The neuroprotective effect of praeruptorin C against NMDA-induced apoptosis through down-regulating of GluN2B-containing NMDA receptors

Toxicology in Vitro 27 (2013) 908–914

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The neuroprotective effect of praeruptorin C against NMDA-induced apoptosis through down-regulating of GluN2B-containing NMDA receptors Le Yang 1, Xu-Bo Li 1, Qi Yang 1, Kun Zhang, Nan Zhang, Yan-Yan Guo, Bin Feng, Ming-Gao Zhao ⇑, Yu-Mei Wu ⇑ Department of Pharmacology, School of Pharmacy, The Fourth Military Medical University, Xi’an, Shaanxi Province 710032, PR China

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Article history: Received 29 June 2012 Accepted 1 January 2013 Available online 9 January 2013 Keywords: Praeruptorin C (Pra-C) Neuron N-methyl-D-aspartate (NMDA) GluN2B-containing NMDA receptor Apoptosis Bcl-2 family

a b s t r a c t Praeruptorin C (Pra-C), one of the principal bioactive components derived from the root of Peucedanum praeruptorum Dunn, has been widely used as an antioxidant and a calcium antagonist to treat diseases. The present study investigated the protective effect of Pra-C on cultured cortical neuron injury induced by glutamate. After challenge with 200 lM N-methyl-D-aspartate (NMDA) for 30 min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. Pra-C conferred protective effects against loss of cellular viability in a concentration-dependent manner. Pra-C also significantly inhibited neuronal apoptosis induced by NMDA exposure by reversing intracellular Ca2+ overload and balancing Bcl-2 and Bax expression. Furthermore, Pra-C significantly reversed the upregulation of GluN2B-containing NMDA receptors by exposure to NMDA but did not affect the expression of GluN2A-containing NMDA receptors. These findings suggest that Pra-C partially protects cortical neurons by inhibiting the expression of GluN2B-containing NMDA receptors and regulating the Bcl-2 family. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Accumulation of glutamate and an excessive stimulation of its receptors induce potent excitotoxicity in CNS (Arundine and Tymianski, 2004; Soundarapandian et al., 2007). The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors play a key role in mediating glutamate excitotoxicity because of the high permeability of calcium. NMDA receptors are heteromeric complexes formed by three types of subunits: GluN1, GluN2 (A, B, C, and D), and GluN3 (A and B) (CullCandy et al., 2001; Paoletti and Neyton, 2007). NMDA receptors typically consist of GluN1 and GluN2 subunits, in which GluN1 subunits are essential for the function of NMDA receptor channels (Papadia and Hardingham, 2007). Excitotoxicity triggered by the selective activation of NMDARs containing GluN2B subunit has been suggested to play an important role in the pathogenesis of neurodegenerative disorders associated with glutamate excitotoxicity (Kew and Kemp, 1998). Overactivated NMDA receptors are permeable to Na+, K+, and Ca2+ ions, among which excess Ca2+ ions is linearly correlated with neuronal cell death triggered by

⇑ Corresponding authors. Tel./fax: +86 29 84774552 (M.-G. Zhao), tel.: +86 29 84774555; fax: +86 29 84774552 (Y.M. Wu). E-mail addresses: [email protected] (M.-G. Zhao), [email protected] (Y.-M. Wu). 1 These authors contributed equally to this study. 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.01.001

intracellular Ca2+-dependent cascades. Many researchers have focused on the extensive studies of GluN2B antagonists because of the ability to potentially cure various CNS diseases (Brown et al., 2011; Cho et al., 2010). In recent years, interest in the treatment of glutamate-induced neurotoxicity with plant-based therapy, including traditional Chinese medicine, has considerably grown, and extensive experience has been accumulated over thousands of years (Feigin, 2007; Harvey, 1999). Multiple interconnected mechanisms leading to neuronal cell death are involved in ischemic stroke or other neurodegenerative disorders (Lo et al., 2003). A single neuroprotective agent affecting only one aspect of these cascades seems unlikely to achieve a satisfactory therapeutic outcome (Savitz and Fisher, 2007). Cocktails of drugs are attractive but present difficulties when designing blend proportions. In this regard, composite extracts from natural plants may be effective in preventing neuronal cell death. Praeruptorin C (Pra-C) is a component extracted from the root of Peucedanum praeruptorum Dunn, a traditional Chinese medicine. Previous studies have shown that Pra-C has protective effects against H2O2-induced damage in myocardial cells (Jiang et al., 2005) and calcium antagonistic action (Sun et al., 1997). However, the effect of Pra-C on the CNS has not yet to be reported. Our study investigates the possible neuroprotective properties of Pra-C against excitatory neurotoxicity mediated by NMDA in primary cortical neurons and elucidates its possible mechanisms. We found that Pra-C provides significant protective effects by

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downregulating GluN2B levels and calcium overloading as well as regulating the Bcl-2 family, including Bcl-2 and Bax expression. 2. Materials and methods 2.1. Chemicals and reagents Praeruptorin C (Pra-C) (purity >98%) was purchased from Shanghai PureOne Biotechnology (Shanghai, China). 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly-D-lysine, trypsin, propidium iodide (PI), Hoechst 33258, NMDA and anti-b-actin antibody were purchased from Sigma– Aldrich (St. Louis, MO, USA). Neurobasal medium, B27, and glutamine were provided by Invitrogen (Carlsbad, CA, USA). AntiMAP2, anti-GluN2A, anti-GluN2B, anti-Bax and anti-Bcl-2 antibodies were purchased from Chemicon (Temecula, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Hyclone (Logan, UT, USA). All secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). M-PER Protein Extraction Buffer and enhanced chemiluminescent solution (ECL) were obtained from Pierce (Pierce, Rockford, IL). All of the other chemicals and reagents were standard commercially available biochemical quality. 2.2. Cell culture and treatment All procedures carried out in this study were in accordance with our institutional guidelines, which comply with international rules and policies. Primary cultures of cortical neurons were prepared from the brain of E15–E16 C57BL/6 mouse embryos (obtained from the Experimental Animal Center of the Fourth Military Medical University, Xi’an, China). Briefly, disassociated cortex tissues from embryonic 15–16 day-old mice were incubated with 0.125% trypsin in Ca2+ and Mg2+-free Hank’s balanced salt solution for 10 min at 37 °C. Then the cortices were washed in DMEM supplemented with 10% FBS to stop trypsin activity, and further dissociated by trituration. The single cell suspension was cultured on poly-D-lysine coated plates in Neurobasal media supplemented with 2% B27, 0.5 mM glutamine, 100 U/ml penicillin, and 100 U/ ml streptomycin. It took 7 days for re-incubation, the time required for maturation of cortical neurons and half of the medium was changed every 2 days. The cells were characterized by immunohistochemistry staining for anti-MAP2 antibody, revealing that this

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culture procedure yielded more than 95% neurons. Neurons were seeded at a density of 5  104 cells/well in 96-well plate for the MTT cell survival analysis, 3  105 cells/well in 24-well plate for PI and Hoechst 33258 double staining, and 2  106 cells/well in 6-well plate respectively for Western blot analysis. Pra-C was added 24 h prior to and after the addition of NMDA and were present throughout the excitotoxic injury. 2.3. Cell viability analysis Neuronal cell viability was determined by MTT assay as described before (Yang et al., 2010) with some modifications. Briefly, cortical neurons were cultured in 96-well plates at 5  104 cells/well for 7 days until mature before each treatment. For NMDA-induced injury, cells were incubated with different concentrations of NMDA (0, 50, 100, 200 and 400 lM) for 30 min, then subjected to MTT assay. For Pra-C-mediated protection assay, neurons were pretreated with Pra-C (0, 0.1, 1, and 10 lM) 24 h before subjected to NMDA (200 lM) stimulation for 30 min. At the end of each treatment, the culture medium was replaced with fresh medium containing 0.5 mg/ml MTT for 4 h at 37 °C. After incubation, the medium was replaced by 150 ll/well dimethyl sulfoxide (DMSO) to resolve the formazan crystals. The optical density (OD) was read on a Universal Microplate Reader (Elx 800, Bio-TEK instruments Inc., USA) at 570 nm (630 nm as a reference). The data were expressed as a percent of control value and means ±SEM of three experiments and six wells included in each group. Cell death was determined by PI and Hoechst 33258 double fluorescent staining as described (Shen et al., 2008). Neurons were cultured in 24-well plates at a density of 3  105 cells/well. Neurons were pretreated with Pra-C (1 and 10 lM) for 24 h then subjected to excitotoxic injury with 200 lM NMDA for 30 min. One day later, the cells were stained with PI (1 lg/ml) and Hoechst 33258 (10 lg/ml) for 15 min, and then fixed by 4% paraformaldehyde for 10 min. Cells were observed under a fluorescence microscope (Olympus BX61, Japan). The Hoechst and PI dye were excited at 340 and 620 nm, respectively. For each well, six visual fields were selected randomly. 2.4. Western blot analysis In order to further explore the mechanisms involved in Pra-Cmediated neuroprotection, we examined the effects of Pra-C on

Fig. 1. Pra-C promotes neuron viability upon NMDA injury. (A) Concentration-dependent cytotoxic effects of NMDA on the cell viability of cortical neurons. Primary cultures of mouse neurons in 96-well plates, 6-wells for one group, were treated with NMDA for 30 min, and cell viability was determined by the MTT method. Results are representative of three independent experiments with neurons from three mice. P < 0.05, P < 0.01 versus the control. (B) Effects of Pra-C on the cell viability of cortical neurons after exposure to NMDA. Primary cultures of mouse neurons in 96-well plates, 6-wells for one group, were treated with Pra-C at different concentrations followed by exposure to 200 lM NMDA for 30 min. Cell viability was determined by the MTT method. Results are representative of three independent experiments with neurons from three mice. # P < 0.05 versus NMDA alone.

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signaling pathways related to survival by Western blot analysis as described previously (Wu et al., 2009). Neurons were cultured in 6-well plates at a density of 2  106 cells/well. After each treatment, cells were rinsed twice with PBS and lysed by M-PER Protein Extraction Buffer containing 1  protease inhibitor cocktail. Cell protein was quantified by a BCA Kit and equal amounts of protein (50 lg) separated on 10% polyacrylamide gel followed by transferred onto an Immun-Blot PVDF membrane. The membrane was blocked for 1 h with 5% non-fat milk in Tris-Phosphate buffer containing 0.05% Tween 20 (TBS
T). It was further incubated overnight at 4 °C with primary antibodies including anti-GluN2A (1:1000), anti-GluN2B (1:1000), anti-Bax (1:1000) and anti-Bcl-2 (1:1000), b-actin (1:10,000) served as a loading control. After five washes with TBS
T, membranes were further incubated with HRP-conjugated secondary antibodies for 1–2 h and followed by four TBS
T washes. The target protein signal was detected and digitalized using ECL and Image J program. The density of target protein was expressed as the fold of control and mean ± SEM from five independent experiments.

2.5. Calcium imaging Calcium imaging was performed as previously described (Nishimura et al., 2006; Shen et al., 2008). Neurons were cultured in 3.5 cm plates made especially for laser scanning microscope at a density 3  105 per plate. Cultured cells were washed twice using Mg2+-free extracellular solution (ECS) containing (in mM): NaCl, 140; KCl, 3; CaCl2, 2; HEPES, 10; and glucose, 10. The pH was adjusted to 7.2–7.3 with NaOH and osmotic pressure adjusted to 310 ± 5 with sucrose. Then, the neurons were incubated with 2.5 lM fluo-3/AM at 37 °C. After 30 min, the cultures were washed twice and returned to the original culture medium for an additional 30 min. The dye-loaded cells were measured for fluorescence using a confocal laser scanning microscope (Olympus, Japan). Prior to NMDA application, the dye-loaded cells were scanned for approximately 1 min to obtain a basal level of intracellular Ca2+. Then, 200 lM NMDA was applied to the cultures, and an equal amount of ECS was added as a placebo. Pra-C was added 24 h before the experiments and present in the whole experimental

Fig. 2. Hoechst 33258 and PI staining in cultured cortical neurons. (A) Primary neurons were cultured in 24-well plates and representative fluorescence images were obtained after Hoechst 33258 and PI double staining in control, NMDA-treated, and NMDA + Pra-C-treated groups. B. The percentage of apoptotic neurons in total neurons for control, NMDA-treated, and NMDA + Pra-C-treated groups. The cell numbers were counted from the Hoechst 33258 and PI staining in three independent observations. Data are shown as means ± SEM of three independent experiments with neurons from three mice. P < 0.05, P < 0.01 versus the control group and ##P < 0.01 versus NMDA alone.

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process. The change of Ca2+ concentration was estimated by the fluorescence ratio of the fluo-3/AM-loaded neurons for another 4 min. The results are expressed as changes relative to basal levels, and five wells were selected randomly for analysis. 2.6. Statistical analysis Data were presented as means ± SEM for three separate experiments (each in triplicate). Comparisons were analyzed by oneway analysis of variance (Gines et al., 2003) and subsequent Bartlett’s test. Difference were considered statistically significant at P < 0.05. 3. Results 3.1. Effects of Pra-C on cell viability NMDA has been proven to be involved in the pathogenesis of neurodegenerative disorders associated with glutamate excitotoxicity. We initially evaluated the effects of Pra-C on MTT induction in cultured cortical neurons to determine whether or not Pra-C confers neuroprotection. MTT assay showed no significant changes in the viability of cortical neurons treated with Pra-C compared with the control, indicating that Pra-C itself exerted no toxicity on the cultured neurons (data not shown). The neurons were exposed to increasing concentrations of NMDA (0, 50, 100, 200, and 400 lM) for 30 min. As expected, NMDA decreased cell viability in a concentration-dependent manner, as measured by the MTT assay (Fig. 1A). The neurons were exposed to 200 lM NMDA for 30 min in subsequent experiments because cell injury was significant in this paradigm (cell viability in NMDA treatment:

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73.2 ± 4.2%, P < 0.01 versus control alone). Pretreatment with 10 lM Pra-C for 24 h showed effective neuroprotection against NMDA injury, as illustrated in Fig. 1B (cell viability in 10 lM PraC treatment: 92.5 ± 7.5%, P < 0.05 versus NMDA alone). Morphological observation provided further evidence of the neuroprotection of Pra-C (data not shown). We observed that cortical neurons cultured in the vehicle (control) exhibited a normal cell shape with a round cell body, intact cell membrane and well-extended neurites. Pra-C treatment even at the highest concentration (10 lM) failed to alter the cell shape of the cortical neurons. However, neurons exposed to NMDA exhibited a cell injury morphology, featuring disappearance of neurites and emergence of vacuoles around the cell body; neurons pretreated with Pra-C partly showed a normal cell shape. We performed Hoechst 33258 and PI double-staining to further determine the neuroprotective effects of Pra-C. Data demonstrated that 9.9 ± 4.1% of the cells in the control group underwent cell death or apoptosis compared with 55.1 ± 2.6% of the cells in the NMDA injury group (P < 0.01 versus control; Fig. 2A and B). Pra-C (1 and 10 lM) significantly attenuated the excitotoxicity of NMDA on the cortical neurons. The percentage of cells in 1 lM and 10 lM Pra-C groups that underwent apoptosis decreased to 17.9 ± 5.3% and 12.1 ± 6.1%, respectively (P < 0.01 versus NMDA alone; Fig. 2A and B). These data suggest that Pra-C protects neurons from excitotoxicity mediated by NMDA. 3.2. Effects of Pra-C on Bcl-2/Bax Expression Bcl-2 family members include both anti-apoptotic (e.g., Bcl-2 and Bcl-xl) and pro-apoptotic (e.g., Bax, Bad, Bak, and Bid) proteins. The ratio between the two subsets determines the susceptibility of

Fig. 3. Effects of Pra-C on Bax and Bcl-2 protein expression. (A) Primary neurons were cultured in 6-well plates and representative Western blot results showed the levels of Bax and Bcl-2 protein after different treatments. (B) The intensity of Bax was quantified by scanning densitometry, normalized with respect to endogenous b-actin, and expressed as fold change. (C) The intensity of the Bcl-2 was quantified by scanning densitometry, normalized with respect to endogenous b-actin, and expressed as fold change. (D) The ratio of Bax/Bcl-2 was expressed as fold change compared with the control. Results are representative of five independent experiments with neurons from five mice. P < 0.05,  P < 0.01 versus the control group and ##P < 0.01 versus NMDA alone. Data are expressed as means ± SEM of five independent experiments.

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3.3. Effects of Pra-C on the expression of GluN2A- and GluN2Bcontaining NMDARs GluN2A- and GluN2B-containing NMDARs are suggested to be linked to different intracellular cascades and participate in different functions in neuronal cell survival or death (Liu et al., 2007). Blockades of GluN2B-containing NMDA receptors promote neuronal survival, exerting a protective action against NMDA receptor-mediated neuronal damage (Liu et al., 2007; Shen et al., 2008; Stanika et al., 2009). In contrast, activation of either synaptic or extra synaptic GluN2Acontaining NMDARs promotes neuronal survival and exerts neuroprotection against either NMDAR-mediated or non-NMDAR-mediated neuronal damage (Liu et al., 2007). We performed Western blot analysis on the primary cultures to further examine Pra-C effects on the expression of NMDAR subtypes. GluN2B subtype expression notably increased in cultured cortical neurons after exposure to NMDA (182.3 ± 14.0% of the control; P < 0.05; Fig. 4A and B), whereas GluN2A subtype expression was unchanged (92.0 ± 19.1% of the control; P > 0.05; Fig. 4A and C). Upregulation of the GluN2B subtype by NMDA stimuli was significantly reduced in the presence of 1 lM and 10 lM Pra-C, as shown in Fig. 4A and B (94.5 ± 6.1% and 58.0 ± 14.3% of the control, respectively; P < 0.05 versus NMDA alone). However, Pra-C had no effects on the GluN2A subtype expression (103.0 ± 9.1% and 89.0 ± 11.1% of the control; P > 0.05 versus NMDA alone; Fig. 4A and C). Thus, downregulated GluN2B subtype expression by Pra-C is suggested to be partly responsible for the neuroprotective effects of Pra-C against NMDA-induced excitotoxic injury. 3.4. Pra-C inhibited NMDA-induced increase in Ca2+ overload in cortical neurons

Fig. 4. Effects of Pra-C on GluN2A and GluN2B expression. (A) Primary neurons were cultured in 6-well plates and representative Western blot results showing the levels of GluN2A and GluN2B subtypes after different treatments. (B) The intensity of GluN2B subtypes level was normalized with endogenous b-actin and expressed as percentage change compared with control. P < 0.01 versus the control group and ## P < 0.01 versus NMDA alone. (C) The intensity of GluN2A subtypes level was normalized with endogenous b-actin and expressed as fold change compared with the control. No difference was found between each group. Data are expressed as means ± SEM of three independent experiments.

the cells to a death signal (Zeng et al., 2011). Western blot analysis showed that Bcl-2 and Bax were both expressed in non-injured cortical neurons, and Pra-C (10 lM) treatment alone did not alter the expression of these proteins (data not shown). NMDA stimulation led to Bcl-2 downregulation (0.59 ± 0.63 fold of the control, P < 0.01; Fig. 3A and C) and Bax upregulation (2.23 ± 0.29 fold of the control; P < 0.01; Fig. 3A and B), thus increasing the Bax/Bcl-2 ratio (3.58 ± 0.42 fold of the control; P < 0.01; Fig. 3D). NMDAinduced increase in the Bax/Bcl-2 ratio was significantly reversed by Pra-C (1 or 10 lM) pretreatment (0.92 ± 0.32 or 0.81 ± 0.29 fold of the control, respectively; P < 0.01; Fig. 3D). The effects of Pra-C on the Bax/Bcl-2 ratio may constitute an important element responsible for neuroprotection.

Considering that NMDAR activation increases cytoplasmic Ca2+ concentration in the cultured neurons (MacDermott et al., 1986) and Ca2+ overload triggers multiple intracellular catabolic processes, followed by the irreversible death of neuronal cells in the brain (Liu et al., 2004), we focused on the effects of Pra-C on Ca2+ overload. Fluorescence intensity can be regarded as an indicator of cytoplasmic Ca2+ concentration (Matsumoto et al., 2004). We found that the Ca2+ concentration in cultured neurons was stable during detection (Fig. 5A and B), and NMDA (200 lM) induced a rapid elevation in Ca2+ concentration in cultured neurons (Fig. 5A and C). A slow reduction was observed over the next 4 min, after which the Ca2+ concentration stayed at a relatively stable elevated stage. Pra-C (1 lM or 10 lM) could attenuate the amplitude and speed of elevation in Ca2+ concentration (Fig. 5A and C). 4. Discussion Pra-C, one of the active ingredients of P. praeruptorum Dunn, significantly attenuates neuronal injury induced by NMDA stimuli, as shown by the results from the MTT assay, apoptotic staining, and Western blot analysis. Our results suggest that this protection may be correlated with the suppression of the cell death and apoptosis by inhibition of GluN2B-containing NMDARs and regulation of the Bcl-2 family. Glutamate is clearly responsible for basal excitatory synaptic transmission and synaptic plasticity, including long-term potentiation and long-term depression associated with cognitive processes (Mark et al., 2001). Excessive glutamate accumulation, however, induces neuronal death both in vitro and in vivo, and whether cells undergo apoptosis or necrosis depends on the dosage and duration of glutamate stimulation (Chen et al., 2008; Li et al., 2007). Most investigators agree that the pathologic activation of subtype NMDARs contributes to neuronal death after acute excitotoxic trauma, such as brain ischemia (Arundine and Tymianski, 2004; Guo et al.,

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Fig. 5. Effects of Pra-C on intracellular calcium overload in cultured cortical neurons. (A) Cultured neurons were loaded with fluo-3, followed by successive perfusion of NMDA for 4 min, and fluorescence images were obtained; extracellular solution (ECS) served as a control. (B) Fluorescence intensity of ECS-perfused neurons was normalized according to the fluorescence detected and values represent the means ± SEM in three separate experiments. (C) Fluorescence intensity of neurons in NMDA-treated (red), NMDA + 1 M Pra-C-treated (blue), NMDA + 10 M Pra-C-treated (green) was normalized according to the fluorescence detected and values represent the means ± SEM in three separate experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2011) and acts as a major mediator. NMDARs consist of a GluN2A subunit that promotes neuron protection, whereas GluN2Bcontaining NMDARs mediate excitotoxicity (Cho et al., 2010). In the present study, the data showed that Pra-C reverses the upregulation of GluN2B induced by NMDA, implying that the neuroprotection of Pra-C is likely to antagonize a particular NMDAR subunit. This phenomenon is quite beneficial for neurodegenerative diseases relevant to glutamate excitotoxicity. The key step in NMDA-induced neuronal cell apoptosis is the overload of intracellular Ca2+, followed by overstimulation of NMDARs (Sattler and Tymianski, 2000). Ca2+ overload triggers several downstream lethal reactions, including nitrosative stress, oxidative stress, and mitochondrial dysfunction (Meldrum, 2000). In this study, the elevation of Ca2+ stimulated by NMDA is inhibited by Pra-C in a concentration-dependent manner to support neuroprotection.

Glutamate clearly evokes different intracellular cytotoxic signals, among which the Bcl-2 family proteins play critical roles in apoptotic cell death (Zeng et al., 2011). The Bcl-2 family proteins consist of anti- and pro-apoptotic molecules. Anti-apoptotic protein, Bcl-2, has been reported to inhibit caspase activation in cell apoptosis, whereas the pro-apoptotic protein, Bax, promotes cell apoptosis via translocation to the mitochondrial membrane as one of the major causes of neurological disorders (Vila et al., 2001). Accordingly, the balance between Bcl-2 and Bax determines the fate of survival or death of cells in response to insults (Zha and Reed, 1997). In this study, we found that Pra-C treatment increased the ratio of Bcl-2/Bax in NMDA-injured neurons, suggesting that Pra-C rescues cortical neurons from cell apoptosis, possibly through the regulation of apoptosis-related proteins. In summary, our results suggest that the neuroprotective effect of Pra-C is partially associated with the downregulation of

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