Dried hooks and stems of U. sinensis were purchased from Hwalim Natural Drug (Busan, Korea) in September 2010. A voucher specimen (accession number PDRLCW-1) has been deposited in the Plant Drug Research Laboratory of Pusan National University (Miryang, Korea). The dried hooks and stems of U. sinensis (2.0 kg) were ground to a fine powder and then successively extracted at room temperature with n-hexane, ethyl acetate and methanol. Briefly, hexane extracts of U. sinensis were filtered and evaporated under reduced pressure at 45℃ and then lyophilized, which yielded a white powder of hexane extract (14.54 g). The hexane extract (11.31 g) was subjected to chromatography on a silica gel (40 µm; J.T. Baker, Phillipsburg, NJ, USA) column (70×8.0 cm) with a step gradient of 50% CHCl₂ in hexane, 100% CHCl₂, 5% and 20% acetone in CHCl₂ and 5%, 25% and 50% MeOH in CHCl₃ to obtain 62 fractions. Of these extracts, the solid form of faction 43 (23.1 mg, JGH43IA) was dissolved with dimethyl sulfoxide for further experiments.

Cytosine-β-D-arabino furanoside, Hochest 33342, L-glutamate, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), poly-L-lysine, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neurobasal medium, B27, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and other neuronal cell culture reagents were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). For our investigations, the following antibodies were used: α-fodrin, calpain 1, calpain 2, CREB, phospho-CREB (pCREB, ser133), phospho-GluN2A (pGluN2A, Tyr1246) and phospho-GluN2B (pGluN2B, ser1303) were purchased from Cell Signaling Technology (Danvers, MA, USA). GluN2B and phospho-STEP (pSTEP, ser221/ser49) were purchased from Millipore (Billerica, MA, USA). GluN2A antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA). STEP antibody was purchased from Novus Biologicals (Littleton, CO, USA). Secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC Annexin V apoptosis dectection kit was purchased from BD Bioscience (San Diego, CA, USA). A lactate dehydreogenase (LDH) cytotoxicity assay kit and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit were purchased from Promega (Madison, WI, USA).

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. Time-pregnant Sprague-Dawley rats (E18-19) (Dooyeol Biotech, Seoul, Korea) were anesthetized and sacrificed by cervical dislocation. The cerebral cortex was carefully dissected out and transferred in ice cold stage; tissues were gently minced using a sterile razor blade and digested in 0.25% trypsin for 15 minutes in Ca²⁺ and Mg⁺-free Hanks' balanced salt solution, 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 biscarbonate, 20 mM HEPES, 0.3 g/l bovine serum albumin, and 1% penicillin/streptomycin at a density of 1×10 cells/ml and grown on poly-L-Lysine-coated plates in a 5% CO₂ humidified incubator at 37℃. After 48 hours, β-D-arabinofuranoside (5 µM) was added in order to prevent proliferation of non-neuronal cells. After 3 days in culture, the medium was exchanged to neurobasal medium containing 2% B27 supplement, 0.5 mM glutamine, and 1% penicillin/streptomycin. Cortical neurons were maintained in primary culture for 9-10 days for use in experiments.

The MTT assay was used for determination of cell viability. Cortical neurons were cultured at a density of 5×10⁴/well in 96 well plates and treated with JGH43IA at different concentration for 24 hours, after treatment with 200 µM of glutamate for 6 hours, the medium was replaced with 0.5 mg/ml MTT solution for 4 hours at 37℃. After incubation, formazan crystals produced in cells were solubilized in dimethyl sulfoxide. Absorbance was measured at 570 nm using a Spectra MAX 190 spectrometer (Molecular Devices, Sunnyvale, CA, USA). Results were expressed as a percentage of control.

Measurement of LDH released from damaged neurons was performed for assessment of cytotoxicity. After treatment, cortical neurons were lysed by addition of lysis solution, followed by incubation at 37℃ for 45-60 minutes. Supernatant samples were transferred to a 96 well enzymatic assay plate, followed by addition of substrate to each sample. The enzymatic reaction was allowed to proceed for 30 minutes at room temperature, protected from light. After the enzymatic reaction was stopped, the plate was read at 490 nm using a Spectra MAX 190 spectrometer (Molecular Devices). Data represent the percentage of LDH released relative to controls.

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% paraformalehyde for 20 minutes at 4℃. Fixed cells were washed three times with phosphate buffered saline (PBS), followed by staining with 10 mg/ml Hoechst 33342 in PBS for 10 minutes at 37℃. The cells were washed three times with PBS and mounted using the medium for fluorescence (Vector Laboratories Inc., Burlingame, CA, USA), followed by observation under a laser scanning confocal microscope (LSM 510, Carl Zeiss, Oberkohen, 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.) 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 minutes at 4℃. Fixed cells were then washed with PBS, followed by incubation with DNA-labeling solution at 37℃ for 60 minutes inside a humidified chamber to allow the tailing reaction to occur. The DNA-labeling reaction was terminated by addition of 2× SSC, followed by washing for removal of unincorporated fluorescein-12-dUTP. The cells were then counterstained with 1 mg/ml propidium iodide (PI) solution in PBS for 15 minutes 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). Data are presented as apoptotic cells as a percentage of total cells.

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 1×10⁵ cells/ml; 100 ml of the solution were transferred to a flow cytometric tube, followed by double staining with Annexin V-FITC and PI in the dark at room temperature for 15 minutes. Subsequently, 400 ml of binding buffer was added and analysis of samples was performed using a flow cytometer (FACS CantoTM II, Becton Dickinson, San Jose, CA, USA).

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% NP40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃Vo₄, and protease inhibitor cocktail). Equal amounts of proteins were then separated by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the resolved proteins were then transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked with 5% skim milk, followed by exposure to the appropriate antibodies. Using an enhanced chemiluminescence system (Pierce Biotech, Rockford, IL, USA), all bands were visualized using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Results of Western blot assay reported here are representative of at least three experiments.

All data were expressed as mean±SEM and the SigmaStat statistical program version 11.2 (Systat Software, San Jose, CA, USA) was used for data analysis. The paired student's t test was used for analysis of data. A value of P<0.05 was considered to indicate a statistically significant result.

We investigated the protective effects of JGH43IA from U. sinensis against glutamate-induced toxicity using MTT and LDH assay. Treatment with glutamate alone led to reduce in cell viability to approximately 68% of the control group, but pretreatment with JGH43IA exhibited in a significant gradual decrease of glutamate-induced toxicity in a dose-dependent manner (Fig. 1A). These data were further verified by the LDH assay. The level of LDH release increased to approximately 58% after exposure to glutamate. However, pretreatment with JGH43IA showed a significant decrease of this release in a dose-dependent manner (Fig. 1B). These results suggest that JGH43IA pretreatment has a significant protective effect against glutamate-induced toxicity in primary cortical neurons.

We performed Hoechst 33342 staining and TUNEL assay in order to characterize the types of neuronal death by JGH43IA treatment. Results of Hoechst staining indicated that treatment with glutamate alone exhibited high levels of condensed chromatin and apoptotic bodies. But pretreatment with JGH43IA showed a significant decrease of these apoptotic features at both concentrations of 0.01 and 0.1 µg/ml (Fig. 2). DNA fragmentation was also evaluated by TUNEL assay as another method for detection of apoptosis. TUNEL-positive cells increased to 29.6% in total cells after exposure to glutamate alone. However, pretreatment with JGH43IA resulted in significantly decreased of TUNEL-positive cells to nearly 19.7% and 9.4% at a concentration of 0.01 and 0.1 µg/ml, respectively (Fig. 3).

We further confirmed these results by use of flow cytometry using Annexin V FITC/PI staining. The percentage of apoptotic cells reached up to 21.4% in glutamate alone-treated neurons, whereas, the increase in necrotic cells was only 4.8%. But, treatment with JGH43IA showed a markedly decrease in the number of glutamate-induced cells death to 10.8% and 5.7% at a concentration of 0.01 and 0.1 µg/ml, respectively (Fig. 4). These results suggest that cortical neurons are to mainly undergo apoptotic neuronal death after exposure to glutamate and pretreatment with JGH43IA exerts a neuroprotective effect by abrogating apoptotic cell death.

The NMDAR and calpain-mediated STEP play a dominant role in mediating the toxic signal under glutamate exposure in our previous study [20]. Thus we attempted to determine whether JGH43IA could inhibit both activation of NMDAR and calpain and subsequent STEP cleavage caused by glutamate. Our previous study shows that NMDAR GluN2A and GluN2B subunit shows a rapid increase in expression of phosphorylation at early time points with a rapid increase in the active form of calpain 1 (~75 kDa), subsequently resulting in a marked increase in a breakdown product of fodrin (~145/150 kDa), indicative of cleavage by activated calpain. The STEP, substrate for Ca²⁺-dependent calpain, is also rapidly cleaved to a smaller isoform, STEP₃₃ following exposure to glutamate alone. Pretreatment with JGH43IA showed a marked selective decrease in glutamate-stimulated phosphorylation of the NMDAR GluN2B subunit. Pretreatment with JGH43IA reduced calpain1 activity and also reduced the accumulation of fodrin breakdown product caused by glutamate with significant blockage of STEP₃₃ production. Especially, pretreatement of JGH43IA resulted in reduced ~145 kDa fodrin production of 30.2% compared with glutamate-treated cells at 360 minutes after glutamate exposure. And lower subsequent STEP₃₃ production, 28.4% compared with glutamate-treated cells, also observed at early phases after glutamate exposure in JGH43IA-treated cells [20]. Pretreatment with JGH43IA increased in the levels of phosphorylated CREB compared to glutamate alone treated cells (Fig. 5). These results suggest that inhibition of NMDAR GluN2B subunit activation and subsequent calpain-mediated STEP cleavage are involved in the neuroprotective mechanisms of JGH43IA.