All procedures used in this study complied with the Guide for the Care and Use of Laboratory Animals of the Xijing Hospital. Cultures of mouse cortical neurons were prepared using methods similar to those previously reported (27). Timed-pregnant (13–15 days) Balb/C mice were anesthetized with halothane and sacrificed by cervical dislocation. After dissection, cortical neurons were dispersed by trituration and digestion in 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C. Then, the cell suspension was centrifuged at 4°C for 5 min at 250 g, and resuspended in dissociating medium [Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA), 2 mM L-glutamine, 10 mM HEPES and 44 mM glucose (all from Sigma-Aldrich)]. Cells were plated on poly-L-lysine-coated culture plates at a density of 1×10⁶ cells/ml. Twenty-four hours later, the medium was replaced by Neurobasal medium consisting of 2% B27^® Supplement, 0.5 mM L-glutamine, and 25 μM glutamate (all from Sigma-Aldrich) to minimize glial growth. At 7 days of growth, one-half of the medium was replaced with new Neurobasal medium. Experiments were performed on cultures following 14–16 days of incubation.

OGD was used as an in vitro model of ischemia. For OGD, the medium was removed and stored separately. Cultures were rinsed three times with phosphate-buffered saline (PBS), and low-glucose DMEM with 2% B27 ^® Supplement was added. Cultures were then transferred to a humidified chamber kept in a 37°C incubator and subjected to an anaerobic environment of 95% N₂-5% CO₂ for 3 h. Oxygen concentration was maintained at 0.5–1.0%, which was monitored by an oxygen analyzer (MSA, Pittsburgh, PA, USA), throughout the experiment. OGD was terminated by the replacement of stored medium and by returning the cultures to a standard incubator maintained at 37°C in a 5% CO₂ atmosphere for 21 h of reoxygenation. Control cells were not subjected to OGD and were grown at 37°C in an atmosphere containing 5% CO₂.

A fresh solution of Res was prepared from a stock of 100 mM Res in 50% dimethylsulfoxide (DMSO) (both from Sigma-Aldrich), and was diluted in PBS to reach the desired final concentration (10, 25, 50 and 100 μM) for treatment. Res treatment controls (vehicle group) received the same amount of DMSO without Res. The duration of treatment was from OGD until the end of the experiment.

Pyrrolidine dithiocarbamate (PDTC) is a specific NF-κB inhibitor. PDTC (10 μM; Sigma-Aldrich) was added 15 min prior to Res treatment. Next, Res was added to cultures at a final concentration of 50 μM. The vehicle group cultures received the same amount of the carrier solvent (0.1% DMSO) without Res. The duration of the treatment was from OGD until the end of the experiment.

NOC-18 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) is a diazeniumdiolate (NONOate) NO donor designed to release NO at a slower rate. NOC-18 (500 μM) was added during Res treatment.

The effects of Res on OGD-induced cytotoxicity were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake assay, with a commercial kit purchased from Sigma-Aldrich. Neuronal primary cultures were grown on 96-well plates at a density of 2×10⁵ cells/cm². At 14 days of growth, cells were subjected to OGD and reoxygenation. Different concentrations of Res were added to the medium. After 21 h of reoxygenation, MTT was added to the cells at a final concentration of 0.5 mg/ml, and the plates were incubated for 4 h at 37°C. The insoluble formazan product was then precipitated by centrifugation, the supernatant removed, and the crystals were dissolved in 100 μl DMSO. Absorbance at 570 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). The ratio of absorbance of treated cells to that of the control cells was calculated, and was used to represent the percentage of growth inhibition.

Cell apoptosis was assayed by flow cytometry with the Annexin V-FITC Apoptosis Detection kit (Sigma-Aldrich). Cells were subjected to OGD and reoxygenation, and different concentrations of Res were added to the medium. Specifically, 1×10⁶ single cells per sample were collected following a 3-h OGD, were reoxygenated for 21 h and were washed twice with PBS buffer; Annexin V/FITC was then added. After incubation for 10 min at room temperature in the dark, the cells were washed and resuspended; propidium iodide was then added to a final concentration of 1 mg/l. Stained cells were analyzed using a FACSCalibur instrument (Becton Dickinson, Mountain View, CA, USA).

The level of MMP-3 mRNA was semi-quantified using RT-PCR. Total RNA was extracted using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and dissolved in nuclease-free water, according to the manufacturer’s instructions. Reverse transcription was performed with oligo(dT) primers and the First-Strand cDNA Synthesis kit (Invitrogen Life Technologies). The synthesized first-strand cDNA (1 μl) was next subjected to PCR amplification using the following program: 94°C for 30 sec; 56°C for 1 min; 72°C for 1 min. A total of 35 cycles were performed for the amplification of MMP-3 and 30 cycles for the housekeeping gene β-actin, which served as the control. The last cycle was followed by 10 min of elongation at 72°C. Primer pairs for the amplification of the mouse MMP-3 and β-actin genes (231 and 242 bp, respectively) were the following: MMP-3 forward, 5′-GTACCAACCTATTCCTGGTTGC-3′, and reverse, 5′-CCAGAGAGTTAGATTTGGTGGG-3′; β-actin forward, 5′-AACCCTAAGGCCAACCGTGAAAAG-3′, and reverse, 5′-TCATGAGGTAGTCTGTCAGGT-3′. PCR products were visualized on 1.5% agarose gels stained with ethidium bromide ausing a UV Transilluminator 2000 (#170-7942; Bio-Rad). Semi-quantitative analysis was conducted using a computerized densitometric imager (Gel Doc™ XR+, Bio-Rad).

Protein concentrations were determined using the Bradford assay (Bio-Rad). The same amount of total proteins (30 μg/lane) was loaded into each lane, electrophoresed and transferred onto nitrocellulose membranes at 80 V for 1 h. After blocking for 4 h in 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies (dilution, 1:1,000) targeting MMP-3, inducible NO synthase (iNOS), NF-κB, β-actin, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) (all from Sigma-Aldrich), and caspase-3 (monoclonal antibody; Cell Signaling Technology, Inc., Danvers, MA, USA). Next, the membrane was incubated with the corresponding secondary antibody (goat anti-rat IgG; diluted with PBS at 1:1,000; Sigma-Aldrich) at room temperature for 1 h. Finally, the proteins were detected using the standard enhanced chemiluminescence ECL method using a kit purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

NO levels were estimated with the Griess assay. We used the Griess Reagent system (Promega Corp., Madison, WI, USA) and measured the total nitrate and nitrite concentrations at 550 nm in a SmartSpec Plus Spectrophotometer (#170-2525; Bio-Rad).

Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times. Results were presented as the means ± SD. Statistical comparisons between groups were performed using one-way ANOVA followed by Student’s t-tests. P<0.05 or <0.01 was considered to indicate a statistically significant difference.

The MTT assay was used to analyze the viability of cells. In this assay, the number of viable cells is directly proportional to the level of the produced formazan product. Our preliminary experiments demonstrated that treatment with 50 μM Res exerted therapeutic effects. Exposure of the cells to OGD for 3 h followed by 21 h of reoxygenation caused a reduction in cell viability of ~45%. Under these conditions, pretreatment with Res (50 μM) increased cell viability (as opposed to OGD-induced cell death) by 75% (Fig. 1).

Flow cytometry was used to quantify neuronal apoptosis induced by OGD. As shown in Fig. 2, in the control (no OGD) cells, there was a very low level (9.8%) of neuronal apoptosis, but the percentage of apoptosis was significantly increased to 49% (P<0.01) upon OGD, and was reversed to 29.1% when 50 μM of Res were applied during OGD (P<0.05). The vehicle solution (DMSO) had no effect on cell apoptosis induced by OGD (P>0.05).

To gain insights into the mechanisms by which Res attenuates OGD-induced cell apoptosis, we studied the expression of pro- and anti-apoptotic proteins following OGD and Res treatment. As shown in Fig. 3, OGD induced the cleavage of the pro-apoptotic protein caspase-3, and this effect was reversed by treatment with 50 μM Res (P<0.05); similarly, OGD-induced Bax expression was also reduced by Res. By contrast, the expression of the anti-apoptotic protein Bcl-2 was reduced by OGD, and this effect was reversed by Res treatment. These results suggest that Res achieves its anti-apoptotic effects through canonical apoptosis signaling pathways.

The expression of MMP-3 is regulated by the transcription factor NF-κB. NF-κB is an ubiquitous transcription factor that resides in the cytoplasm but, when activated, is translocated to the nucleus, where it induces gene transcription. The activated NF-κB induces the expression of >400 genes, some of which are intimately involved in regulation of apoptosis, proliferation and inflammation. Hence, we examined whether Res can modulate OGD-induced expression of NF-κB. As shown in Fig. 4, OGD induced the expression of NF-κB, while Res significantly inhibited its expression.

To further investigate the mechanisms of Res-mediated neuroprotection, we studied its effect on MMP-3 expression. In both RT-PCR and western blot analyses, the exposure of cells to OGD significantly induced the expression of MMP-3 compared to the control cells, while Res treatment inhibited the OGD-induced expression of MMP-3 (Fig. 5). The level of the housekeeping protein β-actin remained unaffected.

Excessive ROS production leads to activation of the transcription factor NF-κB, which is associated with cell death during cerebral ischemia (28). OGD, excessive ROS production or glutamate toxicity induce iNOS expression (29,30). We evaluated the expression of iNOS by western blot analysis, which showed that OGD increases the iNOS level compared to the control, and that this effect is reversed by treatment with 50 μM Res (Fig. 6A). NO is a signaling molecule that regulates numerous biological processes in the nervous system, including neurotransmitter release, plasticity, and apoptosis, and can modulate the biological activity of numerous proteins, including MMPs. Cerebral ischemia and reperfusion injury result in nitrosative stress and hence the production of NO (11). OGD increased the NO level compared to the control, and Res treatment reversed this effect (Fig. 6B). To investigate whether Res attenuates OGD-induced iNOS and NO production via affecting the expression of NF-κB, cells were pretreated with the NF-κB inhibitor PDTC 15 min prior to Res treatment. PDTC treatment reduced OGD-induced iNOS and NO expression, similar to the effect of Res (Fig. 6C). This result suggests that Res inhibits iNOS and NO expression via inhibiting the NF-κB expression.

To investigate the mechanism by which Res regulates OGD-induced cell apoptosis, we studied the exprerssion of apoptosis-related molecules in the presence of Res and NOC-18. Cells were treated with NOC-18 (500 μM) during Res treatment (50 μM). As shown in Fig. 7, supplementing NO via NOC-18 addition partly reversed the effect of Res on MMP-3, Bax, Bcl-2 and activated caspase-3 expression. This suggests that Res inhibits MMP-3 expression and cell apoptosis via iNOS/NO.