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Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro
ARTICLE IN PRESS Neurochemistry International ■■ (2015) ■■–■■
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Neurochemistry International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n c i
Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro Xiao-Long Hu a,b, Li-Ping Guo b, Qi Song b, Qiao Zhang b, Ying Chen a, Jian Wang b, Wei-Hong Meng a, Qing-Chun Zhao a,* a b
Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E
I N F O
Article history: Received 28 December 2014 Received in revised form 19 May 2015 Accepted 1 June 2015 Available online Keywords: Kukoamine B NMDA receptors Oxidative stress NADPH oxidase SH-SY5Y cells Molecular docking
A B S T R A C T
A major cause of cerebral ischemia is overactivation of the N-methyl-D-aspartate receptors (NMDARs). Therefore, NMDAR antagonists are needed for the treatment of cerebral ischemia. In our research, KuB protected the SH-SY5Y cells against NMDA-induced injury, apoptosis, LDH release and MMP loss. In addition, KuB could decrease MDA levels while increasing SOD activity. Meanwhile, KuB decreased NADPH oxidase-mediated ROS production, inhibited Ca2+ influx, and increased the Bcl-2/Bax ratio. Furthermore, KuB not only down-regulated expression of the NR2B subunit of NMDAR but also actively modulated expression of the signaling molecules downstream of NR2B, including p-ERK, p-CREB, p-AKT and SAPKs. Finally, docking results showed that KuB had a high affinity for NR2B-containing NMDARs. Therefore, we conclude that KuB protected the SH-SY5Y cells from NMDA-induced injury likely by antagonizing NMDARs and reducing oxidative stress. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Cerebral ischemia is the third leading cause of death around the world. Furthermore, neurodegenerative disorders such as Alzheimer’s, Huntington’s and Parkinson’s diseases often affect the elderly and devastatingly affect their quality of life. An important question is still unanswered – what is the main factor causing these diseases? Collective evidence shows that N-methyl-D-aspartate receptors (NMDARs), a major glutamate receptor highly expressed in the central nervous system (CNS), play a key role in these diseases (Barkus et al., 2010). Abnormal expression of NMDARs can cause some of the diseases states earlier and also influences learning and memory (Barkus et al., 2010). Therefore, finding effective agents as new NMDAR antagonists is impending. Accumulated evidence demonstrates that excessive stimulation of NMDARs causes a high intracellular Ca 2+ influx. The overloaded Ca2+, which is the main factor in ischemic stroke and neurodegenerative disorders, results in neuron injury following a series of apoptotic events (Hardingham and Bading, 2003; Kruman and Mattson, 1999). NMDARs are heteromeric complexes, composed of two obligatory NR1 subunits and two NR2A-D subunits
* Corresponding author. Department of Pharmacy, General Hospital of Shenyang Military Area Command, 83 Wenhua Road, Shenyang 110016, China. Tel.: +86 24 28856205; fax: +86 24 28856205. E-mail address: [email protected] (Q.-C. Zhao).
(Nakanishi, 1992). Importantly, NR2B-containing NMDARs are the most important of all the subunits when it comes to excessive Ca2+ influx and dysfunctions mentioned earlier (Paul and Connor, 2010). Therefore, NMDAR antagonists, especially NR2B-selective ones, play a critical role in the treatment of these diseases. Computational methods are a promising tool in the area of drug design and screening; they can speed up the process of screening potentially active therapeutics with a desired biological activity (Deuschl et al., 2006). Combining computational tools with proper in vitro experimental data is very important, and could help us understand better a drug’s mechanisms of action (Delgado et al., 2014). In previous studies, NMDAR expression was confirmed in the SHSY5Y cells and these cells were used to study the functional NMDARs in vitro (Akundi et al., 2003; Fang et al., 2014; Naarala et al., 2006; Petroni et al., 2013; Zhou et al., 2014). Compounds such as N-methylD-aspartate (NMDA) could activate NMDARs by allowing a high calcium influx (Wenthold et al., 2003), which is similar to pathologic events in cerebral ischemia. Therefore, the NMDA-induced SHSY5Y injury model was chosen to study the neuroprotective effects of Kukoamine B (KuB, chemical structure shown in Fig. 1A). Discovery Studio (DS), one of the commonly used docking software for structure-based screening of bioactive compounds and prediction of their underlying mechanisms of action (Sugunadevi et al., 2011), was used in our study to predict the potential relationship between KuB and NR2B-containing NMDARs. Cortex lycii radix, a traditional Chinese herb, has many pharmacological properties, including hypotensive, hypoglycemic, hypolipidemic, antipyretic and anti-microbial properties. KuB, a major
http://dx.doi.org/10.1016/j.neuint.2015.06.001 0197-0186/© 2015 Elsevier Ltd. All rights reserved.
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Fig. 1. Protective effects of KuB against NMDA-induced SH-SY5Y cells. (A) Chemical structure of KuB. (B) SH-SY5Y cells were treated with NMDA at concentrations of 1–8 mM. (C) Effects of KuB on cell viability in NMDA-treated SH-SY5Y cells. (D) Effects of KuB on LDH release in NMDA-treated SH-SY5Y cells. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
bioactive component of cortex lycii radicis, was found to exert neuroprotective effects for the first time. In this study, we investigated whether KuB could reduce the SHSY5Y cell injury caused by NMDA. The results showed that KuB could prevent the NMDA-induced SH-SY5Y injury. Results of molecular docking prompted us to investigate the potential intracellular pathways underlying KuB’s mechanisms of action. 2. Materials and methods 2.1. Materials Kukoamine B (>98%) was purchased from Chengdu Biopurify Phytochemicals Ltd. (China). Fluo-3/AM, ROS, Hoechst 33342 assay Kit and Membrane Protein Extraction Kit were purchased from Beyotime (Nanjing, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 0.25% trypsin–EDTA and dimethyl sulfoxide (DMSO) were purchased from Amresco (Solon, OH, USA). Apocynin (Santa Cruz, CA), Annexin V-PI apoptosis detection kit and lactate dehydrogenase (LDH) were purchased from Nanjing Key-Gen Biotech Co. Ltd. (Nanjing, China). Rabbit antibodies to ERK, p-ERK (p44/p42), CREB, p-CREB (Ser133), AKT, p-AKT, p38, p-p38, JNK, p-JNK and NR2B were purchased from Cell Signalling Technology (Inc. USA). Rabbit antibody to p47phox and mouse antibodies to β-actin, Bax, Bcl-2 and HRP-conjugated goat antirabbit (mouse) IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MK801 and Rhodamine 123 were purchased from Sigma-Aldrich (Missouri, USA). NMDA was purchased from Sigma (Sigma, USA). 2.2. Cell culture and drug treatments SH-SY5Y cells were routinely cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (Hyclone) in a humidified atmosphere of 5% CO2 at 37 °C, and the cells were seeded in different
kinds of the culture dish for 24 h. Then pretreated with different concentrations of KuB (final concentrations: 5, 10, 20 μΜ) for 2 h. After these treatments, the supernatant was discarded and the cells were incubated with ECS (in mM: NaCl 140, KCl 3, CaCl2 2, HEPES 10, glucose 10, adjusted to PH 7.2–7.3 with NaOH) with or without containing NMDA (final concentration: 2 mM) for 30 min. And then replaced the ECS with fresh DMEM for another 12 h or 2 h (westernblot for p-p38, p-ERK, p-JNK, p-AKT and p-CREB). After that, the cells or the supernatant liquid was used for next experiments. In all experiments, control group was also parallel administered in the same amount of ECS or DMEM. KuB was dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was less than 0.1% (v/v). All the data were obtained from three independent experiments. 2.3. MTT assay SH-SY5Y cells were seeded in 96-well culture plates at a density of 1 × 104 cells per well. At the end of indicated treatments, MTT (0.5 mg/ml) was added to the medium and incubated for another 4 h at 37 °C. After the removal of the culture medium, the insoluble dark blue formazan crystal was dissolved with 150 ml DMSO. The absorbance was measured at 490 nm with a microplate reader (ELX 800; Bio-TEK instruments, Inc.). Cell viability was expressed as a percentage with the control group as 100%. 2.4. LDH release assay The cytotoxicity of NMDA and the protective effect of KuB on SHSY5Y cells were further evaluated by LDH assay. SH-SY5Y cells were seeded in 96-well at a density of 1 × 104 cells per well. At the end of indicated treatments, the supernatant was used for measuring LDH activity according to the manufacturer protocols. The absorbance was detected by a microplate reader (ELX 800; Bio-TEK instruments, Inc.) using a wavelength of 450 nm.
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2.5. Hoechst 33342 staining SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well); the cells were treated as described earlier. After these treatments, the cells were washed with PBS buffer, and loaded with Hoechst 33342 dye (final concentration: 10 μg/ml) for 15 min in the dark (Tan et al., 2013). Then the cells were visualized under a fluorescence microscope (Olympus, Japan) and recorded images. 2.6. Measurement of intracellular ROS The intracellular reactive oxygen species (ROS) level was monitored by using the 2′, 7′-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe (Chen et al., 2012). SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well), after different treatments, cells were collected and incubated with DCFH-DA (final concentration: 10 mM) for 30 min at 37 °C, then washed with PBS buffer. The fluorescence intensity of DCF was analyzed with flow cytometry (FACScan, Becton Dickinson, USA) with excitation at 488 nm and emission at 530 nm. 2.7. Rhodamine 123 staining for mitochondria membrane potential (MMP) The mitochondria membrane potential was detected by fluorescent dye Rho123 (Sigma-Aldrich, St. Louis, MO, USA). SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well). After the described treatments, cells were incubated with Rho 123 (final concentration: 5 μΜ) at 37 °C for 30 min in the dark and then washed with PBS buffer three times. Each sample was detected by flow cytometry (FACScan, Becton Dickinson, USA) with excitation and emission wavelengths of 485 and 530 nm, respectively. 2.8. Flow cytometry (FCM) analysis of Annexin V and PI double staining Apoptotic rate was quantified by FITC-Annexin V/propidiumiodide (PI) apoptosis assay kit. SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well). At the end of indicated treatments, the cells were harvested and washed twice with cold PBS buffer, and suspended in binding buffer to adjust the density to 1 × 106 cells/ml. Finally, incubated with FITC-Annexin V (5 μl, final concentration: 6 μg/ml) and propidium iodide (5 μl, final concentration: 50 μg/ml) for 15 min at room temperature. The samples were examined with flow cytometry (FACScan, Becton Dickinson, USA). 2.9. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level assay The effects of KuB on SOD activity and MDA level were detected by commercial assay kit (Beyotime, Nanjing, China). SH-SY5Y cells were cultured in 6-well plates at a density of 2 × 105 cells/well. At the end of indicated treatments, the cells were harvested from the plates into ice-cold RIPA analysis buffer (1% NP40, 0.5% deoxycholate, 0.1% SDS). Then the protein concentrations were determined by using the BCA protein assay kit (Beyotime, Nanjing, China). The measurement of MDA and SOD were conducted according to the manufacturer’s protocol (Beyotime Biotechnology, Nanjing, China). The absorbance was detected by a microplate reader (ELX 800; Bio-TEK instruments, Inc.) using wavelengths of 532 nm and 450 nm, respectively. 2.10. Intracellular Ca2+ measurement The laser scanning confocal microscope (LSCM) was used to measure the intracellular calcium concentration. SH-SY5Y cells were
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cultured in three 3.5 mm plates (2 × 105 cells/plate) for 24 h. After that, KuB (20 μM) was added to one of the plates and the other two plates added equal DMEM culture fluid for 2 h. Then the supernatant was discarded and the cells were incubated with 2.5 μM fluo-3/AM (Beyotime, China) at 37 °C for 30 min in the dark. Then the cells were washed twice with ECS buffer and cultured with the original medium for another 30 min. After these treatments, the dyeloaded cells (green fluorescence) were measured with a laser scanning confocal microscope (Leica). For NMDA-treated and KuBpretreated cells, before exposing to NMDA the dye-loaded cells were scanned for 2 min to obtain a basal level of intracellular Ca2+. Then NMDA (final concentration: 2 mM) was applied to the cultures. And for the control group, an equal amount of ECS as the NMDA in model group was added. The images were recorded every 5 s with a laser scanning confocal microscope.
2.11. Western-blot analysis The potential mechanisms of KuB on NMDA-induced apoptosis were verified by western-blot assay. SH-SY5Y cells were cultured in 6-well plates (2 × 105/well, 2 wells/sample). At the end of indicated described, cells were collected from the wells to RIPA lysis buffer with a membrane protein extraction kit. Protein concentration was determined by BCA assay. The samples containing 50 μg of protein from each treatment condition were separated by SDS/ PAGE and transferred to PVDF membranes (Millipore Corporation). Total proteins were blocked with 5% non-fat milk and phosphorylation proteins were blocked with 5% BSA, which were dissolved in TBST for at least 1 h at room temperature. Finally, the membranes were washed three times and incubated with individual primary antibodies (Bax, Bcl-2, NR2B, CREB, p-CREB, p38, p-p38, p-JNK, JNK, p-AKT and AKT at 1:1000, and p-ERK, ERK at 1:800) shaking overnight at 4 °C. After three times of washing with TBST, the membranes were incubated with the anti-rabbit or anti-mouse IgG secondary antibody (1:12,000) in TBST for 1 h at room temperature, followed by three times of washing with TBST. After these treatments, the binding of antibody was detected with BeyoECL Plus. The results were expressed as the percentage of β-actin (% of control, which was deemed to be 100%).
2.12. Docking studies with Discovery Studio 3.0 (DS 3.0) In order to understand the molecular interaction between KuB and NMDARs, a molecular docking study was carried out with CDOCKER protocol of Discovery Studio 3.0. The X-ray crystal structures of NMDARs (PDB ID: 3QEL) (Karakas et al., 2011) were obtained from the Protein Data Bank (PDB) (http://www.wwpdb.org) and were used as the docking protein. First, ligands, water molecules, and original ligand, ifenprodil of NMDARs, were removed from the crystal structures of the receptor, and hydrogen atoms were added. Then, charges were loaded for protein using the method of CHARMm. A short minimization (100 steepest descent steps with tripos force field) was performed to release internal strain. Meanwhile, the compound KuB selected as the docking antagonism was also optimized with Dreiding-like forcefield using the Clean Geometry menu. The protein NMDARs was rigid, while the ligand KuB was flexible during the docking process. The Input Site Sphere was centered on the original ligand ifenprodil. Top hits, Random conformations and Orientations to refine parameters were all set. The conformation corresponding to the lowest CDOCKER Interaction Energy was selected as the most probable binding conformation. A 2D diagram of their interaction is manifested to confirm, and their docking pose was presented for the analysis of the interactions, including hydrogen bond, Van der Waals’ force, electrostatic interaction, and so on.
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2.13. Statistical analysis Data were represented as the mean ± S.E.M. Statistical significance was analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s HSD-post hoc test. Differences with P value less than 0.05 were considered statistically significant.
3. Results 3.1. Option of optimum concentrations of KuB and NMDA by MTT assay To choose appropriate concentrations of NDMA, SH-SY5Y cells were exposed to various concentrations of NMDA. As shown in Fig. 1B, NMDA (1–8 mM) gradually reduced the cell viability, and with 2 mM NMDA incubating for 30 min, the cell viability decreased to 48.37%. Hence, 2 mM NMDA was chosen for subsequent assays. However, pretreatment with KuB (5, 10, 20 μM) recovered the cell viability to 54.1%, 67.2% and 83.3%, respectively (Fig. 1C).
3.2. Effect of KuB on NMDA-induced LDH release LDH release is an indirect measure of dead cells, to further assess the protective effects provided by KuB, the leakage of LDH was measured. Pretreatment with KuB (5, 10, 20 μM) remarkably reduced the LDH activity (225.82 ± 8.36 U/l, 147.33 ± 3.18 U/l and 120.45 ± 6.29 U/l, respectively) compared with the NMDA-treated group (391.90 ± 6.29 U/l) (Fig. 1D). 3.3. Effect of KuB on NMDA-induced apoptosis Hoechst 33342 staining assay and Annexin V-PI double staining assay were used to quantitatively examine the effect of KuB on NMDA-induced SH-SY5Y cells apoptosis. As shown in Fig. 2, SHSY5Y cells showed apoptotic nuclei after treatment with NMDA (2 mM) for 30 min. However, pretreatment with KuB (5, 10, 20 μM) could reverse this tendency. Moreover, as shown in Fig. 3A, the lower left represents viable cells, the upper right represents late apoptotic and lower right represents early apoptotic. The flow cytometry can
automatically calculate the cell number of these areas. The apoptotic rate of the NMDA treatment alone group was increased from 6.58% to 21.48% compared with the control group. However, pretreatment with KuB (5, 10, 20 μM) decreased the apoptosis rate to 16.03%, 10.43% and 8.92% in a dose-dependent manner. These results suggested that KuB decreased NMDA-induced apoptosis in SH-SY5Y cells. 3.4. Effect of KuB on NMDA-induced MMP collapse Mitochondrial membrane potential (MMP) reduction leads to activating a series of apoptosis signaling pathways. In order to clarify the changes in mitochondrial function induced by NMDA or KuB treatment, MMP was detected using a flow cytometry after incubating with Rho 123 probe. As shown in Fig. 4A and B, the MMP was significantly decreased in NMDA-treated alone group compared with the control group. However, pretreatment with KuB could effectively increase the MMP in a dose-dependent manner. 3.5. Effects of apocynin on NMDA-induced p47phox translocation and ROS production It has been reported that NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation. Therefore, apocynin, an inhibitor of NADPH oxidase, was used to further study the NMDAR antagonism of KuB. As shown in Fig. 5A, after exposing to NMDA (2 mM) for 30 min, ROS production (213 ± 7.55%) was 2.04-fold increment compared with control group (100 ± 3.46%). However, apocynin (500 μM) could almost entirely suppress the ROS production. And apocynin can restrain p47phox translocation, which induced by NMDA, from the cytoplasm to the membrane (Fig. 5B). These results indicated that NADPH oxidase induced ROS generation is the primary source in SH-SY5Y cells. 3.6. Effects of KuB and MK801 on NMDA-induced ROS production, MDA level and SOD activity As shown in Fig. 5C, after exposing of NMDA (2 mM) for 30 min, ROS production (213 ± 7.55%) was 2.04-fold increment compared with control group (100 ± 3.46%). However, MK801 (20 μM) and KuB
Fig. 2. Effects of KuB on NMDA-induced nuclear condensation in SH-SY5Y cells. Cells were pretreated with KuB (5, 10, 20 μΜ) for 2 h and then exposed to NMDA (2 mM) for 30 min and continued to cultured for another 12 h with fresh medium. Representative fluorescence images were obtained after Hoechst 33342 staining in SH-SY5Y cells (200×).
Please cite this article in press as: Xiao-Long Hu, et al., Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.001
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Fig. 3. Effects of KuB on NMDA-induced apoptosis in SH-SY5Y cells. (A) Effects of KuB on NMDA-induced early and late apoptosis and representative images were obtained after AV-PI double staining from FCM. (B) The quantitative analysis of three independent experiments. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
(5, 10, 20 μM) obviously decreased NMDA-induced ROS generation to 143 ± 4.97%, 147 ± 2.72%, 118 ± 1.76% and 108 ± 3.28%, respectively. Especially, there was a significant difference (p < 0.05) at the same concentration (20 μM) between MK801 and KuB. In addition, MDA, a marker of oxidant-mediated lipid peroxidation in cells, was measured after exposure to NMDA (2 mM) for 30 min. Treatment with NMDA could result in significant increase in the MDA level (4.54 ± 0.13 μmol/g) compared with control group (2.85 ± 0.08 μmol/g). However, pretreatment with MK801 (20 μM) and KuB (5, 10, 20 μM) could obviously attenuate NMDA-induced increasing of MDA level to 3.95 ± 0.07 μmol/g, 3.87 ± 0.06 μmol/g, 3.27 ± 0.10 μmol/g and 3.01 ± 0.06 μmol/g, respectively (Fig. 5D). Finally, the SOD, an important antioxidase in cells, was measured after the same condition idem. NMDA could obviously lower SOD activity to 0.52 ± 0.02 units compare with the control group (0.85 ± 0.05 units). However, pretreatment with MK801 (20 μM) and KuB (5, 10, 20 μM) could effectively enhance the SOD activity to 0.71 ± 0.01, 0.55 ± 0.02, 0.85 ± 0.02 and 1.32 ± 0.06, respectively (Fig. 5E). Especially, there was a significant difference (p < 0.01) at the same concentration (20 μM) between MK801 and KuB (Fig. 5C–E).
3.7. Effect of KuB on the expression of Bcl-2 and Bax in the cytoplasm of SH-SY5Y cells The ratio of Bcl-2/Bax is often used to measure the state of mitochondria. As shown in Fig. 6A and B, the expression level of Bcl-2 protein stimulated with NMDA alone was attenuated obviously, while the expression of Bax was increased compared to control group in SH-SY5Y cells. However, pretreatment with KuB (5, 10, 20 μM) could dramatically reverse this tendency and naturally result in the ratio of Bcl-2/Bax increasing compared with control cells (p < 0.05 or 0.01). These results suggested that KuB provided neuroprotection by increasing the Bcl-2/Bax ratio. 3.8. Effect of KuB on NMDA-induced Ca2+ influx in SH-SY5Y cells A previous study has shown that NMDA could induce Ca2+ overloaded in SH-SY5Y cells. In our study, compared with the control group, the fluorescence intensity of Fluo-3/AM increased markedly at about 3 min after incubation with NMDA, while pretreatment with KuB (20 μM) obviously attenuated the increased fluorescence
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Fig. 4. Effects of KuB on MMP in SH-SY5Y cells. (A) The representative pictures were obtained from FCM after incubation with Rho 123 dye. (B) The quantitative analysis of three independent experiments. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
intensity that induced by NMDA (Fig. 7A–C). The results indicated that KuB might via inhibiting the extracellular calcium influx to provide neuroprotection. 3.9. Binding affinity study through docking with NR2B subunit of NMDARs Molecular docking of KuB into the active site cavity was employed to gain better understanding of the potential relationship between KuB and NR2B-NMDARs. As shown in Fig. 8A, the threedimensional panoramic view, KuB could present commendably binding mode within the active pocket (Fig. 8B). Analysis of docking results highlighted a peculiar ligand–receptor interaction motif represented by a hydrogen bonding network involving the residue: Glu235, Glu236, Try175, Arg115, Glu106, Ala107, Gln110, Thr110, Pro106, Ile111, Phe114 and Pro78. As shown in Fig. 7B, KuB has a well combination with active pocket of NR2B-NMDARs. The binding energies of ifenprodil and KuB are −10.784 kcal/mol and −17.343 kcal/mol, respectively.
3.10. Effects of NMDA and KuB on the expression of NR2B, p-ERK1/2 and p-CREB In order to further make clear if KuB influence the downstreams of NMDARs, the expression of NR2B, p-ERK1/2 and p-CREB were investigated by western-blot analysis. NMDA-induced NR2B expression was detected at 0 h, 3 h, 6 h, 9 h and 12 h, and the results showed that NMDA could increase the NR2B expression in a time-dependent manner, especially at 12 h (Fig. 9A). The NR2B expression of NMDAtreated cells was significantly increased to 4.10 ± 0.11 compared with control group (relative value = 1). However, pretreatment with different concentrations of KuB (5, 10, 20 μM) could downregulate the NR2B expression to 2.86 ± 0.14, 2.13 ± 0.20 and 1.30 ± 0.06, respectively (Fig. 9B). Meanwhile, the expression ratio of p-ERK/t-ERK in NMDA-treated cells was dramatically downregulated to 0.25 ± 0.03 compared with the control cells (relative value = 1). Nevertheless, pretreatment with KuB (5, 10, 20 μM) could upregulate p-ERK/tERK expression ratio to 0.26 ± 0.03, 0.6 ± 0.06 and 0.89 ± 0.03, respectively (Fig. 9C). Finally, p-CREB/t-CREB expression ratio was
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Fig. 5. Effects of Apocynin on the p47phox translocation as well as ROS generation and effects of KuB and MK801 on ROS generation, MDA level and SOD activity. (A) Apocynin can decrease the NMDA-induced ROS generation and the intracellular ROS production was analyzed by FCM. (B) Effects of Apocynin on the p47phox translocation by Western-blot analysis. (C) Effects of KuB on the intracellular ROS production was analyzed by FCM. (D) Effect of KuB on the SOD activity. (E) Effects of KuB on the level of MDA. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
markedly decreased to 0.28 ± 0.04 by NMDA stimulation compared with control group (relative value = 1). However, pretreatment with KuB (5, 10, 20 μM) could effectively increase the expression ratio of p-CREB/t-CREB to 0.43 ± 0.03, 0.75 ± 0.03 and 0.91 ± 0.05, respectively (Fig. 9D). These results suggested that KuB could modulate the NR2B-ERK-CREB pathway. 3.11. Effects of KuB on the expression of SAPKs (p-p38, p-JNK) and p-AKT Other signal pathways of NMDAR downstreams, including SAPKs and PI3K-AKT that related to NR2B subunit, were detected by
western-blot assay. The expression ratio of p-AKT/t-AKT of NMDA-treated cells was significantly decreased to 0.27 ± 0.04 compared with control group (relative value = 1). However, pretreatment with KuB (5, 10, 20 μM) could increase the ratio to 0.44 ± 0.55, 0.62 ± 0.05 and 0.77 ± 0.04, respectively (Fig. 10A). At the same time, the expression ratio of p-JNK/t-JNK was dramatically upregulated in NMDAtreated cells (2.97 ± 0.15) compared with control cells (relative value = 1). However, pretreatment with KuB (5, 10, 20 μM) could downregulate this ratio to 2.73 ± 0.09, 1.76 ± 0.09 and 1.20 ± 0.06, respectively (Fig. 10B). Finally, the p-p38/t-p38 expression ratio was markedly increased to 2.53 ± 0.09 by NMDA compared with control group (relative value = 1). Whereas, pretreatment with KuB (5, 10, 20 μM) could effectively de-
Fig. 6. The expression level of Bcl-2 and Bax. (A) Protein expressions of Bcl-2 and Bax using β-actin as the loading control. (B) The quantitative analysis of Bcl-2/Bax ratio. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
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Fig. 7. Calcium imaging of SH-SY5Y cells. (A) The green fluorescence under laser scanning microscope at different times showed the concentration of calcium in SH-SY5Y cells. (B) The fluorescence intensity showing the Ca2+ concentration was stable in control group during detection time (n = 15 cells). (C) NMDA (2 mM) is able to evoke strong fluorescence intensity (n = 15 cells) and pretreatment with KuB (20 μM) significantly reduced fluorescence intensity in SH-SY5Y cells (n = 15 cells).
crease this expression ratio to 2.23 ± 0.09, 1.86 ± 0.12 and 0.93 ± 0.03, respectively (Fig. 10C). These results suggested that KuB could modulate the phosphorylation of SAPKs and AKT. 4. Discussion This study revealed that NMDA resulted in cell death and apoptosis in SH-SY5Y cells by increasing intracellular Ca2+ concentration
as well as aggravating oxidative stress. However, KuB treatment protected the cells against NMDA-induced injury. These protective effects of KuB were possibly associated with its actions as an NMDAR antagonist and an anti-oxidative agent. NMDAR-mediated neuro-excitotoxicity leads to neuronal apoptosis and death (Aamodt and Constantine-Paton, 1998; Bliss and Collingridge, 1993). Apoptotic cells are characterized by morphological changes, including cell shrinkage and DNA condensation,
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Fig. 8. Computational molecular docking of KuB with NMDARs. (A) A close-up view of the low energy pose of KuB in the NR2B subunit of NMDARs generated by molecular docking. (B) The space matching of KuB and NR2B subunit. The purple domain represents the H-bond donor and the green domain represents the H-bond acceptor.
which further lead to degradation of chromosomal DNA, and cell death (Enari et al., 1998). Our data revealed that KuB could prevent cell death, LDH release and reduce apoptosis induced by NMDA. Cell morphological studies further demonstrated the protective effects of KuB (Figs. 1–3). These data have proven that KuB had a strong anti-apoptotic effect against NMDA. Our results elucidate that KuB exerts neuroprotective effects in vitro. In order to further explore the mechanisms of action of KuB as a potential NR2B-selective antagonist, we mainly focused on the
following questions: (1) Can KuB decrease the NADPH oxidasemediated ROS generation and Ca2+ overload induced by NMDA? (2) Does KuB bind to NR2B-containing NMDARs in silico? If so, (3) Does KuB participate in activating the NR2B subunit of NMDARs and its down-stream molecules involved in the NR2B–ERK–CREB, NR2B–PI3K–AKT, or NR2B–p38/SAPKs pathways? First, how are ROS generated and cause neuronal death? Evidence has pointed to the fact that massive accumulation of glutamate induces oxidative stress. ROS is one of the crucial etiological factors
Fig. 9. Western-blot analysis of NR2B, p-ERK and p-CREB. Special emphasis is that P-ERK and p-CREB were cultured for only 2 h after NMDA treatment (Section 2.2). (A) Time-course study of the expression of NR2B induced by NMDA. (B) The expressions of NR2B (β-actin as the loading control). (C) The expression of p-ERK/t-ERK (t-ERK as the loading control). (D) The expression of p-CREB/t-CREB (t-CREB as the loading control). Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
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Fig. 10. Western-blot analysis of SAPKs (JNK and p38) and p-AKT. Special emphasis is that P-JNK, p-p38 and p-AKT were cultured for only 2 h after NMDA treatment (Section 2.2). (A) The expressions of p-AKT/t-AKT (using t-AKT as the loading control). (B) The expression of p-JNK/t-JNK (using t-JNK as the loading control). (D) The expression of p-p38/t-p38 (using t-p38 as the loading control). Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
of oxidative stress, which results in MMP collapse, and induces activation of the apoptotic cascades (Halestrap and Brenner, 2003). NMDARs could activate the NADPH oxidase, which is the primary source of superoxide induced by NMDAR activation (Brennan et al., 2009). Membrane translocation of p47phox is necessary for activating the NADPH oxidase. Therefore, we used acopynin, an inhibitor of the NADPH oxidase, to detect the NMDA-induced membrane expression of p47phox in the SH-SY5Y cells. Our results showed that acopynin inhibited the translocation of p47phox induced by NMDA and also decreased ROS production (Fig. 5A and B). It also found that KuB could decrease ROS production induced by NMDA signaling (Fig. 5C). Therefore, we can conclude that NMDA could activate the NMDARs in the SH-SY5Y cells and further activate the NADPH oxidase. In addition, high levels of ROS in turn initiate lipid peroxidation, which results in the accumulation of reactive aldehyde products, such as MDA (Adibhatla and Hatcher, 2006). In contrast to MDA, activity of the antioxidant enzyme, SOD, is decreased in these cells when subjected to ROS (Nita et al., 2001). To our delight, our results showed that KuB could decrease the MDA levels and increase SOD activity (Fig. 5D and E). Taken together, these results showed that NMDA could activate NMDARs in the SHSY5Y cells and KuB could alleviate the NADPH oxidase-mediated ROS production, that is, NMDAR-mediated ROS production. NMDARs are the primary receptors activating excitotoxicity because of increasing Ca2+ permeability to cells. Increase of intracellular Ca2+ levels occurs as a response to NMDAR overactivation which could cause Ca2+ entry into the mitochondria. The Ca2+overloaded mitochondria could cause MMP loss and inhibit ATP production. In addition, MMP is regulated by the Bcl-2 protein family and the expression ratio of Bcl-2/Bax is regarded as an indicator of mitochondrial function and considered to be crucial for determining cell apoptosis (Miquel and Serge, 2003). Consisting with previous studies, our data showed that KuB could efficaciously suppress Ca2+ influx (Fig. 7) and increase the Bcl-2/Bax ratio (Fig. 6). These findings demonstrate that KuB may suppress the NMDARs and restrain the Ca2+ influx, which can attenuate apoptosis down the stream. Second, we used a refined docking experiment using the Discovery Studio 3.0 (DS 3.0) to further predict the potential relationship between KuB and NR2B-containing NMDARs. Ifenprodil, an NMDAR ligand, has been reported to dock in a domain composed of four amino acids, including leucine, isoleucine, and valine-binding protein (LIVBP) subunits. It has been demonstrated that the LIVBP domain is an active pocket for binding of the NR2B antagonists (Malherbe
et al., 2003; Marinelli et al., 2007). Phe 114, Gln 110, Ile 111, and Ile 82 were confirmed to be the key residues in the NR2B subunit (Karakas et al., 2011). Importantly, we found that KuB could form hydrogen bonds with the residues Phe 114, Gln 110, and Ile 111 (Fig. 8A) and form a good match within the active pocket (Fig. 8B). These results indicate that KuB was likely to be a potential NR2Bselective antagonist. Finally, some important pathways related to NR2B-containing NMDARs, including the NR2B–ERK–CREB pathway, the PI3K–AKT pathway and the SAPKs pathway, were detected by western-blot analysis. It has been reported that ERK, a key signaling protein involved in NMDAR-dependent synaptic excitotoxicity, was downregulated once the NR2B-containing NMDARs activated and subsequently inactivated the p-CREB (Banko et al., 2004; Hardingham et al., 2001; Kaphzan et al., 2006; Paul et al., 2007). Our results show that over-expression of the NR2B subunit induced by NMDA decreased p-ERK and p-CREB expression. However, pretreatment with KuB effectively reversed this tendency in a dose-dependent manner (Fig. 9). The PI3K-AKT is a pathway downstream of NMDARs. Activated AKT promotes cell survival by phosphorylation, while exposure of the SH-SY5Y cells to NMDA results in deactivation of the AKT kinase (Corasaniti et al., 2007; Meijer et al., 2003). In contrast to AKT, the SAPKs (p38 and JNK) are generally implicated in cell death in ischemic stroke (Anderson and Tolkovsky, 1999; Borsello and Forloni, 2007). It has been demonstrated that JNK and p38 play a key role in oxidative stress (Lasa et al., 2002; Zhao et al., 2013). Our study presented that the phosphorylation of JNK and p38 after NMDA exposure was suppressed by pretreatment with KuB, whereas the expression of AKT was increased (Fig. 10).
5. Conclusion This study verified that KuB can have a potent neuroprotective activity against NMDA-induced apoptosis in the SH-SY5Y cells. KuB could also suppress the NADPH oxidase-mediated ROS production. Molecular docking results predicted that KuB can potentially interact with the NR2B–NMDAR active sites. In addition, KuB could modulate the expression of the NR2B subunit and its downstream molecules, including the NR2B–ERK–CREB, the NR2B–SAPKs and the NR2B–PI3K–AKT pathways. Consequently, KuB may become a potential NMDAR antagonist. However, in vivo effects of KuB and its NR2B selectivity should be confirmed in future studies.
Please cite this article in press as: Xiao-Long Hu, et al., Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.001
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Conflict of interest There is no conflict of interest.
Acknowledgements This work was supported by the National Science and Technology Major Project, China. (Project number: 2014ZX09J14101-05C).
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.neuint.2015.06.001.
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Contents lists available at ScienceDirect
Neurochemistry International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n c i
Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro Xiao-Long Hu a,b, Li-Ping Guo b, Qi Song b, Qiao Zhang b, Ying Chen a, Jian Wang b, Wei-Hong Meng a, Qing-Chun Zhao a,* a b
Department of Pharmacy, General Hospital of Shenyang Military Area Command, Shenyang 110840, China Department of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E
I N F O
Article history: Received 28 December 2014 Received in revised form 19 May 2015 Accepted 1 June 2015 Available online Keywords: Kukoamine B NMDA receptors Oxidative stress NADPH oxidase SH-SY5Y cells Molecular docking
A B S T R A C T
A major cause of cerebral ischemia is overactivation of the N-methyl-D-aspartate receptors (NMDARs). Therefore, NMDAR antagonists are needed for the treatment of cerebral ischemia. In our research, KuB protected the SH-SY5Y cells against NMDA-induced injury, apoptosis, LDH release and MMP loss. In addition, KuB could decrease MDA levels while increasing SOD activity. Meanwhile, KuB decreased NADPH oxidase-mediated ROS production, inhibited Ca2+ influx, and increased the Bcl-2/Bax ratio. Furthermore, KuB not only down-regulated expression of the NR2B subunit of NMDAR but also actively modulated expression of the signaling molecules downstream of NR2B, including p-ERK, p-CREB, p-AKT and SAPKs. Finally, docking results showed that KuB had a high affinity for NR2B-containing NMDARs. Therefore, we conclude that KuB protected the SH-SY5Y cells from NMDA-induced injury likely by antagonizing NMDARs and reducing oxidative stress. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Cerebral ischemia is the third leading cause of death around the world. Furthermore, neurodegenerative disorders such as Alzheimer’s, Huntington’s and Parkinson’s diseases often affect the elderly and devastatingly affect their quality of life. An important question is still unanswered – what is the main factor causing these diseases? Collective evidence shows that N-methyl-D-aspartate receptors (NMDARs), a major glutamate receptor highly expressed in the central nervous system (CNS), play a key role in these diseases (Barkus et al., 2010). Abnormal expression of NMDARs can cause some of the diseases states earlier and also influences learning and memory (Barkus et al., 2010). Therefore, finding effective agents as new NMDAR antagonists is impending. Accumulated evidence demonstrates that excessive stimulation of NMDARs causes a high intracellular Ca 2+ influx. The overloaded Ca2+, which is the main factor in ischemic stroke and neurodegenerative disorders, results in neuron injury following a series of apoptotic events (Hardingham and Bading, 2003; Kruman and Mattson, 1999). NMDARs are heteromeric complexes, composed of two obligatory NR1 subunits and two NR2A-D subunits
* Corresponding author. Department of Pharmacy, General Hospital of Shenyang Military Area Command, 83 Wenhua Road, Shenyang 110016, China. Tel.: +86 24 28856205; fax: +86 24 28856205. E-mail address: [email protected] (Q.-C. Zhao).
(Nakanishi, 1992). Importantly, NR2B-containing NMDARs are the most important of all the subunits when it comes to excessive Ca2+ influx and dysfunctions mentioned earlier (Paul and Connor, 2010). Therefore, NMDAR antagonists, especially NR2B-selective ones, play a critical role in the treatment of these diseases. Computational methods are a promising tool in the area of drug design and screening; they can speed up the process of screening potentially active therapeutics with a desired biological activity (Deuschl et al., 2006). Combining computational tools with proper in vitro experimental data is very important, and could help us understand better a drug’s mechanisms of action (Delgado et al., 2014). In previous studies, NMDAR expression was confirmed in the SHSY5Y cells and these cells were used to study the functional NMDARs in vitro (Akundi et al., 2003; Fang et al., 2014; Naarala et al., 2006; Petroni et al., 2013; Zhou et al., 2014). Compounds such as N-methylD-aspartate (NMDA) could activate NMDARs by allowing a high calcium influx (Wenthold et al., 2003), which is similar to pathologic events in cerebral ischemia. Therefore, the NMDA-induced SHSY5Y injury model was chosen to study the neuroprotective effects of Kukoamine B (KuB, chemical structure shown in Fig. 1A). Discovery Studio (DS), one of the commonly used docking software for structure-based screening of bioactive compounds and prediction of their underlying mechanisms of action (Sugunadevi et al., 2011), was used in our study to predict the potential relationship between KuB and NR2B-containing NMDARs. Cortex lycii radix, a traditional Chinese herb, has many pharmacological properties, including hypotensive, hypoglycemic, hypolipidemic, antipyretic and anti-microbial properties. KuB, a major
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Fig. 1. Protective effects of KuB against NMDA-induced SH-SY5Y cells. (A) Chemical structure of KuB. (B) SH-SY5Y cells were treated with NMDA at concentrations of 1–8 mM. (C) Effects of KuB on cell viability in NMDA-treated SH-SY5Y cells. (D) Effects of KuB on LDH release in NMDA-treated SH-SY5Y cells. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
bioactive component of cortex lycii radicis, was found to exert neuroprotective effects for the first time. In this study, we investigated whether KuB could reduce the SHSY5Y cell injury caused by NMDA. The results showed that KuB could prevent the NMDA-induced SH-SY5Y injury. Results of molecular docking prompted us to investigate the potential intracellular pathways underlying KuB’s mechanisms of action. 2. Materials and methods 2.1. Materials Kukoamine B (>98%) was purchased from Chengdu Biopurify Phytochemicals Ltd. (China). Fluo-3/AM, ROS, Hoechst 33342 assay Kit and Membrane Protein Extraction Kit were purchased from Beyotime (Nanjing, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 0.25% trypsin–EDTA and dimethyl sulfoxide (DMSO) were purchased from Amresco (Solon, OH, USA). Apocynin (Santa Cruz, CA), Annexin V-PI apoptosis detection kit and lactate dehydrogenase (LDH) were purchased from Nanjing Key-Gen Biotech Co. Ltd. (Nanjing, China). Rabbit antibodies to ERK, p-ERK (p44/p42), CREB, p-CREB (Ser133), AKT, p-AKT, p38, p-p38, JNK, p-JNK and NR2B were purchased from Cell Signalling Technology (Inc. USA). Rabbit antibody to p47phox and mouse antibodies to β-actin, Bax, Bcl-2 and HRP-conjugated goat antirabbit (mouse) IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MK801 and Rhodamine 123 were purchased from Sigma-Aldrich (Missouri, USA). NMDA was purchased from Sigma (Sigma, USA). 2.2. Cell culture and drug treatments SH-SY5Y cells were routinely cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (Hyclone) in a humidified atmosphere of 5% CO2 at 37 °C, and the cells were seeded in different
kinds of the culture dish for 24 h. Then pretreated with different concentrations of KuB (final concentrations: 5, 10, 20 μΜ) for 2 h. After these treatments, the supernatant was discarded and the cells were incubated with ECS (in mM: NaCl 140, KCl 3, CaCl2 2, HEPES 10, glucose 10, adjusted to PH 7.2–7.3 with NaOH) with or without containing NMDA (final concentration: 2 mM) for 30 min. And then replaced the ECS with fresh DMEM for another 12 h or 2 h (westernblot for p-p38, p-ERK, p-JNK, p-AKT and p-CREB). After that, the cells or the supernatant liquid was used for next experiments. In all experiments, control group was also parallel administered in the same amount of ECS or DMEM. KuB was dissolved in dimethylsulphoxide (DMSO). The final concentration of DMSO was less than 0.1% (v/v). All the data were obtained from three independent experiments. 2.3. MTT assay SH-SY5Y cells were seeded in 96-well culture plates at a density of 1 × 104 cells per well. At the end of indicated treatments, MTT (0.5 mg/ml) was added to the medium and incubated for another 4 h at 37 °C. After the removal of the culture medium, the insoluble dark blue formazan crystal was dissolved with 150 ml DMSO. The absorbance was measured at 490 nm with a microplate reader (ELX 800; Bio-TEK instruments, Inc.). Cell viability was expressed as a percentage with the control group as 100%. 2.4. LDH release assay The cytotoxicity of NMDA and the protective effect of KuB on SHSY5Y cells were further evaluated by LDH assay. SH-SY5Y cells were seeded in 96-well at a density of 1 × 104 cells per well. At the end of indicated treatments, the supernatant was used for measuring LDH activity according to the manufacturer protocols. The absorbance was detected by a microplate reader (ELX 800; Bio-TEK instruments, Inc.) using a wavelength of 450 nm.
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2.5. Hoechst 33342 staining SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well); the cells were treated as described earlier. After these treatments, the cells were washed with PBS buffer, and loaded with Hoechst 33342 dye (final concentration: 10 μg/ml) for 15 min in the dark (Tan et al., 2013). Then the cells were visualized under a fluorescence microscope (Olympus, Japan) and recorded images. 2.6. Measurement of intracellular ROS The intracellular reactive oxygen species (ROS) level was monitored by using the 2′, 7′-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe (Chen et al., 2012). SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well), after different treatments, cells were collected and incubated with DCFH-DA (final concentration: 10 mM) for 30 min at 37 °C, then washed with PBS buffer. The fluorescence intensity of DCF was analyzed with flow cytometry (FACScan, Becton Dickinson, USA) with excitation at 488 nm and emission at 530 nm. 2.7. Rhodamine 123 staining for mitochondria membrane potential (MMP) The mitochondria membrane potential was detected by fluorescent dye Rho123 (Sigma-Aldrich, St. Louis, MO, USA). SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well). After the described treatments, cells were incubated with Rho 123 (final concentration: 5 μΜ) at 37 °C for 30 min in the dark and then washed with PBS buffer three times. Each sample was detected by flow cytometry (FACScan, Becton Dickinson, USA) with excitation and emission wavelengths of 485 and 530 nm, respectively. 2.8. Flow cytometry (FCM) analysis of Annexin V and PI double staining Apoptotic rate was quantified by FITC-Annexin V/propidiumiodide (PI) apoptosis assay kit. SH-SY5Y cells were seeded into 6-well plates (2 × 105 cells/well). At the end of indicated treatments, the cells were harvested and washed twice with cold PBS buffer, and suspended in binding buffer to adjust the density to 1 × 106 cells/ml. Finally, incubated with FITC-Annexin V (5 μl, final concentration: 6 μg/ml) and propidium iodide (5 μl, final concentration: 50 μg/ml) for 15 min at room temperature. The samples were examined with flow cytometry (FACScan, Becton Dickinson, USA). 2.9. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level assay The effects of KuB on SOD activity and MDA level were detected by commercial assay kit (Beyotime, Nanjing, China). SH-SY5Y cells were cultured in 6-well plates at a density of 2 × 105 cells/well. At the end of indicated treatments, the cells were harvested from the plates into ice-cold RIPA analysis buffer (1% NP40, 0.5% deoxycholate, 0.1% SDS). Then the protein concentrations were determined by using the BCA protein assay kit (Beyotime, Nanjing, China). The measurement of MDA and SOD were conducted according to the manufacturer’s protocol (Beyotime Biotechnology, Nanjing, China). The absorbance was detected by a microplate reader (ELX 800; Bio-TEK instruments, Inc.) using wavelengths of 532 nm and 450 nm, respectively. 2.10. Intracellular Ca2+ measurement The laser scanning confocal microscope (LSCM) was used to measure the intracellular calcium concentration. SH-SY5Y cells were
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cultured in three 3.5 mm plates (2 × 105 cells/plate) for 24 h. After that, KuB (20 μM) was added to one of the plates and the other two plates added equal DMEM culture fluid for 2 h. Then the supernatant was discarded and the cells were incubated with 2.5 μM fluo-3/AM (Beyotime, China) at 37 °C for 30 min in the dark. Then the cells were washed twice with ECS buffer and cultured with the original medium for another 30 min. After these treatments, the dyeloaded cells (green fluorescence) were measured with a laser scanning confocal microscope (Leica). For NMDA-treated and KuBpretreated cells, before exposing to NMDA the dye-loaded cells were scanned for 2 min to obtain a basal level of intracellular Ca2+. Then NMDA (final concentration: 2 mM) was applied to the cultures. And for the control group, an equal amount of ECS as the NMDA in model group was added. The images were recorded every 5 s with a laser scanning confocal microscope.
2.11. Western-blot analysis The potential mechanisms of KuB on NMDA-induced apoptosis were verified by western-blot assay. SH-SY5Y cells were cultured in 6-well plates (2 × 105/well, 2 wells/sample). At the end of indicated described, cells were collected from the wells to RIPA lysis buffer with a membrane protein extraction kit. Protein concentration was determined by BCA assay. The samples containing 50 μg of protein from each treatment condition were separated by SDS/ PAGE and transferred to PVDF membranes (Millipore Corporation). Total proteins were blocked with 5% non-fat milk and phosphorylation proteins were blocked with 5% BSA, which were dissolved in TBST for at least 1 h at room temperature. Finally, the membranes were washed three times and incubated with individual primary antibodies (Bax, Bcl-2, NR2B, CREB, p-CREB, p38, p-p38, p-JNK, JNK, p-AKT and AKT at 1:1000, and p-ERK, ERK at 1:800) shaking overnight at 4 °C. After three times of washing with TBST, the membranes were incubated with the anti-rabbit or anti-mouse IgG secondary antibody (1:12,000) in TBST for 1 h at room temperature, followed by three times of washing with TBST. After these treatments, the binding of antibody was detected with BeyoECL Plus. The results were expressed as the percentage of β-actin (% of control, which was deemed to be 100%).
2.12. Docking studies with Discovery Studio 3.0 (DS 3.0) In order to understand the molecular interaction between KuB and NMDARs, a molecular docking study was carried out with CDOCKER protocol of Discovery Studio 3.0. The X-ray crystal structures of NMDARs (PDB ID: 3QEL) (Karakas et al., 2011) were obtained from the Protein Data Bank (PDB) (http://www.wwpdb.org) and were used as the docking protein. First, ligands, water molecules, and original ligand, ifenprodil of NMDARs, were removed from the crystal structures of the receptor, and hydrogen atoms were added. Then, charges were loaded for protein using the method of CHARMm. A short minimization (100 steepest descent steps with tripos force field) was performed to release internal strain. Meanwhile, the compound KuB selected as the docking antagonism was also optimized with Dreiding-like forcefield using the Clean Geometry menu. The protein NMDARs was rigid, while the ligand KuB was flexible during the docking process. The Input Site Sphere was centered on the original ligand ifenprodil. Top hits, Random conformations and Orientations to refine parameters were all set. The conformation corresponding to the lowest CDOCKER Interaction Energy was selected as the most probable binding conformation. A 2D diagram of their interaction is manifested to confirm, and their docking pose was presented for the analysis of the interactions, including hydrogen bond, Van der Waals’ force, electrostatic interaction, and so on.
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2.13. Statistical analysis Data were represented as the mean ± S.E.M. Statistical significance was analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s HSD-post hoc test. Differences with P value less than 0.05 were considered statistically significant.
3. Results 3.1. Option of optimum concentrations of KuB and NMDA by MTT assay To choose appropriate concentrations of NDMA, SH-SY5Y cells were exposed to various concentrations of NMDA. As shown in Fig. 1B, NMDA (1–8 mM) gradually reduced the cell viability, and with 2 mM NMDA incubating for 30 min, the cell viability decreased to 48.37%. Hence, 2 mM NMDA was chosen for subsequent assays. However, pretreatment with KuB (5, 10, 20 μM) recovered the cell viability to 54.1%, 67.2% and 83.3%, respectively (Fig. 1C).
3.2. Effect of KuB on NMDA-induced LDH release LDH release is an indirect measure of dead cells, to further assess the protective effects provided by KuB, the leakage of LDH was measured. Pretreatment with KuB (5, 10, 20 μM) remarkably reduced the LDH activity (225.82 ± 8.36 U/l, 147.33 ± 3.18 U/l and 120.45 ± 6.29 U/l, respectively) compared with the NMDA-treated group (391.90 ± 6.29 U/l) (Fig. 1D). 3.3. Effect of KuB on NMDA-induced apoptosis Hoechst 33342 staining assay and Annexin V-PI double staining assay were used to quantitatively examine the effect of KuB on NMDA-induced SH-SY5Y cells apoptosis. As shown in Fig. 2, SHSY5Y cells showed apoptotic nuclei after treatment with NMDA (2 mM) for 30 min. However, pretreatment with KuB (5, 10, 20 μM) could reverse this tendency. Moreover, as shown in Fig. 3A, the lower left represents viable cells, the upper right represents late apoptotic and lower right represents early apoptotic. The flow cytometry can
automatically calculate the cell number of these areas. The apoptotic rate of the NMDA treatment alone group was increased from 6.58% to 21.48% compared with the control group. However, pretreatment with KuB (5, 10, 20 μM) decreased the apoptosis rate to 16.03%, 10.43% and 8.92% in a dose-dependent manner. These results suggested that KuB decreased NMDA-induced apoptosis in SH-SY5Y cells. 3.4. Effect of KuB on NMDA-induced MMP collapse Mitochondrial membrane potential (MMP) reduction leads to activating a series of apoptosis signaling pathways. In order to clarify the changes in mitochondrial function induced by NMDA or KuB treatment, MMP was detected using a flow cytometry after incubating with Rho 123 probe. As shown in Fig. 4A and B, the MMP was significantly decreased in NMDA-treated alone group compared with the control group. However, pretreatment with KuB could effectively increase the MMP in a dose-dependent manner. 3.5. Effects of apocynin on NMDA-induced p47phox translocation and ROS production It has been reported that NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation. Therefore, apocynin, an inhibitor of NADPH oxidase, was used to further study the NMDAR antagonism of KuB. As shown in Fig. 5A, after exposing to NMDA (2 mM) for 30 min, ROS production (213 ± 7.55%) was 2.04-fold increment compared with control group (100 ± 3.46%). However, apocynin (500 μM) could almost entirely suppress the ROS production. And apocynin can restrain p47phox translocation, which induced by NMDA, from the cytoplasm to the membrane (Fig. 5B). These results indicated that NADPH oxidase induced ROS generation is the primary source in SH-SY5Y cells. 3.6. Effects of KuB and MK801 on NMDA-induced ROS production, MDA level and SOD activity As shown in Fig. 5C, after exposing of NMDA (2 mM) for 30 min, ROS production (213 ± 7.55%) was 2.04-fold increment compared with control group (100 ± 3.46%). However, MK801 (20 μM) and KuB
Fig. 2. Effects of KuB on NMDA-induced nuclear condensation in SH-SY5Y cells. Cells were pretreated with KuB (5, 10, 20 μΜ) for 2 h and then exposed to NMDA (2 mM) for 30 min and continued to cultured for another 12 h with fresh medium. Representative fluorescence images were obtained after Hoechst 33342 staining in SH-SY5Y cells (200×).
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Fig. 3. Effects of KuB on NMDA-induced apoptosis in SH-SY5Y cells. (A) Effects of KuB on NMDA-induced early and late apoptosis and representative images were obtained after AV-PI double staining from FCM. (B) The quantitative analysis of three independent experiments. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
(5, 10, 20 μM) obviously decreased NMDA-induced ROS generation to 143 ± 4.97%, 147 ± 2.72%, 118 ± 1.76% and 108 ± 3.28%, respectively. Especially, there was a significant difference (p < 0.05) at the same concentration (20 μM) between MK801 and KuB. In addition, MDA, a marker of oxidant-mediated lipid peroxidation in cells, was measured after exposure to NMDA (2 mM) for 30 min. Treatment with NMDA could result in significant increase in the MDA level (4.54 ± 0.13 μmol/g) compared with control group (2.85 ± 0.08 μmol/g). However, pretreatment with MK801 (20 μM) and KuB (5, 10, 20 μM) could obviously attenuate NMDA-induced increasing of MDA level to 3.95 ± 0.07 μmol/g, 3.87 ± 0.06 μmol/g, 3.27 ± 0.10 μmol/g and 3.01 ± 0.06 μmol/g, respectively (Fig. 5D). Finally, the SOD, an important antioxidase in cells, was measured after the same condition idem. NMDA could obviously lower SOD activity to 0.52 ± 0.02 units compare with the control group (0.85 ± 0.05 units). However, pretreatment with MK801 (20 μM) and KuB (5, 10, 20 μM) could effectively enhance the SOD activity to 0.71 ± 0.01, 0.55 ± 0.02, 0.85 ± 0.02 and 1.32 ± 0.06, respectively (Fig. 5E). Especially, there was a significant difference (p < 0.01) at the same concentration (20 μM) between MK801 and KuB (Fig. 5C–E).
3.7. Effect of KuB on the expression of Bcl-2 and Bax in the cytoplasm of SH-SY5Y cells The ratio of Bcl-2/Bax is often used to measure the state of mitochondria. As shown in Fig. 6A and B, the expression level of Bcl-2 protein stimulated with NMDA alone was attenuated obviously, while the expression of Bax was increased compared to control group in SH-SY5Y cells. However, pretreatment with KuB (5, 10, 20 μM) could dramatically reverse this tendency and naturally result in the ratio of Bcl-2/Bax increasing compared with control cells (p < 0.05 or 0.01). These results suggested that KuB provided neuroprotection by increasing the Bcl-2/Bax ratio. 3.8. Effect of KuB on NMDA-induced Ca2+ influx in SH-SY5Y cells A previous study has shown that NMDA could induce Ca2+ overloaded in SH-SY5Y cells. In our study, compared with the control group, the fluorescence intensity of Fluo-3/AM increased markedly at about 3 min after incubation with NMDA, while pretreatment with KuB (20 μM) obviously attenuated the increased fluorescence
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Fig. 4. Effects of KuB on MMP in SH-SY5Y cells. (A) The representative pictures were obtained from FCM after incubation with Rho 123 dye. (B) The quantitative analysis of three independent experiments. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
intensity that induced by NMDA (Fig. 7A–C). The results indicated that KuB might via inhibiting the extracellular calcium influx to provide neuroprotection. 3.9. Binding affinity study through docking with NR2B subunit of NMDARs Molecular docking of KuB into the active site cavity was employed to gain better understanding of the potential relationship between KuB and NR2B-NMDARs. As shown in Fig. 8A, the threedimensional panoramic view, KuB could present commendably binding mode within the active pocket (Fig. 8B). Analysis of docking results highlighted a peculiar ligand–receptor interaction motif represented by a hydrogen bonding network involving the residue: Glu235, Glu236, Try175, Arg115, Glu106, Ala107, Gln110, Thr110, Pro106, Ile111, Phe114 and Pro78. As shown in Fig. 7B, KuB has a well combination with active pocket of NR2B-NMDARs. The binding energies of ifenprodil and KuB are −10.784 kcal/mol and −17.343 kcal/mol, respectively.
3.10. Effects of NMDA and KuB on the expression of NR2B, p-ERK1/2 and p-CREB In order to further make clear if KuB influence the downstreams of NMDARs, the expression of NR2B, p-ERK1/2 and p-CREB were investigated by western-blot analysis. NMDA-induced NR2B expression was detected at 0 h, 3 h, 6 h, 9 h and 12 h, and the results showed that NMDA could increase the NR2B expression in a time-dependent manner, especially at 12 h (Fig. 9A). The NR2B expression of NMDAtreated cells was significantly increased to 4.10 ± 0.11 compared with control group (relative value = 1). However, pretreatment with different concentrations of KuB (5, 10, 20 μM) could downregulate the NR2B expression to 2.86 ± 0.14, 2.13 ± 0.20 and 1.30 ± 0.06, respectively (Fig. 9B). Meanwhile, the expression ratio of p-ERK/t-ERK in NMDA-treated cells was dramatically downregulated to 0.25 ± 0.03 compared with the control cells (relative value = 1). Nevertheless, pretreatment with KuB (5, 10, 20 μM) could upregulate p-ERK/tERK expression ratio to 0.26 ± 0.03, 0.6 ± 0.06 and 0.89 ± 0.03, respectively (Fig. 9C). Finally, p-CREB/t-CREB expression ratio was
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Fig. 5. Effects of Apocynin on the p47phox translocation as well as ROS generation and effects of KuB and MK801 on ROS generation, MDA level and SOD activity. (A) Apocynin can decrease the NMDA-induced ROS generation and the intracellular ROS production was analyzed by FCM. (B) Effects of Apocynin on the p47phox translocation by Western-blot analysis. (C) Effects of KuB on the intracellular ROS production was analyzed by FCM. (D) Effect of KuB on the SOD activity. (E) Effects of KuB on the level of MDA. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
markedly decreased to 0.28 ± 0.04 by NMDA stimulation compared with control group (relative value = 1). However, pretreatment with KuB (5, 10, 20 μM) could effectively increase the expression ratio of p-CREB/t-CREB to 0.43 ± 0.03, 0.75 ± 0.03 and 0.91 ± 0.05, respectively (Fig. 9D). These results suggested that KuB could modulate the NR2B-ERK-CREB pathway. 3.11. Effects of KuB on the expression of SAPKs (p-p38, p-JNK) and p-AKT Other signal pathways of NMDAR downstreams, including SAPKs and PI3K-AKT that related to NR2B subunit, were detected by
western-blot assay. The expression ratio of p-AKT/t-AKT of NMDA-treated cells was significantly decreased to 0.27 ± 0.04 compared with control group (relative value = 1). However, pretreatment with KuB (5, 10, 20 μM) could increase the ratio to 0.44 ± 0.55, 0.62 ± 0.05 and 0.77 ± 0.04, respectively (Fig. 10A). At the same time, the expression ratio of p-JNK/t-JNK was dramatically upregulated in NMDAtreated cells (2.97 ± 0.15) compared with control cells (relative value = 1). However, pretreatment with KuB (5, 10, 20 μM) could downregulate this ratio to 2.73 ± 0.09, 1.76 ± 0.09 and 1.20 ± 0.06, respectively (Fig. 10B). Finally, the p-p38/t-p38 expression ratio was markedly increased to 2.53 ± 0.09 by NMDA compared with control group (relative value = 1). Whereas, pretreatment with KuB (5, 10, 20 μM) could effectively de-
Fig. 6. The expression level of Bcl-2 and Bax. (A) Protein expressions of Bcl-2 and Bax using β-actin as the loading control. (B) The quantitative analysis of Bcl-2/Bax ratio. Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
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Fig. 7. Calcium imaging of SH-SY5Y cells. (A) The green fluorescence under laser scanning microscope at different times showed the concentration of calcium in SH-SY5Y cells. (B) The fluorescence intensity showing the Ca2+ concentration was stable in control group during detection time (n = 15 cells). (C) NMDA (2 mM) is able to evoke strong fluorescence intensity (n = 15 cells) and pretreatment with KuB (20 μM) significantly reduced fluorescence intensity in SH-SY5Y cells (n = 15 cells).
crease this expression ratio to 2.23 ± 0.09, 1.86 ± 0.12 and 0.93 ± 0.03, respectively (Fig. 10C). These results suggested that KuB could modulate the phosphorylation of SAPKs and AKT. 4. Discussion This study revealed that NMDA resulted in cell death and apoptosis in SH-SY5Y cells by increasing intracellular Ca2+ concentration
as well as aggravating oxidative stress. However, KuB treatment protected the cells against NMDA-induced injury. These protective effects of KuB were possibly associated with its actions as an NMDAR antagonist and an anti-oxidative agent. NMDAR-mediated neuro-excitotoxicity leads to neuronal apoptosis and death (Aamodt and Constantine-Paton, 1998; Bliss and Collingridge, 1993). Apoptotic cells are characterized by morphological changes, including cell shrinkage and DNA condensation,
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Fig. 8. Computational molecular docking of KuB with NMDARs. (A) A close-up view of the low energy pose of KuB in the NR2B subunit of NMDARs generated by molecular docking. (B) The space matching of KuB and NR2B subunit. The purple domain represents the H-bond donor and the green domain represents the H-bond acceptor.
which further lead to degradation of chromosomal DNA, and cell death (Enari et al., 1998). Our data revealed that KuB could prevent cell death, LDH release and reduce apoptosis induced by NMDA. Cell morphological studies further demonstrated the protective effects of KuB (Figs. 1–3). These data have proven that KuB had a strong anti-apoptotic effect against NMDA. Our results elucidate that KuB exerts neuroprotective effects in vitro. In order to further explore the mechanisms of action of KuB as a potential NR2B-selective antagonist, we mainly focused on the
following questions: (1) Can KuB decrease the NADPH oxidasemediated ROS generation and Ca2+ overload induced by NMDA? (2) Does KuB bind to NR2B-containing NMDARs in silico? If so, (3) Does KuB participate in activating the NR2B subunit of NMDARs and its down-stream molecules involved in the NR2B–ERK–CREB, NR2B–PI3K–AKT, or NR2B–p38/SAPKs pathways? First, how are ROS generated and cause neuronal death? Evidence has pointed to the fact that massive accumulation of glutamate induces oxidative stress. ROS is one of the crucial etiological factors
Fig. 9. Western-blot analysis of NR2B, p-ERK and p-CREB. Special emphasis is that P-ERK and p-CREB were cultured for only 2 h after NMDA treatment (Section 2.2). (A) Time-course study of the expression of NR2B induced by NMDA. (B) The expressions of NR2B (β-actin as the loading control). (C) The expression of p-ERK/t-ERK (t-ERK as the loading control). (D) The expression of p-CREB/t-CREB (t-CREB as the loading control). Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
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Fig. 10. Western-blot analysis of SAPKs (JNK and p38) and p-AKT. Special emphasis is that P-JNK, p-p38 and p-AKT were cultured for only 2 h after NMDA treatment (Section 2.2). (A) The expressions of p-AKT/t-AKT (using t-AKT as the loading control). (B) The expression of p-JNK/t-JNK (using t-JNK as the loading control). (D) The expression of p-p38/t-p38 (using t-p38 as the loading control). Data were presented as means ± S.E.M. (n = 3). &p < 0.05 and &&p < 0.01 compared with control group. *p < 0.05 and **p < 0.01 compared with NMDA-treated group.
of oxidative stress, which results in MMP collapse, and induces activation of the apoptotic cascades (Halestrap and Brenner, 2003). NMDARs could activate the NADPH oxidase, which is the primary source of superoxide induced by NMDAR activation (Brennan et al., 2009). Membrane translocation of p47phox is necessary for activating the NADPH oxidase. Therefore, we used acopynin, an inhibitor of the NADPH oxidase, to detect the NMDA-induced membrane expression of p47phox in the SH-SY5Y cells. Our results showed that acopynin inhibited the translocation of p47phox induced by NMDA and also decreased ROS production (Fig. 5A and B). It also found that KuB could decrease ROS production induced by NMDA signaling (Fig. 5C). Therefore, we can conclude that NMDA could activate the NMDARs in the SH-SY5Y cells and further activate the NADPH oxidase. In addition, high levels of ROS in turn initiate lipid peroxidation, which results in the accumulation of reactive aldehyde products, such as MDA (Adibhatla and Hatcher, 2006). In contrast to MDA, activity of the antioxidant enzyme, SOD, is decreased in these cells when subjected to ROS (Nita et al., 2001). To our delight, our results showed that KuB could decrease the MDA levels and increase SOD activity (Fig. 5D and E). Taken together, these results showed that NMDA could activate NMDARs in the SHSY5Y cells and KuB could alleviate the NADPH oxidase-mediated ROS production, that is, NMDAR-mediated ROS production. NMDARs are the primary receptors activating excitotoxicity because of increasing Ca2+ permeability to cells. Increase of intracellular Ca2+ levels occurs as a response to NMDAR overactivation which could cause Ca2+ entry into the mitochondria. The Ca2+overloaded mitochondria could cause MMP loss and inhibit ATP production. In addition, MMP is regulated by the Bcl-2 protein family and the expression ratio of Bcl-2/Bax is regarded as an indicator of mitochondrial function and considered to be crucial for determining cell apoptosis (Miquel and Serge, 2003). Consisting with previous studies, our data showed that KuB could efficaciously suppress Ca2+ influx (Fig. 7) and increase the Bcl-2/Bax ratio (Fig. 6). These findings demonstrate that KuB may suppress the NMDARs and restrain the Ca2+ influx, which can attenuate apoptosis down the stream. Second, we used a refined docking experiment using the Discovery Studio 3.0 (DS 3.0) to further predict the potential relationship between KuB and NR2B-containing NMDARs. Ifenprodil, an NMDAR ligand, has been reported to dock in a domain composed of four amino acids, including leucine, isoleucine, and valine-binding protein (LIVBP) subunits. It has been demonstrated that the LIVBP domain is an active pocket for binding of the NR2B antagonists (Malherbe
et al., 2003; Marinelli et al., 2007). Phe 114, Gln 110, Ile 111, and Ile 82 were confirmed to be the key residues in the NR2B subunit (Karakas et al., 2011). Importantly, we found that KuB could form hydrogen bonds with the residues Phe 114, Gln 110, and Ile 111 (Fig. 8A) and form a good match within the active pocket (Fig. 8B). These results indicate that KuB was likely to be a potential NR2Bselective antagonist. Finally, some important pathways related to NR2B-containing NMDARs, including the NR2B–ERK–CREB pathway, the PI3K–AKT pathway and the SAPKs pathway, were detected by western-blot analysis. It has been reported that ERK, a key signaling protein involved in NMDAR-dependent synaptic excitotoxicity, was downregulated once the NR2B-containing NMDARs activated and subsequently inactivated the p-CREB (Banko et al., 2004; Hardingham et al., 2001; Kaphzan et al., 2006; Paul et al., 2007). Our results show that over-expression of the NR2B subunit induced by NMDA decreased p-ERK and p-CREB expression. However, pretreatment with KuB effectively reversed this tendency in a dose-dependent manner (Fig. 9). The PI3K-AKT is a pathway downstream of NMDARs. Activated AKT promotes cell survival by phosphorylation, while exposure of the SH-SY5Y cells to NMDA results in deactivation of the AKT kinase (Corasaniti et al., 2007; Meijer et al., 2003). In contrast to AKT, the SAPKs (p38 and JNK) are generally implicated in cell death in ischemic stroke (Anderson and Tolkovsky, 1999; Borsello and Forloni, 2007). It has been demonstrated that JNK and p38 play a key role in oxidative stress (Lasa et al., 2002; Zhao et al., 2013). Our study presented that the phosphorylation of JNK and p38 after NMDA exposure was suppressed by pretreatment with KuB, whereas the expression of AKT was increased (Fig. 10).
5. Conclusion This study verified that KuB can have a potent neuroprotective activity against NMDA-induced apoptosis in the SH-SY5Y cells. KuB could also suppress the NADPH oxidase-mediated ROS production. Molecular docking results predicted that KuB can potentially interact with the NR2B–NMDAR active sites. In addition, KuB could modulate the expression of the NR2B subunit and its downstream molecules, including the NR2B–ERK–CREB, the NR2B–SAPKs and the NR2B–PI3K–AKT pathways. Consequently, KuB may become a potential NMDAR antagonist. However, in vivo effects of KuB and its NR2B selectivity should be confirmed in future studies.
Please cite this article in press as: Xiao-Long Hu, et al., Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.001
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Conflict of interest There is no conflict of interest.
Acknowledgements This work was supported by the National Science and Technology Major Project, China. (Project number: 2014ZX09J14101-05C).
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.neuint.2015.06.001.
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Please cite this article in press as: Xiao-Long Hu, et al., Kukoamine B, an amide alkaloid, protects against NMDA-induced neurotoxicity and potential mechanisms in vitro, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.001