Protection by genistein on cortical neurons against oxidative stress injury via inhibition of NF-kappaB, JNK and ERK signaling pathway

Abstract

Context: Genistein, one of the isoflavones derived from soybean seeds, has been reported to exert multiple bioactivities. However, the mechanism of its action on the central nervous system is not fully understood.

Objective: To investigate the cytoprotection of genistein and its molecular mechanism against H₂O₂-induced cell death in primary rat cortical neurons.

Materials and methods: Genistein (0.01, 0.1, and 1 μM) were added into the primary rat neurons 24 h before and co-cultured with 500 μM H₂O₂ for 1 h. Neuronal injury was assessed by MTT, lactate dehydrogenase (LDH) assay, and Hoechst33258 staining. Intracellular reactive oxygen species (ROS) generation induced by H₂O₂ was determined. Neuronal apoptosis was evaluated by Bcl-2/Bax ratio as well as by caspase-9 and caspase-3 activities. The protein levels and phosphorylation of NF-κB/p65, IκB, JNK, and ERK were detected by western blots.

Results: Genistein pretreatment attenuated H₂O₂-mediated neuronal viability loss, nuclear condensation, and ROS generation in a concentration-dependent manner. Genistein exerted anti-apoptotic effects by reversing the apoptotic factors Bcl-2 and Bax ratio, along with the suppression of caspase-9 and caspase-3 activities. In addition, genistein down-regulated the expression of NF-κB/p65, and suppressed the phosphorylation of p65 and IκB. Genistein also inhibited H₂O₂-induced activation of the MAPK-signaling pathway including JNK and ERK.

Discussion and conclusion: The results indicated that genistein effectively protects cortical neurons against oxidative stress at least partly via inactivation of NF-κB as well as MAPK-signaling pathways, and suggested the possibility of this antioxidant for the prevention and treatment of stroke.

• Apoptosis
• MAPK
• NF-κB
• reactive oxygen species

Introduction

Cerebral ischemic injury is considered as a serious insult because of its high mortality and disability in many countries. Among factors involved in cerebral ischemia, oxidative stress resulted from dysfunction of the antioxidant defense system as well as an excessive production of reactive oxygen species (ROS) plays a crucial role in the pathogenesis of the disease. The intracellular ROS generation during ischemia and reperfusion can affect a large amount of biomolecules and disturb cellular integrity and function, leading to neurotoxicity and cell death by triggering a cascade of detrimental cellular pathways (Crack & Taylor, 2005; Doyle et al., 2008; Sugawara et al., 2004). To date, antioxidant therapies have been used to protect against ischemia/reperfusion injury by blocking deleterious pathways both in vitro and in vivo (Clark et al., 2001; Clemens, 2000; Wang et al., 2009), and much attention has been focused on the potential application of natural substances for a possible strategy to prevent ischemic stroke.

Genistein (4′,5,7-trihydroxyisoflavone), one of the nutraceutical molecules found in soybean seeds, is a phytoestrogen that has been traditionally consumed at usual dietary intakes by Asians (Setchell, 2001). The epidemiological evidence and animal model studies indicate that dietary genistein ingestion lowers the risk of cancers, cardiovascular disease, and post-menopausal ailments due to its antioxidant effects (Henderson et al., 2012; Hussain et al., 2012; Li et al., 2010). Our previous study showed that pretreatment with genistein at 2.5–10 mg/kg per day for two consecutive weeks protected mice from ischemia/reperfusion-induced brain injury, as evidenced by reduction of infarct volume, improvement of the neurological deficit, and prevention of neuronal apoptosis (Qian et al., 2012). However, the underlying signal transduction pathways by which genistein exerts its neuroprotection have not been systematically elucidated.

Here, we established an experimental condition of hydrogen peroxide (H₂O₂) exposure which is extensively referred to produce an increase in ROS and lipid peroxidation, cause DNA damage, induce differential protein activation, and consequently lead to neuron injury (Chandra et al., 2002; Liu et al., 2007). Then, we employed this experimental cell culture model to investigate genistein’s neuroprotective activities and explore the possible molecular mechanisms involved in the preventive effects of this compound.

Materials and methods

Materials

Genistein (>98% pure) was purchased from Sigma Chemical (St. Louis, MO) and was dissolved in dimethyl sulfoxide and further diluted in culture medium containing 0.5% solvent. Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), except that the β-actin antibody was obtained from Bioss (Beijing, China). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Boster (Wuhan, China). Minimum essential medium (MEM), neurobasal medium (without phenol red and estrogen free), and B-27 were purchased from Gibco (Grand Island, NY).

Primary neuron cultures

Cerebral cortical neurons were prepared essentially as previously described (Xiong et al., 2004), with some modifications. In brief, cortices were dissected from embryonic day 16 (E16) Sprague–Dawley rat embryos and incubated with 0.05% EDTA–trypsin then passed through a nylon sieve (80 μm pore size) into MEM supplemented with glucose (0.6% wt./vol.), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% (vol/vol) horse serum. The cells were adjusted to approximately 1 × 10⁹/l and planted into 24-well or 6-well plates, which were previously coated with 10 mg/l poly-l-lysine for 24 h, at 37 °C in an atmosphere of 5% CO₂ and 95% O₂. After 4 h, the medium was removed, and neurons were maintained in the neurobasal medium supplemented with 0.02% B-27 and l-glutamine (0.5 mM). Under these conditions, the cultures typically contained more than 95% neurons as assessed by MAP2-specific immunocytochemical detection.

Cell culture treatment

To evaluate the vulnerability of neurons to H₂O₂ exposure, various concentrations of H₂O₂ were added to neuron cultures for 1 h. Neuronal viability was quantified using the 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction test. At the end of H₂O₂ treatment, MTT was added at a final concentration of 0.5 mg/ml for 4 h at 37 °C and the insoluble formazan crystals were dissolved in 100 μl of dimethyl sulfoxide. The cell viability corresponded to the value of the optical density read at 492 nm with a background subtraction at 630 nm. Results were expressed as percentage of the optical density measured in vehicle-treated cells.

Genistein (0.01, 0.1, and 1 μM) was added into the cultures 24 h before and co-cultured with 500 μM H₂O₂ for another 1 h. Control cultures were treated in an identical way without inducing H₂O₂ exposure. Neuronal viability was quantitatively assessed by MTT assay. Cytotoxicity was measured by lactate dehydrogenase (LDH) release into the culture medium, and the total LDH was determined after cell lysis, according to the following equation: LDH release = (LDH activity in the medium/total LDH activity) × 100%, using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China).

ROS measurement

The production of ROS was monitored fluorimetrically 1 h after H₂O₂ exposure, using the fluorescent probe 2′–7′-dichlorofluorescein diacetate (DCFH-DA, Sigma, St. Louis, MO), which is converted into highly fluorescent 2′–7′-dichlorofluorescein (DCF) in the presence of ROS (Wang et al., 2010). Samples from different groups were collected, rinsed with PBS, and incubated with 10 μM DCFH-DA at 37 °C for 30 min. Fluorescence was observed using an Axiovert 40 fluorescence microscope (Zeiss, Germany). For the quantitative assay, the fluorescence intensity was measured by a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The ROS level in each group was presented as the percentage of increase as compared with the control group. Protein analysis was performed in accordance with the procedure described previously, using bovine serum albumin (BSA) as the standard (Lowry et al., 1951).

Hoechest 33258 staining

DNA staining by Hoechst33258 fluorescence was employed to evaluate the nuclear condensation and characteristic features of apoptotic cells. Neurons were plated in 6-well plates. At the end of the treatment, cells were rinsed with phosphate-buffered saline (PBS, pH 7.4) three times and fixed with 4% paraformaldehyde for 30 min at room temperature, followed by incubation with Hoechst33258 (5 μM, final concentration) at 37 °C for 20 min. Fluorescence images were examined under the fluorescence microscope, and were obtained using Image-Pro Plus software. Total cells and damaged cells were counted, and the percentage of apoptotic cells was calculated.

Caspase-3 and caspase-9 activity assay

Caspase-3 and caspase-9 activities were measured in lysates of cortical neurons using the CaspACE^™ Assay System, Colorimetric (Promega, Madison, WI) and Caspase-Glo® 9 Assay kit (Promega, Madison, WI), following the instructions of the manufacturer. Briefly, neurons were lysed by freeze-thaw, and then incubated on ice for 20 min to ensure complete cell lysis. Cell lysates were centrifuged at 12 000 rpm for 10 min at 4 °C, and the supernatant fraction was collected for the determination. For caspase-3 activity assay, an aliquot of culture supernatant was incubated with 200 μM of DEVDpNA substrate at 37 °C for 4 h. The absorbance was measured at 405 nm. Caspase-9 activity was measured using the luminescent assay method. The samples were mixed with the aliquot of Caspase-Glo® 9 reagent (Promega, Madison, WI) and incubated for 3 h at room temperature. The luminescence was measured in a plate-reading. The protein levels in the lysates were determined by the method of Bradford (1976). Results were expressed as a percentage of vehicle-treated cells.

Western blot

The cells were rinsed twice in PBS and lysed in RIPA lysis buffer for 20 min. Twenty micrograms of protein were loaded into each lane, separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY) in Tris–glycine buffer (48 mM Tris, 39 mM glycine, pH 9.2) containing 20% methanol. The membranes were blocked with skimmed milk for 1 h, washed in Tris-buffered saline containing 0.1% Tween-20 (TBST), and incubated overnight with the primary antibodies. After washing three-times with TBST, nitrocellulose membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG. Bands were visualized using the SuperSignalWest Pico Chemiluminescent Substrate Trial Kit (Pierce, Rockford, IL). Images were taken using the ChemiDoc XRS system with Quantity One software (Bio-Rad, Richmond, CA). The expression of target proteins were normalized to β-actin expression and compared with the control.

Statistical analysis

All values are expressed as the mean ± SD of at least three independent preparations. Differences among the groups were compared using one-way ANOVA analysis followed by a Tukey post hoc test. A difference with p < 0.05 was considered statistically significant.

Results

Genistein attenuates H₂O₂-induced cytotoxicity in neurons

In order to determine the optimal concentration of H₂O₂ to induce cytotoxicity in cortical neuronal cultures, cells were incubated with various concentrations of H₂O₂ for 1 h. Result showed that H₂O₂ induced neuron toxicity with an IC₅₀ value of 547.77 μM (Figure 1A). Therefore, 500 μM H₂O₂ was used in all subsequent experiments to induce neurotoxicity in primary-cultured cortical neurons.

Figure 1. Hydrogen peroxide toxicity and the effects of genistein on H₂O₂-induced neuron injury. (A) Neuron viability was analyzed with an IC₅₀ value of 547.77 μM. (B) Neurons pretreated with the indicated concentrations of genistein showed no viability loss. Genistein pretreatment reduced neuron injury induced by H₂O₂ in a concentration-dependent manner by (C) MTT reduction assay and by (D) measuring LDH release. ##p < 0.01 compared with the control; **p < 0.01 compared with the model.

To test whether genistein itself has negative effects on neuronal viability, graded concentrations from 0.01 to 10 μM of genistein were added to neuronal cells for 24 h incubation. The result showed that genistein alone, up to 10 μM, did not affect cell viability (p > 0.34, Figure 1B), indicating that genistein had no toxic effect under basal conditions.

According to the previous work, the neuroprotective efficacy of physiological concentrations (0.1–1 μM) of genistein was observed in the plasma of individuals on a high soy diet (Setchell, 2001). Therefore, we chose 0.01, 0.1, and 1 μM of genistein for its concentration response assay against H₂O₂ toxicity. Cortical neurons were incubated with genistein for 24 h, and then treated with H₂O₂ for an additional 1 h. The viability of cortical neurons exposed to H₂O₂ was reduced to 52.7 ± 7.3% compared with the control group as determined by the MTT assay. But pretreatment with genistein (0.01–1 μM) significantly reduced H₂O₂-induced cell death in a concentration-dependent manner, with the maximal protection (90.0 ± 8.2%) at 1 μM (Figure 1C).

The neuroprotective effect of genistein was also confirmed using the LDH assay. Exposure to H₂O₂ alone induced a significant increase in LDH release approximately 4-fold compared with control. Genistein pretreatment significantly reduced LDH efflux in a concentration-dependent manner (Figure 1D). These data indicated that genistein was effective for the protection of neurons against H₂O₂-induced injury.

Genistein prevents ROS production evoked by H₂O₂ in cultured neuronal cells

Figure 2(A) illustrates DCF fluorescent changes in response to H₂O₂. In the control group, only a small amount of fluorescent cells in each visual field was observed. H₂O₂-treatment provoked an elevation of DCF fluorescence, with a 2.2-fold increase compared with the control (Figure 2B). In the presence of genistein, ROS generation was effectively alleviated by 21.5% and 37.3% in 0.1 and 1 μM groups, respectively, as compared with the model.

Figure 2. Effects of genistein on H₂O₂-induced ROS production in neuronal cultures. (A) The DCF fluorescence was observed under an inverted-microscope. H₂O₂ caused more extensive green fluorescence as compared with the control group, which was partly ameliorated by genistein pretreatment. (B) Quantitative analysis of ROS production in different groups, and the fluorescence intensity was presented as the percentage of increase as compared with the control group. ##p < 0.01 compared with the control; *p < 0.05, **p < 0.01 compared with the model. Scale bar = 50 μm.

Genistein counteracts H₂O₂-induced neuronal apoptosis

ROS induced by H₂O₂ leads to an increase in apoptotic cells (Jiang et al., 2009). To test whether genistein exerts neuroprotection by virtue of anti-apoptosis effects, we employed nuclear staining with Hoechst33258. Control cells showed intact, light blue nuclei whereas neurons exposed to H₂O₂ displayed typical nuclear apoptotic morphology, as indicated by bright, condensed and rounded nuclei, nuclear fragmentation, and apoptotic bodies, which could be partly counteracted by genistein pretreatment (Figure 3A). The incidence of apoptotic neurons exposed to H₂O₂ increased to 42.92 ± 9.77% of the total counted cells, and genistein lowered the number of apoptotic cells (32.5 ± 13.42%, 26.36 ± 10.33%, and 13.09 ± 4.81% in 0.01, 0.1, and 1 μM groups, respectively) compared with the H₂O₂-treated group (Figure 3B). These result showed the potent anti-apoptotic effect of genistein in cortical neurons.

Figure 3. Effects of genistein on H₂O₂-induced neuron apoptosis in neuronal cultures. (A) Nuclear condensation and morphology was visualized by Hoechest 33258 staining. Nuclei showing clearly bright chromatin condensation and/or fragmentation were regarded as apoptotic cells (indicated by white arrows). (B) Neuron apoptosis was calculated and expressed as the percentage of total cell count. ##p < 0.01 compared with control; **p < 0.01 compared with the model. Scale bar = 50 μm.

Genistein regulates apoptotic-related proteins during oxidative stress

Considering that Bcl-2/Bax ratio is a well-established determinant in the regulation of apoptosis (Korsmeyer, 1995), we tested changes in the protein levels of Bcl-2 and Bax after genistein treatment, using western blot analysis. H₂O₂ led to an induction of Bax protein but a down-regulation of Bcl-2 expression. Genistein treatment could enhance the expression of Bcl-2 and reduce that of Bax, thereby significantly increasing the Bcl-2/Bax ratio in H₂O₂-treated neurons (Figure 4A).

Figure 4. Effects of genistein on the expression or activity of apoptotic-related proteins after H₂O₂ insult in neuronal cultures. (A) Genistein treatment increased the Bcl-2/Bax ratio as determined by western blot. Semi-quantitative analyses of protein levels were normalized to β-actin expression and compared with control. Genistein treatment reduced the activity of both (B) caspase-3 and (C) caspase-9 induced by H₂O₂. ## p < 0.01 compared with control; *p < 0.05, **p < 0.01 compared with the model.

We further investigated the influence of genistein upon downstream of the apoptotic signaling pathway by measuring caspase-9 and caspase-3 activities following H₂O₂ exposure. Increases in caspase-9 and caspase-3 activities have been correlated with increased apoptosis in response to H₂O₂ treatment (161 ± 39% and 194 ± 55% of control, respectively). Genistein attenuated the activity of both caspase-9 and caspase-3 significantly in our model. In 0.01, 0.1, and 1 μM genistein treatment groups, the caspase-9 activity was 176 ± 41%, 134 ± 29%, and 124 ± 25% compared with cultures not exposed to H₂O₂, respectively (Figure 4B). The inhibitory effect of genistein on caspase-3 activity was 147 ± 32%, 123 ± 30%, and 116 ± 15% compared with the control (Figure 4C). These results indicated that genistein is quite efficient in protecting neurons from apoptosis via inhibiting the caspase-dependent pathways.

Genistein inhibits NF-κB signals activated by H₂O₂ in neurons

To explore the mechanisms underlying the protective effect of genistein against H₂O₂-induced cortical neurons death, the involved pathways during oxidative stress was investigated. NF-κB is a nuclear factor that has been implicated in oxidative stress-induced apoptosis (Peng et al. 2005; Velez-Pardo et al. 2002. Our observation reveals that treatment of cortical neurons with H₂O₂ caused an increase in NF-κB p65 expression, which was concentration dependently inhibited by genistein treatment (Figure 5A). Coincident with p65 expression, phospho-p65 was markedly detected during the 1 h exposure to H₂O₂ and restored nearly to the normal level by 1 μM genistein treatment (Figure 5A). In addition, H₂O₂ triggered the phosphorylation of IκBα in cortical neurons. Pretreatment with 0.1 and 1 μM genistein significantly attenuate the level of phospho-IκBα (Figure 5B). The total IκBα level was not obviously altered and genitein showed little effect on its expression (Figure 5B). These data suggest that genistein suppressed NF-κB signal pathway by inactivation of p65 and IκBα.

Figure 5. Effects of genistein on NF-κB signal pathway after H₂O₂ insult in neuronal cultures. (A) Genistein down-regulated the expression and phosphorylation of NF-κB/p65 subunit and (B) prevented the phosphorylation of IκBα. Semi-quantitative analyses of protein levels were normalized to β-actin expression and compared with control. #p < 0.05, ##p < 0.01 compared with control; *p < 0.05, ** p < 0.01 compared with the model.

Genistein suppresses phosphorylation of JNK1/2 and ERK1/2 during oxidative stress in neurons

The activation of MAPK signal pathways was previously proved to be implicated in ROS-induced cell death (Ravindran et al., 2011; Zhou et al., 2005). Therefore, we further investigated whether these signal pathways also participate in genistein’s action. After 1 h treatment with H₂O₂, ERK1/2 phosphorylation was dramatically up-regulated compared with the control group. This up-regulation was reversed by 1 μM genistein (Figure 6A). Similarly, JNK1/2 was highly activated after H₂O₂ insult, increasing about 1.9-folds to the control group, whereas the treatment with 1 μM genistein could obviously inhibit the phosphorylation of JNK1/2 (Figure 6B). These results suggested that MAPK inhibition contributed in part to genistein’s cytoprotection on neurons.

Figure 6. Effects of genistein on JNK1/2 and ERK1/2 signal pathways after H₂O₂ insult in neuronal cultures. Genistein down-regulated the phosphorylation of (A) JNK1/2 and (B) ERK1/2 levels. Semi-quantitative analyses of phosphorylation levels were normalized to total JNK1/2 and ERK1/2 expression, respectively, and compared with control. #p < 0.05 compared with control; *p < 0.05 compared with the model.

Discussion

Genistein, an active component from soybean seeds, exhibits many bioactive effects. In this study, we analyzed the effect of genistein in a model of H₂O₂-induced oxidative stress in neuronal cells. We demonstrated that genistein exerted a potent protection on primary-cultured neurons. The mechanisms of this action may be involved in the alleviation of intracellular ROS generation, amelioration of apoptotic parameters such as Bax, Bcl-2 and caspase pathway, as well as inactivation of NF-κB, ERK, and JNK signals.

Previously, we reported that genistein exerted neuroprotection in a cerebral ischemia mouse model. Administration of genistein remarkably reduced the infarct volume, improved the neurological deficit, and prevented cell apoptosis after ischemia. Moreover, genistein modified the antioxidant enzyme system and reversed the mitochondria dysfunction in the mice brain (Qian et al., 2012). Thus, we hypothesized that genistein would be effective on H₂O₂-induced oxidative stress in primary cortical neurons. On the basis of the current data, pretreatment with genistein alleviated the loss of cell viability, LDH release, and DNA condensation following H₂O₂ exposure. These results indicate that genistein may have neuroprotective effects both in vitro and in vivo.

Oxidative stress caused by increased intracellular ROS generation has been implicated in acute cerebral ischemia/reperfusion injury as well as neurodegenerative diseases (Crack & Taylor, 2005; Peters et al., 1998; Slemmer et al., 2008). Excessive production of ROS may induce lipid peroxidation, damage the mitochondrial membrane, and eventually result in cell damage via activation of apoptotic mediators. Therefore, pharmacological modification aimed at oxidative damage is considered one of the most promising avenues for stroke therapy (Bortner & Cidlowski, 2002; Chinopoulos & Adam-Vizi, 2006; Gasparova et al., 2009; Graham et al., 2009). The phenylpropanoid structure with a phenolic hydroxyl permits genistein to exert the antioxidant property (Hodnick et al., 1986). To confirm the ability of genistein to scavenge ROS in neuronal cultures, cells were pre-treated with different concentrations of genistein before exposure to H₂O₂. As we expected, incubation of neurons with H₂O₂ for 1 h strikingly enhanced the formation of intracellular ROS, which could be statistically attenuated by 0.1–1 μM genistein treatment. The results were in good agreement with previous studies, which reported that genistein could protect neuronal PC12 cells against d-galactose-induced oxidative damage (Hsieh et al., 2011). These observations strongly suggest that genistein can act as a potent antioxidant by scavenging cellular-free radicals in neuronal cells.

There are reports suggesting that oxidative stress like H₂O₂ treatment play a critical role in the induction of programmed cell death (Ratan et al., 1994; Zamzami et al., 1995). In the present study, the Hoechst33258 result clearly showed that neuronal cells undergo apoptosis following H₂O₂ treatment, conforming that apoptosis can be triggered by direct oxidative stress. H₂O₂-induced apoptosis is accompanied by a series of biochemical changes such as the expression of Bcl-2 family, cytochrome c release, and subsequent caspase activation. The Bcl-2 family proteins, including the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, are considered to be critical regulators of programmed cell death in neurons (Chao & Korsmeyer, 1998; Korsmeyer, 1995). Bcl-2 prevents disruption of the mitochondria and inhibits the accumulation of cytochrome c in the cytosol, thereby preventing caspase-3 activation and blocking the apoptotic cascade, whereas Bax was identified as the proapoptotic member that triggers the release of caspases. Therefore, the relative ratio of Bcl-2 and Bax is a determining factor in the regulation of apoptotic cell death. To explore whether or not genistein prevents H₂O₂-induced neuronal injury by regulating the Bcl-2 family proteins, the Bcl-2/Bax ratio was determined. We found that genistein enhanced the expression of neuronal Bcl-2 and decreased that of Bax after H₂O₂ exposure, suggesting that genistein shifts the balance of cell death/survival effectors favoring the apoptotic demise of neurons. In addition, the cysteine protease caspase-9 and caspase-3, also activated by H₂O₂, are key mediators in the regulation and execution of neuronal apoptosis (Matsura et al., 1999). The release of cytochrome c from the mitochondria into the cytosol activates caspase-3 by activating apoptotic protease-activating factor and complexes of procaspase-9 (Green & Reed, 1998). Caspase-3 then causes degradation of cytoskeleton, DNA fragmentation, and eventually cell death. In the present study, when cortical neurons were exposed to H₂O₂, an increase in the activity of caspase-9 and caspase-3 was clearly observed, but this activation was suppressed remarkably by treatment with genistein in a dose-dependent manner at 0.01, 0.1, and 1 μM. These results indicate that genistein may inhibit H₂O₂-induced cell apoptosis through antagonizing the caspase-9-dependent pathway. Taken together, we propose that genistein can protect against H₂O₂-induced apoptosis and this effect is predominantly modulated by the changes in the expression of apoptosis related proteins, thereby attenuating activation of the caspase cascade.

ROS are known to regulate multiple cellular processes and trigger the activation of a cascade of intracellular pathways by phosphorylation/dephosphorylation reactions (Dalton et al., 1999; Lopez-Neblina & Toledo-Pereyra, 2006). Here, we focused on the activation of NF-κB, ERK1/2, and JNK1/2 signal pathways in the presence of H₂O₂. NF-κB is known to function as a key factor activated in response to oxidative stress. NF-κB, once activated, increases the expression of many target genes relevant to apoptosis in a variety of cells, and antioxidants treatment blocks both the activation of the factor and the apoptotic cell death (Aoki et al., 2001; Del Rio & Velez-Pardo, 2002; Tarabin & Schwaninger, 2004). NF-κB activation involves the phosphorylation and subsequent degradation of the inhibitory protein IκBα, and the released NF-κB dimmer translocate into the nucleus and activate target genes. Here we showed the effects of genistein on NF-κB activation by the protein measurement of NF-κB/p65, phosphor-p65 as well as IκBα and phosphor-IκBα. We found that H₂O₂ stimulation induced dramatically phosphorylation of p65 subunits and IκBα. Exposure of cells to genistein before the H₂O₂ insult resulted in inhibition of p65 and IκBα phosphorylation. These effects may be responsible, at least in part, for the abortion of the H₂O₂-induced apoptotic program.

The MAPK signaling has been considered as a stress-activated protein kinase pathway, which transduces extracellular stimuli into a series of intracellular phosphorylation cascades (Satoh et al., 2000; Wada & Penninger, 2004). The members of MAPK family, including JNK and ERK, are involved in regulating ROS-mediated cell death (Torres, 2003). Oxidative stress stimuli lead to increased levels of phosphorylated JNK and ERK MAPKs and subsequent cell apoptosis. In our study, we observed that cells exposed to H₂O₂ exhibited increased levels of phospho-JNK1/2 and phospho-ERK1/2. Pretreatment with genistein dramatically attenuated the levels of phosphorylated JNK1/2 and ERK1/2 MAPKs in H₂O₂-treated neuronal cells, suggesting that blocking the phosphorylation of JNK and ERK is obligatory for genistein-induced protection.

Conclusions

In conclusion, we have provided evidence that genistein can protect cultured cortical neurons against oxidative stress injury through multifunctional cytoprotective pathways, including the alleviation of intracellular ROS production, the up-regulation of Bcl-2/Bax ratio, and the following depression of caspase-dependent apoptosis. The inactivation of NF-κB and MAPK pathways may at least partly participate genistein’s neuroprotection. Future studies aiming at the detailed understanding on the cellular mechanisms of this antioxidant may offer additional insights into the molecular basis of its neuroprotection. Overall, the present study suggests the possibility that nutritional therapies incorporating antioxidants could thus open new avenues for the prevention and treatment of stroke.

Declaration of interest

This work was supported in part by the National Key Program for Transgenic Breeding (2008ZX08004-003) and the National Natural Science Foundation of China (31000718). All authors declare that they have no financial and personal relationships with other people or organizations that could inappropriately influence their work. The authors alone are responsible for the content and writing of the paper.