Salidroside attenuates glutamate-induced apoptotic cell death in primary cultured hippocampal neurons of rats

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Salidroside attenuates glutamate-induced apoptotic cell death in primary cultured hippocampal neurons of rats Xia Chen, Jie Liu, Xiaosong Gu, Fei Ding⁎ Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001, PR China

A R T I C LE I N FO

AB S T R A C T

Article history:

Salidroside, a compound of natural origin, has displayed a broad spectrum of

Accepted 9 July 2008

pharmacological properties. This study aimed to evaluate the inhibitory effects of

Available online 22 July 2008

salidroside on glutamate-induced cell death in a primary culture of rat hippocampal neurons as compared to brain-derived neurotrophic factor (BDNF), a usual positive control.

Keywords:

MTT and LDH assays, together with Hoechst 33342 staining, terminal deoxynucleotidyl

Salidroside

transferase dUTP-mediated nicked end labeling (TUNEL) assay and flow cytometric analysis

Glutamate

using annexin-V and propidium (PI) label, indicated that salidroside pretreatment

Apoptosis

attenuated glutamate-induced apoptotic cell death in primary cultured hippocampal

Hippocampal neurons

neurons, showing a dose-dependent pattern. Furthermore, caspase-3 activity assay and calcium measurements with Fluo 4-AM, respectively, revealed that salidroside pretreatment antagonized activation of caspase-3 and elevation of intracellular calcium level, both of which were induced by glutamate stimulation, thus adding to the understanding of how salidroside offered neuroprotection against glutamate excitotoxicity. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Glutamate is a major excitatory amino acid neurotransmitter in the central nervous system (CNS) and its interactions with specific membrane receptors are responsible for many neurological functions, including cognition, memory, movement, and sensation. However, high concentration accumulation of glutamate in CNS and the resulting excessive stimulation of glutamate receptors induce potent neurotoxic action, which, specifically referred to as excitotoxicity, is involved in neuronal damage and degenerative disorders in CNS (Choi, 1988; Rothman and Olney, 1986; Shang et al., 2006). For the prevention and treatment of these diseases, the studies of neuroprotection against glutamate excitotoxicity, especially the search for neuroprotective drugs of natural origin, have attracted increasing research interests.

The hippocampus is responsible for many CNS functions including cognition, learning, and memory, but it is also one of the most vulnerable brain regions as regards to various neurological insults such as hypoxia–ischemia, seizure and prolonged stress. These insults contribute to excessive synaptic-glutamate accumulation, triggering a series of intracellular biochemical changes and finally inducing neuron degeneration even death (Wang et al., 2006). Based on these considerations, the primary cultured hippocampal neurons are commonly used as a culture system for the in vitro study of protection against glutamate-induced neurotoxicity. Rhodiola rosea L. is a popular medicinal plant found in mountains at high altitudes and has long been used in traditional Tibetan medicine system as an adaptogen to enhance the body's resistance to fatigue and to extend human life. The plant displays a range of pharmacological

⁎ Corresponding author. Fax: +86 513 85511585. E-mail address: [email protected] (F. Ding). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.07.051

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Scheme 1 – Chemical structure of salidroside (p-hydroxyphenethyl-β-D-glucoside).

properties, including anti-inflammation, anti-hypoxia, antioxidative, anti-aging, anti-cancer, and hepatoprotection activities (Díaz Lanza et al., 2001; Iaremii and Grigor'eva, 2002; Kanupriya et al., 2005; Kucinskaite et al., 2004). Salidroside, a compound with a chemical structure of phenol glycosides (see Scheme 1), is extracted from the root of Rhodiola rosea as one of its main active ingredients and responsible for all the documented pharmacological effects of the medicinal plant. Recently, salidroside was reported to be capable of protecting the PC12 cells against glutamate-induced excitotoxicity (Cao et al., 2006) or protecting SH-SY5Y human neuroblastoma cells against H2O2-induced cell apoptosis (Zhang et al., 2007). We have also found that salidroside protects PC12 cells against hypoglycemia/serum limitation-induced cytotoxicity in a previous report (Yu et al., 2008). In order to provide a new window into the pharmacological properties of salidroside, the present study was designed to investigate neuroprotection of primary cultured hippocampal neurons of rats, induced by salidroside, against glutamateinduced neurotoxicity, another cell insult different from hypoglycemia/serum limitation-induced cytotoxicity. We hope to expand the understanding of the potential therapeutic value of salidroside for cerebral neurodegenerative diseases.

2.

Results

2.1. Effects of salidroside pretreatment on glutamate-induced decrease of cell viability in hippocampal neurons MTT assay revealed the dose-dependent excitotoxicity of glutamate (31.25–500 μM) on cultured hippocampal neurons (Fig. 1A). In subsequent experiments, an exposure to 125 μM glutamate for 15-min was used to induce cell insult. As illustrated in Fig. 1B, glutamate stimulation decreased the cell viability in hippocampal neurons to 69.31 ± 1.49%, and salidro-

side at very low concentrations (e.g. 30 μM) was not effective for neuroprotection. Salidroside at 60 or 120 μM, however, significantly prevented cultured hippocampal neurons from glutamate-induced damage, and restored the cell survival to 75.33 ± 2.08 or 84.18 ± 1.28%, respectively, displaying dosedependent protective effects. On the other hand, glutamate stimulation significantly increased LDH release of hippocampal neurons from 18.66 ± 1.53 to 43.64 ± 3.05%. The protective effect of salidroside was shown by the changes in LDH release, with the most pronounced effect occurring at 120 μM salidroside that led to the decrease in LDH release from 43.64 ± 3.05 to 36.70 ± 8.94% (Fig. 1C). Brain-derived neurotrophic factor (BDNF, 100 ng/ml), as a usual positive control, also significantly inhibited glutamate-induced cytotoxicity according to either MTT or LDH assay (Figs. 1B and C). In addition, salidroside pretreatment alone resulted in neither cell viability loss nor LDH release change in hippocampal neurons (Figs. 1B and C). The light micrographs confirmed the neuroprotective effects of salidroside. The untreated hippocampal neurons (control) exhibited uniformly dispersed chromatin, normal organelles and intact cell membrane. After exposure to glutamate hippocampal neurons exhibited a significant cell insult evidenced by the disappearance of cellular processes, and decrease of the refraction. However, the cell damage in cultured hippocampal neurons was greatly antagonized by salidroside pretreatment according to micrographic observation (Fig. 1D). Taken together, the results collectively suggest that salidroside pretreatment attenuates glutamate-induced mitochondrial dysfunction and cell membrane damage in cultured hippocampal neurons.

2.2. Effects of salidroside pretreatment on glutamate-induced apoptosis of hippocampal neurons Considering that MTT or LDH release assay failed to distinguish between necrosis and apoptosis, we carried out morphological examinations for determining the type of cell death induced by glutamate. Hoechst staining showed that after the excitotoxic insult of 125 μM glutamate, about 30–35% of hippocampal neurons displayed an apoptotic morphology, characterized by the condensation of chromatin, the nuclear shrinkage, and the formation of a few apoptotic bodies. Pretreatment with 60, 120 μM of salidroside or 100 ng/ml BDNF, however, reduced the excitotoxic effect of glutamate on hippocampal neurons by about 30%, 54% and 60%, respectively (Figs. 2A and B).

Fig. 1 – Cell viability determined using the conventional MTT assay and LDH release assay. Cell viability was assessed 18 h after glutamate stimulation. Although salidroside alone caused no significant cytotoxicity compared to control (P > 0.05), salidroside attenuated glutamate-induced reduction in cell viability of cultured hippocampal neurons. (A) Dose-dependent cytotoxic effects of glutamate on the cell viability of hippocampal neurons. *P < 0.05, **P < 0.01 vs control. (B) Effects of salidroside on the cell viability of hippocampal neurons after exposure to glutamate stimulation. *P < 0.05, **P < 0.01 and ***P < 0.001 vs glutamate stimulation alone. ###P < 0.001 vs control. (C) Effects of salidroside on LDH release of hippocampal neurons after exposure to glutamate stimulation. Here the LDH release percentage was calculated using the formula: (absorbance of sample ÷ absorbance of maximum enzyme activity) × 100. *P < 0.05, **P < 0.01, and ***P < 0.001 vs hippocampal neurons stimulated with glutamate alone. ###P < 0.001 vs control. (D) The light micrographs showing the cell morphology of hippocampal neurons. All data were expressed as mean ± S.D. of four experiments and each included sextuplet.

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Glutamate-induced apoptotic cell death was also confirmed by TUNEL staining in terms of DNA fragmentation (Gavrieli et al., 1992). Exposure to 125 μM glutamate alone yielded 36% TUNEL-positive cells in total hippocampal neurons. Pretreatment with 120 μM salidroside or 100 ng/ml BDNF decreased the ratio of TUNEL-positive cells in total cells to 21 and 19%, respectively (Figs. 2C and D). Similarly, flow cytometric analysis with a detection apoptotic kit was used for distinguishing necrotic from apoptotic cell death. The data demonstrated that stimulation with 125 μM glutamate alone induced a 375% elevation of apoptotic cells and a 218% increase of necrotic cells in hippocampal neurons as compared to control. In pretreatment with 60, 120 μM salidroside and 100 ng/ml BDNF, however, glutamateinduced cell apoptosis was significantly suppressed by 24, 36 and 41.8% respectively, while necrosis showed no significant alternations (Figs. 3A and B).

2.3. Inhibition of caspase-3-like protease activity in cell lysates of hippocampal neurons on exposure to glutamate by salidroside pretreatment Exposure to glutamate has been shown to induce activation of pro-apoptotic proteins including caspases (Li et al., 2007). In this study, we found that a 15-min exposure of hippocampal neurons to 125 μM glutamate followed by incubation for different times induced the time-dependent elevation in the enzymatic activity of caspase-3 (Fig. 4A). Preincubation of hippocampal neurons with Z-DEVD-FMK, a cell-permeable irreversible inhibitor of caspase-3-like enzymes, (Ekert et al., 1999) significantly attenuated the decrease in the cell viability induced by glutamate stimulation (Fig. 4B). This suggests that caspase-3-like proteases are involved in the glutamateinduced apoptotic death of hippocampal neurons. Moreover, we also found that pretreatment with salidroside (60 or 120 μM) or 100 ng/ml BDNF, respectively, led to a significant decrease in caspase-3 activity compared to stimulation with glutamate alone (Fig. 4C), suggesting the suppressive effect of salidroside on glutamate-induced cell death.

2.4. Effects of salidroside pretreatment on expression of Bcl-2 and Bax in cultured hippocampal neurons after exposure to glutamate According to Western blot analysis, the total amount of anti-apoptotic protein Bcl-2 or pro-apoptotic protein Bax in cultured hippocampal neurons was not significantly changed after a 15-min exposure to 125 μM glutamate followed

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by incubation for 6-, 12-, or 24-h (Fig. 5A), and the total amount of both proteins in cultured hippocampal neurons subjected to pretreatment with different concentrations of salidroside plus glutamate stimulation was not significantly different from that subjected to glutamate stimulation alone (Fig. 5B).

2.5. Inhibition of glutamate-induced intracellular calcium increase in cultured hippocampal neurons by salidroside pretreatment We measured intracellular calcium levels ([Ca2+]i) in cultured hippocampal neurons after exposure to 125 μM glutamate, because the intracellular calcium overload is considered to be a prominent feature of glutamate-mediated excitotoxicity. As shown in Figs. 6A and B, glutamate stimulation triggered a rapid elevation in [Ca2+]i by about 2.5-fold. However, pretreatment with 120 μM salidroside or 100 ng/ml BDNF significantly lowered [Ca2+]i in hippocampal neurons compared to glutamate stimulation alone.

3.

Discussion

A high concentration of glutamate induces neuronal cell damage under in vitro (Ankarcrona et al., 1995b; Choi, 1987; Li et al., 2007) or in vivo conditions (Koh et al., 1990; PorteraCailliau et al., 1997; Turski et al., 1991). The type of cell death (apoptosis versus necrosis) changes with the timing and extent of the insult (Liu et al., 2004; Nicotera et al., 1999). In this study, exposure to glutamate resulted in the cell viability loss of hippocampal neurons in a dose-dependent manner. The morphological examinations indicated that exposure to glutamate led to extensive apoptotic-like cell death in primary cultured rat hippocampal neurons. These results are consistent with the previously reported findings that stimulation with a certain concentration of glutamate within a delayed time period induces neuronal death in a prevailing form of apoptosis under in vitro conditions (Ankarcrona et al., 1995a; Cheung et al., 1998; Chihab et al., 1998), despite the underlying mechanism that remains unclear (Bonfoco et al., 1995; Dessi et al., 1994; Portera-Cailliau et al., 1997). Salidroside, a major active ingredient isolated from the plant Rhodiola rosea, has been reported to have a wide range of pharmaceutical properties. This study aimed to explore the neuroprotective effects of salidroside against glutamateinduced cell damage in hippocampal neurons. Our data indicated that salidroside itself caused no conspicuous

Fig. 2 – Hoechst 33342 staining and TUNEL assay in cultured rat hippocampal neurons. Cells were stained with fluorescent dye 18 h after glutamate stimulation. Salidroside pretreatment significantly decreased glutamate-induced nuclear condensation and DNA fragmentation of cultured hippocampal neurons. (A) Morphological apoptosis was determined by staining with Hoechst 33342. (B) The percentage of nuclear condensation in the cultured hippocampal neurons was counted in response to glutamate stimulation alone. (C) Morphological apoptosis was determined by TUNEL assay. Green-stained cells (a) were TUNEL-positive cells. All nuclei were stained with propidium iodide (b). The merge of a and b is c. (D) The ratio of TUNEL-positive hippocampal neurons in the total hippocampal neurons. ###P < 0.001 vs control. *P < 0.05, **P < 0.01 and ***P < 0.001 vs glutamate stimulation alone. All data were expressed as mean ± S.D. of three experiments and each included sextuplet. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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pretreatment enhances the ability of hippocampal neurons to counteract glutamate-induced mitochondrial dysfunction and cell membrane damage. Activation of caspase-3 is a hallmark of apoptotic cell death and precedes the changes in nuclear morphology (Almeida et al., 2005; Degterev et al., 2003). In the present study, exposure of cultured hippocampal neurons to glutamate was shown to induce the elevation of caspase-3 activity, which was inhibited by the addition of Z-DEVD-FMK, thus providing further evidence that cell apoptosis was the prevailing type of

Fig. 3 – Flow cytometric analyses with annexin-V-FITC and PI label in cultured rat hippocampal neurons. Cells was stained with fluorescent dye 18 h after glutamate stimulation. Salidroside pretreatment significantly suppressed glutamate-induced cell apoptosis, while necrosis showed no significant alternations. (A) Apoptosis determined by staining with annexin-V + PI. (B) The percentage of apoptotic or necrotic hippocampal neurons in total hippocampal neurons, ##P < 0.01, ###P < 0.001 vs control. *P < 0.05, **P < 0.01 and ***P < 0.001 vs glutamate stimulation alone. All data were expressed as mean ± S.D. of three experiments and each included sextuplet.

alterations in the neuronal viability. However, pretreatment with different concentrations (30, 60 or 120 μM) of salidroside decreased the cell viability loss and LDH release induced by glutamate, which was in parallel with the morphological analyses obtained by Hoechst 33342 staining, Flow cytometry and TUNEL assay. These results suggest that salidroside

Fig. 4 – Salidroside inhibited caspase-3-like activity induced by glutamate. (A) The caspase-3-like protease activity in hippocampal neurons at different incubation times following glutamate stimulation as measured using colorimetric substrate, Ac-DEVD-pNA. (B) Glutamate-induced cell death were inhibited by z-DEVD-fmk. Hippocampal neurons were pretreated with 100 μM z-DEVD-fmk for 12 h, and then exposed to 125 μM glutamate for 15 min followed by incubation for 6 h and examination for cell viability by MTT assay. Data were presented as mean ± S.D. of four experiments performed in independent preparations. ### P < 0.001 vs Control; **P < 0.01 vs glutamate stimulation alone. (C) Effects of salidroside on the caspase-3-like protease activity in hippocampal neurons after exposure to glutamate stimulation. ###P < 0.001 vs Control; *P < 0.05, **P < 0.01 and ***P < 0.001 vs glutamate stimulation alone.

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Fig. 5 – Effects of salidroside on expression of apoptotic signaling proteins Bcl-2 and Bax in cultured hippocampal neurons after the application of glutamate. (A) Effects of glutamate on expression of total amounts of Bax, and Bcl-2. The hippocampal neurons were challenged with 125 μM glutamate for 15 min, followed by incubation in neuronal culture medium for 6, 12 or 24 h. Total proteins (10 μg) were analyzed by Western blotting with anti-Bax, anti-Bcl-2, and anti-actin antibodies. There were no changes in the total amounts of these proteins after glutamate stimulation. (B) Effects of salidroside pretreatment on the total amount of Bax or Bcl-2 after glutamate stimulation. Total proteins (10 μg) were analyzed by Western blotting with anti-Bax, anti-Bcl-2 and anti-actin antibodies. The total amount of either protein in hippocampal neurons subjected to salidroside pretreatment plus glutamate stimulation was comparable with that subjected to glutamate stimulation alone.

glutamate-induced cell death in hippocampal neurons. We also observed that salidroside pretreatment significantly antagonized the elevation of caspase-3 activity. This suggests that salidroside protects hippocampal neurons at the level or upstream of the activation of caspase-3-like enzymes. Bcl-2 family proteins play critical roles in apoptotic cell death induced by a wide array of death signals (Antonsson, 2004). Among them, Bcl-2 and Bcl-XL are anti-apoptotic, while Bax, Bcl-Xs, Bad, Bak and Bik are pro-apoptotic. The relative level of Bcl-2 family proteins determines whether cells undergo survival or apoptosis (Kroemer, 1997; Yang and Korsmeyer, 1996). In our study, we found that the total amount of either Bax or Bcl-2 protein was unaffected after glutamate stimulation, regardless of pretreatment with or without salidroside. However, we cannot rule out the possibility that under more severe pathological conditions, such as application of glutamate at higher concentrations and/or for longer periods, salidroside protects hippocampal neurons against glutamate-induced neurotoxicity through regulation of the Bcl-2 family proteins in apoptotic signaling (Ishihara et al., 2005). Although we did not find that salidroside pretreatment followed by glutamate stimulation significantly affected the expression of Bax and Bcl-2 proteins, the examination on the change in [Ca2+]i contributed to the elucidation of the mechanisms responsible for the neuroprotective effect of salidroside against glutamate-induced apoptosis. The view of glutamate-induced [Ca2+]i overload within neurons has been widely accepted as one of the mechanisms of glutamateinduced excitotoxicity (Choi and Rothman, 1990; Kristian and

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Siesjo, 1996; Nishizawa, 2001). Calcium release may impair mitochondrial function and activate certain phospholipase, protease, and endonucleases, leading to irreversible damage to the membrane, organelles, and chromatin, and eventually cell death (Trump and Berezesky, 1995). The results in this study showed that exposure to glutamate evoked the increase of Ca2+ influx, whereas salidroside pretreatment significantly blocked glutamate-induced elevation in [Ca2+]i. It follows that the neuroprotection of salidroside might be associated with the inhibition of the calcium overload.

Fig. 6 – Effects of salidroside on [Ca2+]i in hippocampal neurons. (A) Time course of change in [Ca2+]i. (B) Effects of salidroside pretreatment on peak [Ca2+]i after glutamate stimulation. Data are presented as mean ± S.D. of three experiments performed in independent preparations. ### P < 0.001 vs Control; *P < 0.05 and **P < 0.01 vs glutamate.

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In conclusion, salidroside efficiently protects hippocampal neurons against glutamate-induced apoptotic cell death. The protective effects are mediated by inhibiting the increased caspase-3-like activity and excessive Ca2+ influx triggered by glutamate. The neuroprotective effect of salidroside might be used to develop a potential therapeutic approach for preventing and/or treating neuronal damage and degenerative disorders. Further studies need to be done to explore the underlying mechanisms.

4.

Experimental procedures

4.1.

Materials

Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), trypsin, poly-L-lysine, Neurobasal medium, B27, Hoechst 33342, the monoclonal mouse anti-Bcl-2 antibody, antiBax antibody and anti-β-actin antibody, IRDye 800-conjugated goat anti-mouse IgG, L-glutamate, and Brain-derived neurotrophic factor (BDNF) were purchased from Sigma (St. Louis, MO). Caspase-3/CPP32 colorimetric assay kit and Z-DEVD-FMK were purchased from BioVision (Mountain View, CA). Salidroside were obtained from the National Institute for the control of pharmaceutical and biological product (Beijing, China). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and lactate dehydrogenase (LDH)-Cytotoxic test kit were purchased from Genmed (Westbury, NY). Molecular Probes Fluo-4/acetoxymethyl ester (Fluo-4/AM) were purchased from Molecular Probes (Eugene, OR). AnexinV/PI detection apoptotic kit was purchased from BD BioSciences (San Jose, CA). dUTP-mediated nicked end labeling (TUNEL) assay kit was purchased from Promega (Madison, WI).

4.2.

Cell culture and treatment

Primary cultures of rat hippocampal neurons were prepared from the hippocampi of E18–E19 Sprague–Dawley (SD) rat embryos (obtained from the Experimental Animal Center of Nantong University. China). After treatment with 0.25% trypsin for 15 min at 37 °C in Ca2+ and Mg2+-free Hank's balanced salt solution (Ambrosio et al., 2000), the hippocampi were washed in DMEM with 10% FBS in order to stop trypsin activity. Then the cells were resuspended in DMEM supplemented with 10% FBS and plated onto poly-L-lysine-coated plates for 4 h at 37 °C in a humidified atmosphere of 95% air and 5% CO2. After cells attached to the substrate, the medium was replaced with neuronal culture medium, namely serumfree Neurobasal medium with 2% B27 supplement, 0.5 mM glutamine, 100 U/mL penicillin/100 U/mL streptomycin, followed by re-incubation for 7–8 days, the time required for maturation of hippocampal neurons, with half of the medium being changed every 3 days (Ambrosio et al., 2000; Jiang et al., 2005; Julien and Mushynski, 1998; Pennypacker et al., 1991). Then, the cells were characterized by immunohistochemistry for neurofilament protein and fibrillary acidic protein, revealing that the cell cultures contained about 95% neurons. The primary cultured hippocampal neurons were pretreated with 30, 60, or 120 μM salidroside or 100 ng/ml BDNF, respectively, for 24 h, followed by exposure to 125 μM

glutamate with 10 μM of glycine in supplemented neuronal culture medium for 15 min at 37 °C in a humidified incubator of 5% CO2/95% air. After excitotoxicity was induced, the cells were further incubated with the neuronal culture medium at 37 °C for desired time periods. The hippocampal neurons undergoing neither salidroside pretreatment nor glutamate stimulation served as control.

4.3.

Cell viability test

4.3.1.

MTT assay

The cell viability was determined using the conventional MTT assay based on the cleavage of the yellow tetrazolium salt MTT to purple formazan by mitochondrial enzymes in metabolically active cells (Denizot and Lang, 1986). Briefly, hippocampal neurons were cultured in 96-well plates at a density of 5 × 105 cells per well. After stimulation with glutamate, the cells were further incubated with the neuronal culture medium for a certain time period, and then 10 μl of MTT solution were added to each well for incubation at 37 °C for 4 h. Afterwards, 100 μl of 20% sodium dodecyl sulfide (SDS) solution were added to each well to dissolve the precipitate for 20 h and the absorbance was measured by spectrophotometry at 570 nm with an ElX-800 Microelisa reader (Bio-Tek Inc., Winooski, VT). The data were expressed as a percent of control value.

4.3.2.

LDH release assay

Cytotoxicity was quantified by measurement of LDH released in the medium by using a LDH-Cytotoxic test kit according to the manufacturers' instructions. Briefly, hippocampal neurons were cultured in 96-well plates at a density of 5 × 105 cells per well. Then 10 μl of cell lysis solution was added to untreated cells, which were selected as the control of maximum LDH activity. The treated cells were added with equal volume of balanced solution. After 1 h treatment, 50 μl of the supernatant was transferred to the corresponding well of a fresh 96-well plate and was mixed with 50 μl of the LDH substrate. After incubation for 0.5 h at room temperature, the reaction was stopped by adding 100 μl of stop buffer and the absorbance was measured at 490 nm with an ElX-800 Microelisa reader (BioTek, Inc.). The LDH release percentage was calculated using the formula: (absorbance of sample ÷ absorbance of maximum enzyme activity) × 100.

4.4.

Cell apoptosis assessment

4.4.1.

Hoechst 33342 staining

The hippocampal neurons were cultured on poly-L-lysinecoated glass coverslips at a density of 5 × 104 cells/cm2. Then the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. After stained with 10 μg/ml Hoechst 33342 for 10 min, the cells were observed under a DMR fluorescence microscope (Leica Microsystems, Wetzlar, Germany) on ultraviolet illumination. The Hoechst dye was excited at 340 nm, and fluorescence emission was filtered with a 510 nm barrier filter.

4.4.2.

TUNEL assay

The hippocampal neurons were grown on poly-L-lysine-coated glass coverslips, at a density of 5 × 104 cells/cm2. After treated as

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described above, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. Then fixed cells were washed and fragmented DNA was detected in apoptotic cells by adding fluorescein 12-dUTP to nicked ends of DNA. Slides were incubated for 1 h at 37 °C, and the reaction was terminated with 2 × SSC (300 mM sodium chloride and 30 mM sodium citrate, pH 7.4). Then the cells were stained with propidium iodide (PI). Apoptotic cells were detected as localized bright green cells (positive cells) in a red background by scanning laser confocal microscopy (Leica, Heidelberg, Germany). Data were expressed as the ratio of apoptotic neurons to total neurons.

4.4.3.

Flow cytometric analysis

The hippocampal neurons that had been treated as described above were harvested and washed with Ca2+ and Mg2+-free PBS. After centrifugation at 1500 rpm for 5 min, the cells was resuspended in 1 × binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) at a density of 1 × 106 cell/ml. Cells (100 μl) were transferred to a culture tube, and 5 μl of annexin-V-FITC and 5 μl of PI were added. Following gentle vortex, the tube was incubated for 15 min at room temperature (20–25 °C) in the dark, 400 μl of 1 × binding buffer was then added into it. The sample was analyzed using a dual-laser FACS VantageSE flow cytometer (Becton Dickinson, Mountain View, CA) within a 1 h period. The percentages of apoptotic and necrotic cell for each sample were estimated.

4.5.

temperature for another 2 h. The images were scanned with GS800 Densitometer Scanner (Bio-Rad, Hercules, CA), and the data of optical density were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-actin was used as an internal reference.

4.7.

[Ca2+]i measurement

The [Ca2+]i measurements were accomplished with Fluo-4/AM by scanning laser confocal microscopy (Leica, Heidelberg, Germany). After pretreatment with or without salidroside for 24 h, the hippocampal neurons were incubated with 5 μM Fluo-4/AM at 37 °C and 5% CO2 for 45 min in the dark and then washed to remove extracellular Fluo-4/AM dye, followed by stimulation with 125 μM glutamate. After addition of glutamate, laser scanning began to obtain time series of images over a short time period (15 min) at excitation and emission wavelengths of 488 nm and 526 nm, respectively. The obtained images were quantitatively analyzed for changes in fluorescence intensities within cells. The data were expressed as the relative fluorescence intensity.

4.8.

Statistical analysis

Data were expressed as mean ± S.D. Statistical significance was determined by one-way analysis of variance (ANOVA) and subsequent Bartlett's test. Differences were considered significant at p b 0.05.

Caspase-3 activity measurement

Cellular caspase-3 activity was assayed using a caspase-3/ CPP32 colorimetric assay kit. After stimulation with glutamate, the cells were further incubated with the neuronal culture medium for 6 h at 37 °C. The cells were then lysed in a lysis buffer and centrifuged. The supernatant was collected, and a volume containing 500–100 μg of protein was diluted to 50 ml with lysis buffers for each assay. The lysate was then mixed with 50 μl of 2 × reaction buffer (containing 10 mM dithiothreitol), and 200 μM DEVD-pNA substrate was added. The reaction was performed in a 37 °C water bath for 1–2 h. The cleaved pNA, with a light emission at 405 nm, was quantified by using an ElX-800 Microelisa reader (Bio-Tek).

4.6.

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Western blot analysis

After cell treatment, the hippocampal neurons (1 × 106 cells/ml each plate) were collected and subjected to Western blot analysis for Bcl-2 and Bax protein expression. Cell proteins were extracted and quantified by a BCA-100 Protein Quantitative Analysis Kit as described previously (Xu et al., 2006). After addition of sample loading buffer, protein samples were electrophoresed on a 12% SDS-PAGE and subsequently transferred to PVDF membrane (Millipore, Bedford, MA, USA). The membrane was incubated in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4, containing 5% nonfat dried milk) at room temperature for 30 min and then probed with the monoclonal mouse anti-Bcl-2 antibody, anti-Bax antibody and anti-β-actin antibody in blocking buffer at 4 °C overnight. The membrane was washed three times for 5 min each using PBST (PBS and 0.1% Tween 20). Afterwards it was incubated in the IRDye 800-conjugated goat anti-mouse IgG at room

Acknowledgment This study was supported by Hi-Tech Research and Development Program of China (973 Program, Grant No. 2003CB515306).

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