The dried hooks and stems of U. sinensis were purchased in September 2010 from Hwalim Natural Drugs (Busan, Korea) and were authenticated by Professor J.W. Hong, Division of Clinical Medicine I, School of Korean Medicine, Pusan National University. A voucher specimen (accession number PDRLJG-1) was deposited at the Plant Drug Research Laboratory of Pusan National University (Miryang, Korea). The dried hooks and stems of U. sinensis (1.0 kg) were ground to a fine powder, followed by successive extraction at room temperature with n-hexane, ethyl acetate and methanol. Briefly, filtration and evaporation of hexane extracts of U. sinensis were performed under reduced pressure at 45°C, followed by lyophilization. A white powder of HEUS (7.27 g) was obtained. Sequential extraction of the remaining powder was performed using ethyl acetate and methanol to yield ethyl acetate extracts of U. sinensis (10.68 g) and methanol extracts of U. sinensis (31.66 g), respectively. Finally, the solid form of the extract was dissolved with dimethyl sulfoxide (DMSO) for use as HEUS in experiments.

Cytosine-β-D-arabinose furanoside, Hoechst 33342, L-glutamate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly-L-lysine, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), neurobasal medium, B27, and other neuronal cell culture reagents were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). For western blot analysis, antibodies recognizing DR4, DR5, Bcl-2, Bax, Bid, XIAP, caspases-3, -8, -9, and poly (ADP-ribose) polymerase (PARP) were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA), and cIAP-1 and cIAP-2 were supplied by Calbiochem (Cambridge, MA, USA). Secondary antibodies were supplied by Santa Cruz Biotechnology. Caspase colorimetric assay kits for caspases-3, 8 and 9 were purchased from R&D Systems (Minneapolis, MN, USA) and a FITC Annexin V apoptosis dectection kit was purchased from BD Bioscience (San Diego, CA, USA). A lactate dehydrogenase (LDH) cytotoxicity assay kit and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit were purchased from Promega (Madison, WI, USA).

A previously described protocol with some modifications was used for preparation of primary cultures of cortical neurons from fetal rats (17). Briefly, E18-E19 Sprague-Dawley (SD) rat fetuses (DooYeol Biotech, Seoul, Korea) were sacrificed by cervical dislocation under anesthesia. All procedures used in these studies followed the guidelines of protocols approved by the Pusan National University Animal Care and Use Committee in accordance with the National Institutes of Health Guidelines. The cerebral cortex was dissected and chopped into small pieces and digested in 0.25% trypsin for 15 min in Ca²⁺ and Mg⁺-free Hanks’ balanced salt solution (HBSS), followed by mechanical dissociation using a pasteur pipette. After centrifugation, cells were re-suspended in DMEM supplemented with 10% FBS, 1 mM pyruvate, 4.2 mM sodium bicarbonate, 20 mM HEPES, 0.3 g/l bovine serum albumin, and 1% penicillin/streptomycin at a density of 1×10⁶ cells/ml and grown on poly-L-lysine-coated plates in a 5% CO₂ humidified incubator at 37°C. Forty eight hours after plating, cytosine-β-D-arabinose furanoside was added in order to inhibit proliferation of non-neuronal cells. After three days in culture, the medium was replaced with neurobasal medium containing 2% B27 supplement, 0.5 mM glutamine, and 1% penicillin/streptomycin. Cortical neurons were maintained for 9–10 days in primary culture for use in the experiments.

The MTT assay was used for assessment of primary cortical cell viability. Cortical neurons were seeded in 96-well plates at a density of 5×10⁴/well and incubated for 24 h prior to experimental treatments. After incubation, cells were treated with 200 μM of glutamate for 6 h, followed by treatment with 0.5 mg/ml MTT for 4 h at 37°C. Formazan crystals formed in cells were solubilized in DMSO and the absorbance was measured at 570 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Results were expressed as a percentage of the control.

Cytotoxicity was measured quantitatively according to release of LDH in the culture medium of cell samples. After treatment, cortical neurons were lysed by addition of lysis solution, followed by incubation at 37°C for 45–60 min. Sample supernatants were transferred to a 96-well enzymatic assay plate, followed by addition of substrate to each supernatant sample. The enzymatic reaction was allowed to proceed for 30 min at room temperature, protected from light. After the enzymatic reaction was stopped, the plate was read at 490 nm using a SpectraMax 190 spectrophotometer. Data represent the percentage of LDH released relative to controls.

After treatment, cortical neurons were harvested and cells were washed twice with cold PBS, followed by re-suspension in binding buffer at a concentration of 1×10⁶ cells/ml; 100 μl of the solution was transferred to a flow cytometric tube, followed by double staining with Annexin V-FITC and propidium iodide (PI) in the dark at room temperature for 15 min. Subsequently, 400 μl of binding buffer was added and analysis of the samples was performed using a flow cytometer (FACSCanto™ II; Becton-Dickinson, San Jose, CA, USA).

Cortical neurons were grown on poly-L-lysine-coated glass coverslips at a density of 5×10⁴ cells/ml. After treatment, the cells were fixed in 4% paraformalehyde for 20 min at 4°C. Fixed cells were washed with PBS three times, followed by staining with 10 μg/ml Hoechst 33342 in PBS for 10 min at 37°C. The cells were washed with PBS three times and mounted in the medium for fluorescence (Vector Laboratories, Inc.), followed by observation under a laser scanning confocal microscope (LSM 510; Carl Zeiss, Inc., Oberkochen, Germany). Data are presented as the ratio of chromosomal condensation and morphological change as a percentage of total cells.

Cortical neurons were grown on Lab-Tek^® chamber slides (Thermo Fisher Scientific, Inc., Rochester, NY, USA) at a density of 1×10⁵ cells/ml. After treatment, the cells were fixed in 4% methanol-free formaldehyde solution in PBS (pH 7.4) for 25 min at 4°C. Fixed cells were then washed with PBS, followed by incubation with DNA-labeling solution at 37°C for 60 min inside a humidified chamber to allow the tailing reaction to occur. The DNA-labeling reaction was terminated by 2X SSC, and followed by washing for removal of unincorporated fluorescein-12-dUTP. The cells were then counterstained with 1 μg/ml PI solution in PBS for 15 min at room temperature in the dark. Apoptotic neurons were determined by localization of green fluorescent cells (fluorescein-12-dUTP) on a red background using a fluorescence microscope (Carl Zeiss, Inc.). Data are presented as apoptotic cells as a percentage of total cells.

After treatment, cortical neurons were washed in cold PBS buffer, followed by homogenization in lysis buffer [200 mM Tris (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1 mM NaF, 1% NP-40, 1 mM PMSF, 1 mM Na₃Vo₄, protease inhibitor cocktail]. Equal amounts of proteins were then separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which the resolved proteins were transferred to a nitro-cellulose membrane (Whatman GmbH, Dassel, Germany). The membrane was blocked with 5% skim milk, followed by exposure to the appropriate antibodies. All bands were visualized using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence system (Pierce Biotechnology, Inc., Rockford, IL, USA). Results of the western blot assay reported here are representative of at least three experiments.

After treatment, quantification and homogenization of cortical neurons were performed in order to recognize activation of cellular caspase; 150 μg protein in cortical neurons was mixed with extraction buffer [40 mM HEPES (pH 7.4), 20% glycerol (v/v), 1 mM EDTA, 0.2% NP-40, and 10 mM DL-DTT] containing 100 μM fluorogenic peptide substrate, followed by dilution with extraction buffer, setting a volume of 100 μl/sample in a microtiter plate. Caspase-3, -8 and -9 substrates were used with Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA), Ile-Glu-Thr-Asp (IETD)-pNA and Leu-Glu-His-Asp (LEHD)-pNA, respectively. Caspase activities were determined by measurement of absorbance (405 nm) of prepared plates after incubation for a period of 3 h.

All data are expressed as means ± SEM and analyzed using the SigmaStat statistical program version 11.2 (Systat Software, Inc., San Jose, CA, USA). The paired Student’s t-test was used for analysis of data. A P-value <0.05 was considered to indicate a statistically significant result.

In order to determine whether HEUS is protective against glutamate-induced toxicity, cell viability was measured by MTT assay. Cortical neurons were pretreated with various concentrations of HEUS (0.1–10 μg) for 24 h, followed by exposure to glutamate at a concentration of 200 μM for a period of 6 h. Stimulation with glutamate alone resulted in reduced cell viability, ∼34% of that of the control, however, pretreatment with HEUS resulted in significantly reduced glutamate-induced toxicity in a dose-dependent manner (Fig. 1A). Next, the protective effects of HEUS were also estimated according to changes in the intracellular release of LDH. The level of LDH release showed a significant increase, to 47.6%, after exposure to glutamate, while treatment with HEUS resulted in a marked decrease in the release of LDH, to 38.4 and 24.9%, at a concentration of 1 and 5 μg/ml, respectively (Fig. 1B). The protective effects of HEUS were also confirmed by morphological observation using a phase-contrast microscope. Exposure to glutamate resulted in significant cell insult to the cortical neurons, including diminution of cellular processes and decrease in refraction; these morphological features showed marked recovery when cells were pretreated with 5 μg/ml of HEUS (Fig. 1C). These results indicated that pretreatment with HEUS can exert a protective effect against glutamate-induced toxicity in primary cortical neurons.

Findings from recent studies suggest an association of glutamate-triggered cell death with both apoptotic and necrotic processes (18). In order to determine the type of cell death induced by glutamate, we performed several morphological assessments under our experimental conditions. Upon nuclear staining with Hoechst 33342, cortical neurons showed typical morphological signs of apoptosis, including nuclear and cytoplasmic shrinkage, and chromatin condensation, after exposure to glutamate, compared with control cells, which had regular and round-shaped nuclei. However, treatment with HEUS resulted in a significant reduction in these apoptotic features by ∼30.8, 22.6, 13.2 and 7.0% from 33.2% as observed in the glutamate-treated cells at a concentration of 0.1, 0.5, 1 and 5 μg/ml, respectively (Fig. 2A). Next, we performed TUNEL staining, which is designed for detection of DNA strand breaks and quantitation of apoptosis. Consistent with the results of Hoechst staining, after exposure to glutamate, cortical neurons showed a ratio of nuclear DNA fragmentation in total cells to 37.3%. Treatment with HEUS resulted in a marked reduction in TUNEL-positive cells to 18.0 and 7.0% at a concentration of 1 and 5 μg/ml, respectively (Fig. 2B). We further confirmed our results by performance of flow cytometry with Annexin V FITC/PI staining. The percentage of apoptosis in glutamate-treated neurons was up to 38.2%, while the proportion of necrotic cells showed a slight increase to 10.9% (Fig. 3). These results indicated that after exposure to glutamate, cortical neurons underwent extensive apoptotic-like deaths under our experimental conditions. However, treatment with HEUS resulted in a significant decrease in the number of glutamate-induced apoptotic cells, from 19.7 to 7.9%, at a concentration of 1 and 5 μg/ml, respectively (Fig. 3A). These results suggest that pretreatment with HEUS results mainly in protection of cortical cells against glutamate-induced neuronal apoptosis.

Mediation of apoptosis occurs primarily through activation of death receptors and/or mitochondrial signaling (19). Next, using western blot analysis, we evaluated the levels of DRs and apoptosis-related gene expression. Exposure to glutamate resulted in markedly enhanced expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor DR4, whereas, its expression was reduced by pretreatment with HEUS (Fig. 4). Attenuation of TRAIL-induced apoptosis is also known to occur through upregulation of numerous anti-apoptotic proteins, including IAPs, which are potent suppressors of apoptosis (19). Thus, we attempted to determine whether the neuroprotective effect of HEUS was involved in expression of IAP family members. Pretreatment with HEUS led to significantly increased expression of glutamate-attenuated XIAP protein, but not cIAPs. Next, we investigated the effect of HEUS on members of the pro- and anti-apoptotic Bcl-2 family, a key regulator of apoptosis. After exposure to glutamate, cortical neurons showed significantly decreased levels of Bcl-2, compared with the control. However, treatment with HEUS resulted in markedly increased glutamate-attenuated anti-apoptotic Bcl-2 expression without alteration of pro-apoptotic Bax expression. Another pro-apoptotic member of the Bcl-2 family, Bid, is cleaved to its truncated active form; tBid induces permeabilization of the outer mitochondrial membrane, leading to promotion of apoptosis (19). Treatment with HEUS resulted in slight recovery of glutamate-attenuated whole Bid expression at a concentration of 5 μg/ml. These results suggest that pretreatment with HEUS inhibits neuronal apoptosis via both inhibition of DR4 and activation of anti-apoptotic proteins XIAP and Bcl-2.

Eventually, activation of the caspase cascade was induced during the process of glutamate-induced neuronal apoptosis (5,20). Thus we investigated expression and activities of caspase-8, -9 and -3, which could act either as initiator caspases or as effector caspases, with involvement in both death receptor- and mitochondrial-initiated apoptosis. Western blotting data showed that treatment with HEUS resulted in significantly decreased expression of the cleaved active form of caspase-3 (Fig. 5A). Neither of the active forms of caspase-8 and -9 was detected in our studies, however, treatment with HEUS induced an increase in their proforms. To further confirm involvement of caspases, we assessed their activities in the lysates of the cells. Exposure to glutamate to cortical neurons resulted in markedly increased activities of caspase-8, -9 and -3, whereas these activities were significantly attenuated by treatment with HEUS (Fig. 5B). In addition, pretreatment with HEUS resulted in significantly decreased glutamate-enhanced cleavage of PARP, a downstream target of the activated caspase-3 (Fig. 5A). These results suggest that pretreatment with HEUS results in inhibition of neural apoptosis via inhibition of caspase-8, -9 and -3 activation.