In the present study, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and antibiotics (penicillin and streptomycin) were all purchased from Welgene, Inc. (Daegu, Korea). Baicalein (5,6,7-trihydroxyflavone; purity 98%), H₂O₂, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazo-lylcarbocyanine-iodide (JC-1), zinc protoporphyrin (ZnPP) IX and auranofin were all purchased from Sigma Chemical Co. (St. Louis, MO, USA); 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes, Inc. (Eugene, OR, USA); primary antibody against phosphorylated histone variant H2A.X (p-γH2A.X; #9718) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); β-actin (sc-1616), poly(ADP-ribose) polymerase (PARP; sc-7150), X-linked inhibitor of apoptosis protein (XIAP; sc-11426), Nrf2 (sc-13032), Keap1 (sc-15246), HO-1 (sc-7696) and TrxR1 (sc-28321) antibodies were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The secondary antibodies against goat anti-rabbit IgG-HRP (sc-2004), goat anti-mouse IgG-HRP (sc-2005) and bovine anti-goat IgG-HRP (sc-2350) were all purchased from Santa Cruz Biotechnology, Inc.

C6 glial cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotics (100 µg/ml streptomycin, 100 U/ml penicillin) at 37°C in a humidified incubator in an atmosphere of 5% CO₂ in air. Baicalein was dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 100 mM, which was then diluted with DMEM to the desired concentration prior to use. H₂O₂ was diluted in DMEM to a final concentration of 0.5 mM.

C6 cells were seeded in 6-well plates at a density of 2x10⁵ cells/well. After incubation for 24 h, the cells were pretreated with various concentrations of baicalein for 1 h prior to incubation in the absence or presence of H₂O₂ for 24 h. An MTT working solution (0.5 mg/ml) was added to the culture plates and incubated for 3 h at 3°C. The culture supernatant was removed from the wells, and DMSO was added to dissolve the formazan crystals completely. The absorbance of each well was measured at 540 nm using a microplate reader (Dynatech Laboratories, Chantilly VA, USA). The effect of baicalein on cell growth was assessed as the percentage of cell viability, in which the vehicle-treated cells (0.05% DMSO) were considered 100% viable.

The cell suspension was mixed with 0.5% low melting agarose (LMA) at 37°C, and the mixture was spread on fully frosted microscopic slides pre-coated with 1% normal melting agarose. After solidification of the agarose, the slides were covered with 0.5% LMA and then immersed in a lysis solution (2.5 M NaCl, 100 mM Na-ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 1% Triton X-100, and 10% DMSO, pH 10.0) for 30 min at 4°C. The slides were then placed in a CometAssay Electrophoresis System Starter Kit (KORMED, Seongnam, Korea) containing 300 mM NaOH and 10 mM Na-EDTA (pH 13.00) for 40 min to allow for DNA unwinding and examination of alkali-labile damage. An electrical field was then applied (300 mA, 20 V) for 20 min at 4°C to draw the negatively charged DNA toward the anode. After electrophoresis, the slides were washed three times for 5 min at 4°C in a neutralizing buffer (0.4 M Tris, pH 7.5). The slides were then stained with 20 µg/ml PI and observed using a fluorescence microscope (Carl Zeiss, Inc., Oberkochen, Germany). The images were also analyzed using an image analysis system (Komet 5.5; Kinetic Imaging, Liverpool, UK) to evaluate the degree of DNA damage. The tail length and the tail moment were used as measures of the extent of DNA damage. One hundred cells were randomly selected from one sample (two slides were made for one sample, 50 randomly selected cells per slide) and then measured. We calculated the values of the mean tail length for each sample and the percentage values of the cells in five ranges of tail length: undamaged cells without a tail, cells with a tiny tail, cells with a dim tail (28), cells with a clear tail, and only tail. The ranges of tail length and tail moment were divided arbitrarily in the present study.

To assess the generated ROS, the cells were incubated with 10 µM DCFH-DA for 20 min at room temperature in the dark to monitor ROS production. ROS levels in the cells were monitored with a flow cytometer (Becton-Dickinson, San Jose, CA, USA) using CellQuest Pro software, as previously described (29).

For quantitative assessment of the induced cell apoptotic rate, a fluorescein-conjugated Annexin V (Annexin V-FITC) staining assay was performed according to the manufacturer's instructions (BD Biosciences Pharmingen, San Jose, CA, USA). Briefly, the cells in each sample were stained with 5 µl Annexin V-FITC and 5 µl PI. After incubation for 15 min at room temperature in the dark, the degree of apoptosis was quantified by flow cytometer as a percentage of the Annexin V-positive and PI-negative cells (Becton-Dickinson) using CellQuest Pro software (29).

The mitochondrial transmembrane electrochemical gradient was measured using JC-1 staining. Briefly, the cells were collected and incubated with 10 µM JC-1 solution for 15 min at 37°C in the dark. The fluorescence intensity of the red/green ratio was quantified by flow cytometer (Becton-Dickinson) using CellQuest Pro software, as previusly described (30).

After removing the media, the cells were washed with ice-cold phosphate-buffered saline (PBS) and gently lysed for 20 min in an ice-cold lysis buffer (40 mM Tris, pH 8.0, 120 mM, NaCl, 0.5% nonidet-P40, 0.1 mM sodium orthovanadate, 2 µg/ml leupeptin, and 100 µg/ml phenymethylsulfonyl fluoride). The supernatants were collected, and the protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For western blot analysis, equal amounts of protein extracts were denatured by boiling at 95°C for 5 min in a sample buffer [0.5 M Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.1% bromophenol blue and 10% β-mercaptoethanol] at a ratio of 1:1. The samples were stored at −80°C or immediately used for western blot analysis. Aliquots containing 30 µg of total protein were separated by denaturing SDS-polyacrylamide gel electrophoresis and transferring electrophoretically to nitrocellulose membranes (Amersham Biosciences, Arlington Heights, IL, USA). The membranes were then blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies, probed with enzyme-linked secondary antibodies for 1 h at room temperature, and detected using an enhanced chemiluminescence (ECL) detection system (both from Amersham Biosciences).

Nrf2 siRNA and control siRNA were both purchased from Santa Cruz Biotechnology, Inc. The siRNAs were transfected into C6 cells according to the manufacturer's instructions using Lipofectamine^® RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA). The cells were seeded in 6-well culture plates for transfection and incubated with 50 nM control or Nrf2 siRNA for 24 h in serum-free OPTI-MEM media (Invitrogen). After 24 h transfection, the cells were incubated under the experimental conditions.

Data are expressed as the means ± standard error of the mean (SEM). The comparison between groups was undertaken using ANOVA, and significance was analyzed using Duncan's multiple range test. A P-value <0.05 was considered to indicate a statistically significant difference.

We first determined the effect of baicalein on the viability of C6 cells using the MTT assay. As shown in Fig. 1A, the results demonstrated that baicalein (25–300 µM) alone for 24 h had no detectable effect on C6 cell survival. To determine the protective effects of baicalein on H₂O₂-induced cytotoxicity in C6 cells, the cells were pre-treated with baicalein for 1 h and exposed to H₂O₂ for an additional 24 h. As shown in Fig. 1B, the treatment of C6 cells with 0.5 mM H₂O₂ for 24 h resulted in approximately a 43% loss of cellular viability compared with the control cells. However, the cytotoxic effect of H₂O₂ was blocked by pretreating cells with baicalein (25–100 µM).

To examine the inhibitory effect of baicalein on H₂O₂-induced ROS production, C6 cells were stimulated with 0.5 mM H₂O₂ for 30 min in the presence and absence of baicalein, and the intracellular ROS level was then determined. As expected, increased ROS generation was detected in cells after stimulation with H₂O₂ alone (Fig. 2A). However, pretreatment with baicalein significantly reduced H₂O₂-induced ROS production. We further examined the effects of baicalein on DNA damage caused by H₂O₂ using a Comet assay and western blot analysis. As indicated in Fig. 2B, treatment with H₂O₂ alone significantly increased the number of DNA breaks, resulting in an increase in fluorescence intensity in the tails of the comet-like structures, which was associated with an increase in the tail length and tail moment (Table I). However, these phenomena were prevented by pretreatment with baicalein. In addition, the western blot analysis revealed that the level of p-γH2A.X, a classic marker of DNA double-strand break formation (31), in C6 cells treated with H₂O₂ alone was markedly increased. However, pretreatment with baicalein was found to inhibit the increase in p-γH2A.X expression caused by treatment with H₂O₂ (Fig. 2C).

As mitochondrial permeability is critical for the oxidative stress-induced apoptotic pathway (32), we evaluated the effect of baicalein on the MMP of C6 cells using flow cytometry. After incubation with H₂O₂ alone, the loss of MMP was markedly increased compared to the untreated control (Fig. 3A), which indicated mitochondrial damage and dysfunction. By contrast, pretreatment with baicalein effectively prevented the loss of MMP induced by H₂O₂ in a concentration-dependent manner. We also examined the ability of baicalein to protect against H₂O₂-triggered C6 cell apoptosis using Annexin V/PI-staining. The flow cytometry results indicated that the percentage of apoptotic cells treated with 0.5 mM H₂O₂ was approximately 51% (Fig. 3B), which was significantly reduced by pretreatment with baicalein. Furthermore, we determined the effects of baicalein on the expression of apoptosis-related proteins PARP and XIAP. As illustrated in Fig. 3C, the degradation of PARP and the downregulation of XIAP were observed in H₂O₂-treated C6 cells. However, pretreatment with baicalein effectively protected against these changes.

The fact that Nrf2 signaling regulates cellular antioxidant response has been well documented (33). We sought to determine whether signaling was associated with baicalein-mediated neuroprotection. Western blot analysis indicated that treating C6 cells with baicalein induced the expression of Nrf2 protein in a concentration- and time-dependent manner, which was associated with the downregulation of Keap1 (Fig. 4). Concomitant with the induction of Nrf2, the levels of HO-1 and TrxR1 were also markedly increased after treatment with baicalein in C6 cells.

In order to investigate the role of HO-1 induction in the baicalein-mediated neuroprotective effects exerted against oxidative stress, we inhibited HO-1 activity using ZnPP, a specific inhibitor of HO-1. As shown in Fig. 5A and B, in the presence of ZnPP, the protective effects of baicalein on H₂O₂-induced loss of MMP and reduction of cell viability were significantly attenuated. Furthermore, ZnPP blocked the protection provided by baicalein against H₂O₂-induced degradation of PARP, phosphorylation of γH2A.X, and downregulation of XIAP as well as apoptosis (Fig. 5C and D). To determine whether the protective effect of baicalein was related to its inductive effect on TrxR1 expression, we blocked TrxR1 activity using auranofin, a selective TrxR1 inhibitor. The results indicated that auranofin also reversed the inhibition of loss of MMP and apoptotic activity caused by baicalein in H₂O₂-stimulated C6 cells (Fig. 6).

It has previously been reported that HO-1 and TrxR1 are regulated through the Nrf2 cascade (19,34–36). We developed an Nrf2 gene knockdown model using siRNA transfection to demonstrate the contribution of Nrf2 signaling to the negative effects of baicalein on H₂O₂-induced cytotoxicity. Western blot analysis indicated that Nrf2 siRNA reduced the baicalein-induced expression of Nrf2 compared with untransfected control and control siRNA-transfected cells) (Fig. 7A). The baicalein-induced expression of HO-1 and TrxR1 was also blocked by Nrf2 siRNA, which is evidence that the augmentation of HO-1 and TrxR1 was mediated by Nrf2. Furthermore, Nrf2 siRNA significantly attenuated the protective effects of baicalein against the H₂O₂-induced reduction of cell viability and apoptosis (Fig. 7B and C), which was associated with the disappearance of the potential of baicalein to protect against H₂O₂-induced PARP degradation, γH2A.X phosphorylation and XIAP reduction (Fig. 7D).