and 70–75-nm-
thick sections were cut with an ultramicrotome, picked up
on 200-mesh copper grids, double-stained with uranyl
acetate and lead citrate, and examined with H7700 trans-
mission electron microscopes (Hitachi High-Technologies
Corporation, Beijing, China).
Asymmetric spine synapses were counted according to
the rules of the dissector technique within an unbiased
counting frame superimposed onto each electron micro-
graph. The average volumetric density (synapse/μm3) of
spine synapses within each sampling area was then
determined by dividing the sum of spine synapses
counted in all samples taken from that particular sam-
pling area by the dissector volume. The dissector volume
was calculated by multiplying the area of the unbiased
counting frame (79 μm2) by ultrasection thickness (aver-
age 75 nm) and by the number of dissectors. Finally, the
volumetric density of spine synapses was multiplied by
the volume of the sampling area, determined earlier, to
arrive at the total estimated number of spine synapses.
Statistical analysis
All data were normally distributed and are presented as
mean ± SEM. Statistical analyses were performed using
SPSS statistical software, version 17.0 (SPSS Inc,
Chicago, Illinois, USA). The data were analyzed by one-
way analysis of variance, followed by Duncan’s test. P value
less than 0.05 was considered significant.
Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved.
Results
Effects of folic acid on sucrose preference
Chronic stress significantly reduced sucrose preference com-
pared with the control group (P < 0.01). After the adminis-
tration of folic acid (50 and 75 mg/kg), sucrose preference
significantly increased in stressed rats (P < 0.05) (Fig. 1a).
Effects of folic acid on forced swimming test
The stress-related despairing status of rats in the FST is
evaluated by immobility time. The immobility time was
significantly increased in the CUMS group compared with
the control group (P < 0.01, Fig. 1b). In contrast, the immo-
bility time of the folic acid treatment group (50 and 75 mg/kg)
was significantly decreased (P < 0.05, Fig. 1b). Immobility
time compared with the CUMS group and the effect of folic
acid (75 mg/kg) was more obvious (P < 0.01, Fig. 1b).
Effects of folic acid on tail suspecting test
The duration of immobility was measured in the TST to
evaluate the stress-related despairing status in rats. The
immobility time of the CUMS group was significantly
longer than that of the control group (P < 0.01, Fig. 1c).
After drug treatment, the immobility time of the folic
acid (50 and 75 mg/kg) group was significantly decreased
compared with the CUMS group (50 mg/kg, P < 0.05;
75 mg/kg, P < 0.01, Fig. 1c).
Effects of folic acid on 5-hydroxytryptamine
concentration in the brain
5-HT plays a crucial role in the pathophysiology of
depression, and brain 5-HT is always associated with
happiness and reward. As shown in Fig. 2, the brain
5-HT concentration, especially 5-HT in the hippo-
campus and association cortex, significantly decreased in
the rats subjected to 4 weeks of CUMS compared with
the control group (P < 0.01, Fig. 2). After the treatment
with folic acid (50 and 75 mg/kg), 5-HT concentration
significantly increased compared with the CUMS group
(50 mg/kg, P < 0.05; 75 mg/kg, P < 0.01).
Effects of folic acid on corticosterone concentration in
the blood
Corticosterone plays a crucial role in the pathophysiology of
depression. As shown in Table 1, the serum corticosterone
concentration significantly increased in the rats subjected to
CUMS compared with the control group (P < 0.01, Table 1).
After the treatment with folic acid (50 and 75 mg/kg), cor-
ticosterone concentration significantly decreased compared
with the CUMS group (P < 0.01, Table 1).
Effects of folic acid on brain-derived neurotrophic factor
expression in the brain
As shown in Fig. 3, high-performance liquid chromato-
graphy analysis revealed that there is a decrease of BDNF
expression in the CUMS-induced rats, especially in the
hippocampus and association cortex (P < 0.01, Fig. 3). After
folic acid (50 and 75 mg/kg) treatment, the expression level
 Folic acid exerts antidepressant effects Gao et al. 1081

Fig. 1

Effects of folic acid (FA) on the percentage of sucrose consumption in the sucrose preference test (a). Effects of FA on the immobility time in the
forced swimming test (b) and effects of FA on the immobility time in the tail suspension test (c). Data are expressed as mean ± SEM (n = 10 mice/
group). **P < 0.01 vs. CONT group, #P < 0.05 vs. chronic unpredictable mild stress (CUMS) group, ##P < 0.01 vs. CUMS group. FL, fluoxetine.

Table 1 Effect of folic acid on the glucocorticoids in the blood

Fig. 2
of rats
Groups Dose (mg/kg) Content of glucocorticoids (ng/µl)

CONT – 0.3861 ± 0.1182

CUMS – 1.0627 ± 0.0606**
FA 50 0.6702 ± 0.1275#
FA 75 0.4018 ± 0.1342##
FL 20 0.4235 ± 0.0661##

The results were represented as mean ± SEM from three independent experi-
ments in each group (n = 10 mice/group).
CONT, control; CUMS, chronic unpredictable mild stress; FA, folic acid;
FL, fluoxetine.
**P < 0.05 versus control group.
#
P < 0.05 vs. chronic unpredictable mild stress (CUMS) group.
##
P < 0.01 versus CUMS group.

of BDNF increased compared with the CUMS group

(50 mg/kg, P < 0.05; 75 mg/kg, P < 0.01).

Effects of folic acid on glutamate receptor 1 expression

Effects of folic acid (FA) on 5-hydroxytryptamine (5-HT) concentration in the
hippocampus (a) and effects of FA on 5-HT concentration in the combined
cortex (b). Data are expressed as mean ± SEM (n = 10 mice/group).
**P < 0.01 vs. CONT group, #P < 0.05 vs. chronic unpredictable mild stress
(CUMS) group, ##P < 0.01 vs. CUMS group. FL, fluoxetine.
in the brain
After 4 weeks of CUMS procedures, the GluR1 depression
levels in the hippocampus and association cortex (P < 0.01,
Fig. 4) were decreased in the CUMS group. The adminis-
tration of folic acid (50 and 75 mg/kg) increased the expres-
sion of GluR1 (50 mg/kg, P < 0.05; 75 mg/kg, P < 0.01).
Effects of folic acid on the structure of mitochondria in
hippocampal cells
In observed cells of CUMS rats (mostly with ultrastructural
peculiarities of pyramidal neurons), except for the changes

Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved.

1082 NeuroReport 2017, Vol 28 No 16
Fig. 3
Effects of folic acid (FA) on brain-derived neurotrophic factor (BDNF)
expression in the hippocampus (a) and effects of FA on BDNF
expression in combined cortex (b). Data are expressed as means ± S.E.
M (n = 10 mice/group). **P < 0.01 vs. control (CONT) group, #P < 0.05
vs. chronic unpredictable mild stress (CUMS) group, ##P < 0.01 vs.
CUMS group. FL, fluoxetine.
on the number of spine synapses, several ultrastructural
changes were detected compared with the cells of control
group rats. Mitochondrial damage was the most commonly
seen abnormality in large dendrites and synaptic terminals.
Thus, moderate swelling of some of them, disruption of
several cristae, vacuolar degeneration, or even interruption
of mitochondrial membrane were observed. After the
administration of folic acid, the structure of most mito-
chondria returned to normal (Fig. 5a) and the number of
spine synapses significantly increased (Fig. 5b).
Discussion
In the present study, the ability of CUMS-induced rats to
effectively mimic the depressive state is shown in the
sucrose intake assay by reduction in SPT and by immo-
bility time increasing in TST and FST. Biochemical
characterization demonstrated a concomitant reduction of
5-HT concentration in the brain, increased corticosterone
levels in blood, and downregulated expression of BNDF
and GluR1 in CUMS rats. Strikingly, folic acid treatment
significantly reversed the depressive status in the
CUMS-induced rats, and this antidepressant effect may
be mediated by increasing brain 5-HT concentration and
BDNF signaling pathway activity.
Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved.
Fig. 4
Effects of folic acid (FA) on glutamate receptor 1 (GluR1) expression in
the hippocampus (a) and effects of FA on GluR1 expression in
combined cortex (b). Data are expressed as mean ± SEM (n = 10 mice/
group).*P < 0.05 vs. control group,**P < 0.01 vs. control (CONT) group,
#
P < 0.05 vs. chronic unpredictable mild stress (CUMS) group,
##
P < 0.01 vs. CUMS group. FL, fluoxetine.
Folic acid may be directly involved in the regulation of
the serotonergic function in depression [26]. Alterations
in the serotonergic function by folate deficiency have
been reported to be associated with impaired 5-HT
metabolism [26,27]. In a previous study, the involvement
of the 5-HT system with the antidepressant-like effect of
folic acid was investigated by inhibiting 5-HT synthesis
with the tryptophan hydroxylase inhibitor p-chlor-
ophenylalanine and by using 5-HT1A and 5-HT2A/2C
receptor agonists or antagonists to examine the beha-
vioral responses to folic acid in the FST [28].
The CUMS-induced depression model, a well-assured
animal model that resembles human depression, has
been widely used to investigate the molecular mechan-
isms of depression and evaluate the antidepressant
effects of drugs [29]. In our study, rats exposed to CUMS
exhibited a reduction of sucrose solution and a significant
prolongation of immobility time in the TST and FST as
compared with the control group. However, treatment
with folic acid significantly reversed the behavioral
changes, implying that folic acid reversed the depression-
like symptoms of CUMS rats.
The monoamine deficiency hypothesis of depression
argues that the depression is caused by a deficiency of
 Folic acid exerts antidepressant effects Gao et al. 1083

Fig. 5

Effects of folic acid (FA) on the structure of mitochondria (a) and number of spine synapses in a hippocampal cell (n = 10 mice/group) (b). CONT,
control; CORT, corticosterone; CUMS, chronic unpredictable mild stress; FL, fluoxetine. **P < 0.01 vs. (CONT) group, #P < 0.05 vs. chronic
unpredictable mild stress (CUMS) group, ##P < 0.01 vs. CUMS group.The (black arrows) marks the mitochondria.

monoamine transmitters (5-HT, noradrenaline, and
dopamine) in the brain, and the therapeutic effects of
antidepressants are mediated by increasing the levels of
monoamines [30,31]. The decrease of 5-HT concentra-
tion in the brain occurred in the depressed patients [11].
Consistent with these findings, our results showed a
decrease of 5-HT level in the CUMS-induced rats.
However, folic acid treatment restored 5-HT concentra-
tion, suggesting that the amelioration of depressive
behaviors after folic acid treatment was relevant to an
increase of 5-HT concentration in the brain.
BDNF plays several prominent roles in synaptic plasti-
city. Evidence from animal models of depression
demonstrates that chronic stress impairs hippocampal
BDNF expression and antidepressant drug effects cor-
relate with increased BDNF synthesis and activity in the
hippocampus [32]. BDNF signaling is negatively regu-
lated by the stress hormones glucocorticoids that impair
synaptic plasticity in the brain by downregulating spine
density, neurogenesis, and long-term potentiation.
Growing evidence suggests that downregulated clearance
of glutamate and signaling pathways involving BDNF
and its receptor TrkB are intimately involved in mor-
phological changes in the hippocampus of patients with
depression. BDNF-TrkB signaling regulates glutamate
transporter 1 on astrocytes, which are responsible for
most glutamate reuptake from the synapse [33].
Alterations of the metabotropic GluR1, metabotropic
GluR5, and BDNF mRNA have been shown to con-
tribute to depression-like and anxiety-like behaviors of
prenatally stressed offspring rats [34].
Our results implied that BDNF protein expression in the
brain was reduced in the CUMS-induced rats, thus leading
to the downregulation of GluR1 and the increase of glu-
cocorticoids, whereas the antidepressant-like effects of folic
acid were accompanied with an increase of BDNF in the
hippocampus and association cortex. In addition, folic acid
can also reduce the destruction of the mitochondrial

Copyright r 2017 Wolters Kluwer Health, Inc. All rights reserved.

1084 NeuroReport 2017, Vol 28 No 16
structure induced by CUMS. This study suggested that a
decrease of BDNF in the brain may be involved in the
pathogenesis of CUMS, and an increase of BDNF may be
related to the antidepressant-like effects of folic acid.
The roles of functional effects of BDNF and GluR1
activation on antidepressant-like effects of folic acid need to
be assessed in future studies. Such studies could address the
ability of a TrkB receptor antagonist to block the BDNF-
TrkB signaling pathway, and an α-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid receptor antagonist to
examine the role of GluR1 in the antidepressant-like effects
of folate.
Acknowledgements
L.G., X.L., and L.Y. designed the study; Y.L. analyzed
the behavioral test data; X.L., J.W., and M.X. analyzed
the biochemical data; X.L. drafted the manuscript. All
authors revised and approved the manuscript.
This work is supported by Shanghai Committee of
Science and Technology. (no. 16DZ2348900), National
Training Programs of Innovation and Entrepreneurship
for Undergraduates (201510269136, 201610269085). This
work is also supported by DaXia student research project
(2015DX-169).
Conflicts of interest
There are no conflicts of interest.
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