Journal of Behavioral and Brain Science, 2011, 1, 37-46
doi:10.4236/jbbs.2011.12006 Published Online May 2011 (http://www.scirp.org/journal/jbbs)
Copyright © 2011 SciRes. JBBS
Neuroprotective Effects of Sodium Ferulate and Its
Antidepressant-Like Effect Measured by Acute and
Chronic Experimental Methods in Animal Models of
Depression
Yongping Zhang1, Lijian Yu1, Yanping Wang1, Mingneng Liao1,
Xia Zhang1, Rundi Ma1, Xiaoyu Zhang1,2, Tingxi Yu1,3
1Key Laboratory of Marine Materia Medica, Guangdong Ocean University, Zhanjiang, China
2Department of Otorhinolaryn go logy-Head and Neck S urgery, University of Maryland School of Medicine,
Baltimore, USA
3Department o f S urg e ry, Depart ment of Pat h ol ogy, Universit y of Maryla n d Scho ol of Me di ci ne an d Baltimore
Veterans Affairs Medical Center, Baltimore, USA
E-mail: {yulj, mard}@gdou.edu.cn, yutingxi@yahoo.com
Received February 21, 2011; revised March 25, 2011; accept ed M ar ch 28, 2011
Abstract
Antidepressants with novel targets and without side effects are in great demand. Ferulic acid (FA) is a ubiq-
uitous phenolic acid of low toxicity, and sodium ferulate (SF) is its sodium salt. Our previous studies have
revealed that FA and SF show significant protective effect on excitotoxicity, we now test its potential neuro-
protective and antidepressant-like effects. MTT assay and morphological analysis by fluorescence micros-
copy were adopted to measure the neuroprotective effects of SF; forced-swimming, tail-suspension, and
chronic mild stress (CMS) tests were performed to assess its antidepressant-like activity. The results showed
that SF had protection against H2O2-induced oxidative damage and dexamethasone (DXM)-induced neuro-
toxicity pheochromocytoma (PC12) cells. Acute administration of SF markedly decreased the duration of
immobility during forced-swimming in rats and mice and tail-supension tests in mice. However, SF has no
any effects on reserpine-induced hypothermia, 5-hydroxytryptophan-induced head-twitch response, and po-
tentiation of noradrenaline toxicity in mice. Chronic administration of SF reversed the effects of CMS on
consumption of food and sucrose solution, weight gain, and histopathology of hippocampus by light micros-
copy, and potently shortened the immobility time during forced-swimming test following CMS in rats. This
study provides evidence that SF possesses obviously antidepressant-like activity, and the antidepressant-like
effect may result from its neuroprotective effects.
Keywords: Sodium Ferulate, Neuroprotective Effect, PC12 Cells, Animal Models of Depression
1. Introduction
Despite the advances in the treatment of depression with
selective serotonin reuptake inhibitors (SSRIs) and sero-
tonin and norepinephrine reuptake inhibitors (SNRIs),
there continue to be many unmeet clinical needs with
respect to both efficacy and side effects. To address these
needs, antidepressants with novel mechanisms and tar-
gets of action and without side effects are in great de-
mand. The increased desire to use in vitro and in vivo
techniques in depression has resulted in the search for
clonal cell lines and animal models which may be useful
for screening antidepressant drugs and for studying the
mechanism of action of antidepressant drugs.
FA, 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid
(Figure 1A), was isolated from several traditional Chi-
nese medicines and herbs, such as Felula assafoetida L.,
Ligusticum chuanxiong Hort., Equisetum hiemale L.,
Allium cepa L., Raphanus sativus L., rice bran and other
plants [1]. FA is water-insoluble, but its sodium salt, SF
(Figure 1B), is water-soluble, stable, and can be pre-
pared by chemical synthesis [2]. FA shows a lot of bio-
38 Y. P. ZHANG ET AL.
Figure 1. Chemical structure of ferulic acid (A) and sodium
ferulate (B).
logical activities, in cluding antioxidant [3,4], anti-in-
flammatory [5,6], and hypotensive effect [7,8]. Chinese
scientists have contributed a lot to its investigation, and
as an available blood-activating and stasis-eliminating
component, it has been extensively applied to the treat-
ment of vascular diseases of heart and brain, and ob-
tained excellent efficiency in China [9].
Long-term administration of FA induces resistance to
beta-amyloid peptide toxicity in the brain, suggesting
that FA may be a useful chemopreventive agent against
Alzheimer’s disease [10]. FA have sedative effect [11],
and the plants containing FA were employed, for in-
stance, in the treatment of headache and irritability, apo-
plexy, stasis, and etc. [12,13]. FA is a metabolite of
5-caffeoylquinic acid found abundantly in coffee [7], this
origin may be evoked the relationships between coffee
and depression considered. Our previous work demon-
strates that the maternal ig excessive administration of
monosodium glutamate (MSG) at a late stage of preg-
nancy results in a series of behavioral disturbance, in-
cluding MSG-induced hyperactivity that is reminiscent
of what is seen in the children suffered from attention
deficit hyperactivity disorder, and obvious histopa-
thological lesion in hippocampus in the filial mice [14],
and the administration of SF not only has no obvious
effects on behavior and histopathology, but also reverses
the effects of MSG on them [15]. The results suggest that
SF is a novel competitive N-methyl-D-aspartate (NMDA)
receptor antagonist and neuroprotective agent. Since ad-
ministration of SF has potent protective effects against
Glu-induced neurotoxicity in adult mice [16], it is inter-
esting to further investigate the neuroprotective effects of
SF and antidepressant-like effects of SF.
2. Materials and Methods
2.1. Drugs and Chemical
SF was purchased from Yaoyou Pharmaceutic Co. Ltd
(Chongqing, China), and fluoxetine (FLU) hydrochloride
purchased from Eli Lilly and Company Limited (USA).
Dexamethasone (DXM) sodium phosphate was pur-
chased from Xinzheng Pharmaceutic Co. Ltd (Tianjin,
China); H2O2 purchased from Guangzhou Chemical
Factory (China). Dulbecco’s Modified Eagle Medium
(DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT), Triton X-100, and sodium do-
decyl sulfate (SDS) were purchased from Sigma Chemi-
cal Co.; and newborn calf serum (NCS) obtained from
Sijiqing Biological Material Co. (Hangzhou, China). All
other chemicals used were of reagent grade.
2.2. Cell Line and Cell Culture
A clonal cell line derived from a pheochromocytoma of
the rat adrenal medulla [17] was purchased from Shang-
hai Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences. Cells were grown in DMEM sup-
plemented with 10% NCS, 5% horse serum and antibiot-
ics (100 units/ml penicillin, 100 μg/ml streptomycin) in
flasks precoated with collagen [18]. The culture was re-
placed after 2 months of passage by thawing a fresh ali-
quot of frozen cells. The culture were maintained in a
humidified atmosphere containing 5% CO2 at 37˚C.
Cells in log phase growth were used in the experiments.
2.3. Animal
Adult male Sprague-Dawley (SD) strain rats weighing
220 - 280 g and Kunming (KM) mice weighing 23 - 26 g
(specific pathogen free) purchased from the experimental
animal center of Guangdong Medical College (experi-
mental experimental animal license SCXKyue 2007-
2008A034No.0 001909Zhanjiang, China), were used
across all the experiments. They had free access to tap
water and standard laboratory food unless otherwise
stated. Housing conditions were controlled, temperature
was maintained at 22˚C ± 1˚C with approximately 60%
relative humidity. They were kept on a reversed 12/12 h
light/dark cycle (light 07:00 - 19:00 h). Animals were
acclimated to the animal quarters for 1 week (for rats) or
3 days (for mice) before any experimental procedure. All
the animals were treated in compliance with “Guidance
Suggestion for the Care and Use of Laboratory Animals”
issued by The Ministry of Science and Technology of
People’s Republic of China.
2.4. MTT Assay
The mitochondrial metabolism of MTT to its insoluble
blue formazan was used for enumerating cells to assess
the effects of H2O2, and DXM on the growth of PC12
cells and the protective effects of SF against oxidative
damage and glucocorticoid-induced neurotoxicity ac-
Copyright © 2011 SciRes. JBBS
Y. P. ZHANG ET AL.
39
cording to the methods of Hansen et al. [19]. Briefly,
single-cell suspensions were prepared and seeded into 96
well microculture plates with 1.0 × 105 cells/ml (90
µl/well). Cells were cultured for 12 h before addition of
drugs. Drugs were diluted into DMEM and added to each
well in a volume of 10 µl. Cells were incubated at 37˚C
for the time indicated. MTT solution (5 mg/ml) was ali-
quoted to each well in a volume of 20 µl, and 5 h later
100 µl of the solubilization solution (10% SDS-5% iso-
butyl alcohol-0.012 M HCl (w/v/v)) was added into each
well. The plates were allowed to stand overnight in the
incubator in a humidified atmosphere. Absorbance at 570
nm was determined for each well using an ELISA reader.
Control wells contained all of the agents presented in the
treated wells except the drug (s). Each experimental
point was performed in three replicates. Data were ex-
pressed as a percentage of untreated control cultures.
2.5. Effect of SF on H2O2-Induced PC12 Cell
Damage
2.5.1. Cell Viability Assay
The indicated concentration of SF was added 1 h prior to
H2O2 (150 µM) stimulation. Following 24-h incubation,
the viability of cells treated with or without H2O2 or SF +
H2O2 was evaluated by assessing the reduction of MTT.
2.5.2. Morphological Analysis by Fluorescence
Microscopy
Cells were incubated in the presence of SF (40 μM) for 1
h, and 100 μM H2O2 for additional incubation of 24 h.
H2O2-induced cell apoptosis was analyzed by acridine
orange/ethidium (AO/EB) (Sino-American Biotechnol-
ogy Co.) double fluorescent staining. Cells were washed
with phosphate buffered saline (PBS), rinsed in PBS, and
then AO/EB solution (one part of 100 µg/ml AO in PBS,
one part of 100 µg/ml EB in PBS) was added [20]. Cells
were analyzed in a fluorescence microscope (DMIRB,
Leica) using a fluorescein filter.
2.6. Effect of SF on DXM-Induced PC12
Cell Damage
The indicated concentration of SF was added 1 h prior to
DXM (200 µM). Following 24-h incubation, the viability
of cells treated with or without DXM or SF + DXM was
evaluated by assessing the reduction of MTT.
2.7. Forced-Swimming Test
Measurement of immobility time was carried out by ob-
serving the motoric activity of the mice or rats, which
were placed in a pool of water. A glass cylinder, 15 cm
(for mice) or 30 cm (for rats) in diameter, height 20 cm
(for mice) or 40 cm (for rats), was filled with water to a
height of 12 cm (for mice) or 24 cm (for rats). The tem-
perature of water was 25˚C ± 1˚C. Each mouse or rat
received twice a respective dose of FLU (20 mg/kg, ig)
or SF (20 mg/kg, 40 mg/kg, 80 mg/kg, ip) 17 h and 7 h
(for FLU) or 7 minutes (for SF) before the forced-
swimming test. This dose of FLU was chosen because it
had been shown to be differentially effective in HR vs.
LR animals [21]. The mice or rats in controls received ip
injection of physiological saline instead of SF. And then
the animals were subjected to the test. Measurement was
carried out for six (for mice) or five minutes (for rats).
For mice, the first two minutes the animal was allowed to
adjust to the new conditions; after these two minutes, the
immobility time that alternated with conditions of en-
hanced motor activity was measured. Immobility time
was measured with a stopwatch for the next four minutes
[22]. For rats, no time the animal was allowed to adjust
to the new conditions. Immobility time is the time during
which the animal floated on the surface with front paws
together and made only those movements which were
necessary to keep afloat. Shorter immobility time is an
indicator of the stronger antidepressant effect of the
tested substance [22-25].
2.8. Tail-Suspension Test
Each mouse received twice a respective dose of FLU (20
mg/kg, ig) or SF (20 mg/kg, 40 mg/kg, 80 mg/kg, ip) 17
h and 7 h (for FLU, ig) or 7 minutes (for SF, ip) before
the tail-suspension test, the mice in control received ip
injection of physiological saline instead of SF, and then
the animals were subjected to the test. A cord of about 50
cm in length was stretched between two metal tripods at
a height of ca 70 cm, to which the mice were attached by
the tail with sticky tape. After the initial period (the first
two minutes) of vigorous motor activity, the mice be-
came still and the immobility time was measured with a
stopwatch for a total duration of 4 minutes [25,26]. Mice
were considered immobile when they hung passively and
completely motionless.
2.9. CMS Procedures
The animals were assigned randomly into six matched
groups (n = 11 animals in each group) based on weight,
sucrose consumption (1% sucrose solution), and loco-
motor behavior in an open field test before onset of CMS:
control, CMS, positive control (CMS + FLU), CMS + SF
(20 mg/kg/d, 40 mg/kg/d, 80 mg/kg/d) groups. The
stressed rats were exposed to CMS for 28 days; The rats
in CMS + FLU group were exposed to CMS and re-
ceived administration of FLU (2.0 mg/kg/d, ig,
Copyright © 2011 SciRes. JBBS
40 Y. P. ZHANG ET AL.
once-daily) for 28 days; The rats in CMS + SF groups
were exposed to CMS and received administration of SF
(20 mg/kg/d, 40 mg/kg/d, 80 mg/kg/d, ip, once-daily)
respectively for 28 days. The control rats were given
ordinary daily care and received ip administration of
normal saline simultaneously for 28 days. The stressed
and control rats were kept in different rooms to allow
independent manipulation of their environments during
the duration of the stress procedure. Control rats were
housed together, while the stressed rats were housed sin-
gly.
Most of the stressors were adapted from the procedure
described by Willner and collaborators [27] and some
stressors were included from Moreau and collaborators
(e.g. empty water bottle, restricted food) [28]. Each week
included 2 h of paired caging, 3 h of tilted cage (45 de-
grees), 18 h of food deprivation immediately followed by
1 h of restricted access to food (5 micropellets), 2 × 18 h
of water deprivation immediately followed by 1 h expo-
sure to an empty bottle, 21 h of wet cage (200 ml water
in 100 g sawdust bedding), and 36 h of continuous light.
Stressors were presented both during the rats’ active
(dark) period and during the inactive (light) period. The
same stressors were used in all experiments.
2.9.1. Food and Fluid Consumption, Sucrose Intake
and Body Weight
Food and fluid consumption, sucrose intake (1% sucrose
solution) and body weight were measured once a week.
During a one-hour window after 23 hours of food and
water deprivation, the fluid consumption and sucrose
intake were measured by comparing bottle weight before
and after the one-hour window, and expressed in relation
to the animal’s body weight (ml/kg). Baseline was
measured five days before the start of CMS. The food
and water deprivation period preceding sucrose intake
measurement may be considered as a further stress ap-
plied on top of the CMS protocol. However, control rats
were also exposed to the food and water deprivation, as a
part of the sucrose test. A percent preference for sucrose
was calculated by determining the percentage of total
fluid consumption accounted for by ingestion of the 1%
sucrose solution.
2.9.2. Open Field Test during CMS
The open-field test was conducted on the same day each
week (Thursday) in a quiet room. The open-field appa-
ratus consisted of a raised plastic platform (80 × 80 × 40
cm). The floor was marked with a grid dividing it into 25
equal-size squares. Each animal was tested in the appa-
ratus once. It was placed in the central square and ob-
served for 3 min. A record was kept of the time each rat,
the amount of time it spent rearing (defined as standing
upright on its hind legs), and the number of grid lines it
crossed with at least three paws. Between animal tests
the apparatus was cleaned [29].
2.9.3. Forced-Swimming Test Following CMS in Rats
The rats exposed to CMS and received SF or FLU for 28
days were subjected to forced-swimming, and the meas-
urement of immobility time was carried out by observing
the motoric activity of the rats as described above. The
final administration of SF was given 24 h prior to testing.
2.9.4. Examination of Histopathology
The rats exposed to CMS and received SF or FLU for 28
days, 4 mice of each group were anaesthetized ip with
sodium pentobarbital (60 mg/kg) and then sacrificed by
perfusion fixation of the central nervous system (CNS)
with 40 g/L formaldehyde. The whole brain was excised
carefully, and was further fixed in 40 g/L formaldehyde
for 1 week. The hippocampal region of each animal was
sectioned, and 10-µm-thick sections were cut and stained
with hematoxylin and eosin (HE). 4 sections cutting
across hippocampal region were examined by light mi-
croscopy. A representative section was presented in re-
sults.
2.10. Data Analyses
Values are expressed as the means ± SEM or means ±
SD of 11 animals per group. Data were analyzed with
SPSS 10.0 software. A probability of P < 0.05 was con-
sidered significant.
3. Results
3.1. Protective Effect of SF against H2O2-Induced
Cytotoxicity in PC12 Cells
To determine the effect of SF on H2O2-induced cytotox-
icity, PC12 cells were pretreated with varying concentra-
tions of SF for 1 h. 150 M H2O2 was added for addi-
tional 24 h incubation. Cell viability then was examined
using an MTT mitochondrial function assay. As shown
in Figure 2, H2O2 (150 M) significantly decreased cell
viability, which was concentration-dependently attenu-
ated by SF treatment.
Morphologic alanalysis of H2O2-induced cytotoxicity
was investigated using AO/EB staining for fuorescence
microscopy. Obvious difference was observed in the
number of apoptotic and necrotic cells between the
H2O2- and SF + H2O2-treated groups (Figure 3B, 3C);
obvious differences were also observed in the nuclei of
H2O2-, SF + H2O2-treated and untreated PC12 cells after
staining with AO/EB. AO/EB dyes stained morphologi-
cally normal nuclei green (Figure 3A), whereas 100 M
Copyright © 2011 SciRes. JBBS
Y. P. ZHANG ET AL.
41
Figure 2. Protective effect of SF on H2O2-induced cytotoxic-
ity in PC12 cells. The indicated concentrations of SF was
added 1 h prior to H2O2 (150 µM) stimulation. Following
24-h in cubation, the viability of cells treated with or with-
out H2O2 or SF + H2O2 was examined by MTT assay. The
data of one representative experiment from three inde-
pendent experiments were expressed as mean ± SEM (n =
4). ## P < 0.01 as compared with control group; *P < 0.05,
** P < 0.01 as compared with H2O2 corticosterone group.
H2O2-treated cells demonstrated red or orange, smaller
and shrunken nuclei (Figure 3B). These changes in nu-
clear morphology, which were observed after 24 h of 100
M H2O2 treatment, reflected chromatin condensation
and nuclearshrinkage. However, in comparison with
H2O2, PC12 cells treated with SF (20 M) and then with
H2O2 (100 M) demonstrated lighter yellow, and slight
nuclear shrinkage (Figure 3C). The results indicated that
SF partly protected PC cells from apoptotic induction by
H2O2.
3.2. Protective Effect of SF against DXM-Induced
Neurotoxicity in PC12 Cells
To determine the effect of SF on DXM-induced cytotox-
icity, PC12 cells were pretreated with varying concentra-
tions of SF for 1 h. 200 μM DXM was added for an addi-
tional 24 h incubation. Cell viability then was examined
using an MTT mitochondrial function assay. As shown
in Figure 4, DXM (200 M) significantly decreased cell
viability, which was concentration-dependently attenu-
ated by SF treatment.
3.3. Effects of Acute Administration of SF on the
Immobility Time in Forced-Swimming and
Tail-Suspension Animal Models of Depression
The results showed that acute administration of SF po-
tently decreased the duration of immobility during
Figure 3. Protective effect of SF on H2O2-induced apoptosis
in PC12 cells by double staining with AO/EB. PC12 cells
were incubated in the presence of SF (40 μM) for 1 h, and
100 μM H2O2 for additional incubation of 24 h.
H2O2-induced cell apoptosis was analyzed by AO/EB double
fluorescent staining as described in “Materials and meth-
ods”. Cells in which nuclei were red or orange (yellow-red)
indicate apoptotic cells (original magnification, × 200). a.
control; b. H2O2 (100 μM); c. SF (20 μM) + H2O2 (100 μM).
forced-swimming in mice and rats (P < 0.05) (Table 1, 2)
and tail-supension (P < 0.01) tests in mice (Table 3),
suggesting that SF has the acute antidepressant-like effect.
It shoud be noted that rat is more sensitive to SF than
mouse is, because SF (20 mg/kg) significantly decreased
the duration of immobility in rat, but the same amount of
SF was not effective in mice.
Copyright © 2011 SciRes. JBBS
42 Y. P. ZHANG ET AL.
Figure 4. Protective effect of SF against DXM-induced
neurotoxicity in PC12 cells. The indicated concentrations of
SF were added 1 h prior to DXM (200 M). Following 24-h
incubation, the viability of cells treated with or without
DXM or SF + DXM was evaluated by assessing the reduc-
tion of MTT. The data of one representative experiment
from three independent experiments were expressed as
mean ± SEM (n = 4). ## P < 0.01 as compared with control
group; *P < 0.05, **P < 0.01 as compared with DXM group.
Table 1. Effect of acute ip administration of SF on the im-
mobility time during forced-swimming test in mice.
Ggroups Dose
(mg/kg)
Immobility time (s)

x
SD
Change (%)
Control 0 164.8 ± 28.7
FLU 20 × 2 123.5 ± 30.4* 25.1
SF 20 × 2 142.6 ± 29.6 13.5
40 × 2 117.3 ± 21.5* 28.8
80 × 2 112.4 ± 24.9* 31.8
*P < 0.05 Student’s t-test (n = 10) versus control.
Table 2. Effect of acute ip administration of SF on the im-
mobility time during forced-swimming test in rats.
Ggroups Dose
(mg/kg)
Immobility time (s)

x
SD Change (%)
Control 0 152.5 ± 28.2
FLU 20 × 2 109.8 ± 30.8* 33.4
SF 20 × 2 103.4 ± 41.2* 9.7
40 × 2 108.5 ± 48.32* 35.6
80 × 2 125.9 ± 53.5 39.7
*P < 0.05 Student’s t-test (n = 8) versus control.
Table 3. Effect of acute ip administration of SF on the im-
mobility time during tail-suspension test in mice.
Ggroups Dose
(mg/kg)
Immobility time (s)

x
SD Change (%)
Control 0 168.2 ± 22.6
FLU 20 × 2 112.1 ± 39.4** 28.0
SF 20 × 2 151.9 ± 32.3 32.2
40 × 2 108.3 ± 40.7** 28.9
80 × 2 101.5 ± 23.8** 17.4
**P < 0.01 Student’s t-test (n=10) versus control.
3.4. Effects of Chronic Administration of SF on
CMS Animal Model of Depression
3.4.1. Food Consumption and Body Weight Gain
The mean food consumption and body weight of rats in
the six experimental groups did not differ significantly
initially. Over the four weeks of the experiment, the
mean body weight increased in the six groups. Compared
to the control animals, the weight gain was significantly
less in the group exposed to CMS (P < 0.05), but the
weight gain in the CMS + SF (20, 40, 80 mg/kg/d)- and
the CMS + FLU (2.0 mg/kg/d)-treated rats was more
than that in the group exposed to CMS. At the end of
fourth week, the control rats had gained 111.1 g, the
CMS-treated rats had gained only 77.1 g, while the CMS
+ SF (20, 40, 80 mg/kg/d)- and CMS + FLU (2.0 mg/kg/
d)-treated rats had gained 83.7, 92.5, 88.0 and 79.6 g
(Figure 5), respectively. Over the four weeks of the ex-
periment, the mean food consumption (%) decreased in
the six groups. Compared to the control animals, the food
consumption was significantly less in the group exposed
to CMS (P < 0.01), but food consumption in the CMS +
SF (20, 40, 80 mg/kg/d)- and the CMS + FLU (2.0
mg/kg/d)-treated rats was significantly more than that in
the group exposed to CMS (P < 0.01). At the end of
fourth week, the control rats consumed food of 8.2 g/100
g/d, the CMS-treated rats consumed food of only
Figure 5. Time course of body weight gain. Data are repre-
sented as the means ± SEM. n = 11 animals in each group;
*P < 0.05 vs. control; Control (), CMS (), CMS + FLU
(), CMS + SF (20 mg/kg) (), CMS + SF (40 mg/kg) ()
and CMS + SF (80 mg/kg) ().
6.3 g/100 g/d, while the CMS + SF (20, 40, 80 mg/kg/d)- and
CMS + FLU(2.0 mg/kg/d)-treated rats consumed food of
7.0, 7.2, 7.2 and 7.1 g/100 g/d (Figure 6), respectively.
3.4.2. Consumption of Sucrose Solution
The stressed animals consumed significantly less sucrose
solution by the end of the second week (P < 0.01) and
continued to do so at the end of the third and the fourth
weeks (P < 0.01) (Figure 7). However, the consumption
of sucrose solution by CMS + SF (20 - 80 mg/kg/d)-
Copyright © 2011 SciRes. JBBS
Y. P. ZHANG ET AL.
43
Figure 6. Time course of food consumption. Data are repre-
sented as the means ± SEM. n = 11 animals in each group; **P
< 0.01 vs. control; Control (), CMS (), CMS + FLU (),
CMS + SF (20 mg/kg) (), CMS + SF (40 mg/kg) () and CMS
+ SF (80 mg/kg) ().
Figure 7. Time course of sucrose consumption (a), and su-
crose percentage (b). Prior to each test, rats were food- and
water-deprived for 23 h. They were then exposed to both a
1% sucrose solution and tap water. Sucrose preference =
amount of sucrose solution consumption / total fluid con-
sumption × 100%. Data are represented as means ± SEM. n
= 11 animals in each group; *P < 0.05, **P < 0.01 vs. con-
trol; Control (), CMS (), CMS + FLU (), CMS + SF
(20 mg/kg) (), CMS + SF (40 mg/kg) () and CMS + SF (80
mg/kg) ().
treated rats was significantly more than that by the rats
exposed to CMS (P < 0.01), and was comparable with
the control and positive control (P > 0.05).
3.4.3. Locomotion and Exploration Behavior
Open field testing is used to assess locomotion, explora-
tion, and anxiogenic-like behavior of rats. Compared to
stressed rats, the control animals (P < 0.01), the CMS +
SF (80 mg/kg/d)- and CMS + FLU (2.0 mg/kg/d)-treated
rats (number of squares crossed: P < 0.05, number of
rearing: P < 0.01) were more active in the open field
(Figure 8).
3.4.4. Immobility Time during Forced-Swimming Test
Following CMS
As shown in Figure 9, CMS potently increased the dura-
tion of immobility (P < 0.05), and long-term administra-
tion of SF potently decreased the immobility time during
forced-swimming test in rat model of depression (P <
0.05), suggesting that SF has the chronic antidepressant
effect.
3.4.5. Histopathology of Hippocampus
The results showed CMS-induced hippocampal lesions
characterized by slight intracellular edema, degeneration
and hyperplasia (Figure 10B) in comparison with the
control (Figure 10A). It is interesting that significant
neuronal damage was not detected in the hippocampi of
the rats treated with CMS + FLU or CMS + SF (Figure
10C, 10D, 10E, 10F). These findings suggest that stress-
induced damage and loss of hippocampal neurons may
contribute to the pathophysiology of depression, and
treatment with SF overcomes the stress-induced damage
and loss of hippocampal neurons.
Figure 8. Time course of locomotion and exploratory behavior.
Rats were placed in the central square of the open field and
observed for 3 min; Data are represented as the means ±
SEM. n = 11 animals in each group; *P < 0.05, **P < 0.01; a.
Number of squares crossed (times); b. Number of rearing
(times); Control (), CMS (), CMS + FLU (), CMS +
SF (20 mg/kg) (), CMS + SF (40 mg/kg) () and CMS + SF
(80 mg/kg) () rats.
Copyright © 2011 SciRes. JBBS
44 Y. P. ZHANG ET AL.
Figure 9. Effect of long-term administration of SF on
theimmobility time during the forced-swim test in CMS
rats. The rats exposed to CMS and received SF (20 - 80
mg/kg, ip, once-daily) for 28 days were subjected
forced-swimming test, and the measurement of immobility
time was carried out by observing the motoric activity of
the rats as described in “Materials and methods”. The final
administration of SF was given 24 h prior to testing. Values
are expressed as the means ± SEM of 11 animals per group.
Data were analyzed with SPSS 10.0 software. #P < 0.05 vs.
control; *P < 0.05 vs. CMS.
4. Discussion
The result of in vitro tests showed that SF can protect
PC12 cells from the apoptosis induced by H2O2 and
DXM. The results of in vivo tests reveal that acute ad-
ministration of SF significantly decrease the duration of
immobility during forced-swimming test and tail-suspen-
sion test, suggesting that SF has an acute antidepres-
sant-like effect. However, SF has no any effects on re-
serpine-induced hypothermia, 5-hydroxytryptophan-in-
duced head-twitch response, and potentiation of noradr-
enaline toxicity in mice (data not shown). These experi-
mental facts suggest that the mechanisms of SF antide-
pressant-like effects are different from those of main
kinds of antidepressants that have been employed in the
treatment of depression for several decades. Chronic
administration of SF reverses the effects of CMS on
consumption of food and sucrose solution, weight gain,
and histopathology of hippocampus, and significantly
shortens the immobility time during forced-swimming
test following CMS in rats, suggesting that SF has a
chronic antidepressant-like effect. The present behavioral
and histopathological data demonstrate that acute ad-
ministration of SF (also FLU) exhibits a rapid onset of
antidepressant effects during forced-swimming and tail-
suspension tests, and that chronic administration of SF
(20 - 80 mg/kg, ip, for 28 days ) (also FLU) shows a
gradual onset of action. A notable fact is that chronic
administration of SF decreases rat aggression (data not
shown), this contrasts sharply with the common ability of
chronic antidepressive treatment to increase rodent ag-
gression.
Figure. 10 Sections through hippocampal regions of CMS
rats showing morphorlogical effects of chronic administra-
tion of SF. (a). Control, × 50; (b). CMS, ×50; (c). CMS +
FLU (2.0 mg/kg), × 50; (d). CMS + SF (20 mg/kg), × 50; (e).
CMS + SF (40 mg/kg), × 50; (f). CMS + SF (80 mg/kg), × 50;
Arrow a. slight intracellular edema, degeneration and hy-
perplasia .
The deficiencies in our understanding of the etiology
of psychiatric disorders are the major obstacle in creating
animal models. The only approach that can be used is to
try to reproduce one or more symptoms of that disorder
in animals. The CMS model considers the reduction of
sucrose intake as a measure indicative of anhedonia,
which is one symptom of depression. The forced-swim-
ming test as antidepressant screening test relies on acute
response to drug treatment. The CMS plus forced-
swimming animal model of depression used in the pre-
sent study may be better able to mimic aspects of the
clinical situation of human depression, therefore, may be
better used as screening tests for elucidating antidepres-
sant activity.
From what has been discussed above, we may safely
draw the conclusion that acute and chronic exposure to
SF has an antidepressant-like effect that may involve its
neuroprotective actions. Our views to deal with the
problem of mechanisms of antidepressant-like effects of
SF are as follows. To begin with, SF (or FA), as a con-
formationally constrained Glu analogue, may be a com-
petitive antagonist at the Glu receptor [15], and thus in-
Copyright © 2011 SciRes. JBBS
Y. P. ZHANG ET AL.
45
hibits the uptake of Glu in brain and Glu-induced [Ca2+]i
through receptor operated Ca2+ channel [30]. H2O2, one
of the main reactive oxygen species, is known to cause
lipid peroxidation and DNA damage in cells [31]. Al-
though the specific cause of depression remains un-
known, recent studies have provided evidence that oxi-
dative stress plays a role in the pathogenesis of the dis-
ease [32,33]. NextSF (or FA), therefore, as a free radi-
cal scavenger, may reduce production of ROSs. These
two events strictly interconnected eventually result in
reversal of Glu-induced neurotoxicity. In fact, long-term
administration of FA per se induces transient activation
of astrocytes in hippocampus [10]. Astrocytes are source
of various neurotrophic factors, therefore, activation of
astrocytes with accompanying expression of neurotro-
phic factors was postulated to be responsible for brain
repair [34,35]. The results obtained from the present
study also demonstrate that chronic administration of SF
reverses the effect of CMS on histopathology of hippo-
campus. Preclinical and clinical investigations have
shown the involvement of dysregulation of hypotha-
lamic-pituitary-adrenal (HPA) axis in the pathogenesis of
depression. Hypercortisolemia and the associated hippo-
campal atrophy were observed in patients with depres-
sion, which could be ameliorated by the treatment with
antidepressants [36]. And lasttherefore, SF (or FA) may
act by mitigating the DXM-induced neurotoxicity.
Last but not least, it should be mentioned that there are
some limitations of our study. Firstly, the dose-depen-
dency between 20 mg/kg and 80 mg/kg of SF is not clear,
indicating that it should be explored for doses lower than
20 mg/kg of SF. Evidencing of an efficacy for doses
lower than 20 mg/kg would reinforce the interest for
such a drug, even if acutely its effect on forced swim-
ming or tail suspension tests appears only from 20 mg/kg.
Secondly, effect of ig administration of SF on depression
should be assayed, because oral intake of drugs is more
common and convenient. Thirdly, the side effects of SF
should be further investigated, even though no obvious
side effect was detectcted. Finally, an important point to
be stressed is that the neuroprotective effects of SF are
not sufficient to explain the mechanisms of antidepres-
sive-like effect of SF. Recent studies suggest that in-
creased cell proliferation and increased neuronal number
may be a mechanism by which antidepressant treatment
overcomes the stress-induced atrophy and loss of hippo-
campal neurons and may contribute to the therapeutic
actions of antidepressant treatment [37]. Therefore, a
study on pontential neurogenesis-enchancing action of
SF is now in progress in our laboratory.
5. Acknowledgements
The authors would like to thank Dr. Depu Yu for his
great encouragement and continuous promotion.
6. References
[1] Y. B. Ji, “Pharmacological Action and Application of
Blood-Activating and Stasis-Eliminating Available Com-
position of Traditional Chinese Medicine,” Heilongjiang
Science and Technique Press, Harbin, 1999, pp. 118-121.
[2] H. Jing, L. Y. Yao, J. S. Li, Y. Q. Song and W. Chao,
“Research Progress of Pharmacology of Sodium
Ferulate,” Northwest Pharmaceutical Journal, Vol. 17,
No. 5, 2002, pp. 236-238.
[3] E. Grafe, “Antioxidant Potential of Ferulic Acid,” Free
Radical Biology and Medicine, Vol. 13, No. 4, 1992, pp.
435-448. doi:10.1016/0891-5849(92)90184-I
[4] B. C. Scott, J. Butler, B. Halliwell and O. I. Aruoma,
“Evaluation of the Antioxidant Actions of Ferulic Acid
and Catechins,” Free Radical Research Communications,
Vol. 19, No. 4, 1993, pp. 241-253.
doi:10.3109/10715769309056512
[5] M. A. Fernandez, M. T. Saenz and M. D. Garcia, “Anti-
inflammatory Activity in Rats and Mice of Phenolic Ac-
ids Isolated from Scrophularia Frutescens,” Journal of
Pharmacy and Pharmacology, Vol. 50, No. 10, 1998, pp.
1183-1186. doi:10.1111/j.2042-7158.1998.tb03332.x
[6] Y. Ozaki, “Antiinflammatory Effect of Tetramethylpyra-
zine and Ferulic Acid,” Chemical and Pharmaceutical
Bulletin, Vol. 40, No. 4, 1992, pp. 954-956.
[7] A. Suzuki, D. Kagawa, R. Ochiai, I. Tokimitsu and I.
Saito, “Green Coffee Bean Extract and Its Metabolites
have a Hypotensive Effect in Spontaneously Hyperten-
sive Rats,” Hypertension Research, Vol. 25, No. 1, 2002,
pp. 99-107. doi:10.1291/hypres.25.99
[8] A. Suzuki, D. Kagawa, A. Fujii, R. Ochiai, I. Tokimitsu
and I. Saito, “Short- and Long-Term Effects of Ferulic
Acid on Blood Pressure in Spontaneously Hypertensive
Rats,” American Journal of Hypertension, Vol. 15, No. 4,
2002, pp. 351-357. doi:10.1016/S0895-7061(01)02337-8
[9] J. Zhang, Q. Z. Jin and X. G. Wang, “Research Pro-
gresses on Synthesis and Pharmacological Activities of
Ferulic Acid and Its Derivatives,” Grain and Oil, Vol. 13,
No. 1, 2007, pp. 43-45.
[10] J. J. Yan, J. Y. Cho, H. S. Kim, K. L. Kim, J. S. Jung, S.
O. Huh, H. W. Suh, Y. H. Kim and D. K. Song, “Protec-
tion against Beta-Amyloid Peptide Toxicity in vivo with
Long-Term Administration of Ferulic Acid,” British Jour-
nal of Pharmacology , Vol. 133, No. 1, 2001, pp. 89-96.
[11] Y. P. Zhang, R. D. Ma and L. J. Yu, “Sedative and Hyp-
notic Effect of Sodium Ferulate in Mice,” Nei Mongol
Journal of Traditional Chinese Medicine, Vol. 27, No. 7,
2008, pp. 9-11.
[12] S. Z. Li, “Compendium of Materia Medica,” (reprinted
from 1590 Jinling wood-engraved edition), People’s
Health Press, Beijing, 2004, pp. 683-686.
[13] Jiangsu College of New Medicine, “Dictionary of Tradi-
tional Chinese and Herbal Medicine,” Shanghai Science
and Technology Publishing House, Shanghai, 1986, pp.
220-222.
[14] T. X. Yu., Y. Zhao, W. C. Shi, R. D. Ma and L. J. Yu,
Copyright © 2011 SciRes. JBBS
Y. P. ZHANG ET AL.
Copyright © 2011 SciRes. JBBS
46
“Effects of Maternal Oral Administration of Monosodium
Glutamate at a Late Stage of Pregnancy on Developing
Mouse Fetal Brain,” Brain Research, Vol. 747, No. 2,
1997, pp. 195-206. doi:10.1016/S0006-8993(96)01181-X
[15] L. J. Yu, Y. P. Zhang, R. D. Ma, L. Bao, J. Z. Fang and T.
X. Yu, “Potent Protection of Ferulic Acid against Excito-
toxic Effects of Maternal Intragastric Administration of
Monosodium Glutamate at a Late Stage of Pregnancy on
Developing Mouse Fetal Brain,” European Neuropsy-
chopharmacology, Vol. 16, No. 3, 2006, pp. 170-177.
[16] Y. P. Zhang, L. J. Yu, R. D. Ma, L. Bao, R. Zeng, J. Z.
Fang, X. Y. Zhang and T. X. Yu, “Potent Protective Ef-
fect of Ferulic Acid on Glutamat-Induced Neurotoxicity
in Adult Mice,” Chinese Journal of Neuromedicine, Vol.
7, No. 6, 2008, pp. 596-599.
[17] T. J. Shafer and W. D. Atchison, “Methylmercury Blocks
N- and L-Type Ca++ Channels in Nerve Growth Fac-
tor-Differentiated Pheochromocytoma (PC12) Cells,”
Journal of Pharmacology and Experimental Therapeutics,
Vol. 258, No. 1, 1991, pp. 149-157.
[18] M. Yoshizumi, T. Kogame and Y. Suzaki, Ebselen At-
tenuates Oxidative Stress-Induced Apoptosis via the In-
hibition of the c-Jun N-Terminal Kinase and Activator
Protein-1 Signalling Pathway in PC12 Cells,” British
Journal of Pharmacology, Vol. 136, No. 2, 2002, pp.
1023-1032. doi:10.1038/sj.bjp.0704808
[19] M. B. Hansen, S. E. Nielsen and K. Berq, “Re-exami-
nation and Further Development of a Precise and Rapid
Dye Method for Measuring Cell Growth/Cell Kill,”
Journal of Immunological Methods, Vol. 119, No. 2,
1989, pp. 203-210. doi:10.1016/0022-1759(89)90397-9
[20] V. Chigancas, E. N. Miyaji, A. R. Muotri, J. de Fatima
Jacysyn, G. P. Amarante-Mendes, A. Yasui and C. F.
Menck, “Photorepair Prevents Ultraviolet-induced Apop-
tosis in Human Cells Expressing the Marsupial
Photolyase Gene,” Cancer Research, Vol. 60, No. 2,
2000, pp. 2458-2463.
[21] K. Taghzouti, S. Lamarque, M. Kharouby and H. Simon,
“Interindividual Differences in Active and Passive Be-
haviors in the Forced-Swimming Test: Implications for
Animal Models of Psychopathology,” Biological Psy-
chiatry, Vol. 45, No. 6, 1999, pp. 750-758.
[22] R. D. Porsolt, A. Bertin and M. Jalfre, “Behaviour De-
spair Models in Mice: A Primary Screening Test for An-
tidepressants,” Archives Internationales de Pharmacody-
namie et de Thérapie, Vol. 229, No. 2, 1977, pp. 327-336.
[23] R. D. Porsolt, M. Lepichon and M. Jalfre, “Depression: A
New Animal Model Sensitive to Antidepressant Treat-
ments,” Nature, Vol. 266, No. 21, 1977, pp. 730-732.
doi:10.1038/266730a0
[24] R. D. Porsolt, G. Anton and N. Blavet, “Behaviour De-
spair in Rats: A New Model Sensitive to Antidepressant
Treatments,” European Journal of Pharmacology, Vol.
47, No. 4, 1978, pp. 379-391.
doi:10.1016/0014-2999(78)90118-8
[25] R. D. Porsolt, “Animal Models of Depression: Utinity for
Transgenic Research,” Reviews in the Neurosciences, Vol.
11, No. 1, 2000, pp. 53-58.
[26] J. M. Vaugeois, G. Passera, F. Zuccaro and J. Costentin,
“Individual Differences in Response to Imipramine in the
Tail Mouse Suspension Test,” Psychopharmacology, Vol.
134, No. 4, 1997, pp. 387-391.
[27] P. Willner, A. Towell, D. Sampson, S. Sophokleous and
R. Muscat, “Reduction of Sucrose Preference by Chronic
Unpredictable Mild Stress, and Its Restoration by a Tri-
cyclic Antidepressant,” Psychopharmacology, Vol. 93,
No. 3, 1987, pp. 358-364.
[28] J. L. Moreau, F. Jenck, J. R. Martin, P. Mortas and W. E.
Aefely, “Antidepressant Treatment Prevents Chronic Un-
predictabl Mild Stress-Induced Anhedonia as Assessed
by Ventral Tegmentum Self-Stimulation Behavior in
Rats,” European Neuropsychopharmacology, Vol. 2, No.
1, 1992, pp. 43-49. doi:10.1016/0924-977X(92)90035-7
[29] K. T. Hallam, J. E. Horgan, C. McGrath and T. R. Nor-
man, “An Investigation of the Effect of Tacrine and Phy-
sostigmine on Spatial Working Memory Deficits in the
Olfactory Bulbectomised Rat,” Behavioural Brain Re-
search, Vol. 153, No. 2, 2004, pp. 481-486.
doi:10.1016/j.bbr.2004.01.005
[30] J. J. Zhang, A. Q. Chen, Y. Z. Gao, H. X. Liu and Y.
Zhou, “Effect of Sodium Ferulate on Free Intracellular
Calcium in Cortical Neuronal Cultures,” Chinese Journal
of Clinical Neuros ciences, Vol. 10, No. 4, 2002, pp. 342-344.
[31] B. Halliwell and O. I. Aruoma, “DNA Damage by Oxy-
gen-Derived Species. Its Mechanism and Measurement in
Mammalian Systems,” FEBS Letters, Vol. 281, No. 1-2,
1991, pp. 9-19. doi:10.1016/0014-5793(91)80347-6
[32] G. Lucca, C. M. Comim, S. S. Valvassori, G. Z. Réus, F.
Vuolo, F. Petronilho, F. Dal-Pizzol, E. C. Gavioli and J.
Quevedo, “Effects of Chronic Mild Stress on the Oxida-
tive Parameters in the Rat Brain,” Neurochemistry Inter-
national, Vol. 54, No. 5-6, 2009, pp. 358-362.
doi:10.1016/j.neuint.2009.01.001
[33] Y. C. Wei, F. L. Zhou, D. L. He, J. R. Bai, L. Y. Hui, X.
Y. Wang and K. J. Nan, “The Level of Oxidative Stress
and the Expression of Genes Involved in DNA-Damage
Signaling Pathways in Depressive Patients with Colorec-
tal Carcinoma,” Journal of Psychosomatic Research, Vol.
66, No. 3, 2009, pp. 259-266.
doi:10.1016/j.jpsychores.2008.09.001
[34] H. Kato, K. Kogure, T. Araki and Y. Itoyama, “Astroglial
and Microglial Reactions in the Gerbil Hippocampus with
Induced Ischemic Tolerance,” Brain Research, Vol. 664,
No. 1994, pp. 69-76.
[35] N. Kawahara, C. A. Ruetzler, G. Mies and I. Klatzo,
“Cortical Spreading Depression Increases Protein Syn-
thesis and Upregulates Basic Fibroblast Growth Factor,”
Experimental Neurology, Vol. 158, No. 1, 1999, pp. 27-36.
[36] PGIS, “Global Terrorism Database 1998-2004. GTD2,
Codebook, Draft 1.0,” Pinkerton Global Intelligence Ser-
vices (PGIS), Washington DC, 2007.
[37] J. E. Malberg, A. J. Eisch, E. J. Nestler and R. S. Duman,
“Chronic Antidepressant Treatment Increases Neuro-
genesis in Adult Rat Hippocampus,” Journal of Neuro-
science, Vol. 20, No. 24, 2009, pp. 9104-9110.