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Neuroprotection of the leaf and stem of vids amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons.


Vitis amurensis (Vitaceae) has been reported to have anti-oxidant and anti-inflammatory activities. The present study investigated a methanol extract from the leaf and stem of V. amurensis for neuroprotective effects on cerebral ischemic damage in rats and on excitotoxicity induced by glutamate in cultured rat cortical neurons. Transient focal cerebral ischemia was induced by 2h middle cerebral artery occlusion followed by 24-h reperfusion (MCAO/reperfusion) in rats. Orally administered V. amurensis (25-100 mg/kg) reduced MCAO/reperfusion-induced infarct and edema formation, neurological deficits, and neuronal death. Depletion of glutathione (GSH) level and lipid peroxidation induced by MCAO/reperfusion was inhibited by administration of V. amurensis. The increase of phosphorylated mitogen-activated protein kinases (MAPKs), cyclooxygenase-2 (COX-2), and pro-apoptotic proteins and the decrease of anti-apoptotic protein in MCAO/reperfusion rats were significantly inhibited by treatment with V. amurensis. Exposure of cultured cortical neurons to 500 [micro]M glutamate for 12 h induced neuronal cell death. V. amurensis (1 -50 fxg/rnl) and (+)-ampelopsin A, [gamma]-2-viniferin, and frans-c-viniferin isolated from the leaf and stem of V. amurensis inhibited glutamate-induced neuronal death, the elevation of intracellular calcium ([[Ca.sup.2+].sub.i]), the generation of reactive oxygen pecies (ROS), and changes of apoptosis-related proteins in cultured cortical neurons, suggesting that the neuroprotective effect of V. amurensis may be partially attributed to these compounds. These results suggest that the neuroprotective effect of V. amurensis against focal cerebral ischemic injury might be due to its anti-apoptotic effect, resulting from anti-excitotoxic, anti-oxidative, and anti-inflammatory effects and that the leaf and stem of V. amurensis have possible therapeutic roles for preventing neurodegeneration in stroke.

[c] 2011 Elsevier GmbH. All rights reserved.


Keywords: Vitis amurensis Neuvoprotection Focal ischemia Excitotoxicity Antiapoptosis


Acute ischemic stroke induced by a transient or permanent reduction of cerebral blood flow is one of the leading causes of death and long lasting disability in adults. The reduction of blood flow to brain tissue causes the loss of various neurological functions (Khan et at, 2009). The major pathological mechanisms of ischemic brain injury include excitotoxicity, oxidative stress, inflammation, and apoptosis, which are associated with mitocondrial dysfunction and a rapid decrease of ATP (Candelario-Jalil, 2009). With energy depletion, disrupted membrane potential causes massive neuronal depolarization and an increase of intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) resulting in an extracellular buildup of excitatory amino acids and consequent excitotoxicity (Katsura et al., 1994; Heiss and Graf, 1994). Depletion of glutathione (GSH) in ischemic brain leads to neuronal cell apoptosis via lipid peroxidation (Liu et al., 2010). Phosphorylation of mitogen-activated protein kinases (MAPKs) including c-jun N-terminal kinase (JNK), extracellular signaling-regulating kinases (ERK), and P38 MAPK activates inflammatory cascades; elevates generation of free radicals including superoxide anion ([O.sub.2]-), hydroxyl radical (OH-), and hydrogen peroxide ([H.sub.2][O.sub.2]); and subsequently triggers neuronal cell apoptosis in ischemic brain (Kishimoto et al., 2010; Choi et al., 2004). The BcL-2 family proteins (e.g. anti-apoptotic BcL-2 and BCL-XL, pro-apoptotic BAK and BAX) are involved in the apoptotic pathway cascade (Allsopp et al., 1993; Hetz et al., 2005).

Vitis species, from the family Vitaceae, have been traditionally used world-wide as medicinal herbs with anti-inflammatory, analgesic, and antitoxic properties (Nassiri-Asl and Hosseinzadeh, 2009). Grape seed extract and grape polyphenols suppress hypoxic ischemic brain injury in rats (Huang et al.f 2009). Vitis amurensis is one of the most common wild grapes in Korea, Japan, and China. Its fruits are used to make juice and wine, while the root and stem have been used as traditional medicine for the treatment of pain, such as stomach ache, neuralgic pain, and abdominal pain and cancer (Huang and Lin, 1999). Recently, the root and stem of V. amurensis have been reported to have anti-oxidant and anti-inflammatory activities and neuroprotective effects in PCI2 cells (Huang et al., 2009; Jang et al., 2007). Studies on the chemical substances and pharmacological activities of the leaf and stem of V. amurensis have isolated 11 resveratrols, including stilbenes and oligostil-benes, which have antioxidant and anti-inflammatory effects and cytotoxic activity in cancer cell lines (Ha do et al., 2009a, 2009b; Yim et al., 2010). In a recent study, we demonstrated that the leaf and stem of V. amurensis protected against (3-amyloid protein-induced memory deficit in mice and cultured neuronal cell death (jeong et al., 2010). The present study was conducted to further verify the neuroprotective effect of the leaf and stem of V. amurensis on cerebral ischemic injury and excitotoxicity using both in vivo and in vitro studies. We investigated the protective effect of the leaf and stem of V. amurensis against neuronal injury induced by middle cerebral artery occlusion followed by reperfusion (MCAO/reperfusion) in rats, and against excitotoxicity induced by glutamate in cultured rat cortical neurons. In addition, we identified (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin as active components contributing to the neuroprotective effect of V. amurensis against glutamate-induced neurotoxicity (Fig. 1).

Materials and methods

Plant material preparation, and isolation of active compounds

The leaf and stem of V. amurensis were gathered in Keryong Mountain in Daejeon, Korea, in July 2007. Botanical identification was performed by Professor KiHwan Bae, and the voucher specimen (CNU-1552) was deposited at the herbarium of the College of Pharmacy, Chungnam National University, Korea. Dried leaf and stem of V. amurensis (4.6 kg) were extracted using methanol (MeOH) (151 x 24h x 3) at room temperature, filtered, and concentrated to yield a MeOH extract (658 g), which was stored at room temperature until needed. The MeOH extract was suspended in water, and the suspension was consecutively partitioned with hexane (1.51 x 3), ethylacetate (EtOAc) (1.51 x 3), and butanol (BuOH) (1.51 x 3) for the activity-guided purification. Since the EtOAc-soluble fraction exhibited considerable activity for inhibiting glutamate-induced neuronal death, this fraction was extensively investigated. The EtOAc fraction (185g) was subjected to a silica gel column (Merck, 0.063-0.200 mm) eluted with hexane-EtOAc, increasing EtOAc gradually to supply seven fractions (Fr. 1-7). Fr. 4 (50.8 g) was further chromatographed on a Sephadex LH-20 column and eluted with MeOH-[H.sub.2]O (2:1) to give four sub-fractions (Fr. 4.1-4.4). [gamma]-2-Viniferin (214 mg) and trans-[epsilon]-viniferin (1148mg) were yielded from Fr. 4.2 (2.8g) after purification by silica gel column chromatography [(Merck, 0.040-0.063 mm); CHCl3-EtOAc-acetone-MeOH (20:1:1:1)]. Fr. 6 (56g) was chromatographed on a silica gel column [(Merck, 0.040-0.063 mm)] using a gradient of CH[Cl.sub.3]-MeOH mixture (from 7:1 to 1:1) to yield eight fractions (Fr. 6.1-6.8). Fr. 6.2 (47 g) was repeatedly subjected to silica gel column chromatography [(Merck, 0.040-0.063 mm); CHCl3-EtOAc-acetone-MeOH (7:1:1:1)] to give nine fractions (Fr. 6.2.1-6.2.9). The purification of Fr. 6.2.2 (4.2 g) using an RP column [YMC C-18; MeOH-H20 (1:2)] yielded (+)-ampelopsin A (300 mg). (Ha do et al.,2009a). The structures of (+)-ampelopsin A, 7-2-viniferin, and frans-[epsilon]-viniferin are shown in Fig. 1.

A HPLC was performed using two Waters 515 pumps, a 2996 photodiode array detector, and a Chemcobond 5-ODS-H column (4.6 mm x 150 mm I.D.). The mobile phase was composed of acetonitrile (A) and water (B) with a linear gradient elution: Omin, 20% A; 40 min, 100% A. The crude MeOH extract of leaf and stem of V. arnurensis was filtered on membrane filters with a prose size 0.45mm (Millipore) and the injection volume was lOfxl. As shown in Fig. 2, the MeOH extract contained (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin along with seven other known compounds, which were also isolated from leaf and stem of V. arnurensis.

Chemicals and reagents

Glutamate, verapamil, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MIT), Dulbecco's modified Eagle's medium (DMEM), 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin (H&E), L-glutathione (GSH), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), and [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluo-4 AM and 2',7'-dichlorodihydrofluorescin diacetate ([H.sub.2]DCF-DA) were from Molecular Probes Inc. (Eugene, OR, USA), fetal bovine serum, from JRS scientific Inc. (Woodland, CA, USA); (5R,10s)-(+)-5-methyl-10,ll-dihydro-5H-dibezo[a, d]cyclohepten-5, 10-imine (MK-801), from RBI (Natick, MA, USA); thiobarbituric acid (TBA), from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan); proprep buffer. from iNtRONBio. Inc. (Gyunggi-Dot Korea); and sodium dodecyl sulfate (SDS) was from Bioshop Canada hie. (Ontario, Canada). Antibodies recognizing rabbit polyclonal antibody against BcL-2, BAK, BAX, cleaved-caspase-3 (c-caspase-3), phosphorylated-ERK 1/2 (p-ERK 1/2) MAPK, phosphorylated-P38 (p-P38) MAPK, and cyclooxygenase-2 (COX-2); mouse polyclonal antibody against [3-actin; and horseradish peroxidase-conjugated anti-rabbit secondary antibody were purchased from Millipore Inc. (Bedford, MA, USA). Horseradish peroxidase-conjugated anti-mouse secondary antibody was purchased KOMA Biotech Inc. (Seoul, Korea). All other chemicals used were of the highest grade available.

Experimental animals

Pregnant Sprague-Dawley (SD) rats (Daehan BioLink Co., Ltd., Chungbuk, Korea) and male SD rats (280-300 g) (Sam: TacN(SD)fBR) (Samtako, Inc., Gyunggi-Do, Korea) were housed in environmentally controlled rooms at 22[+ or -]2[degrees]C, with a relative humidity of 55 [+ or -]5%, a 12 h light/dark cycle, and food and water ad libitum. The procedures involving experimental animals complied with the regulations for the care and use of laboratory animals of the animal ethical committee of Chungbuk National University and the "Guide Principles in the Use of Animals in Toxicology", which was adopted by the Society of Toxicology in 1989.

MCAO/reperfusion-induced focal cerebral ischemia in rats and evaluation of ischemic brain damage

Before surgery, male SD rats weighing 280-300g were fasted overnight with free access to water. Focal cerebral ischemia was produced by MCAO for 2h, followed by reperfusion for 24 h, as previously described (Ban et al, 2008). The sham-operated rats did not suffer MCAO, except with exposure to external and internal carotid arteries. Following neurological score evaluation and motor coordination test after MCAO/reperfusion, rats were sacrificed by decapitation under anesthesia (diethyl ether), and brains were quickly removed. The infarct and edema volumes of brain tissue were measured using TTC staining, as previously described (Ban et al., 2008). Histological examination of brain tissue was performed using H&E staining (Ban et al., 2008). The neurological status of animals was evaluated according to the Menzies method after MCAO/reperfusion (Menzies et al., 1992). The rotarod test was performed to evaluate motor coordination of animals as previously described (Kim et al., 2011). V. amurensis (25, 50, and l00 mg/kg, p. o.) was orally administered three times: 0.5 h before and 1 h after occlusion and lh after reperfusion. MK-801 (1 mg/kg, i.p.) was administered two times immediately after occlusion and reperfusion.

Biochemical assays

Additionally, ipsilateral and contralateral cortical tissue was dissected after 24 h of reperfusion and maintained at -80[degrees]C The homogenates with a four-fold volume of 100 mM phosphate buffer (pH 7.4) were prepared freshly in an ice bath. Reduced GSH was estimated spectrophotometrically, according to the method described by Ellman et al. (1961). Values were expressed as nmol per mg protein. The extent of lipid peroxidation was assayed by the measurement of thiobarbituric acid reactive substance (TBARS) at 532 nm, as described by Yoshioka et al. (1979) using 1,1,3,3-tetramethoxypropane as a standard. TBARS values were expressed as nmol per mg protein. Protein concentration was determined according to Lowry's method (Lowry etal., 1951).

Western blot

After 24 h reperfusion, brain tissue in penumbral areas was isolated and homogenized with proprep buffer, and the total protein was extracted. In cultures, neurons treated with 500 [micro]M glutamate for 12 h on dishes were lysed with RIPA buffer containing 150mM NaCl, 1 mM Na-EDTA, protease inhibitor cocktail, and 50 mM Tris-HCl, pH 7.4 to extract protein. The amount of protein was measured by the Bradford method (Bradford, 1976). Approximately 50 [micro]g of total protein were loaded on 12.5% SDS-PAGE and transferred to a PVDF membrane (Perkin Elmer Co., MA, USA). The membranes were incubated with primary antibodies against BcL-2, BAK, BAX, c-caspase-3, p-ERK 1/2 MAPK, p-P38 MAPK, COX-2, or [beta]-actin (1:1000), followed by horseradish-peroxidase conjugated anti-rabbit (1:1500) or anti-mouse (1:1000) secondary antibodies. Protein expression was detected with an enhanced chemilumi-nescence detection reagent (Santa Cruz Biotechnology Inc., CA, USA). Images were quantified using image analysis software (a freely available application in the public domain for image analysis and process, developed and maintained by Wayne Rasband at the Research Services Branch, National Institutes of Health, USA).

Induction and measurement of neurotoxicity in cultured cerebral cortical neurons

Primary cortical neuron cultures were prepared using embryonic day 15-16 SD rat fetuses, as previously described (Ban et al., 2008). Neurotoxicity experiments were performed using neurons after 4-6 days in culture. Neurons were exposed to 500 [micro]M glutamate in a HEPES-buffered solution containing 8.6 mM HEPES, 154mM NaCl, 5.6 mM KC1, and 2.4 mM Ca [Cl.sub.2], pH 7.4 at 37[degrees]C for 12 h to produce neuronal death and 4h for ROS generation. V. amurensis and the compounds, (+)-ampelopsin A, [gamma]-2-viniferin, and trans-e-viniferin, were dissolved in methanol at concentrations of 50mg/ml and 5mM, respectively, and they were further diluted in experimental buffers. The final concentration of methanol was [less than or equal to] 0.1%, which did not affect cell viability. For each experiment, V. amurensis, (+)-ampelopsin A, [gamma]-2-viniferin, trans-e-viniferin, MK-801 (10 [micro]M),verapamil (20 [micro]M),and L-NAME(l mM)were applied 20 mm prior to treatment with glutamate; they also were present in the medium during glutamate incubation. Neuronal viability was measured using the colorimetric MTT assay. Changes in [[Ca.sup.2+].sub.j] and reactive oxygen species (ROS) generation were measured with fluorescent dyes, as previously described (Ban et al., 2008).

Statistical analysis

Data were expressed as the mean [+ or -] S.E.M., and statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's tests. P values of < 0.05 were considered significant.


Inhibitory effect of V.amurensis on MCAO/reperfusion-induced histopathologicai damage

Representative brain slices from sham-, vehicle-, V. amurensis-, and MK-801-treated groups are shown in Fig. 3A. After MCAO/reperfusion, a large ipsilateral cerebral infarction was observed in the rat brain. A TTC-stained coronal section, in which normal brain tissue stains deep red, was used to determine the volume of a cerebral infarction; infarct tissues did not stain. Orally administered V. amurensis (25, 50, and 100mg/kg) significantly reduced cerebral infarct and edema volumes induced by MCAO/reperfusion (Fig. 3B). In addition, MK-801 (1 mg/kg, i.p.), an N-methyl-D-aspartate (NMDA) receptor antagonist, markedly reduced the infarct and edema volumes. Rat body temperatures were monitored for 6h after cerebral reperfusion started, and no significant differences were observed between the groups (data not shown). Thus, the observed neuroprotective effect of V. amurensis could not be attributable to hypothermic effects.

H&E staining showed the presence of histological changes in the ischemic regions of injured brains with the following characteristics: condensate nuclei, loss of affinity for hematoxylin, perinuclear vacuole, pyknotic nuclei, and cytoplasmic eosinophilia in the subcortical and hippocampal CA1 regions (Fig. 4B, E, and H); however, such changes were rarely detected in the contralateral hemispheres or in sham animals (Fig. 4A, D, and G). V. amurensis (l00mg/kg) inhibited MCAO/reperfusion-induced changes (Fig. 4C, F, and I).

Inhibitory effect ofV. amurensis on MCAO/reperfusion-induced behavioral deficits in rats

MCAO/reperfusion rats showed neurological deficits, such as circling movement and decreased grip of the contralateral fore-limb. The neurological deficit score was reduced significantly to 2.3 [+ or -]0.4 in the V. amurensis-treated (l00mg/kg) group compared to that of the vehicle-treated control group, which scored 3.4 [+ or -] 0.2 (Table 1). Rotarod duration of MCAO/reperfusion rats also decreased to 66.8 [+ or -] 2.2 s, compared to 255.3 [+ or -] 10.2 s for the sham group. Decrease of rotarod duration was significantly prevented by V. amurensis treatment, showing 145.5 [+ or -] 6.0 s for 100 mg/kg treatment (Table 1).

Table 1
Effect of V. omurensis on MCAO/reperfusion-induced
behavioral deficits in rats.
Group         Dose(mg/kg)  Neurological score  Rotarod duration (s)

Sham                    -                   0    255.3 [+ or -]10.2
Vehicle                 -     3.8 [+ or -]0.2   66.8 [+ or -] 2.2##
V. amurensis           25     2.9 [+ or -]0.2      87.9 [+ or -]6.6
V. amurensis           50   2.3 [+ or -] 0.4*   127.8 [+ or -]5.7**
V. amurensis          100   2.0 [+ or -]0.3**   145.5 [+ or -]6.0**

After MCAO/reperfusion, neurological status and rotarod duration of
the animals used in Fig. 2 were evaluated. Five categories of
neurological finding according to Menzies were scored: 0, no apparent
deficits; 1, contralateral forelimb flection; 2, decreased grip of
contralateral forelimb; 3, contralateral circling of pulled by tail;
and 4, spontaneous contralateral circling. Values are
expressed as mean [+ or -] S.E.M.
* P < 0.05.
** P < 0.01 vs. vehicle.
** P < 0.01 vs. sham control.

Inhibitory effect of V. amurensis on MCAO/reperfusion-induced decrease of GSH content and lipid peroxidation

Levels of GSH and TBARS, which is a marker of lipid peroxidation, were estimated 24 h after MCAO/reperfusion in the ipsilateral brain. The GSH content significantly decreased in vehicle animals compared to that of sham animals. However, V. amurensis dose-dependently inhibited the decrease of GSH level (Table 2). The level of TBARS was significantly elevated in vehicle animals compared to that of sham animals. Rats treated with V. amurensis exhibited significant attenuation of TBARS level (Table 2). The contralateral tissues in all groups showed no significant changes in GSH and TBARS concentrations (data not shown).

Table 2
Effect of V. amurensis on MCAO/reperfusion-induced change of
GSH and TBARS contents in rat brains.

Group         Dose (mg/kg)     GSH (nmol/mg           TBARS
                                 protein)            (nmol/mg

Sham                     -    35.3 [+ or -]3.0     11.2[+ or -]1.0
Vehicle                  -    333[+ or -]1.7##  17.8 [+ or -]1.4##
V. amurensis            25    24.1 [+ or -]1.5    12.9[+ or -]1.1*
V. amurensis            50   28.7 [+ or -]1.0*   13.2 [+ or -]0.8*
V. amurensis           100  30.5 [+ or -]1.1**  10.8 [+ or -]0.7**

GSH and TBARS concentrations of ipsilateral brains were measured after
24 h of reperfusion. Values are expressed as mean [+ or -] S.E.M,
of data obtained from six rats.
* P < 0.05.
* P < 0.01 vs. vehicle.
** P < 0.01 vs. sham control.

Inhibitory effect of V. amurensis on MCAO/reperfusion-induced expression of apoptosis-associated proteins, MAPKs, and COX-2

The expression level of BcL-2, an anti-apoptotic protein, significantly decreased due to MCAO/reperfusion compared to that of the sham-operated group. In contrast, in animals treated with V. amurensis (25, 50, and l00mg/kg), BcL-2 expression level was markedly upregulated. MCAO/reperfusion also caused increases in the expression of pro-apoptotic proteins, BAK, BAX, and c-caspase-3. V. amurensis (25, 50, and l00 mg/kg) prevented the MCAO/reperfusion-induced increase of the pro-apoptotic protein expression (Fig. 5).

Animals that had undergone MCAO/reperfusion displayed higher expression levels of p-ERK 1/2 MAPK, and p-P38 MAPK than sham-operated animals. However, V. amurensis (25, 50, and l00mg/kg) decreased the expression levels of MAPKs in the MCAO/reperfusion brain. The expression of COX-2 also increased after MCAO/reperfusion, which was significantly inhibited by treatment with V. amurensis (Fig. 5).

Inhibitory effects of V. amurensis and active compounds on glutamate-induced apoptotic neuronal cell death

To elucidate the neuroprotective mechanism, we investigated the effect of the leaf and stem of V. amurensis on neuronal damage induced by glutamate, which is released from ischemic tissues to cause neuronal excitotoxicity, using primarily cultured rat cortical neurons. In preliminary experiments, it was confirmed that gJutamate in concentrations of 250-1000 [micro]M produced a concentration-dependent reduction of cell viability in cultured cortical neurons. Therefore, the concentration of 500 [micro]M was used for determination of glutamate-induced neuronal cell death in the present experiments. Neuronal cell viability after treatment with glutamate was measured using a MTT assay. When cerebral cortical neurons were exposed to 500 [micro]M glutamate for 12 h, MTT absorbance was 68.8 [+ or -]1.4% of untreated controls, showing that glutamate induced neuronal cell death. In cultures treated with V. amurensis extract (1, 10, or 50 [micro]/ml), glutamate-induced neuronal death was significantly reduced (absorbance, 103.2 [+ or -]3.2% of control for 50 [micro]/ml V. amurensis) (Fig. 6A).

To determine which components of V. amurensis exhibit neuroprotective activity, we examined all of the stilbenes and oligostilbenes isolated from V. amurensis in our previous study (Ha do et al., 2009a, 2009b), which were shown in the HPLC chromatogram of Fig. 2. Of these components, (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin protected against glutamate-induced neurotoxicity in cultured cortical neurons. Pretreatment of cortical neurons with 5 [micro]M of (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin reduced the neuronal death induced by 500 [micro]M glutamate (absorbance, 92.1 [+ or -]1.9%, 115.0 [+ or -]2.4%, and 97.8 [+ or -]3.4% of control, respectively; Fig. 6A). MK-801 (10 [micro]M), an NMDA glutamate receptor antagonist; verapamil (20 [micro]M), a voltage-dependent [Ca.sup.2+]-channel blocker; and L-NAME (1 mM), a nitric oxide synthase (NOS) inhibitor, also inhibited glutamate-induced neuronal death (Fig. 6A).

To elucidate the apoptotic neuronal cell death, immunoreactivities of BcL-2, an anti-apoptotic protein, and BAX, a pro-apoptotic protein, were measured after treatment with 500 [micro]M glutamate for 12h. In 500 [micro]M glutamate-treated neurons, BCL-2 activity decreased and BAX markedly increased compared to that of control cultures. V. amurensis (1, 10, and 50 [micro]/ml), (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin (5 [micro]M) significantly blocked the glutamate-induced decrease of BcL-2 and increase of BAX expression (Fig. 6B).

Inhibitory effects of V. amurensis and active compounds on glutamate-induced elevation of [[Ca.sup.2+].sub,i].

Glutamate-induced neuronal death is usually associated with the elevation of [[Ca.sup.2+].sub,i] following NMDA receptor activation. As shown in Fig. 7 [[Ca.sup.2+].sub,i] was rapidly elevated in response to treatment with 500 [micro]M glutamate, which was maintained over l0 min. In contrast, V. amurensis (1, 10, and 50 [micro]/ml) showed a concentration-dependent inhibition of glutamate-induced increase of [[Ca.sup.2+].sub.i]. (+)-AmpeIopsin A, [gamma]-2-viniferin, and frans-8-viniferin at concentrations of 5 [micro]M showed significant inhibition of glutamate-induced [[Ca.sup.2+].sub.i] elevation, even though these compounds failed to exhibit complete inhibition in the earlier part of the elevations. The increase of [[Ca.sup.2+].sub.i] by 500 [micro]M glutamate was remarkably blocked by treatment with MK-801 (10 [micro]M), verapamil (20 [micro]M), and l-NAME (1 mM). V. amurensis extract and the other compounds did not affect basal [[Ca.sup.2+].sub.i] (Fig. 7).

Inhibitory effects of V, amurensis and active compounds on glutamate-induced ROS generation

The involvement of oxidative stress in glutamate toxicity was investigated by measurement of ROS accumulation after the exposure of the neurons to glutamate for 4h. In [H.sub.2]DCF-DA-loaded cortical neurons, 500 [micro]M glutamate increased the fluorescence intensity, indicating that ROS were generated- In neurons treated with 500 [micro]M glutamate, the relative fluorescence increased ~3.5-fold to 231.0 [+ or -]4.3, compared to the value in control neurons (65.8[+ or -]1.6; Fig. 8). V. amurensis extract (1, 10, and 50[micro]/ml) and (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin (0.5, 1, and 5 [micro]M) showed concentration-dependent inhibition of the glutamate-induced ROS generation. MK-801 (10 [micro]M), verapamil (20 [micro]M), and L-NAME (1 mM) also significantly inhibited glutamate-induced ROS generation (Fig. 8).


The current study demonstrated the neuroprotective effect of the leaf and stem of V. amurensis on transient focal ischemia-induced brain damage using a MCAO/reperfusion model in rats as well as glutamate-induced neurotoxicity in cultured cortical neurons. Since the focal cerebral ischemia model with transient MCAO followed by reperfusion in experimental animals is generally accepted as the most appropriate model for human stroke (Crack and Taylor, 2005), we used the MCAO/reperfusion model to induce ischemic injury in rats. Ischemia leads to the release of cellular toxic mediators and increases the permeability of the blood brain barrier (BBB) (Demougeot et al., 2004). Hyperper-meability of the BBB leads to brain cellular swelling and causes brain infarction and edema (Gursoy-Ozdemir et al., 2000; Janigro et al., 1994). The size of brain infarct and edema correlates with severity of the neurological deficits (Tominaga and Ohnishi, 1989). MCAO/reperfusion produced significant impairments in neurological function and coordination, and brain infarction and edema were observed in the present study. V. amurensis, however, dose-dependently inhibited MCAO/reperfusion-induced brain infarct and edema formation, neurological deficits, and loss of coordinated movements. Furthermore, V. amurensis significantly prevented MCAO/reperfusion-induced neuronal death in the cerebral cortex and hippocampus.

It has been reported that excessive production of ROS and impairment of the ROS removal system are associated with behavioral, neurochemical, and histological abnormalities induced by MCAO/reperfusion (Yousuf et al, 2009; Ahmad et al., 2006; Khan et al., 2010). GSH is considered the most prevalent and important free radical scavenger for the antioxidant defense of cells (Wang et al., 2010). Depletion of GSH triggers peroxidation of lipids, proteins, and carbohydrates and DNA damage, consequently causing neuronal apoptosis (Khan et al, 2010). MCAO/reperfusion increases lipid peroxidation in the brain because of a high lipid content (Muralikrishna Adibhatla and Hatcher, 2006). ROS produce malondialdehyde, an end product of lipid peroxidation, which reacts with TBA and is estimated by TBARS (Ponist et al, 2070). In the present study, the GSH and TBARS contents significantly decreased and increased, respectively, in MCAO/reperfusion rats. V. amurensis, however, showed a significant protection against MCAO/reperfusion-induced collapse of these anti-oxidative mechanisms.

Damage to neurons caused by excessive ROS generation is associated with mitochondrial impairment, which leads to activation of an apoptosis cascade under ischemia/reperfusion conditions (Chan, 2004; Matsuda et al., 2009). Members of the BcL-2 family, such as BcL-2, BcL-xs, BAK, and BAX, regulate the progress of apoptosis (Adams and Cory, 1998). BcL-2, a well-known anti-apoptotic protein prevents apoptosis by preserving mitochondrial integrity and inhibiting the release of cytochrome c into cytosol (Graham and Chen, 2001). Pro-apoptotic proteins, such as BAK and BAX, are inserted into the outer mitochondrial membrane and trigger the release of cytochrome c (Chao and Korsmeyer, 1998). Released cytochrome c into cytosol forms the apoptosome to activate caspase-9 and caspase-3 (Hetz et al., 2005). Activation of caspase-3 cleaves numerous proteins, triggering biochemical cascades that lead to cell death (Chan and Mattson, 1999). MCAO/reperfusion caused the decrease of BcL-2 and overexpression of BAK, BAX, and c-caspase-3 in rat brains; these changes were significantly suppressed by treatment with V. amurensis in the present study. These results suggest that the neuroprotective effect of V. amurensis on MCAO/reperfusion-induced cerebral ischemic damage could be attributed to its anti-apoptotic activity.

MAPKs play important roles in apoptosis and neurodegeneration (Miloso et al., 2008) as well as in the survival, proliferation, and differentiation of neuronal cells (Chang and Karin, 2001; Crack and Taylor, 2005). In ischemic injury, phosphorylated ERK 1/2 MAPK translocate into the nucleus from the cytoplasm, resulting in the stimulation of a large number of downstream signaling events associated with apoptosis (Alessandrini et al., 1999; Namura et al., 2001). Activation of the P38 MAPK pathway and the resultant inflammatory processes, such as COX-2 expression and [PGE.sub.2] production, play crucial roles in post-ischemic damage in the brain (Walton et al., 1998). Therefore, inhibition of ERK 1/2 MAPK and P38 MAPK phosphorylation can reduce ischemic brain injury by preventing inflammation-mediated cell death (Irving and Bamford, 2002; Sugino et al., 2000). V, amurensis significantly prevented the increase of p-ERK 1/2 MAPK and p-P38 MAPK expression in MCAO/reperfusion brain in this study. The expression of an inflammatory enzyme, COX-2, also was inhibited by V. amurensis. These results suggest that the anti-inflammatory effect of V. amurensis might be responsible for its neuroprotection against MCAO/reperfusion-induced brain damage. Taken together, it is concluded that V. amurensis inhibited apoptotic neuronal death in MCAO/reperfusion-induced ischemic brain via anti-oxidative and anti-inflammatory activities.

Excitotoxicity through overactivation of NMDA receptors by the extensive release of glutamate is well established as an important trigger of tissue damage in focal cerebral ischemia (Coyle and Puttfarcken, 1993; Upton, 1999). Our data also showed that MCAO/reperfusion-induced infarct and edema were blocked by the NMDA receptor antagonist MK-801. Activation of NMDA receptors elevates the influx of [Ca.sup.2+] and that of non-NMDA glutamate receptors promotes the influx of [Na.sup.+], both of which can lead to membrane depolarization. In turn, depolarization can activate plasma membrane voltage-dependent [Ca.sup.2+] channels, leading to additional [Ca.sup.2+] influx (Liu et al, 2009; Upton, 1999). Furthermore, increased [[Ca.sup.2+].sub.i] activates enzymes, such as xanthine oxidase and NOS, that are involved in ROS generation, leading to lipid peroxidation and neuronal damage (Sun and Chen, 1998; White and Reynolds, 1996). In the present study using cultured cortical neurons, glutamate produced neuronal cell death through an apoptotic pathway, which was confirmed by the decrease of BcL-2 and increase of BAX expressions. Glutamate also increased [[Ca.sup.2+].sub.i] and ROS generation. Glutamate-induced neuronal cell death and increase of [[Ca.sup.2+].sub.i] and ROS generation were significantly inhibited by MK-801, verapamil, and L-NAME. Treatment of neurons with the leaf and stem of V. amurensis significantly suppressed glutamate-induced neuronal cell death, change of pro- and anti-apoptotic protein expression, [[Ca.sup.2+]]j increase, and ROS generation in cultured cortical neurons. Therefore, it might be concluded that the favorable effects of V. amurensis on MCAO/reperfusion-induced brain damage can be attributed to blockade of the apoptotic cascade after inhibition of [[Ca.sup.2+].sub.i] (increase and ROS generation, which were primarily triggered by excessively released glutamate under ischemic conditions.

Phytochemically, we isolated 11 resveratrol derivatives and resveratrol oligomers possessing antioxidant and antiinflammatory activities from the leaf and stem of V. amurensis (Ha do et al., 2009a, 2009b). Resveratrol derivatives have been demonstrated to inhibit [Ca.sup.2+] entry through [Ca.sup.2+] channels and to possess ROS scavenging activity (Ban et al., 2008; jakab et al., 2008). (+)-Ampelopsin A, [gamma]-2-viniferin, and frans-[epsilon]-viniferin isolated from the leaf and stem of V. amurensis could inhibit glutamate-induced increases of [[Ca.sup.2+].sub.i] ROS generation, and resultant apoptotic neuronal death in cultured neurons in the present work. These active principles, thus, are probably involved in the mechanisms of anti-oxidative and additional neuroprotective effects of V. amurensis against MCAO/reperfusion-induced brain injury. Although there previously has been no evidence to show that (+)-ampelopsin A, [gamma]-2-viniferin, and trans-[epsilon]-viniferin antagonize glutamate-induced excitotoxicity, the present study demonstrated their novel pharmacological activity in neurons.

In conclusion, the present study demonstrated that the leaf and stem of V. amurensis exhibit neuroprotective effects against MCAO/reperfusion-induced cerebral ischemic damage. It might be concluded that these protective effects are related to the anti-apoptotic effects of V. amurensis via anti-excitotoxic, anti-oxidative, and anti-inflammatory actions, and that the leaf and stem of V. amurensis might be used to treat ischemic stroke.


This work was supported by a grant (PJ007102201003 and PJ0071482011006) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.


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* Corresponding author. Tel.: +82 43 261 2968; fax: +82 43 267 3150.

E-mail address: (Y.H. Seong).

1. These authors contributed equally to this work.

0944-7113/$ - see front matter 2011 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2011.06.015

Joo Youn Kima (a), (1) Ha YeonJeong (a), (1) HongKyu Lee (a), SeungHwan Kim (b), Bang Yeon Hwang (c), KiHwan Bae (d), Yeon Hee Seong (a), (*)

(a) College of Veterinary Medicine, Clumgbuk National University, Cheongju 361-763, Republic of Korea

(b) College of Physical Education, Kyunghee University. Yongin 446-701, Republic of Korea

(c) College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea

(d) College of Pharmacy, Chungnam National University, Taejon 305-764, Republic of Korea
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Author:Kima, Joo Youn; Jeong, Ha Yeon; Lee, Hong Kyu; Kim, SeungHwan; Hwang, Bang Yeon; Bae, KiHwan; Seong,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Geographic Code:9SOUT
Date:Feb 15, 2012
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