Water Extract from the Leaves of Withania somnifera
Protect RA Differentiated C6 and IMR-32 Cells against
Hardeep Kataria1, Renu Wadhwa2*, Sunil C. Kaul2, Gurcharan Kaur1*
1Department of Biotechnology, Guru Nanak Dev University, Amritsar, India, 2National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative disorders.
Search for herbal remedies that may possibly act as therapeutic agents is an active area of research to combat these
diseases. The present study was designed to investigate the neuroprotective role of Withania somnifera (Ashwagandha),
also known as Indian ginseng, against glutamate induced toxicity in the retinoic acid differentiated rat glioma (C6) and
human neuroblastoma (IMR-32) cells. The neuroprotective activity of the Ashwagandha leaves derived water extract (ASH-
WEX) was evaluated. Cell viability and the expression of glial and neuronal cell differentiation markers was examined in
glutamate challenged differentiated cells with and without the presence of ASH-WEX. We demonstrate that RA-
differentiated C6 and IMR-32 cells, when exposed to glutamate, undergo loss of neural network and cell death that was
accompanied by increase in the stress protein HSP70. ASH-WEX pre-treatment inhibited glutamate-induced cell death and
was able to revert glutamate-induced changes in HSP70 to a large extent. Furthermore, the analysis on the neuronal
plasticity marker NCAM (Neural cell adhesion molecule) and its polysialylated form, PSA-NCAM revealed that ASH-WEX has
therapeutic potential for prevention of neurodegeneration associated with glutamate-induced excitotoxicty.
Citation: Kataria H, Wadhwa R, Kaul SC, Kaur G (2012) Water Extract from the Leaves of Withania somnifera Protect RA Differentiated C6 and IMR-32 Cells against
Glutamate-Induced Excitotoxicity. PLoS ONE 7(5): e37080. doi:10.1371/journal.pone.0037080
Editor: Rafael Linden, Universidade Federal do Rio de Janeiro, Brazil
Received October 7, 2011; Accepted April 13, 2012; Published May 14, 2012
Copyright: ? 2012 Kataria et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from the Department of Biotechnology, Government of India to Guru Nanak Dev University, under DBT-AIST, Japan
collaboration program. H.K. is supported by a fellowship grant from the Council of Scientific and Industrial Research (CSIR), India. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (GK); firstname.lastname@example.org (RW)
Research into medicinal plants so as to identify the novel,
natural and safe phytotherapies has flourished and recently several
in vitro and in vivo pre-clinical studies validating the therapeutical
value of newly identified phytochemicals have been launched.
Presently, many of the traditional herbal medicines are increas-
ingly being appreciated with Western models of integrative health
sciences and evidence-based approach both in research and clinic
. In contrast to the conventional single-module medicine, the
herbal extracts function through multi-target mechanisms and
hence may hold key to the success where conventional agents fail
. Brain pathologies pose an extra degree of complexity in their
treatment and hence there is a compelling reason to search for
naturotherapeutic ways. Recently, many studies have focused on
the potential of crude extracts and their isolated compounds from
fruits, vegetables and herbs to prevent certain neurological
disorders. Some beneficial phytochemicals from Curcuma longa,
Withania somnifera, Panax ginseng, and Ginkgo bilobae etc. [3,4,5,6] have
been identified that exhibit significant neuroprotective effects in
various experimental models of neurological disorders. The
proposed underlying mechanisms include preconditioning, anti-
oxidation and anti-inflammation effects. Some of the herbs have
been classified as brain tonics or rejuvenators in Ayurveda, the
traditional Indian medicine system. Among these, the most
important plant is Ashwagandha whose extracts make a significant
component to the daily supplements for body and brain health.
Although a variety of Ashwagandha extracts have displayed
neuroprotective, neuroregenerative and anticancer potentials in
recent in vitro studies [7,8,9,10,11] using brain-derived cells,
potentials of water extract of leaves of Ashwagandha (ASH-
WEX) remain largely unexplored. In the present study, we used
glutamate induced excitotoxicity as a model to investigate the
neuroprotective potentials of ASH-WEX.
Glutamate is the major excitatory neurotransmitter in the CNS
where it acts upon ionotropic (N-methyl-D-aspartate (NMDA) and
a-amino-3-hyroxy-5-methylisoxazole proprionic acid (AMPA)) or
metabotropic (mGlu1-mGlu8) receptors [12,13]. Although gluta-
mate plays a central role in excitatory neurotransmission,
alterations in glutamate homeostasis can have significant reper-
cussions on neural cells through the generation of neurotoxic or
excitotoxic cascades [14,15]. Abnormalities in glutamate neuro-
transmitter system are not only involved in acute neural trauma
such as ischemia, spinal cord injury, head trauma, and epilepsy,
but also in neurodegenerative disorders such as Huntington’s,
Alzheimer’s and Parkinson’s diseases, amyotrophic lateral sclero-
sis, AIDS complex, and domoic acid neurotoxicity [16,17,18].
After brain ischemia or traumatic injury to the CNS, there is a
pathological release of glutamate from neurons and glial cells
[19,20]. Glutamate uptake by astrocytes normally prevents
excitotoxic glutamate elevations in brain extracellular space .
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The uncontrolled release of glutamate can lead to a constant
stimulation of glutamate receptors and the deregulation of
intracellular Ca++homeostasis, mainly through NMDA receptor
activation. However, in an excitatory crisis, the potentially
protective functions of reactive astrocytes, such as glutamate
uptake and elimination of free radicals can eventually be reduced
or even reversed and might instead contribute to the development
of neural damage [22,23]. Thus, activated astrocytes might both
protect from and contribute to the glutamate-mediated neuronal
damage. As glutamate neurotoxicity is involved in the pathogen-
esis of various diseases, reduction of glutamate toxicity is one of the
important therapeutic strategy for drug designig [24,25,26] and
several drugs targeting glutamate toxicity are under development.
The molecular mechanisms of cell death induced by glutamate
have not been fully elucidated . Since the free radical-
scavenging agents and antioxidants such as, vitamin E  are
shown to have protective impact on glutamate-toxicity, excessive
accumulation of free radicals has been speculated to be
responsible, at least in part, for glutamate-induced neuropathol-
ogies. Curcumin (a major componet of turmeric) and epicatechin-
gallate (a major component of green tea) have been shown to
protect primary cultured neurons from glutamate-induced cell
The present study was designed to test the hypothesis that
Ashwagandha leaves derived water extract (ASH-WEX) may
confer protection against glutamate induced toxicity. Retinoic acid
(RA) differentiated C6 glioma and IMR32 neuroblastoma cells
have been widely accepted for in vitro studies due to their close
resemblance to glial and neuronal cells, respectively [30,31].
Expression analysis of glial and neuronal cell markers (glial
fibrillary acidic protein (GFAP) and Neurofilament 200 (NF200),
respectively) was made following treatments with glutamate and
ASH-WEX. Upregulation of GFAP is a marker for reactive gliosis,
trauma and degeneration in CNS whereas; HSP 70 is a useful
stress response marker. Experiments using both animal models of
stroke and tissue culture systems have indicated that overexpres-
sion of HSP70 reduced ischemic injury and protected both
neurons and glial cells [32,33]. Further the expression of stress
related marker HSP70 along with plasticity markers NCAM and
PSA-NCAM was evaluated to establish their role in ASH-WEX
mediated neuroprotection. NCAM, a member of the immuno-
globulin superfamily of cell recognition molecules, is widely
expressed on axons and dendrites  and is a key regulator of
neuronal development and function . NCAM is associated
with an unusual glycan, polysialic acid, a highly negatively charged
and voluminous carbohydrate modulating its adhesive and
concomitant signal transduction functions. NCAM and PSA-
NCAM play important roles in the development of the nervous
system and NMDA receptor dependent synaptic plasticity in the
adult . The induction of PSA expression in damaged adult
CNS tissues has been shown to be promising therapeutic target in
repair, remodelling and regeneration . Use of crude leaf water
extract is both eco- and bio-friendly as neither there is a need to
sacrifice whole plant (as in case of roots) nor any organic solvents
are required. Moreover the aqueous extract is easy to prepare and
convenient as well as safe to use.
Materials and Methods
Preparation of the water extract of leaves of
ASH-WEX was prepared as reported earlier . Briefly, 10 g
of dry leaf Ashwagandha powder was suspended in 100 ml of
sterile distilled water. It was incubated at 45uC for overnight with
slow stirring. The slurry was centrifuged at 10,000 rpm and was
then filtered under sterile conditions. The filtrate so obtained was
treated as 100% ASH-WEX.
Cell culture and treatments
C6 (rat glioma) and IMR-32 (human neuroblastoma) cells were
purchased from National Centre for Cell Science (Pune, India).
The cells were routinely grown in DMEM supplemented with
10% Fetal bovine serum and 16PSN mix (Invitorgen) at 37uC in
a humidified atmosphere containing 5% CO2.
Undifferentiated cultures were subcultured by trypsinization
and cultured in 96 and 24 well plates according to the requirement
of the experiment. After 24 hrs of seeding, C6 and IMR-32 cells
were differentiated for 4 and 6 days, respectively by adding
retinoic acid (RA) to the culture medium to a final concentration
of 10 mM. The medium was changed every two days. RA
differentiated cultures were pretreated with 0.05% and 0.1%
ASH-WEX for 24 hrs and then exposed to glutamate (0.06 mM–
10 mM) in the presence of ASH-WEX.
Proliferation and Cytotoxicity assays
ASH-WEX was tested for protective activity against glutamate
on C6 and IMR-32 cells using the 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) test. This method is based
on the reduction of the tetrazolium salt MTT into a crystalline
Table 1. Primer sequences used for semi-quantitative RT-PCR.
No. mRNA Primer Sequence Expected product size
1. GFAPF 59GGCGCTCAATGCTGGCTTCA39
2. NF200F 59CAAGGAACCCAGCAAACCA39
4. NCAM F 59TGAGGGTACTTACCGCTGTG39
5. PSTF 59TAAGGTGCAATCTAGCTCCTGTGGTGG39
b-actin F 59TCACCCACACTGTGCCCATCTACGA39
Withania Protects against Glutamate Excitotoxicity
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blue formazan product by the cellular oxidoreductase. The
amount of formazan produced is considered as a relaible
representation of viable cell number. After 24 hrs of treatment
with glutamate, the culture medium was removed and replaced
with fresh culture medium containing MTT (0.5 mg/ml). After
4 h incubation at 37uC, this solution was removed, and the
resulting blue formazan was solubilized in 100 ml of DMSO and
the optical density was read at 595 nm using microplate reader
(Multiskan PLUS, Thermo Scientific).
In order to assay the cytotoxicity, Lactate dehydrogenase (LDH)
assay was done as described by Abe and Matsuki . Briefly,
LDH substrate mixture (1 ml) was prepared as follows; 2.5 mg l-
lactate lithium salt and 2.5 mg NAD were dissolved in 0.9 ml of
0.2 M Tris–HCl buffer (pH 8.2) with 0.1% (v/v) Triton X-100,
and 0.1 ml of MTT stock solution (2.5 mg/ml) and 1 ml of MPMS
stock solution (100 mM) were added. 50 ml of the culture
supernatant was transferred to 96-well culture plates, and mixed
with 50 ml of the LDH substrate mixture. The reaction was
stopped by adding 100 ml of a solution containing 50%
dimethylformamide and 20% sodium dodecyl sulfate (DMF/
SDS, pH 4.7). The absorbance was measured at 570 nm with
Multiskan PLUS reader (Thermo Scientific).
Figure 1. The morphological changes in C6 (a) and IMR-32 (f) cells were studied using phase contrast images. Cell viability and toxicity
of various concentrations of glutamate was assayed by MTT and LDH assays in RA differentiated C6 (b,c) and IMR-32 (g,h) cells. (d) and (i) histograms
represents the relative percentage viability of glutamate and ASH-WEX treated C6 and IMR-32 cells, respectively, as compared to the control cells. (e)
and (j) histograms represents the relative LDH activity when the control and ASH-WEX pretreated cells were exposed to different glutamate
concentrations. ‘‘*’’ represents the statistical significant difference between all the treatment groups (glutamate alone or glutamate + ASH-WEX
groups) with respect to control group. ‘‘#’’ represents the statistical difference between ‘‘glutamate + ASH-WEX’’ treated groups with their respective
‘‘glutamate’’ treatment groups. ‘‘*’’ and ‘‘#’’=p,0.05.
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All cells, control and treated, were fixed with 4% paraformal-
dehyde followed by permeabilization with 0.3% Triton X-100 in
Phosphate buffer saline (PBST). Cells were incubated with anti-
GFAP (1:500, Sigma), anti-HSP70 (1:1000, Sigma), anti NF200
(1:500, Sigma), anti-NCAM (1:500, Sigma) and anti PSA-NCAM
(1:250, AbCys) diluted in 0.1% PBST, for 24 h at 4uC in humid
chamber. Secondary antibody (Alexa Fluor 488, 546, Invitrogen)
was applied for 2 h at room temperature. Cells were incubated
with (DAPI, 1:5000 in 0.1%PBST) for 10 minutes for nuclear
staining and then mounted with anti-fading reagent (Fluoromount,
Sigma) and observed under the microscope (Nikon A1RConfocal).
Images were captured under (406) and were analyzed using image
pro-plus software version 4.5.1 from the media cybernetics.
Protein assay and Western blotting
Cells grown and treated in 10 cm dishes were harvested with
PBS-EDTA (1 mM). Cell pellet was lysed in homogenizing buffer
(50 mM Tris, 150 mM NaCl, 1 mM EDTA, 100 mM NaVO4,
1 mM PMSF and 0.5 mM DTT, Protease Inhibitor cocktail) and
protein content in the supernatant was determined by the
Cell lysate (20–30 mg) was resolved on 8–10% SDS-PAGE
followed by transfer onto a PVDF membrane (Hybond-P) using
the semidry Novablot system (Amersham Pharmacia). Subse-
Figure 2. Representative Western blots and their densitometry analysis for GFAP (a) and NF200 (e) for RA differentiated C6 and
IMR-32 cells, respectively. RT-PCR results for GFAP and NF200 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry
analysis was represented by histograms. The expression of GFAP in C6 (c) and NF200 in IMR-32 (g) cells was analysed by immunocytostaining and
relative intensity was plotted as histogram as analysed by Image pro-plus software. ‘‘*’’ represents the statistical significant difference between all the
treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. ‘‘#’’ represents the statistical
difference between ‘‘glutamate + ASH-WEX’’ treated groups with their respective ‘‘glutamate’’ treatment groups. ‘‘*’’ and ‘‘#’’=p,0.05.
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quently, membranes were probed with mouse anti-GFAP (1:3000),
anti-HSP70 (1:1000),Anti-NF200 (1:3000), anti-NCAM (1:2500)
or anti-PSA-NCAM (1:2000) antibody. Immunoreactive bands
were visualized using ECL Plus Western blot detection system
(Amersham Biosciences). In order to account for potential
variations in protein estimation and sample loading, expression
of each protein was compared to that of a-tubulin.
Total RNA was extracted from cells by the TRI reagent (Sigma)
according to manufacturer’s instruction. Equal amounts of RNA
were used for cDNA synthesis in 20 ml reactions containing 200 U
M-MLV reverse transcriptase, 4 ml 56first strand buffer (Fermen-
tas), 5 mg of total RNA, 1 mM each of dNTPs (Fermentas),
20 units of ribonuclease inhibitor (Sigma), and 250 ng pd(N)6
random hexamers (Fermentas). 2 ml of cDNA was amplified in a
50 ml PCR reaction mixture containing two units Taq polymerase,
5 ml 106 PCR buffer, 3.0 ml of 25 mM MgCl2(Sigma), 1 ml of
10 mM dNTP mix (Fermentas), and 20 picomoles of respective
primers as listed in Table 1. Cycling conditions comprised of an
initial denaturation of 3 min at 94uC followed by 35 cycles of
amplification (at 94uC for 40 sec, 55uC for 45 sec and 72uC for
1 min) and final elongation step at 72uC for 10 min. To control
the PCR reaction components and the integrity of the RNA, 2 ml
of each cDNA sample was amplified separately for b-actin specific
Figure 3. Representative Western blots and their densitometry analysis for HSP70 in RA differentiated C6 (a) and IMR-32 (e) cells,
respectively. RT-PCR results for HSP70 mRNA in C6 (b) and IMR-32 (f) cells, respectively and their relative densitometry analysis was represented by
histograms. The expression of HSP70 in C6 (c) and IMR-32 (g) cells was analysed by immunocytostaining and relative intensity was plotted as
histogram as analysed by Image pro-plus software. ‘‘*’’ represents the statistical significant difference between all the treatment groups (ASH-WEX
alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. ‘‘#’’ represents the statistical difference between ‘‘glutamate
+ ASH-WEX’’ treated groups with their respective ‘‘glutamate’’ treatment groups. ‘‘*’’ and ‘‘#’’=p,0.05.
Withania Protects against Glutamate Excitotoxicity
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Gelatinase Zymography is simple, sensitive, quantifiable, and
functional assays to analyze MMPs in biological samples which
identifies MMPs by the degradation of their preferential substrate
gelatine and by their molecular weight. Gelatin zymography is
mainly used for the detection of the gelatinases, MMP-2 and
MMP-9 as gelatin is the specific substrate for these two MMPs.
Gelatinase zymography was performed in 10% SDS Polyacryl-
amide Gel in the presence of 0.1% gelatin under nonreducing
conditions. Culture media (20 ml) were mixed with sample buffer
and loaded for SDS-PAGE. Samples were not boiled before
electrophoresis. Following electrophoresis the gels were washed
twice in 16 Renaturing Buffer (Invitrogen) for 30 min at room
temperature to remove SDS. The gels were then incubated at
37uC for 48–72 hrs in Developing Buffer (Invitrogen) stained with
0.5% Coomassie Blue R250 in 50% methanol and 10% glacial
acetic acid for 30 min and destained. Upon renaturation of the
enzyme, the gelatinases digest the gelatin in the gel and give clear
bands against an intensely stained background.
Figure 4. Representative Western blots and their densitometry analysis for NCAM in RA differentiated C6 (a) and IMR-32 (d) cells,
respectively. RT-PCR results for NCAM mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented by
histograms. The expression of NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. ‘‘*’’ represents the statistical significant difference
between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. ‘‘#’’ represents
the statistical difference between ‘‘glutamate + ASH-WEX’’ treated groups with their respective ‘‘glutamate’’ treatment groups. ‘‘*’’ and ‘‘#’’=p,0.05.
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The captured images were analyzed using Image Pro-Plus
software version 4.5.1 from Media Cybernetics. The extent of
GFAP, NF 200, HSP70 immunoreactivity was quantified by the
overall density of their respective immunoreactivity each in 5–6
randomly selected fields on each image using the count/size
command of the Image Pro-Plus software. 15 different images
were used from three different experiments and the data were
averaged and expressed as percentage with respect to control.
Data was analyzed statistically using Sigma Stat for Windows
(version 3.5). The results were analyzed using One-way ANOVA
to determine the significance of the mean between the groups.
Figure 5. Representative Western blots and their densometery analysis for PSA-NCAM in RA differentiated C6 (a) and IMR-32 (d)
cells, respectively. RT-PCR results for PST mRNA in C6 (b) and IMR-32 (e) cells, respectively and their relative densometery analysis was represented
by histograms. The expression of PSA-NCAM in C6 (c) and IMR-32 (f) cells was analysed by immunostaining. ‘‘*’’ represents the statistical significant
difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to control group. ‘‘#’’
represents the statistical difference between ‘‘glutamate + ASH-WEX’’ treated groups with their respective ‘‘glutamate’’ treatment groups. ‘‘*’’ and
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Values of p#0.05 were considered significant. The means of the
data are presented together with the standard error mean (SEM).
ASH-WEX attenuated glutamate-induced cytotoxicity
To determine whether glutamate can induce the death of RA
differentiated C6 and IMR-32 cells, differentiated cultures were
treated with various doses of glutamate (0.06 mM–10 mM).
Cultures treated with glutamate concentrations (0.5 mM and
1 mM for C6 cells and 0.25 mM and 0.5 mM for IMR-32 cells)
for 24 h exhibited cell shrinkage and rounding (Fig. 1a-iii,v and 1f-
iii,v). MTT assay on glutamate treated cells revealed a decrease of
the number of living cells after treatment with increasing doses of
glutamate (Fig. 1b, g). The extent of glutamate toxicity for RA-
differentiated C6 glial and IMR-32 neuronal cells was different;
whereas 1 mM glutamate exposure induced more than 50% C6
glial cell death, IMR-32 neuronal cells showed similar effect with
0.5 mM glutamate. Using these doses as toxicity models, we next
investigated whether ASH-WEX (0.05% and 0.1%) could protect
differentiated C6 and IMR-32 cells against glutamate-induced cell
death. Pretreatment for 24 hrs with ASH-WEX (0.1%) signifi-
cantly inhibited the death of C6 (Fig. 1b, d) and IMR-32 (Fig. 1g, i)
cells exposed to glutamate. Glutamate-induced changes in the cell
morphology were partially suppressed by treatment with 0.1%
ASH-WEX. Of note, the recovery in the cell morphology was
observed only for the low dose (0.5 mM for C6 and 0.25 mM for
IMR-32) of glutamate treatment (Fig. 1a-iv, f-iv). LDH assay also
confirmed the protective effect of ASH-WEX as the enzyme
activity increased with rise in glutamate dose, implicating
glutamate toxicity, which was significantly reduced in 0.1%
ASH-WEX pretreatment group in C6 (Fig. 1c, e) and IMR-32
(Fig. 1h, j) cells. Based upon MTT and LDH assay a low (0.5 mM
for C6, 0.25 mM for IMR-32) and high dose (1 mM for C6 and
0.5 mM for IMR-32) of glutamate was selected for further
ASH-WEX abolished glutamate induced changes in the
C6 and IMR-32 marker proteins
GFAP is an astrocyte-specific intermediate filament thought to
provide structural support to normal astrocytes. Increase in GFAP
production is a sign of astrogliosis, reactive injury, and neurode-
generation in the differentiated cells. Cells were exposed to
0.5 mM or 1 mM glutamate (24 h) after the pretreatment with
0.1% ASH-WEX (24 h). As shown in Fig. 2a, there was a
significant increase (p,0.05) in GFAP expression in the C6 cells
upon treatment with glutamate which was suppressed with ASH-
WEX pre-treatment in the 0.5 mM glutamate treatment group. In
the 1 mM glutamate treatment group 0.1% ASH-WEX was not
able to normalize the GFAP expression level (Fig. 2a). We
performed RT-PCR in the control and treated groups and found
that there was a significant increase in GFAP mRNA in glutamate
treated groups. ASH-WEX pre-treatment was able to suppress the
upregulation in GFAP mRNA in both low and high dose
glutamate groups (Fig. 2b). Single cell quantitative immunocyto-
fluoroscence for GFAP in these groups further revealed dose
dependent increase in GFAP expression in the glutamate-exposed
as compared to the control cells (Fig. 2c and d). In cells pretreated
Figure 6. Representative Gelatin Zymograms for MMP 2 & 9 from media obtained from different groups of C6 (a) and IMR-32 (b)
cells. The zymograms were analysed using spot –denso method in Alpha Ease software and data was represented as histograms. ‘‘*’’ represents the
statistical significant difference between all the treatment groups (ASH-WEX alone, glutamate alone or glutamate + ASH-WEX groups) with respect to
control group. ‘‘#’’ represents the statistical difference between ‘‘glutamate + ASH-WEX’’ treated groups with their respective ‘‘glutamate’’ treatment
groups. ‘‘*’’ and ‘‘#’’=p,0.05.
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with ASH-WEX, the expression level of GFAP was normalized in
low dose glutamate group but there was no significant difference in
expression of GFAP in high dose group (Fig. 2d).
Neurofilaments (NFs) belong to the family of intermediate
filaments (IFs) and are structural elements of the neuronal
cytoskeleton in an interconnection with actin microfilaments,
microtubules and other IFs. NF-H (NF200) is expressed mainly in
the differentiated neurons while other two forms are abundant in
pre-natal stages. NF200 expression in IMR-32 cells was reduced
upon glutamate exposure, whereas, pretreatment with ASH-WEX
lead to recovery of glutamate induced decrease in NF200 levels
shown by Western blotting (Fig. 2e). These results were supported
by RT-PCR that revealed that ASH-WEX pretreatment resulted
in recovery of NF200 mRNA in glutamate treated cells. There was
20–35% increase in NF200 mRNA expression in ASH-WEX
pretreated groups as compared to their respective 0.25 mM and
0.5 mM glutamate groups (Fig. 2f). NF200 immunostaining
confirmed the changes observed in Western blotting and RT-
PCR results at single cell level (Fig. 2g) that was further
quantitated by intensity analysis (Fig. 2h).
ASH-WEX abolished glutamate-induced increase in
HSP70 is a member of heat shock protein family and serves as a
housekeeper in the cell, assisting in the correct folding, trafficking,
and degradation of many proteins during normal and stressed
conditions. We examined HSP70 expression in control and ASH-
WEX pretreated cells that were challenged with glutamate. As
shown in Fig. 3a and e, HSP70 Western blots revealed significant
increase after exposure to glutamate in a dose dependent manner
in both C6 and IMR-32 cells suggesting that glutamate treatment
evoked stress response in these cells. ASH-WEX treatment both in
C6 and IMR-32 cells resulted in a moderate induction of HSP70
expression and led to normalization of increase in HSP70 induced
by low dose of glutamate (Fig. 3a, e). Upregulation in HSP70
expression (about 60% increase in C6 cells and 40% in IMR-32
cells) induced by higher dose of glutamate was not recovered
significantly upon ASH-WEX pre-treatment (Fig. 3a,e). The RT-
PCR analysis showed increase (p,0.05) in HSP70 mRNA levels at
low dose glutamate treatment group in both cell types; the high
dose glutamate did not cause higher induction of HSP70 mRNA
(Fig. 3b,f). Furthermore, the immunocytostaining for HSP70
showed enhanced intensity in glutamate treated groups as
compared to control (Fig. 3c,g). ASH-WEX pre-treatment resulted
in downregulation of HSP70 in low dose glutamate group in both
the cell lines; high dose glutamate groups remained unaffected
ASH-WEX induces NCAM and PSA-NCAM expression to
reduce excitotoxic cell death in glutamate challenged
NCAM is a glycoprotein of immunoglobulin (Ig) superfamily,
expressed on the surface of neurons and glia cells. It has a role in
cell–cell adhesion, neurite outgrowth, synaptic plasticity, neuro-
protection and learning and memory. We examined NCAM
expression in control and treated groups and found that ASH-
WEX treatment caused a minor increase in NCAM expression
both in C6 and IMR-32 cells (Fig. 4 a, d). The low dose treatment
of glutamate (0.5 mM) led to upregulation of NCAM expression
(p,0.05) which was further increased in the high dose glutamate
(1 mM) treated cells as seen on the Western blots. ASH-WEX
(0.1%) pre-treatment led to normalization of NCAM expression in
the low dose glutamate group but its expression remained
significantly higher (around 45%) in the high dose treatment
group (Fig. 4a,d). These changes were also apparent at mRNA
level. Lower dose of glutamate (0.5 mM) exposure led to increase
in NCAM mRNA level that was normalized by ASH-WEX in C6
cells (Fig. 4b). On the other hand high dose treatment group did
not show normalization of NCAM mRNA in C6 cells when
pretreated with ASH-WEX. Furthermore, ASH-WEX did not
cause any recovery in NCAM mRNA expression in both low and
high dose glutamate groups of IMR-32 (Fig. 4e). Immunocytos-
taining for NCAM was enhanced upon glutamate exposure in case
of C6 cells as well as in IMR-32 cells (Fig. 4 c, f). ASH-WEX pre-
treatment induced normalization was evident by NCAM staining.
Consistent with the protein and mRNA expression data, NCAM
immunocytostaining in IMR-32 cells revealed that ASH-WEX
was not able to recover cells from glutamate-induced changes.
The polysialylated neuronal cell adhesion molecule (PSA-
NCAM) is considered as a marker of developing and migrating
neurons and of synaptogenesis in the immature vertebrate nervous
system. However, it persists in the mature normal brain in some
regions which retain a capability for morphofunctional reorgani-
zation throughout life. We examined PSA-NCAM in control and
treated groups and found that glutamate exposure led to an
increase in the PSA-NCAM expression by about 25% at low
glutamate dose both in C6 and IMR-32 cells which was further
enhanced in ASH-WEX pre-treatment group in the IMR-32 cells
(Fig. 5 a,d). The PSA-NCAM was around 15% (p,0.05) higher at
high dose glutamate treatment group in C6 cells as compared to
control. ASH-WEX pretreated group did not show any significant
change (Fig. 5a). In contrast, there was a dose dependent increase
in PSA-NCAM expression in the IMR-32 cells from 15–45% that
was further enhanced in the ASH-WEX pretreatment (Fig. 5d).
The expression of polysialyltransferase (PST) mRNA was exam-
ined by RT-PCR and was found to be significantly increased both
in glutamate and ASH-WEX treatment groups as compared to
control (Fig. 5 b,e). Immunocytostaining revealed that PSA-
NCAM expression was enriched along the projections of the
differentiated cells in the control group that was further enhanced
by low glutamate treatment both in the C6 and IMR-32 cells.
High dose glutamate led to disruption of surface expression of
PSA-NCAM both in C6 and IMR-32 cells (Fig. 5 c,f).
ASH-WEX modulated MMP-2 and 9 expression after
Matrix metalloproteinase (MMPs) are a family of proteinases
that function to cleave virtually all components of the extracellular
matrix (ECM), making them excellent mediators of early
inflammatory processes, tissue remodeling and scar formation
following a variety of injury types. In particular, the gelatinases,
MMP-2 (gelatinase A) and MMP-9 (gelatinase B) degrade
common ECM components, as well as the major CNS matrix
component, chondroitin sulfate proteoglycans (CSPGs). MMP-2
and MMP-9 have been linked to blood–brain barrier disruption,
inflammation, angiogenesis, remodeling of the ECM and glial scar
formation and are associated with extracellular remodeling that
occurs in injury and repair processes in the CNS. The expression
and activity of MMP-2 and 9 was studied by gelatin zymography.
The expression/activity of both these enzymes was increased in
glutamate treatment groups as apparent by the area of the white
bands. ASH-WEX reduced the enzyme activity significantly upon
treatment in low dose glutamate exposed cultures but was unable
to induce any significant changes in high dose glutamate group in
both the cell lines (Fig. 6 a,b).
Withania Protects against Glutamate Excitotoxicity
PLoS ONE | www.plosone.org9 May 2012 | Volume 7 | Issue 5 | e37080
The current data reveal that the ASH-WEX ameliorated
glutamate induced decrease of cell viability in a dose-dependent
manner. Moreover, glutamate-induced apoptosis/necrosis was
also attenuated after treatment with ASH-WEX as evident from
LDH assay and phase contrast images of cells. Although
Ashwagandha has been reported to improve learning and memory
in rats and as a potent neuroprotectant , but the water soluble
Ashwagandha leaf extract has not been evaluated for its
In the present study, enhanced expression of GFAP upon
glutamate exposure of RA differentiated C6 cells may be
attributed to reactive gliosis and its induction. Upregulation of
intermediate filament proteins, in particular GFAP by reactive
astrocytes is perhaps the best known hallmark of reactive
astrocytes and reactive gliosis. IF upregulation has been found in
CNS trauma, hypoxia, around growing tumors, and in many
neurodegenerative conditions . Recently it has been demon-
strated that a crosstalk between GFAP and glutamate signalling
exists and the expression of GFAP is essential to anchor the
glutamate transporter GLAST in the astrocyte plasma membrane
thus enhancing GLAST-mediated transport . Also GFAP
knockout mice exhibit reduced glutamate clearance . Thus
changes in GFAP gene expression and glutamate homeostasis
might mutually influence each other. Glutamate activates the
GFAP gene promoter of astrocytes through TGF-b pathway .
Normalizaton of GFAP expression with low dose of ASH-WEX
(0.1%) in glutamate (0.5 mM) treatment group depicted possible
cytoprotective effect of the extract in C6 cells.
The expression of NF200 and its phosphorylated form was
reduced upon treatment with glutamate as compared to control
and corresponding ASH-WEX treatment groups. In an adult
neuron, Neurofilaments (NFs) are major cytoskeletal components
of neurons and are composed mainly of three different polypeptide
subunits: NF-L (68 kDa); NF-M (160 kDa); and NF-H (200 kDa)
. Extensive phosphorylation of NFs at the carboxyterminal
domains has been considered one of the means by which
neurofilaments crosslink and stabilize the axonal cytoskeleton
. Therefore, NF200 degradation and dephosporylation in the
glutamate treatment group may be a sign of loss of neuronal
function and eventual neuronal cell death. The ASH-WEX
treatment seems to overcome the glutamate induced adverse
effects in IMR-32 cells with respect to NF200 expression and its
phosphorylation proving its neuroprotective potential. Ashwa-
gandha has been reported to protect against stress induced
neuronal damage in rats due to its antioxidant properties .
Here we propose that the reversal of glutamate mediated changes
in IFs could be partially attributed to its antioxidant mediated
Glutamate exposure lead to increase in HSP70 expression in
dose dependent manner which was reduced in low dose glutamate
exposed cells treated with ASH-WEX. The HSP70 has been
shown to have a neuroprotective role both in animal and cell
culture models of neurotoxicity such as ischaemia , trauma
, seizures  and Alzheimer’s disease . HSPs provide a
line of defense against misfolded aggregation prone proteins and
among the most potent suppressors of neurodegeneration in
animal models . Neurons may rely on their constitutive levels
of HSC70 as a ‘pre-protection’ mechanism for defense against
protein misfolding and aggregation that is induced by stressful
stimuli or associated with neurodegenerative diseases. The
expression of HSP70 was also upregulated in ASH-WEX alone
treated group, thus suggesting that the ASH-WEX treatment
could possibly induce HSP70 expression increasing protective
capacity of cells against glutamate toxicity. In earlier studies,
certain herbal extracts have been reported to induce HSP70
expression . Our current results suggest that ASH-WEX
treatment mediated induction of HSP70 expression may be one of
the mechanisms for its neuroprotective potential against glutamate
toxicity. The increase in expression of HSP70 and cell survival in
the 1 mM glutamate exposed cells treated with ASH-WEX may
be rescuing the cells under stress conditions. Overexpression of
HSP70 has been reported to be associated with a decrease in
apoptotic cell death and a reduction in matrix metalloproteinases
In the present study, we further observed that MMP-2 and
MMP-9 activity is upregulated during glutamate induced damage.
Upon ASH-WEX treatment the expression was significantly
lowered especially in the low dose glutamate treatment group in
C6 and IMR-32 cells. Although MMP-2 is expressed constitutively
in normal nerve cells, its expression is upregulated after injury.
The temporal pattern of this activation coincides with nerve
degeneration and suggests that MMP-2 plays a role in the
regenerative process. Also, MMP-9 is detected in the nerve
immediately following injury and is most abundant at the site of
injury . Another excitotoxic agent Kainic acid (KA) has been
shown to induce neuronal degeneration by up regulation of MMPs
expression. Similarly glutamate mediated upregulation of MMPs
exacerbates neuronal and glial damage . Consistently gluta-
mate led to increase in MMPs expression in dose dependent
manner. The exact mechanisms that trigger glutamate induced
protease synthesis are not clear. It is evident in the present study
that ASH-WEX intervention leads to protection of both the cell
types, at least in low glutamate treated group, which may be
explained by the decrease in expression of MMPs as shown by
NCAM and PSA-NCAM are important cell surface plasticity
markers that play important role in regeneration and repair.
NCAM is developmentally down-regulated but has been shown to
increase after brain injury and this increase has been linked to
potential of brain for regeneration . In the present study, we
observed marked increase in NCAM expression in glutamate
treated group. ASH-WEX treatment further upregulated NCAM
expression besides enhanced cell viability even at high dose of
glutamate. Our study is consistent with the earlier report where
excitotoxic increase in NCAM has been shown in hippocampal
slices . The increase in cell viability could be partially due to
enhanced NCAM expression which is a potent neuroprotection
conferring target as evident from previous studies [5,56,58]. Even
soluble NCAM has been shown to interfere with glutamate-
induced cell death in in vitro excitotoxicity assays. The growth
factor, FGF-2 associated with NCAM signalling has been
described to be neuroprotective against excitotoxicity caused by
glutamate . Control of PSA-NCAM expression by NMDA
receptor activation has been described in several systems,
suggesting a functional link between these two proteins. NMDA
receptors exhibit a dichotomy of signalling with both toxic and
plastic responses. Recent reports from our lab have shown that
exposure to subtoxic concentration of NMDA results in a PSA-
NCAM mediated neuroprotective state that was measured when
these neurons were subsequently challenged with toxic doses of
glutamate [31,60]. Constituents of Withania have been associated
with neuritic regeneration and synaptic reconstruction [3,61].
NCAM and its polysialylated form being important molecules for
CNS repair and regeneration, there may be direct association
between NCAM and PSA-NCAM expression and ASH-WEX
Withania Protects against Glutamate Excitotoxicity
PLoS ONE | www.plosone.org10 May 2012 | Volume 7 | Issue 5 | e37080
mediated regenerative and protective effects towards normaliza-
tion and repair, which needs to be explored further.
PSA-NCAM expression was found to be significantly enhanced
in response to glutamate induced excitotoxicity in C6 and IMR-32
cells, which may represent a compensatory mechanism to combat
stress. In C6 cells, low dose glutamate exposure lead to significant
increase in the PSA-NCAM expression level but percent change
was less than the high dose treated cells. Moreover, the expression
of PST in C6 cells showed dose dependent increase with increase
in glutamate treatment groups and its expression was further
elevated to significant level in the ASH-WEX treatment groups. In
contrast, there was dose dependent increase in PSA-NCAM
expression in glutamate treated IMR-32 cells. ASH-WEX
treatment leads to further increase in expression of PSA-NCAM
as well as PST. These differences could be possibly due to
difference in cell type and therefore the differential expression of
NCAM and degree of polysialylation on neuronal and glial cells.
Several studies have shown that PSA is a potent target to
prevent excitotoxic neuronal cell death during development as well
as under pathological conditions, resulting in glutamate release, at
least in cases when glutamate is accumulated in the extracellular
space at low concentrations. PSA has been proposed to inhibit
activation of GluN2B-containing receptors, possibly by steric
hindrance of the ligand to access the glutamate binding site at low
micromolar concentrations of glutamate . PSA has been
shown to act as a neuroprotective agent, disconnecting overstim-
ulated synapses to protect the relevant circuits from damage
caused by excess glutamtergic input . In another study the
upregulation of HSP70 and PSA-NCAM by hyperthermia has
been correlated and reported to significantly impact the hippo-
campal plasticity, permitting induction of the complex molecular
cascade responsible for neuroprotection [64,65]. In line with these
results, it may be proposed that observations of increase in
expression of HSP70 and PSA-NCAM upon glutamate treatment
protected the cells from excitotoxic cell death. Pharmacological
and biochemical analysis of PSA synthesis have suggested calcium
dependent PST activity . Thus the changes in the expression
of PSA-NCAM in the present study may possibly be attributed to
accumulation of intracellular calcium due to glutamate exposure.
Alteration in PSA-NCAM expression levels on cell surface could
also reflect differential delivery of PSA to cell surface as evident by
a study in oligodendrocyte precursor cells in which NMDA
induced influx of calcium probably enhanced transport of PSA to
the cell surface . PSA inhibits GluN2B-containing receptors at
low micromolar concentrations of glutamate found in the
extracellular space [68,69]. It has been shown that PSA inhibits
NMDAR currents at lower but not at higher concentrations of
glutamate possibly by competing with glutamate in binding to
positively charged amino acids. Furthermore, the expression and
cleavage of the extracellular domain of NCAM/PSA-NCAM is
regulated by metalloproteinase activity resulting in MMP induced
proteolysis resulting in neuronal damage [70,71]. Thus the
decrease in MMP levels upon ASH-WEX treatment could possibly
lead to cellular protection against any such damage. The increase
in NCAM and PSA-NCAM expression upon glutamate exposure
could be protective and regenerative response of the cells towards
glutamate induced damage which is further enhanced by ASH-
WEX treatment possibly leading to recovery of cells from
excitotoxicity. In another study of glutamate-induced excitotoxic-
ity it was revealed that treatment with PSA prevents cell death,
whereas removal of neuronal cell surface-expressed PSA promotes
cell death . Thus, PSA carried by NCAM regulates both
synaptic plasticity and viability via modulation of NMDA
receptors. The increase in PSA levels seen in the current results
may be functionally linked to cell tolerance e.g. protection against
glutamate-induced cell death, which is apparent at lower
concentration of glutamate only.
Withania extracts has been widely studied for their neuropro-
tective properties in animal models and in vitro studies. ASH-WEX
comprises of six different water soluble molecules  which
might be alone or in combination are associated with neuropro-
tective activity of the extract. One of the components of alcoholic
extract of leaves, Withaonone has been shown to impart
protection against Methoxyacetic acid (MAA) induced toxicity
by suppressing the ROS levels, DNA and mitochondrial damage in
vitro . Its bioactive components Sitoindosides VII-X and
withaferin A have been shown to modulate brain functions by
binding with cholinergic receptors . Modulation of release of
three neurotransmitters i.e., acetylcholine, glutamate and seroto-
nin by Withania in all probability contributes to inhibition of
nNOS in extract treated stressed mice . The neuroprotective
properties of Withania have been attributed to neurochemical
alterations of specific neurotransmitter systems and suppression of
glucocorticoid release in chronic stress which could be exploited
for treatment of neurodegenerative diseases . Withania root
extracts have been shown to impart protection against 6-
hydroxydopamine induced rat model and various other animal
models for neurological disorders [75,76,77]. Evidence also
indicate that withanolide A, withanoside IV and withanoside VI
from the Withania extract induced significant regeneration of both
axons and dendrites, in addition to the reconstruction of pre- and
postsynapses in the neurons . The crude ethanolic extract of
Withania roots has been shown to mitigate the effects of
excitotoxicity and oxidative damage in hippocampus and the
underlying mechanism could be attributed to its antioxidative
properties [7,47,78]. Consistent with these neuroprotective prop-
erties of Withania extracts, present study illustrates the neuro-
modulatory role of aqueous extract from leaves of Withania
against glutamate induced stress and upregulation of plasticity
marker proteins such as HSP70, NCAM and PSA-NCAM may
rescue the glial and neuronal cells from glutamate induced
The cytoprotective effects observed in this study could be
attributed to the presence of free radical scavenging compounds in
the water extract of Ashwagandha. In the present study low level
glutamate induced effects were normalized by ASH-WEX but it
could only partially revert the cytotoxic effects when challenged
with high dose of glutamate. The higher expression of HSP70,
NCAM and PSA-NCAM in response to glutamate exposure could
be possibly due to cytoprotective response of cells towards
excitotoxicity in the time frame of these experiments. ASH-
WEX treatment lead to significant increase in viability in
glutamate treated groups implicating its cytoprotective role against
cytotoxicity. As elevated levels of glutamate have been implicated
in a wide range of neurological diseases thus further research into
the molecular mechanism of ASH-WEX mediated neuroprotec-
tion and the search for bioactive component(s) in these extracts
may prove valuable therapeutic agent to combat neurological
Conceived and designed the experiments: GK HK RW SK. Performed the
experiments: HK. Analyzed the data: HK GK RW SK. Contributed
reagents/materials/analysis tools: GK. Wrote the paper: GK HK RW SK.
Withania Protects against Glutamate Excitotoxicity
PLoS ONE | www.plosone.org11 May 2012 | Volume 7 | Issue 5 | e37080
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PLoS ONE | www.plosone.org13 May 2012 | Volume 7 | Issue 5 | e37080