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Introduction

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), are generated during normal cellular metabolism, and they play critical roles in the signal transmission mechanism (1,2). However, H2O2 also exerts genotoxicological effects, as it produces new free radicals and causes damage to the main cellular components (3,4). Moreover, the excess production of ROS increases oxidative damage, leading to cellular dysfunction and cell death (5,6). Oxidative stress is mainly caused by neurodegenerative disorders, including dementia, which has attracted significant attention; as brain cells are damaged, the interaction between neurons is inhibited, resulting in disability of memory and cognitive functions (7,8). Glial cells, which outnumber neurons in the brain, are non-neuronal cells that maintain homeostasis as well as supporting and protecting neurons in the central nervous system (CNS) (9). Therefore, the attenuation of oxidative stress and the inhibition of apoptosis in glial cells are critical for protection from neurodegenerative disorders. Thioredoxin reductase 1 (TrxR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductase that decreases the active site of cytosolic thioredoxin-1 (Trx1) from the disulfide form to the biologically active dithiol form (10). Trx1 contributes to antioxidative activity by donating electrons to peroxiredoxins for the reduction of H2O2 (11,12).

In addition, the heme oxygenase (HO) pathway has been reported to be active in the CNS and to operate as an underlying protective mechanism of cells exposed to an oxidizing agent (13). Moreover, the enhancement of HO-1 protein expression has been associated with protection against stress conditions, such as oxidative stress (14). HO-1 gene expression is mainly regulated by the nuclear factor-erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, and the induction of this enzyme protects cells against oxidative stress-induced damage and apoptosis (15,16). The Kelch-like ECH-associated protein-1 (Keap1) controls Nrf2 activation and nuclear accumulation by binding to the Nrf2 protein and targeting it for proteosomal degradation (17). Nrf2 is released from Keap1 repression under conditions of oxidative stress, and it translocates to the nucleus where it increases several antioxidant genes, such as HO-1 and TrxR1 (15,18,19). Thus, we suggest that the activation of Nrf2 is crucial for the cytoprotective mechanism against oxidative stress.

Baicalein, a flavonoid originally obtained from the roots of a traditional Chinese herb, Scutellaria baicalensis Georgi, has been widely used in the treatment of inflammation, hypertension, cardiovascular disease, bacterial infection and cancer (20,21). Moreover, the neuroprotective effects of baicalein have been demonstrated in several experimental models, such as models of Alzheimer's disease (22,23), ischemic stroke (24,25), and Parkinson's disease (26,27). However, an association between the Nrf2 pathway with the neuroprotective role of baicalein against oxidative stress in glial cells has not previously been demonstrated. Thus, in the present study, we investigated the neuroprotective effect exerted by baicalein and also its mechanisms which protect against H2O2-induced neuronal damage in a model using C6 glial cells.

Materials and methods

Reagents and antibodies

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.

Cell culture and treatment

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.

Cell viability assay

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.

Comet assay (single-cell gel electrophoresis)

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.

Measurement of intracellular ROS

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).

Measurement of apoptosis

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).

Measurement of mitochondrial membrane potential (MMP; ΔΨm)

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).

Protein extraction and western blot analysis

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).

Small interfering RNA (siRNA) transfection

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.

Statistical analysis

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.

Results

Baicalein prevents H2O2-induced growth inhibition in C6 cells

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).

Figure 1 Effects of baicalein on H2O2-induced growth inhibition in C6 cells. The cells were treated with various concentrations of baicalein for 24 h (A) or pretreated with the indicated concentrations of baicalein for 1 h, and further incubated in the presence and absence of 0.5 mM H2O2 for 24 h (B). Cell viability was estimated using an MTT reduction assay. The data are represented as the means ± SEM obtained from three independent experiments (*P<0.05 compared with the untreated control group; #P<0.05 compared with H2O2-treated group).

Baicalein attenuates H2O2-induced ROS generation and DNA damage in C6 cells

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).

Figure 2 Effects of baicalein on H2O2-induced reactive oxygen species (ROS) generation, DNA damage, and γH2A.X phosphorylation in C6 cells. The cells were pretreated with the indicated concentration of baicalein for 1 h and then stimulated with or without 0.5 mM H2O2 for 30 min. The cells were incubated at 37°C in the dark for 20 min with a culture medium containing 10 µM DCFH-DA to monitor ROS production. ROS generation was measured by flow cytometry (A). The cells were pretreated with baicalein for 1 h and further incubated in the presence and absence of 0.5 mM H2O2 for 24 h. The comet assay was performed to detect cellular DNA damage, and representative photographs of the comets were taken using a fluorescence microscope (x200 original magnification) (B). Cellular proteins isolated in cells grown under the same conditions as (B) were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies against p-γH2A.X and β-actin (C), as a loading control.

Table I Preventive effect exerted by baicalein on H2O2-induced DNA damage in C6 cells (means ± SEM).

Table I

Preventive effect exerted by baicalein on H2O2-induced DNA damage in C6 cells (means ± SEM).

Compounds	Scored cells	Tail moment	Tail length
Control	100	2.27±0.81	64.4±4.97
Baicalein (100 µM)	100	1.50±1.23	53.16±8.13
H2O2 (0.5 mM)	100	42.09±4.34	156.55±9.04
Baicalein+H2O2	100	4.75±1.72	97.66±6.21

Baicalein reduces H2O2-induced loss of MMP and apoptosis in C6 cells

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.

Figure 3 Effects of baicalein on H2O2-induced loss of mitochondrial membrane potential (MMP), apoptosis, poly(ADP-ribose) polymerase (PARP) degradation, and X-linked inhibitor of apoptosis protein (XIAP) inhibition in C6 cells. The cells were pretreated with various concentrations of baicalein for 1 h, and then incubated with and without 0.5 mM H2O2 for 24 h (A and B). The cells were collected and incubated with 10 µM JC-1 for 15 min at 37°C in the dark. The cells were then washed once with PBS, and mean JC-1 fluorescence intensity was detected using a flow cytometer (A). The cells were stained with FITC-conjugated Annexin V and PI for flow cytometric analysis (B). The percentage of apoptotic cells was determined by counting the percentage of Annexin V-positive cells. The data are represented as the means ± SEM values obtained from three independent experiments (*P<0.05 compared with untreated control group; #P<0.05 compared with H2O2-treated group). Cells were pretreated with 100 µM baicalein for 1 h and then incubated with and without 0.5 mM H2O2 for 24 h. The cells were lysed, and the cellular proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies against PARP and XIAP (C). β-actin was used as the loading control.

Baicalein upregulates Nrf2, HO-1 and TrxR1 expression in C6 cells

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.

Figure 4 Effects of baicalein on the levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein-1 (Keap1), hemeoxy-genase-1 (HO-1), and thioredoxin reductase 1 (TrxR1) in C6 cells. The cells were incubated for the indicated periods of time with 100 µM baicalein (A) or with various concentrations of baicalein for 6 h (B). Cellular proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies against Nrf2, Keap1, HO-1 and TrxR1. β-actin was used as the loading control.

Induction of HO-1 and TrxR1 is involved in the protective effect exerted by baicalein against H2O2 treatment 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).

Figure 5 Effects of a heme oxygenase-1 (HO-1) inhibitor on the baicalein-mediated attenuation of mitochondrial membrane potential (MMP) loss, growth inhibition, and apoptosis caused by H2O2 in C6 cells. The cells were pretreated for 1 h with 100 µM baicalein and then treated for 24 h with or without 0.5 mM H2O2 in the absence or presence of 2.5 µM zinc protoporphyrin (ZnPP). After incubation with 10 µM JC-1 for 20 min, JC-1 fluorescence intensity was detected using a flow cytometer (A). Cell viability was assessed using an MTT reduction assay (B). Cellular proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies against poly(ADP-ribose) polymerase (PARP), X-linked inhibitor of apoptosis protein (XIAP) and p-γH2A.X (C). β-actin was used as the loading control. To quantify the degree of apoptosis, the cells were stained with FITC-conjugated Annexin V and propidium iodide (PI) for flow cytometric analysis (D). The data are represented as the means ± SEM values obtained from three independent experiments (*P<0.05 compared with untreated group; #P<0.05 compared with H2O2-treated group; $P<0.05 compared with H2O2 and baicalein-treated group).

Figure 6 Effects of a thioredoxin reductase 1 (TrxR1) inhibitor on the baicalein-mediated attenuation of loss of mitochondrial membrane potential (MMP), growth inhibition, and apoptosis by H2O2 in C6 cells. The cells were pretreated for 1 h with 100 µM baicalein and then treated for 24 h with or without 0.5 mM H2O2 in the absence or presence of 0.5 µM auranofin. After incubation with 10 µM JC-1 for 20 min, JC-1 fluorescence intensity was detected using a flow cytometer (A). Cell viability was assessed using an MTT reduction assay (B). Cellular proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with specific antibodies against poly(ADP-ribose) polymerase (PARP), X-linked inhibitor of apoptosis protein (XIAP) and p-γH2A.X (C). β-actin was used as the loading control. To quantify the degree of apoptosis, the cells were stained with FITC-conjugated Annexin V and propidium iodide (PI) for flow cytometry analysis (D). The data are represented as the means ± SEM values obtained from three independent experiments (*P<0.05 compared with the untreated group; #P<0.05 compared with H2O2-treated group; $P<0.05 compared with H2O2- and baicalein-treated group).

Baicalein upregulates HO-1 and TrxR1 expression via Nrf2 activation in C6 cells

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).

Figure 7 Nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated induction of heme oxygenase-1 (HO-1) and thioredoxin reductase 1 (TrxR1) by baicalein in C6 cells. The cells were transfected with a control (Con siRNA, as a negative control for RNA interference) and Nrf2 siRNA. After 24 h, the cells were treated with 100 µM baicalein for 1 h and then stimulated with or without 0.5 mM H2O2 for 24 h. Cellular proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies (A and D). β-actin was used as the loading control. Cell viability was assessed using an MTT reduction assay (B). To quantify the degree of apoptosis, the cells were stained with FITC-conjugated Annexin V and propidium iodide (PI) for flow cytometry analysis (C). The results are in mean ± SEM values obtained from three independent experiments (*P<0.05 compared with the untreated group; #P<0.05 compared with H2O2-treated group; $P<0.05 compared with H2O2- and baicalein-treated group).

Discussion

The excessive production of ROS, which causes oxidative damage to proteins, lipids and DNA, is one of the most prominent factors related to neurodegeneration (37). The mitochondrial electron transport system is a major source of intracellular ROS generation (32), whereby the mitochondria play a pivotal role in the process of ROS-mediated cell death. Moreover, H2O2 directly induces mitochondrial dysfunction, followed by a rapid efflux of intracellular ROS, which increases the permeabilization and depolarization of the mitochondrial membrane. This event likely facilitates the rapid disruption of MMP and the release of apoptosis-inducing factors that activate the caspase-dependent signaling cascades in the induction of apoptosis (38). Therefore, the search for functional food or bioactive compounds that act against oxidative stress is critical for the prevention and cure of neurodegenerative disorders. The purpose of the present study was to determine whether baicalein blocked H2O2-induced oxidative stress in C6 cells or not. The results demonstrated that treatment of C6 cells with H2O2 caused the marked intracellular accumulation of ROS and the loss of MMP, and further inhibited cell survival, leading to apoptosis. However, when the C6 cells were pretreated with baicalein, the H2O2-induced generation of ROS, loss of MMP, reduction of cell viability, and apoptosis were significantly attenuated, as previously reported in studies on other neuron-like cells (27). Thus, we presume that baicalein improves mitochondrial function through eliminating the overproduction of ROS induced by H2O2 and thereby reducing H2O2-induced apoptosis. In addition, our results showed that H2O2 treatment increased DNA tail moment and length in the comet assay and expression of p-γH2A.X, which are widely used markers for the detection of DNA damage (31). However, in the present study, both events were abolished by baicalein, indicating that it protected against H2O2-induced apoptosis in the C6 cells by reducing the amount of DNA damage caused by the destructive impact of oxidative stress in the C6 cells.

Apoptosis, which is programmed cell death, is a tightly regulated cell suicide response that facilitates the correct development and homeostasis of multicellular organisms; in mammalian cells, two major apoptotic pathways (the cell death receptor-mediated and mitochondrial-mediated apoptotic pathways) have been studied (39). Previous research has indicated that the impairment of MMP and ROS generation is closely linked to the initiation of caspase-dependent apoptotic signaling in many types of cells (5). Caspases, which are a family of cysteine acid proteases, are the central regulators of the execution of cell death in response to various apoptotic stimuli (40). In the final stage of apoptosis, both pathways induce the activation of executioner caspases, such as caspase-3 and -7, which results in the degradation of substrate proteins, such as PARP, a biochemical hallmark of cell apoptosis (39). The activation of caspases may also be regulated by a variety of proteins, including members of the inhibitor of apoptosis proteins (IAP) family, which promote cell survival after a wide variety of apoptotic stimuli elicited via intrinsic and extrinsic pathways through selectively binding with caspases, thus inhibiting caspase activity and apoptosis (39). Moreover, previous research has indicated that H2O2 induces apoptosis through the activation of caspases in addition to the inhibition of IAP family proteins (41). In the present study, western blot analyses revealed that baicalein effectively blocked the H2O2-induced cleavage of PARP, a downstream target protein of the activated caspase-3 in C6 cells. Under the same conditions, baicalein also rescued the H2O2-induced downregulation of XIAP, a representative member of the IAP family, compared with the control. Although further molecular studies are needed, our findings indicate that baicalein potentially prevents H2O2-induced apoptosis through the inactivation of caspase cascades in C6 cells.

Previous research has suggested that Nrf2, a master cellular sensor for oxidative stress, and its repressor, Keap1, play indispensable roles in protecting a variety of tissues from a wide array of toxic insults, including oxidative stress. Under normal conditions, Nrf2 is inactive and bound in the cytosol by Keap1 (35). The dissociation of Nrf2 from Keap1 is a prerequisite for nuclear translocation, and the subsequent DNA binding of Nrf2 is necessary to regulate the inducible expression of cytoprotective phase II enzymes (15,18). In the present study, we found that baicalein increased Nrf2 protein expression and decreased Keap1 in a concentration- and time-dependent manner in C6 cells. These results are consistent with those of previous studies (42–44), and were associated with the induction of HO-1 and TrxR1. However, the inhibition of HO-1 function using ZnPP significantly weakened the inhibitory effects of baicalein on H2O2-induced MMP loss, growth inhibition, and apoptosis by blocking PARP cleavage, XIAP inhibition, and γH2A.X phosphorylation. Moreover, pre-treatment with a TrxR1 inhibitor also markedly abrogated the protective effects of baicalein against H2O2-induced oxidative stress. In addition, the knockdown of Nrf2 by Nrf2-targeted siRNA completely cancelled out baicalein-induced HO-1 and TrxR1 expression, suggesting that Nrf2 is a critical upstream regulator of the baicalein-mediated induction of HO-1 and TrxR1. Furthermore, the removal of Nrf2 also halted the baicalein-induced restoration of H2O2-mediated growth inhibition and the apoptosis of C6 cells. These results suggest that the Nrf2-dependent induction of HO-1 and TrxR1 by baicalein, at least in part, may participate in the protection against oxidative stress in C6 cells.

In conclusion, the results of our present study clearly demonstrated that baicalein exerted a protective effect against H2O2-induced DNA damage, growth inhibition, and apoptosis in C6 cells. Baicalein also successfully suppressed the accumulation of intracellular ROS, leading to substantial regain of MMP, at least in part, through the activation of Nrf2 signaling and the induction of HO-1 and TrxR1. These findings suggest that baicalein has a potential neuroprotective value as an antioxidant agent.

Acknowledgments

The present study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea Government (no. 2015R1A2A2A01004633) and the High Value-added Food Technology Development Program (314043–3), Ministry of Agriculture, Food and Rural Affairs.

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