http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(8): 1052–1059 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.877039
ORIGINAL ARTICLE
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Neuroprotective effects of oxysophocarpine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion Qing-Luan Zhu1*, Yu-Xiang Li2*, Ru Zhou1, Ning-Tian Ma1, Ren-Yuan Chang1, Teng-Fei Wang1, Yi Zhang3, Xiao-Ping Chen3, Yin-Ju Hao1, Shao-Ju Jin1, Lin Ma4, Juan Du1, Tao Sun4, and Jian-Qiang Yu1 1
Department of Pharmacology, College of Pharmacy, Ningxia Medical University, Yinchuan, China, 2College of Nursing, Ningxia Medical University, Yinchuan, China, 3Shanghai Pudong New Area Gongli Hospital, Shanghai, China, and 4Ningxia Key Lab of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Yinchuan, China Abstract
Keywords
Context: Oxysophocarpine (OSC), a quinolizidine alkaloid extracted from leguminous plants of the genus Robinia, is traditionally used for various diseases including neuronal disorders. Objective: This study investigated the protective effects of OSC on neonatal rat primary-cultured hippocampal neurons were injured by oxygen–glucose deprivation and reperfusion (OGD/RP). Materials and methods: Cultured hippocampal neurons were exposed to OGD for 2 h followed by a 24 h RP. OSC (1, 2, and 5 mmol/L) and nimodipine (Nim) (12 mmol/L) were added to the culture after OGD but before RP. The cultures of the control group were not exposed to OGD/ RP. MTT and LDH assay were used to evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca2+]i and mitochondrial membrane potential (MMP) were determined to evaluate the degree of neuronal damage. Morphologic changes of neurons following OGD/RP were observed with a microscope. The expression of caspase-3 and caspase12 mRNA was examined by real-time quantitative PCR. Results: The IC50 of OSC was found to be 100 mmol/L. Treatment with OSC (1, 2, and 5 mmol/L) attenuated neuronal damage (p50.001), with evidence of increased cell viability (p50.001) and decreased cell morphologic impairment. Furthermore, OSC increased MMP (p50.001), but it inhibited [Ca2+]i (p50.001) elevation in a dose-dependent manner at OGD/RP. OSC (5 mmol/L) also decreased the expression of caspase-3 (p50.05) and caspase-12 (p50.05). Discussion and conclusion: The results suggested that OSC has significant neuroprotective effects that can be attributed to inhibiting endoplasmic reticulum (ER) stress-induced apoptosis.
Apoptosis, endoplasmic reticulum, ischemic injury, neuron
Introduction Ischemic brain injury in aged populations is a problem of enormous importance. Cerebral ischemia causes an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration (Roman, 2004). Cellular and molecular pathways underlying ischemic neurotoxicity are multifaceted and complex (Mattson et al., 2000). Ischemic brain injury leads to mitochondria dysfunction and altered intracellular calcium homeostasis in many different cell types, apoptotic death caused by hypoxia, and hypoglycemia followed by ischemia (Orrenius et al., 2003). Recently, a new apoptosis pathway was found in the endoplasmic reticulum (ER), and the activation of caspase-12 can result in cell apoptosis or
*These authors contributed equally to this work. Correspondence: Jian-Qiang Yu, College of Pharmacy, Ningxia Medical University, Yinchuan 750004, Ningxia, PR China. Tel: +86 951 4081046. E-mail: [email protected]
History Received 19 July 2013 Revised 4 December 2013 Accepted 16 December 2013 Published online 7 March 2014
necrosis (Nakka et al., 2010). Since the mechanism of ischemic brain injury is very complicated, the protective effects of Chinese traditional medicines are receiving more attention in an effort to find agents for the treatment of ischemic brain vascular diseases. Oxysophocarpine (OSC) (Figure 1) is a quinolizidine alkaloid, isolated from Sophora flavescens Ait. (Leguminosae) and S. alopecuroides Linn. (approved by the State Food and Drug Administration of China, SFDA) and other leguminous plants of the Robinia (approved by the SFDA) (Yang et al., 2006). These herbal plants are widely distributed in China. OSC has been used in traditional Chinese medicine as analgesic, anti-inflammatory, immunosuppressive, etc., therapeutic agents (Zhao & Li, 2009). OSC has high solubility in lipids and has the potential ability to permeate the blood–brain barrier (BBB) (Wang et al., 2000). This study characterized the potential neuroprotective effects of OSC on neonatal rat primary-cultured hippocampal neurons injured by oxygen–glucose deprivation and reperfusion (OGD/RP) in an attempt to explore a new
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Figure 1. Chemical structure of oxysophocarpine (OSC). The molecular formula for OSC is C15H22N2O2 and the molecular weight is 262.35.
multifunctional cytoprotective agent to treat ischemic cerebral vascular diseases.
Materials and methods Drugs and reagents OSC, white or almost white crystalline powder with purity 498.6%, was prepared by Ningxia Institute of Materia Medica (Yinchuan, China) and dissolved in normal saline before use. Nimodipine injection (Nim) (0.2 mg/ml) was obtained from the German Bayer Company (Leverkusen, Germany). The reagents used in this experiment are as follows: Dulbecco’s Modified Eagles Medium (DMEM, Gibco, Paisley, Scotland), Neurobasal-A-Medium (NAM, Gibco), fetal bovine serum (FBS, Gibco), B-27 Supplement (Gibco), 0.25% trypsin (Gibco), poly-L-lysine (PLL, Gibco), HEPESbuffered salt solution (Gibco), Earle’s balanced salt solution (EBSS) (in mg/L: 6800 NaCl, 400 KCl, 264 CaCl2H2O, 200 MgCl27 H2O, 2200 NaHCO3, 140 NaH2PO4H2O, and 1000 glucose, pH 7.2), phosphate-buffered saline (PBS), and 3(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Solarbio, Beijing, China). Rabbit anti-NSE antibody and FITC-labeled goat anti-rabbit IgG were obtained from Boster (Wuhan, China). The commercial kit for the detection of mitochondrial membrane potential (MMP), calcium fluorescence probe (Fluo-3 AM), and Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Jiangsu Province, China). The commercial kit for the detection of lactate dehydrogenase (LDH) was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other commercial kits of the Transcript RT/RI Enzyme Mix and the TransStart Top Green qPCR SuperMix were purchased from the TransGen Biotech (Beijing, China). Hippocampal neuron culture, purity determination, and oxygen–glucose deprivation and reperfusion Primary hippocampal neuronal cells were prepared from newborn Sprague–Dawley rats. After trituration and trypsinization, the viability of hippocampal neurons was above 95% as calculated using conventional trypan blue staining, under an optical microscope. The single-cell suspension was plated at a density of 1 106/mL, seeded in polyL-lysine-coated in 6-well or 96-well plates, and was cultured in (DMEM) supplemented with 10% FBS and 2% HEPESbuffered salt solution. Cells were maintained in a humidified incubator (37 C with 5% CO2) for 1.5 h to achieve the well-adherent cells; DMEM culture medium was removed
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and was replaced with 2% B-27 of Neurobasal-A-Medium culture medium. After that, half of the culture medium was replaced with fresh culture medium for every 2 d (Chen et al., 2012). Neuron-specific enolase (NSE) stain was made to observe the purity of neurons 8 d after incubation. Hippocampal neurons in culture plates were fixed by 4% paraformaldehyde for 20 min, incubated with 0.05% TritonX-100 for permeabilization and incubated with 1% BSA for 15 min to block non-specific binding sites. Then cells were incubated with rabbit anti-NSE antibody for 24 h at 4 C, incubated with FITC-labeled goat anti-rabbit IgG for 1.5 h at room temperature. Sections were washed in PBS after each step and finally visualized by laser scanning confocal microscopy (CLSM, Tokyo, Japan) (Zhao et al., 2012). Neurons were washed with Earle’s balanced salt solution (EBSS) without glucose for three times, and then the culture media were replaced with a glucose-free EBSS incubated for 2 h in oxygen-free N2/CO2 (95%/5%) gas at 37 C. Then cultures were incubated again in normoxic incubator with normal culture medium for an additional 24 h as reperfusion. The cultures of the control group were not exposed to OGD/RP. Other treating processes were as the same as the injury groups. The IC50 of OSC was found to be 100 mmol/L. Cells were randomly divided into six groups: control group; vehicle (OGD/RP) group; Nim group (12 mmol/L); low, middle, and high concentration OSC (1, 2, and 5 mmol/L) groups. OSC was given at the start of reperfusion phase, acting through the processes of reperfusion. Morphological detection The morphology of the neurons was observed and recorded via an inverted phase contrast microscope (Olympus, Tokyo, Japan). The cultured hippocampal neurons were stained with 10 mM Hoechst 33342 dye for 5 min followed by observing nuclear condensation/aggregation under a fluorescence microscope (Olympus, Tokyo, Japan) to obtain morphological evidence for apoptotic nuclei (Ye et al., 2009). Assessment of cortical neuron viability and damage Cell viability was assessed by the measurement of formazan. MTT was added to 96-well plates after the above cell treatment protocol at a final concentration of 5 mg/ml. After 4 h incubation, formazan formed, the medium was removed, and cells were dissolved in DMSO. The formation of formazan was measured by spectrophotometry at 490 nm using an ELISA reader (ELx800uv, Bio Tek Instruments, Winooski, VT). Results of formazan formation were normalized to the control group, and expressed as the percentage of viable cells (Wang et al., 2010). Neuronal damage in cells was also quantitatively assessed by measuring the activity of LDH, released from damaged or dead cells. A previous study has shown that the efflux of LDH occurring from either necrotic or apoptotic cells is proportional to the number of neurons damaged or destroyed (Gwag et al., 1995). LDH activity in the medium and total activity were measured by LDH assay kit (BioVision Inc., Milpitas, CA) at 440 nm. The percentage of LDH leakage was expressed as (culture medium OD values/culture medium
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OD values + cells homogenate OD values) 100% (Hong et al., 2004).
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Measurement of [Ca2+]i of hippocampal neuronal cells The primary hippocampal neurons were cultured in Petri dishes with a coverslip. Cells were washed with Hank’s solution (Sigma-Aldrich, St. Louis, MO) twice and were then loaded with 10 mmol/L Fluo-3/AM for 30 min at 37 C in the dark. After 30 min at 37 C, the cells were rinsed three times with Hank’s solution to remove the extracellular Fluo-3/AM. The fluorescence intensity of [Ca2+]i in hippocampal neurons was determined by laser scanning confocal microscopy. Total images were scanned and the data were stored in disks for analysis (Chen et al., 2012). Determination of MMP We measured MMP by MMP assay kit (Sigma-Aldrich, St. Louis, MO) with JC-1, using the confocal laser scanning microscope. JC-1 is a convenient voltage sensitive probe to monitor MMP (Reers et al., 1991). Cells containing forming J-aggregates have high 4Cm and show red fluorescence. Cells with low 4Cm are those in which JC-1 maintains (or reacquires) monomeric form and show green fluorescence (Zhu et al., 2009). The relative proportions of red and green fluorescence were used to measure the ratio of mitochondrial depolarization. Reverse transcription real-time quantitative PCR assay Expression of caspase-3 and caspase-12 mRNA was detected using real-time quantitative PCR. The total RNA of primarycultured neurons was isolated with Trizol agent (Invitrogen, Carlsbad, CA). Denaturing agarose gel electrophoresis was used for the detection of concentration and purity of RNA. RNA was reversed transcribed with TransScript First-Strand cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Beijing, China). The nucleotide sequences of the primers used in this study were as follows: 50 -GGA GCA GTT TTG TGT GTG TGA T-30 (forward) and 50 -GAA GAG TTT CGG CTT TCC AGT30 (reverse) for caspase-3; 50 -GGA ATG TGT GTT GAG CCA TAG A-30 (forward) and 50 -TGG AAA TGA AGA GAG AGC CAC T-30 (reverse) for caspase-12; 50 -CCC ATC TAT GAG GGT TAC GC-30 (forward) and 50 -TTT AAT GTC ACG CAC GAT TTC-30 (reverse) for b-actin. The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time quantitative PCR analysis (initial template denaturation at 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 57 C for 30 s and 72 C for 1 min). The relative differences in expression among groups were expressed as cycle time (Ct). The Ct values of the interested genes were first normalized with b-actin from the same sample, and then the relative difference between the control group and each treatment group was calculated and expressed as a relative increase.
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treated groups for different parameters was determined by a one-way analysis of variance (ANOVA). The significance of differences between vehicle and control group was evaluated by Student’s t-test. The software package SPSS 18.0 (SPSS Inc., Chicago, IL) was used for calculations. p Values of less than 0.05 were considered statistically significant.
Results Primary-cultured hippocampal neurons purity determination At 8 d after plating, the cultures were used for the experiments. Confirmed by the use of rabbit anti-NSE staining, the majority of cultured cells showed NSE immunoreactivity (Figure 2), which indicated that these cells were neurons and the purity was calculated over 87%. Later on, the cells further showed full of thick and projecting interweaving network. This stage was the best time for testing. Effects of OSC on neuronal viability The effects of OSC on cell viability were determined by the MTT assay. It indicated some protective effects of OSC against the OGD/RP. As shown in Figure 3(A), hippocampal neurons’ viability was markedly decreased after the cell was exposed to OGD/RP as determined by MTT. While pretreated with OSC (1, 2, and 5 mmol/L), OGD/RP-induced cell toxicity was significantly attenuated with a concentration-dependent manner. The viabilities were raised to 46.5 ± 4.1%, 73.7 ± 5.3%, and 76.8 ± 3.9%, respectively, after treated with OSC (1, 2, and 5 mmol/L) compared with the control group (100%) and Nim group (73.1 ± 3.7%). Neuronal damage was also quantitatively assessed by measuring the activity of LDH released from the damaged or dead cells. As shown in Figure 3(B), LDH leakage increased to 48.5% (vehicle-treated group) after OGD/RP and OSC significantly attenuated OGD/RP-induced cell death by
Statistical analysis All quantitative data were expressed as mean ± SD. The significance of differences in means between control and
Figure 2. Hippocamoal neurons fluorescense staining with rabbit antirat neuron-specific enolase (NSE). The purity of hippocamoal neurons was calculated as over 87% (200).
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Figure 3. Effects of OSC on cell viability in primary hippocampal neuronal cell under oxygen–glucose deprivation for 2 h and reperfusion for 24 h, determined by the MTT assay (A), and the extent of LDH release (B). Histograms represent mean ± SD, n ¼ 6. ###p50.001 OGD/RP + vehicle group versus control group; *p50.05, ***p50.001 versus OGD/RP + vehicle group.
reducing LDH leakage from 48.5% to 30.2–26.7% (Figure 3B) while the LDH leakage was 23.3% in the positive drug-controlled group. Consistent results were obtained using the MTT assay. Effects of OSC on neuronal morphological changes Neuronal morphology was examined after the exposure to OGD/RP and neuronal morphological changes were observed by both phase and fluorescent imaging. OGD/RP induced neuronal loss and the damage of neurites. Meanwhile, during the progression of cellular necrosis, some neurons displayed condensed cytoplasm, cell shrinkage with the plasma membrane remaining intact, and formation of apoptotic bodies that stained more densely with Hoechst 33342. However, the situation became much better in the groups treated with OSC (1, 2, and 5 mmol/L) and Nim (12 mmol/L) (Figure 4). This indicated some protective effects of OSC against the OGD/RP. Effects of OSC on [Ca2+]i of hippocampal neurons [Ca2+]i was expressed by green fluorescence intensity (Figure 5), compared with the control group, the values of [Ca2+]i of hippocampal neurons at OGD 2 h/RP 24 h showed significant changes (nearly 4.5-fold versus that of the control group), increased to 0.44 ± 0.03 (n ¼ 6, p50.001, Figure 5G). Compared to the vehicle group, [Ca2+]i in neuron cultures was decreased in the groups treated with OSC (1, 2, and 5 mmol/L) and Nim (12 mmol/L) after OGD 2 h/RP 24 h, especially in the OSC-treated group (5 mmol/L). Green fluorescence intensity was decreased from 0.44 ± 0.03 to 0.22 ± 0.03, 0.29 ± 0.02, 0.25 ± 0.03, and 0.17 ± 0.02, after treatment with Nim (12 mmol/L) and OSC (1, 2, and 5 mmol/ L), respectively. Effects of OSC on MMP of hippocampal neurons JC-1 was used to evaluate the loss of MMP in hippocampal cultures exposed to OGD/RP. MMP was assessed in neurons according to fluorescence ratios (Figure 6). Relative to normal neurons 4.30 ± 0.25, MMP had decreased
to 0.50 ± 0.15 (n ¼ 6, p50.001, Figure 6G) in the vehicle group, similar to the decrease of cell viability and increase of apoptosis ratio. Decreased MMP was recovered to 1.84 ± 0.28, 1.53 ± 0.21, 2.15 ± 0.43, 2.56 ± 0.49, following the treatments with Nim (12 mmol/L) and OSC (1, 2, and 5 mmol/L). Real-time quantitative PCR analysis of mRNA expression To determine whether OSC affected the regulation of apoptosis at the mRNA level, we carried out a real-time quantitative PCR analysis. RNA purity was tested by UV absorbance with 28S:18S RNA approximately at the ratio of 2:1. Caspase-3 and caspase-12 mRNA expression was significantly increased after OGD/RP. However, in the OSC (5 mmol/L) treated neurons, the expressions of caspase-3 and caspase-12 mRNA were reduced significantly (Figure 7).
Discussion Ischemic brain disease, also known as ischemic stroke, at present has become one of the major causes of mortality and disabling disease in a global scale. To treat these diseases is a challenge. Therefore, to explore effective treatment strategies for ischemic cerebrovascular disease has been one of the major topics of common concern in the international medical circles. At present, more and more evidence indicates that Chinese herbal medicines have a beneficial role to play in the treatment of these disorders. OSC is well known to provide anti-inflammatory, anti-tumor effect, etc., but its neuroprotection effect has not been widely studied. In our study, we obtained further evidence demonstrating that OSC has a good protective effect on neonatal rat primary-cultured hippocampal neurons injured by OGD/RP, suggesting that OSC has the potential for becoming an effective drug for the treatment of ischemia brain disease. Nim is a calcium antagonist and has a potent cerebrovascular activity in vitro (Kazda, 1985; Towart & Perzborn, 1981; Towart et al., 1982; Wadworth & McTavish, 1992), so we used Nim as a positive control drug.
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Figure 4. Effects of OSC on morphological changes of OGD/RP injured primary hippocampal neurons. Cellular morphological changes were examined using Olympus optics (Olympus, Tokyo, Japan) (A–F, 400). Nuclei were labeled with Hoechst 33342 (G–L, 400). Representative photomicrographs showing damage of neurites and denote chromatin condensation of hippocampal neurons. (A, G) Control; (B, H) exposure to OGD/ RP; (C, I) treated with Nim (12 mmol/L); (D, J) treated with low concentration of OSC (1 mmol/L); (E, K) treated with a middle concentration of OSC (2 mmol/L); (F, L) treated with a high concentration of OSC (5 mmol/L).
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Figure 5. Effects of OSC on [Ca2+]i in primary hippocampal neuronal cell under OGD 2 h/RP 24 h as indicated by Fluo-3/ AM(100). (A) Control cells. (B) Cells exposed to OGD 2 h/RP 24 h with no treatment of OSC. (C) Nim (12 mmol/L) was added to the culture before the reperfusion. (D–F) OSC (1, 2, and 5 mmol/L) was, respectively, added to the culture before the reperfusion. (G) Effects of OSC on [Ca2+]i in primary hippocampal neuronal cell under OGD 2 h/RP 24 h. Histograms represent mean ± SD, n ¼ 6. ###p50.001 OGD/ RP + vehicle group versus control group; ***p50.001 versus OGD/RP + vehicle group.
In our study, the degree of injury in hippocampal neurons exposed to OGD/RP was evaluated by MTT and LDH release. The results showed that significantly reduced cell viability after neurons exposure to OGD/RP and treatment with OSC (5 mmol/L) significantly attenuated neuronal damage and inhibited LDH release. These experimental findings indicate that OSC has protective effects on OGD/RP in hippocampal neuronal cells and demonstrates a remarkable concentrationdependent relationship. Ischemia or hypoxia can cause cell death by necrosis and apoptosis (MacManus & Linnik, 1997). Numerous studies have shown that OGD/RP induced hippocampal neuron death, mainly through apoptosis (Chu et al., 2010; Wang et al., 2010). OGD/RP insult increased the apoptosis rate of neurons and OSC inhibited the apoptosis induced by OGD/RP, which was consistent with morphological changes examined with an inverted phase contrast microscope and fluorescence microscope. To further explore the anti-apoptotic mechanisms of OSC on OGD/RP injury, we performed an additional study of [Ca2+]i overload, mitochondria function, and mRNA expression of apoptotic enzyme genes.
Calcium plays an important role in signal transduction and neurotransmitter release as a second messenger. Although the mechanisms of OGD/RP disturbing neural functions are not completely understood, cellular calcium dysregulation appears to be a common endpoint in the development of cerebral ischemia injury (Saris & Carafoli, 2005). Mitochondrial dysfunction is an early feature of nervous system ischemia. Stained elevated Ca2+ levels induced by OGD/RP may impair mitochondrial function preceded by the opening of a permeability transition pore in the inner mitochondrial membrane, destabilization of the neuronal cytoarchitecture, eventually leading to neuronal apoptosis and death. From the results of this study, we infer that OSC could alleviate calcium overload and maintain mitochondrial function, which assists the anti-apoptosis action. To further explore the anti-apoptotic effect of OSC at the mRNA level, we examined the expression of caspase-3. Caspases play an essential role during apoptotic cell death (Slee et al., 1999). Among 14 distinct caspases, caspase-3 is crucial during neuronal development and under pathological conditions including cerebral ischemia (Nicholson, 1999). During ischemia, caspase-3 is cleaved and activated
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Figure 6. Effects of OSC on MMP changes in primary hippocampal neuronal cell under OGD 2 h/RP 24 h as indicated by JC1(200). (A) Control cells. (B) Cells exposed to OGD 2 h/RP 24 h with no treatment of OSC. (C) Nim (12 mmol/L) was added to the culture before the reperfusion. (D–F) OSC (1, 2, and 5 mmol/L) was, respectively, added to the culture before the reperfusion. (G) Effects of OSC on MMP changes in primary hippocampal neuronal cell under OGD 2 h/RP 24 h. Histograms represent mean ± SD, n ¼ 6. ###p50.001 OGD/ RP + vehicle group versus control group; **p50.01, ***p50.001 versus OGD/ RP + vehicle group.
Figure 7. Effects of OSC (5 mmol/L) on OGD/RP-induced expression of caspase-3 and caspase-12 mRNA in primary hippocampal neurons as determined by real-time quantitative PCR. Data are mean ± SD, n ¼ 3. #p50.05, ##p50.01 OGD/RP + vehicle group versus control group; *p50.05, **p50.01 versus OGD/RP + vehicle group.
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DOI: 10.3109/13880209.2013.877039
whereupon it degrades multiple substrates in the cytoplasm and nucleus leading to cell death. We also observed the expression of caspase-12 mRNA, another member of the caspase family, it was discovered as the first ER-associated caspase by Nakagawa and Yuan (2000). Caspase-12 is specifically localized on the cytoplasmic side (outer membrane) of the ER. Studies showed that caspase-12 activation was induced by ER stress, like treatment with thapsigarpin and tunicamycin, and executed cell death (Nakagawa et al., 2000). They have found that activation of ER-resident caspase-12 causes activation of cytoplasmic caspase-3 during ER stress-induced apoptosis (Hitomi et al., 2004). Notably, our study also indicated that OGD/RP insult increased the expression of caspase-12 mRNA and the overexpression of caspase-3 mRNA simultaneously (Figure 7). Treatment with OSC could significantly reduce these effects in a concentration-dependent manner, parallel with its effects on [Ca2+]i elevation and apoptosis induced by OGD/ RP. The fact that treatment with OSC prevents caspasedependent cell apoptosis suggests that the molecular mechanisms of OSC effects may involve a caspase-signaling cascade. OSC may act through signal transduction pathways to influence apoptosis. In summary, our results demonstrate the protective effects of OSC on OGD/RP neuronal injury in vitro. This protection is probably associated with the preservation of MMP, inhibiting calcium overload, and lessening neuron apoptosis that is mediated, at least in part, by caspase-3-dependent and caspase-12-dependent pathways.
Acknowledgements We are indebted to the staff in the animal center and the Science & Technology Centre who provided assistance in the study. The authors would like to thank Dr. Ding-Feng Su, Prof. Wan-Nian Zhang, and Yan Zhang for their contributions to development and implementation of this study.
Declaration of interest The authors declare that they have no competing interests. The study was supported by the National Natural Science Foundation of China (Grant no. 81360182), the Personnel training projects of the Chinese Academy of Sciences ‘‘Western Light’’ (Grant no. 2012018), the special talents of Ningxia Medical research and start-up projects (Grant no. XT2012015) and Ningxia Medical University Students’ science and technology innovation project (Grant no. 2012).
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Neuroprotective effects of oxysophocarpine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion Qing-Luan Zhu1*, Yu-Xiang Li2*, Ru Zhou1, Ning-Tian Ma1, Ren-Yuan Chang1, Teng-Fei Wang1, Yi Zhang3, Xiao-Ping Chen3, Yin-Ju Hao1, Shao-Ju Jin1, Lin Ma4, Juan Du1, Tao Sun4, and Jian-Qiang Yu1 1
Department of Pharmacology, College of Pharmacy, Ningxia Medical University, Yinchuan, China, 2College of Nursing, Ningxia Medical University, Yinchuan, China, 3Shanghai Pudong New Area Gongli Hospital, Shanghai, China, and 4Ningxia Key Lab of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Yinchuan, China Abstract
Keywords
Context: Oxysophocarpine (OSC), a quinolizidine alkaloid extracted from leguminous plants of the genus Robinia, is traditionally used for various diseases including neuronal disorders. Objective: This study investigated the protective effects of OSC on neonatal rat primary-cultured hippocampal neurons were injured by oxygen–glucose deprivation and reperfusion (OGD/RP). Materials and methods: Cultured hippocampal neurons were exposed to OGD for 2 h followed by a 24 h RP. OSC (1, 2, and 5 mmol/L) and nimodipine (Nim) (12 mmol/L) were added to the culture after OGD but before RP. The cultures of the control group were not exposed to OGD/ RP. MTT and LDH assay were used to evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca2+]i and mitochondrial membrane potential (MMP) were determined to evaluate the degree of neuronal damage. Morphologic changes of neurons following OGD/RP were observed with a microscope. The expression of caspase-3 and caspase12 mRNA was examined by real-time quantitative PCR. Results: The IC50 of OSC was found to be 100 mmol/L. Treatment with OSC (1, 2, and 5 mmol/L) attenuated neuronal damage (p50.001), with evidence of increased cell viability (p50.001) and decreased cell morphologic impairment. Furthermore, OSC increased MMP (p50.001), but it inhibited [Ca2+]i (p50.001) elevation in a dose-dependent manner at OGD/RP. OSC (5 mmol/L) also decreased the expression of caspase-3 (p50.05) and caspase-12 (p50.05). Discussion and conclusion: The results suggested that OSC has significant neuroprotective effects that can be attributed to inhibiting endoplasmic reticulum (ER) stress-induced apoptosis.
Apoptosis, endoplasmic reticulum, ischemic injury, neuron
Introduction Ischemic brain injury in aged populations is a problem of enormous importance. Cerebral ischemia causes an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration (Roman, 2004). Cellular and molecular pathways underlying ischemic neurotoxicity are multifaceted and complex (Mattson et al., 2000). Ischemic brain injury leads to mitochondria dysfunction and altered intracellular calcium homeostasis in many different cell types, apoptotic death caused by hypoxia, and hypoglycemia followed by ischemia (Orrenius et al., 2003). Recently, a new apoptosis pathway was found in the endoplasmic reticulum (ER), and the activation of caspase-12 can result in cell apoptosis or
*These authors contributed equally to this work. Correspondence: Jian-Qiang Yu, College of Pharmacy, Ningxia Medical University, Yinchuan 750004, Ningxia, PR China. Tel: +86 951 4081046. E-mail: [email protected]
History Received 19 July 2013 Revised 4 December 2013 Accepted 16 December 2013 Published online 7 March 2014
necrosis (Nakka et al., 2010). Since the mechanism of ischemic brain injury is very complicated, the protective effects of Chinese traditional medicines are receiving more attention in an effort to find agents for the treatment of ischemic brain vascular diseases. Oxysophocarpine (OSC) (Figure 1) is a quinolizidine alkaloid, isolated from Sophora flavescens Ait. (Leguminosae) and S. alopecuroides Linn. (approved by the State Food and Drug Administration of China, SFDA) and other leguminous plants of the Robinia (approved by the SFDA) (Yang et al., 2006). These herbal plants are widely distributed in China. OSC has been used in traditional Chinese medicine as analgesic, anti-inflammatory, immunosuppressive, etc., therapeutic agents (Zhao & Li, 2009). OSC has high solubility in lipids and has the potential ability to permeate the blood–brain barrier (BBB) (Wang et al., 2000). This study characterized the potential neuroprotective effects of OSC on neonatal rat primary-cultured hippocampal neurons injured by oxygen–glucose deprivation and reperfusion (OGD/RP) in an attempt to explore a new
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Figure 1. Chemical structure of oxysophocarpine (OSC). The molecular formula for OSC is C15H22N2O2 and the molecular weight is 262.35.
multifunctional cytoprotective agent to treat ischemic cerebral vascular diseases.
Materials and methods Drugs and reagents OSC, white or almost white crystalline powder with purity 498.6%, was prepared by Ningxia Institute of Materia Medica (Yinchuan, China) and dissolved in normal saline before use. Nimodipine injection (Nim) (0.2 mg/ml) was obtained from the German Bayer Company (Leverkusen, Germany). The reagents used in this experiment are as follows: Dulbecco’s Modified Eagles Medium (DMEM, Gibco, Paisley, Scotland), Neurobasal-A-Medium (NAM, Gibco), fetal bovine serum (FBS, Gibco), B-27 Supplement (Gibco), 0.25% trypsin (Gibco), poly-L-lysine (PLL, Gibco), HEPESbuffered salt solution (Gibco), Earle’s balanced salt solution (EBSS) (in mg/L: 6800 NaCl, 400 KCl, 264 CaCl2H2O, 200 MgCl27 H2O, 2200 NaHCO3, 140 NaH2PO4H2O, and 1000 glucose, pH 7.2), phosphate-buffered saline (PBS), and 3(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Solarbio, Beijing, China). Rabbit anti-NSE antibody and FITC-labeled goat anti-rabbit IgG were obtained from Boster (Wuhan, China). The commercial kit for the detection of mitochondrial membrane potential (MMP), calcium fluorescence probe (Fluo-3 AM), and Hoechst 33342 were purchased from Beyotime Institute of Biotechnology (Jiangsu Province, China). The commercial kit for the detection of lactate dehydrogenase (LDH) was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Other commercial kits of the Transcript RT/RI Enzyme Mix and the TransStart Top Green qPCR SuperMix were purchased from the TransGen Biotech (Beijing, China). Hippocampal neuron culture, purity determination, and oxygen–glucose deprivation and reperfusion Primary hippocampal neuronal cells were prepared from newborn Sprague–Dawley rats. After trituration and trypsinization, the viability of hippocampal neurons was above 95% as calculated using conventional trypan blue staining, under an optical microscope. The single-cell suspension was plated at a density of 1 106/mL, seeded in polyL-lysine-coated in 6-well or 96-well plates, and was cultured in (DMEM) supplemented with 10% FBS and 2% HEPESbuffered salt solution. Cells were maintained in a humidified incubator (37 C with 5% CO2) for 1.5 h to achieve the well-adherent cells; DMEM culture medium was removed
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and was replaced with 2% B-27 of Neurobasal-A-Medium culture medium. After that, half of the culture medium was replaced with fresh culture medium for every 2 d (Chen et al., 2012). Neuron-specific enolase (NSE) stain was made to observe the purity of neurons 8 d after incubation. Hippocampal neurons in culture plates were fixed by 4% paraformaldehyde for 20 min, incubated with 0.05% TritonX-100 for permeabilization and incubated with 1% BSA for 15 min to block non-specific binding sites. Then cells were incubated with rabbit anti-NSE antibody for 24 h at 4 C, incubated with FITC-labeled goat anti-rabbit IgG for 1.5 h at room temperature. Sections were washed in PBS after each step and finally visualized by laser scanning confocal microscopy (CLSM, Tokyo, Japan) (Zhao et al., 2012). Neurons were washed with Earle’s balanced salt solution (EBSS) without glucose for three times, and then the culture media were replaced with a glucose-free EBSS incubated for 2 h in oxygen-free N2/CO2 (95%/5%) gas at 37 C. Then cultures were incubated again in normoxic incubator with normal culture medium for an additional 24 h as reperfusion. The cultures of the control group were not exposed to OGD/RP. Other treating processes were as the same as the injury groups. The IC50 of OSC was found to be 100 mmol/L. Cells were randomly divided into six groups: control group; vehicle (OGD/RP) group; Nim group (12 mmol/L); low, middle, and high concentration OSC (1, 2, and 5 mmol/L) groups. OSC was given at the start of reperfusion phase, acting through the processes of reperfusion. Morphological detection The morphology of the neurons was observed and recorded via an inverted phase contrast microscope (Olympus, Tokyo, Japan). The cultured hippocampal neurons were stained with 10 mM Hoechst 33342 dye for 5 min followed by observing nuclear condensation/aggregation under a fluorescence microscope (Olympus, Tokyo, Japan) to obtain morphological evidence for apoptotic nuclei (Ye et al., 2009). Assessment of cortical neuron viability and damage Cell viability was assessed by the measurement of formazan. MTT was added to 96-well plates after the above cell treatment protocol at a final concentration of 5 mg/ml. After 4 h incubation, formazan formed, the medium was removed, and cells were dissolved in DMSO. The formation of formazan was measured by spectrophotometry at 490 nm using an ELISA reader (ELx800uv, Bio Tek Instruments, Winooski, VT). Results of formazan formation were normalized to the control group, and expressed as the percentage of viable cells (Wang et al., 2010). Neuronal damage in cells was also quantitatively assessed by measuring the activity of LDH, released from damaged or dead cells. A previous study has shown that the efflux of LDH occurring from either necrotic or apoptotic cells is proportional to the number of neurons damaged or destroyed (Gwag et al., 1995). LDH activity in the medium and total activity were measured by LDH assay kit (BioVision Inc., Milpitas, CA) at 440 nm. The percentage of LDH leakage was expressed as (culture medium OD values/culture medium
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OD values + cells homogenate OD values) 100% (Hong et al., 2004).
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Measurement of [Ca2+]i of hippocampal neuronal cells The primary hippocampal neurons were cultured in Petri dishes with a coverslip. Cells were washed with Hank’s solution (Sigma-Aldrich, St. Louis, MO) twice and were then loaded with 10 mmol/L Fluo-3/AM for 30 min at 37 C in the dark. After 30 min at 37 C, the cells were rinsed three times with Hank’s solution to remove the extracellular Fluo-3/AM. The fluorescence intensity of [Ca2+]i in hippocampal neurons was determined by laser scanning confocal microscopy. Total images were scanned and the data were stored in disks for analysis (Chen et al., 2012). Determination of MMP We measured MMP by MMP assay kit (Sigma-Aldrich, St. Louis, MO) with JC-1, using the confocal laser scanning microscope. JC-1 is a convenient voltage sensitive probe to monitor MMP (Reers et al., 1991). Cells containing forming J-aggregates have high 4Cm and show red fluorescence. Cells with low 4Cm are those in which JC-1 maintains (or reacquires) monomeric form and show green fluorescence (Zhu et al., 2009). The relative proportions of red and green fluorescence were used to measure the ratio of mitochondrial depolarization. Reverse transcription real-time quantitative PCR assay Expression of caspase-3 and caspase-12 mRNA was detected using real-time quantitative PCR. The total RNA of primarycultured neurons was isolated with Trizol agent (Invitrogen, Carlsbad, CA). Denaturing agarose gel electrophoresis was used for the detection of concentration and purity of RNA. RNA was reversed transcribed with TransScript First-Strand cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Beijing, China). The nucleotide sequences of the primers used in this study were as follows: 50 -GGA GCA GTT TTG TGT GTG TGA T-30 (forward) and 50 -GAA GAG TTT CGG CTT TCC AGT30 (reverse) for caspase-3; 50 -GGA ATG TGT GTT GAG CCA TAG A-30 (forward) and 50 -TGG AAA TGA AGA GAG AGC CAC T-30 (reverse) for caspase-12; 50 -CCC ATC TAT GAG GGT TAC GC-30 (forward) and 50 -TTT AAT GTC ACG CAC GAT TTC-30 (reverse) for b-actin. The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real-time quantitative PCR analysis (initial template denaturation at 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 57 C for 30 s and 72 C for 1 min). The relative differences in expression among groups were expressed as cycle time (Ct). The Ct values of the interested genes were first normalized with b-actin from the same sample, and then the relative difference between the control group and each treatment group was calculated and expressed as a relative increase.
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treated groups for different parameters was determined by a one-way analysis of variance (ANOVA). The significance of differences between vehicle and control group was evaluated by Student’s t-test. The software package SPSS 18.0 (SPSS Inc., Chicago, IL) was used for calculations. p Values of less than 0.05 were considered statistically significant.
Results Primary-cultured hippocampal neurons purity determination At 8 d after plating, the cultures were used for the experiments. Confirmed by the use of rabbit anti-NSE staining, the majority of cultured cells showed NSE immunoreactivity (Figure 2), which indicated that these cells were neurons and the purity was calculated over 87%. Later on, the cells further showed full of thick and projecting interweaving network. This stage was the best time for testing. Effects of OSC on neuronal viability The effects of OSC on cell viability were determined by the MTT assay. It indicated some protective effects of OSC against the OGD/RP. As shown in Figure 3(A), hippocampal neurons’ viability was markedly decreased after the cell was exposed to OGD/RP as determined by MTT. While pretreated with OSC (1, 2, and 5 mmol/L), OGD/RP-induced cell toxicity was significantly attenuated with a concentration-dependent manner. The viabilities were raised to 46.5 ± 4.1%, 73.7 ± 5.3%, and 76.8 ± 3.9%, respectively, after treated with OSC (1, 2, and 5 mmol/L) compared with the control group (100%) and Nim group (73.1 ± 3.7%). Neuronal damage was also quantitatively assessed by measuring the activity of LDH released from the damaged or dead cells. As shown in Figure 3(B), LDH leakage increased to 48.5% (vehicle-treated group) after OGD/RP and OSC significantly attenuated OGD/RP-induced cell death by
Statistical analysis All quantitative data were expressed as mean ± SD. The significance of differences in means between control and
Figure 2. Hippocamoal neurons fluorescense staining with rabbit antirat neuron-specific enolase (NSE). The purity of hippocamoal neurons was calculated as over 87% (200).
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Figure 3. Effects of OSC on cell viability in primary hippocampal neuronal cell under oxygen–glucose deprivation for 2 h and reperfusion for 24 h, determined by the MTT assay (A), and the extent of LDH release (B). Histograms represent mean ± SD, n ¼ 6. ###p50.001 OGD/RP + vehicle group versus control group; *p50.05, ***p50.001 versus OGD/RP + vehicle group.
reducing LDH leakage from 48.5% to 30.2–26.7% (Figure 3B) while the LDH leakage was 23.3% in the positive drug-controlled group. Consistent results were obtained using the MTT assay. Effects of OSC on neuronal morphological changes Neuronal morphology was examined after the exposure to OGD/RP and neuronal morphological changes were observed by both phase and fluorescent imaging. OGD/RP induced neuronal loss and the damage of neurites. Meanwhile, during the progression of cellular necrosis, some neurons displayed condensed cytoplasm, cell shrinkage with the plasma membrane remaining intact, and formation of apoptotic bodies that stained more densely with Hoechst 33342. However, the situation became much better in the groups treated with OSC (1, 2, and 5 mmol/L) and Nim (12 mmol/L) (Figure 4). This indicated some protective effects of OSC against the OGD/RP. Effects of OSC on [Ca2+]i of hippocampal neurons [Ca2+]i was expressed by green fluorescence intensity (Figure 5), compared with the control group, the values of [Ca2+]i of hippocampal neurons at OGD 2 h/RP 24 h showed significant changes (nearly 4.5-fold versus that of the control group), increased to 0.44 ± 0.03 (n ¼ 6, p50.001, Figure 5G). Compared to the vehicle group, [Ca2+]i in neuron cultures was decreased in the groups treated with OSC (1, 2, and 5 mmol/L) and Nim (12 mmol/L) after OGD 2 h/RP 24 h, especially in the OSC-treated group (5 mmol/L). Green fluorescence intensity was decreased from 0.44 ± 0.03 to 0.22 ± 0.03, 0.29 ± 0.02, 0.25 ± 0.03, and 0.17 ± 0.02, after treatment with Nim (12 mmol/L) and OSC (1, 2, and 5 mmol/ L), respectively. Effects of OSC on MMP of hippocampal neurons JC-1 was used to evaluate the loss of MMP in hippocampal cultures exposed to OGD/RP. MMP was assessed in neurons according to fluorescence ratios (Figure 6). Relative to normal neurons 4.30 ± 0.25, MMP had decreased
to 0.50 ± 0.15 (n ¼ 6, p50.001, Figure 6G) in the vehicle group, similar to the decrease of cell viability and increase of apoptosis ratio. Decreased MMP was recovered to 1.84 ± 0.28, 1.53 ± 0.21, 2.15 ± 0.43, 2.56 ± 0.49, following the treatments with Nim (12 mmol/L) and OSC (1, 2, and 5 mmol/L). Real-time quantitative PCR analysis of mRNA expression To determine whether OSC affected the regulation of apoptosis at the mRNA level, we carried out a real-time quantitative PCR analysis. RNA purity was tested by UV absorbance with 28S:18S RNA approximately at the ratio of 2:1. Caspase-3 and caspase-12 mRNA expression was significantly increased after OGD/RP. However, in the OSC (5 mmol/L) treated neurons, the expressions of caspase-3 and caspase-12 mRNA were reduced significantly (Figure 7).
Discussion Ischemic brain disease, also known as ischemic stroke, at present has become one of the major causes of mortality and disabling disease in a global scale. To treat these diseases is a challenge. Therefore, to explore effective treatment strategies for ischemic cerebrovascular disease has been one of the major topics of common concern in the international medical circles. At present, more and more evidence indicates that Chinese herbal medicines have a beneficial role to play in the treatment of these disorders. OSC is well known to provide anti-inflammatory, anti-tumor effect, etc., but its neuroprotection effect has not been widely studied. In our study, we obtained further evidence demonstrating that OSC has a good protective effect on neonatal rat primary-cultured hippocampal neurons injured by OGD/RP, suggesting that OSC has the potential for becoming an effective drug for the treatment of ischemia brain disease. Nim is a calcium antagonist and has a potent cerebrovascular activity in vitro (Kazda, 1985; Towart & Perzborn, 1981; Towart et al., 1982; Wadworth & McTavish, 1992), so we used Nim as a positive control drug.
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Figure 4. Effects of OSC on morphological changes of OGD/RP injured primary hippocampal neurons. Cellular morphological changes were examined using Olympus optics (Olympus, Tokyo, Japan) (A–F, 400). Nuclei were labeled with Hoechst 33342 (G–L, 400). Representative photomicrographs showing damage of neurites and denote chromatin condensation of hippocampal neurons. (A, G) Control; (B, H) exposure to OGD/ RP; (C, I) treated with Nim (12 mmol/L); (D, J) treated with low concentration of OSC (1 mmol/L); (E, K) treated with a middle concentration of OSC (2 mmol/L); (F, L) treated with a high concentration of OSC (5 mmol/L).
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Figure 5. Effects of OSC on [Ca2+]i in primary hippocampal neuronal cell under OGD 2 h/RP 24 h as indicated by Fluo-3/ AM(100). (A) Control cells. (B) Cells exposed to OGD 2 h/RP 24 h with no treatment of OSC. (C) Nim (12 mmol/L) was added to the culture before the reperfusion. (D–F) OSC (1, 2, and 5 mmol/L) was, respectively, added to the culture before the reperfusion. (G) Effects of OSC on [Ca2+]i in primary hippocampal neuronal cell under OGD 2 h/RP 24 h. Histograms represent mean ± SD, n ¼ 6. ###p50.001 OGD/ RP + vehicle group versus control group; ***p50.001 versus OGD/RP + vehicle group.
In our study, the degree of injury in hippocampal neurons exposed to OGD/RP was evaluated by MTT and LDH release. The results showed that significantly reduced cell viability after neurons exposure to OGD/RP and treatment with OSC (5 mmol/L) significantly attenuated neuronal damage and inhibited LDH release. These experimental findings indicate that OSC has protective effects on OGD/RP in hippocampal neuronal cells and demonstrates a remarkable concentrationdependent relationship. Ischemia or hypoxia can cause cell death by necrosis and apoptosis (MacManus & Linnik, 1997). Numerous studies have shown that OGD/RP induced hippocampal neuron death, mainly through apoptosis (Chu et al., 2010; Wang et al., 2010). OGD/RP insult increased the apoptosis rate of neurons and OSC inhibited the apoptosis induced by OGD/RP, which was consistent with morphological changes examined with an inverted phase contrast microscope and fluorescence microscope. To further explore the anti-apoptotic mechanisms of OSC on OGD/RP injury, we performed an additional study of [Ca2+]i overload, mitochondria function, and mRNA expression of apoptotic enzyme genes.
Calcium plays an important role in signal transduction and neurotransmitter release as a second messenger. Although the mechanisms of OGD/RP disturbing neural functions are not completely understood, cellular calcium dysregulation appears to be a common endpoint in the development of cerebral ischemia injury (Saris & Carafoli, 2005). Mitochondrial dysfunction is an early feature of nervous system ischemia. Stained elevated Ca2+ levels induced by OGD/RP may impair mitochondrial function preceded by the opening of a permeability transition pore in the inner mitochondrial membrane, destabilization of the neuronal cytoarchitecture, eventually leading to neuronal apoptosis and death. From the results of this study, we infer that OSC could alleviate calcium overload and maintain mitochondrial function, which assists the anti-apoptosis action. To further explore the anti-apoptotic effect of OSC at the mRNA level, we examined the expression of caspase-3. Caspases play an essential role during apoptotic cell death (Slee et al., 1999). Among 14 distinct caspases, caspase-3 is crucial during neuronal development and under pathological conditions including cerebral ischemia (Nicholson, 1999). During ischemia, caspase-3 is cleaved and activated
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Figure 6. Effects of OSC on MMP changes in primary hippocampal neuronal cell under OGD 2 h/RP 24 h as indicated by JC1(200). (A) Control cells. (B) Cells exposed to OGD 2 h/RP 24 h with no treatment of OSC. (C) Nim (12 mmol/L) was added to the culture before the reperfusion. (D–F) OSC (1, 2, and 5 mmol/L) was, respectively, added to the culture before the reperfusion. (G) Effects of OSC on MMP changes in primary hippocampal neuronal cell under OGD 2 h/RP 24 h. Histograms represent mean ± SD, n ¼ 6. ###p50.001 OGD/ RP + vehicle group versus control group; **p50.01, ***p50.001 versus OGD/ RP + vehicle group.
Figure 7. Effects of OSC (5 mmol/L) on OGD/RP-induced expression of caspase-3 and caspase-12 mRNA in primary hippocampal neurons as determined by real-time quantitative PCR. Data are mean ± SD, n ¼ 3. #p50.05, ##p50.01 OGD/RP + vehicle group versus control group; *p50.05, **p50.01 versus OGD/RP + vehicle group.
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whereupon it degrades multiple substrates in the cytoplasm and nucleus leading to cell death. We also observed the expression of caspase-12 mRNA, another member of the caspase family, it was discovered as the first ER-associated caspase by Nakagawa and Yuan (2000). Caspase-12 is specifically localized on the cytoplasmic side (outer membrane) of the ER. Studies showed that caspase-12 activation was induced by ER stress, like treatment with thapsigarpin and tunicamycin, and executed cell death (Nakagawa et al., 2000). They have found that activation of ER-resident caspase-12 causes activation of cytoplasmic caspase-3 during ER stress-induced apoptosis (Hitomi et al., 2004). Notably, our study also indicated that OGD/RP insult increased the expression of caspase-12 mRNA and the overexpression of caspase-3 mRNA simultaneously (Figure 7). Treatment with OSC could significantly reduce these effects in a concentration-dependent manner, parallel with its effects on [Ca2+]i elevation and apoptosis induced by OGD/ RP. The fact that treatment with OSC prevents caspasedependent cell apoptosis suggests that the molecular mechanisms of OSC effects may involve a caspase-signaling cascade. OSC may act through signal transduction pathways to influence apoptosis. In summary, our results demonstrate the protective effects of OSC on OGD/RP neuronal injury in vitro. This protection is probably associated with the preservation of MMP, inhibiting calcium overload, and lessening neuron apoptosis that is mediated, at least in part, by caspase-3-dependent and caspase-12-dependent pathways.
Acknowledgements We are indebted to the staff in the animal center and the Science & Technology Centre who provided assistance in the study. The authors would like to thank Dr. Ding-Feng Su, Prof. Wan-Nian Zhang, and Yan Zhang for their contributions to development and implementation of this study.
Declaration of interest The authors declare that they have no competing interests. The study was supported by the National Natural Science Foundation of China (Grant no. 81360182), the Personnel training projects of the Chinese Academy of Sciences ‘‘Western Light’’ (Grant no. 2012018), the special talents of Ningxia Medical research and start-up projects (Grant no. XT2012015) and Ningxia Medical University Students’ science and technology innovation project (Grant no. 2012).
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