Anti-apoptotic effects of hyperoside via inhibition of NR2B-containing NMDA receptors.

Abstract

Hyperoside (Hyp) is a flavonoid compound isolated from a folk remedy, Rhododendron ponticum L. leaves. It has been shown to have neuroprotective effects both in vivo and in vitro. However, little is known about the effects of Hyp on the neuronal apoptosis induced by glutamate. The present study showed that Hyp significantly attenuated, in a concentration-dependent manner, the apoptosis induced by the exposure of cultured neurons to NMDA. Western blot analysis revealed that Hyp antagonized the expression of excess NR2B-containing NMDA receptors; however, it had no effect on the expression of NR2A-containing NMDA receptors. Our results demonstrate that the neuroprotective effect of Hyp owes, at least partially, to its differential modulation of NR2A- and NR2B-containing NMDA receptors.

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Short communication
Anti-apoptotic effects of hyperoside via inhibition
of NR2B-containing NMDAreceptors
Xiao-Nan Zhang1*, Jin-Mao Li2*, Qi Yang1*, Bin Feng1, Shui-bing Liu1,
Zhao-Hui Xu1, Yan-Yan Guo1, Ming-Gao Zhao1
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Correspondence: ???????? ????? ??????? ???????????????????
Abstract:
Hyperoside (Hyp) is a flavonoid compound isolated from a folk remedy, Rhododendron ponticum L. leaves. It has been shown to
haveneuroprotectiveeffectsbothinvivoandinvitro.However,littleisknownabouttheeffectsofHypontheneuronalapoptosisin-
ducedbyglutamate.ThepresentstudyshowedthatHypsignificantlyattenuated,inaconcentration-dependentmanner,theapoptosis
induced by the exposure of cultured neurons to NMDA. Western blot analysis revealed that Hyp antagonized the expression of ex-
cessNR2B-containingNMDAreceptors;however,ithadnoeffectontheexpressionofNR2A-containingNMDAreceptors.Ourre-
sults demonstrate that the neuroprotective effect of Hyp owes, at least partially, to its differential modulation of NR2A- and
NR2B-containing NMDAreceptors.
Key words:
hyperoside, apoptosis, NMDAreceptor, neuron
Abbreviations: CNS – central nervous system, DIV – days in
vitro, DMEM – Dulbecco’s Modified Eagle Medium, Hyp – hy-
peroside, NMDA – N-methyl-D-aspartate, NMDARs – NMDA
subtype of glutamate receptors, ROS – reactive oxygen species
Introduction
The accumulation of high concentrations of glutamate
and the excessive stimulation of glutamate receptors
induce potent excitotoxicity in the central nervous
system (CNS) [4]. It is widely accepted that the N-
methyl-D-aspartate (NMDA) subtype of glutamate re-
ceptors (NMDARs) plays a key role in mediating glu-
tamate excitotoxicity due to the high permeability of
calcium [3, 17]. Activation of NMDARs is a critical
early step in the glutamate-evoked excitotoxic injury
of CNS neurons. Native NMDARs are composed of
NR1, NR2 (A, B, C, and D), and NR3 (A and B)
subunits. The formation of functional NMDARs re-
quires a combination of NR1, an essential channel-
forming subunit, and at least one NR2 subunit. It is
well-known that the NR2A and NR2B subunits pre-
dominate in the forebrain, where they determine
many of the functional properties of NMDARs.
NMDARs are thought to underlie important forms of
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synaptic and cellular plasticity and have a docu-
mented role in learning and memory [2]. However,
overactivation of extrasynaptic NMDARs has been
linked to excitotoxic death [7], implying that the loca-
tion of NMDARs plays an important role in specify-
ing neuronal survival or protective actions [17]. In ad-
dition, differences in NMDAR subunit compositions
are responsible for neuronal cell survival or death. It
has been suggested that NR2A- and NR2B-containing
NMDARs are linked to different intracellular cas-
cades and participate in different functions in synaptic
plasticity and pathological conditions, whereas exci-
totoxicity is triggered by the selective activation of
NMDARs containing the NR2B subunit [11, 17].
Hyperoxide (Hyp) is a flavonoid compound iso-
lated from a folk remedy, Rhododendron ponticum L.
leaves (Fig. 1A), one of the most common herbal
medicines used in China. Animal experiments have
revealed its anti-inflammatory and cardiovascular
protective activities [1, 18]. Recently, Hyp has been
investigated for its possible effects on the central
nervous system, including antidepressant, neuropro-
tective, and analgesic activities in mice [5, 12]. Fur-
thermore, Hyp is shown to possess cytoprotective
properties against oxidative stress by scavenging in-
tracellular reactive oxygen species (ROS) and en-
hancing antioxidant enzyme activity [10, 14]. How-
ever, little is known about the effects of Hyp on the
neuronal apoptosis induced by glutamate, the princi-
pal excitatory neurotransmitter in the CNS.
In recent years, there has been a growing interest in
the neuroprotective effects of herbal medicines due to
their NMDAR antagonist properties [6]. In the pres-
ent study, we employed integrative approaches, in-
cluding primary cortical neuron culture, Hoechst/PI
double staining and western blots, to investigate
whether the neuroprotective effects of Hyp involve
the inhibition of NR2B-containing NMDARs.
Materials and Methods
Primary neuron culture
Experiments were carried out on Sprague-Dawley rats
(18-day-old embryos). All animal protocols used were
approved by the Animal Care and Use Committee of
the Fourth Military Medical University. Cultured pre-
frontal cortex neurons were prepared as previously
described [20]. Briefly, the prefrontal cortex was dis-
sected, minced, and trypsinized for 15 min using
0.125% trypsin (Invitrogen). Cells were seeded onto
either 24-well plates containing glass coverslips
(Fisher Scientific), 60-mm dishes, or 96-well plates
precoated with 50 µg/ml poly-D-lysine (Sigma) in
water, and grown in Neurobasal-A medium (Invitro-
gen) supplemented with B27 and 2 mM GlutaMax
(Invitrogen). The cultures were incubated at 37°C in
95% air/5% carbon dioxide with 95% humidity. The
cultures were used for experiments between 10 and 14
days in vitro (DIV).
Cell treatment
Cultures were washed twice using Mg??-free extracel-
lular solution containing (in mM): NaCl 140, KCl 3,
CaCl?2, HEPES 10, and glucose 10; cultures were
adjusted to pH 7.2–7.3 with NaOH and osmotic pres-
sure 290 ± 5 Osm with sucrose [15]. The cortical neu-
rons were exposed to Mg??-free extracellular solution
supplemented with NMDA and 10 µM glycine
(Sigma) for 30 min. The cells were then washed three
times and returned to the original culture medium for
24 h. Hyp (Sigma) was dissolved in 50% ethanol. The
final concentration of ethanol was less than 0.1% in
the culture medium. Hyp was added 24 h prior to the
addition of NMDA and was present throughout the
excitotoxic insult and subsequent 24 h culture.
Cell viability analysis
An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide] assay was used to detect cell vi-
ability as previously described [15]. Briefly, neurons
were cultured in 96-well plates at a density of 5 × 10?
cells per well. MTT was dissolved in Dulbecco’s
Modified Eagle Medium (DMEM) and added to each
well at a final concentration of 0.5 mg/mL for 4 h at
37°C. The medium was then replaced with 150 µl di-
methyl sulfoxide (DMSO). The optical density (OD)
was read on a Universal Microplate Reader (Elx 800,
Bio-TEK Instruments Inc., USA) at 570 nm (with
630 nm as a reference). Cell viability was expressed
as a percentage of the control value.
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Hoechst/PI double staining
Apoptosis was determined by propidium iodide (PI,
Sigma) and Hoechst 33258 (Sigma) double fluores-
cent staining as previously described [15]. Neurons
were cultured in 24-well plates at a density of 600
cells/mm?. After the excitotoxic injury, the cells were
stained with PI (1 µg/ml) and Hoechst 33258 (10 µg/
ml) for 15 min, then fixed by 4% paraformaldehyde
for 10 min. Cells were observed under a fluorescence
microscope (Olympus BX61, Japan). The Hoechst
and PI dyes were excited at 340 nm and 620 nm, re-
spectively. Apoptotic neurons displayed a distinct
morphology characterized by the condensation of
chromatin, nuclear shrinkage, and the formation of
a few apoptotic bodies. For each well, three visual
fields were selected randomly.
Western blot
Equal amounts of protein (50 µg) from the total cell
lysate were separated and electrotransferred onto
PDVF membranes (Invitrogen), which were probed
with anti-NR2A, anti-NR2B, anti-Bax, and anti-Bcl-2
(Chemicon), as well as with ?-actin (Sigma) as
a loading control. The membranes were incubated
with horseradish peroxidase conjugated secondary an-
tibodies (anti-rabbit IgG for the primary antibodies),
and bands were visualized using an ECL system
(Perkin Elmer).
Data analysis
Results were expressed as the mean ± SEM. Statisti-
cal comparisons were performed using a t-test, one-
way analysis of variance (ANOVA), or two-way re-
peated measures ANOVA with the Student’s t-test for
post-hoc analyses. In all cases, values of p < 0.05
were considered statistically significant.
Results
Effects of Hyp on cell viability
Cortical neurons exposed to 100 µM NMDA for 30
min caused a serious neuron loss. As illustrated in
Figure 1B, treatment with Hyp alone had no effects on
the cell viability. However, Hyp prevented NMDA-
mediated cell death in a concentration-dependent
manner (Fig. 1B). NMDA-mediated excitotoxicity
was also blocked by 0.3 µM Ro 25-6981, a specific
NR2B subunit antagonist. These results show that
Hyp may attenuate NMDA-induced excitotoxicity in
cultured cortical neurons.
Neuroprotection of Hyp against NMDA-induced
apoptotic cell death
Considering that the MTT assay failed to distinguish
between necrosis and apoptosis, we carried out
Hoechst/PI double staining to detect the anti-
apoptotic activities of Hyp. The Hoechst 33258 and PI
staining demonstrated that 27.2% ± 2.8% cells under-
went apoptosis in the NMDA insult group, whereas
5.2% ± 0.2% cells in the normal control group under-
went apoptosis (p < 0.01 vs. the NMDA group; Fig.
2A and 2B). However, the presence of 1 µM and
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951
Anti-apoptotic effects of hyperoside
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10 µM Hyp significantly reversed the apoptosis in-
duced by NMDAin the cultured cortical neurons. The
apoptotic rates in NMDA-treated cells were decreased
to 17.2% ± 1.2% and 9.8 % ± 0.8% in the presence of
1 µM and 10 µM Hyp, respectively (p < 0.01 vs. the
NMDAgroup; Fig. 2Aand 2B).
Effects of Hyp on expression of Bcl-2 and Bax
Next, we performed western blot analyses of the
apoptotic-related proteins from the primary cultures
with NMDA stimuli in the absence or presence of
Hyp. Anti-apoptotic proteins of the Bcl-2 family may
exert neuronal protection by multiple mechanisms,
such as a direct antioxidant effect, inhibition of pro-
apoptotic proteins, and mitochondrial membrane sta-
bilization. As shown by the western blots, the total
amount of the anti-apoptotic protein Bcl-2 was sig-
nificantly changed (0.71 ± 0.04-fold of the control,
p < 0.01; Fig. 3A and 3B), as was the pro-apoptotic
protein Bax (3.12 ± 0.06-fold of the control, p < 0.01;
Fig. 3A and 3C), in cultured cortical neurons after
exposure to 100 µM NMDA. However, the presence
of 1 µM and 10 µM Hyp showed an anti-apoptotic ac-
tivity by increasing Bcl-2 (0.89 ± 0.12 and 0.91 ±
0.15-fold of the control, respectively, p < 0.05 vs. the
NMDA group; Fig. 3A and 3B) and inhibiting Bax
(1.71 ± 0.15 and 1.05 ± 0.11-fold of the control, re-
spectively, p < 0.05 or 0.01 vs. the NMDAgroup; Fig.
3Aand 3C) expression.
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Downregulation of NR2B-containing NMDARs
by Hyp
It has been suggested that NR2A- and NR2B-containing
NMDARs are linked to different intracellular cas-
cades and have different functions in cell survival or
death [11]. To further examine the effects of Hyp on
NMDAR subtypes, we performed western blots to ana-
lyze NR2A- and NR2B-containing NMDARs expres-
sion. Our results showed that the NR2B subtype ex-
pression was notably increased in cultured cortical
neurons after exposure to 100 µM NMDA (4.7 ± 1.8-
fold of the control, p < 0.01; Fig. 4A and 4C), whereas
the NR2Asubtype expression was not notably changed
(1.1 ± 0.1-fold of the control; Fig. 4A and 4B). Over-
expression of the NR2B subtype by NMDA stimuli
was significantly reduced in the presence of 1 µM and
10 µM Hyp (2.5 ± 0.3 and 1.4 ± 0.3-folds of the con-
trol, respectively; p < 0.05 vs. the NMDA group; Fig.
4A and 4C). However, Hyp had no effects on the
NR2A subtype expression (1.2 ± 0.23 and 1.2 ± 0.2-
fold of the control, respectively; Fig. 4A and 4B).
These results suggest that downregulation of NR2B-
containing NMDARs expression by Hyp at least par-
tially contributes to its neuroprotection against gluta-
mate-evoked excitotoxic injury.
Discussion
In the present study, we demonstrated that Hyp pre-
vented the injury induced by NMDA stimuli in cul-
tured cortical neurons and increased their cell viabil-
ity, as shown by the results from the MTT assay,
apoptotic staining, and western blot analysis. The re-
sults suggest that the neuronal protection of Hyp is
correlated to the suppression of apoptosis induced by
activation of NR2B-containing NMDARs.
Glutamate is the main excitatory neurotransmitter
in the mammalian central nervous system. NMDAR-
mediated signals are critical for the survival of devel-
oping neurons and for several forms of synaptic plas-
ticity [2, 13]. However, a high concentration of gluta-
mate induces neuronal cell damage under in vitro or in
vivo conditions [3, 17]. Overactivation of NMDARs is
a critical early step in the glutamate-evoked excito-
toxic injury of CNS neurons. It has been documented
that NR2A- and NR2B-containing NMDARs are
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953
Anti-apoptotic effects of hyperoside
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