Scutellarin Attenuates Oxidative Glutamate Toxicity in PC12 Cells

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Materials
• Cell culture and treatment
• MTT assay for cell survival
• Assay of LDH leakage
• Measurement of intracellular ROS accumulation
• Thiobarbituric acid-reactive substance (TBARS) assay
• Glutathione assay
• Estimation of GSSG reductase activity
• Data analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

The present study investigated the protective effects of the antioxidant scutellarin against oxidative toxicity induced by glutamate in PC12 cells. Vitamin E, a classical antioxidant was employed as a comparative agent. Incubation of PC12 cells with 10 mM glutamate resulted in significant cytotoxity as evaluated by the MTT and lactate dehydrogenase (LDH) assays, decreases of GSSG reductase activity, disturbance of the cell redox state as indicated by the GSH/GSSG ratio, and accumulation of intracellular reactive oxygen species (ROS) and lipid peroxidation products. Scutellarin at 0.1, 1 and 10 μM significantly protected against the cytoxicity and production of ROS and lipid peroxidation induced by glutamate. Scutellarin did not prevent the reduction of cellular GSH levels, but it up-regulated GSSG reductase activity, thus preventing an increase in cellular GSSG levels, and concomitantly improved the cell redox status. Our data also show that the protective effects of scutellarin against glutamate-induced oxidative toxicity are more potent than that of vitamin E. These results demonstrate that scutellarin can protect PC12 cells from oxidative glutamate toxicity by scavenging ROS, inhibiting lipid peroxidation and improving the cell redox status, and may reduce the cellular damage in pathological conditions associated with excessive glutamate release.

Abbreviations

MTT:3-(4,5-dimethythiazole-2-yl)-2,5-diphenyl-tetrazo-lium bromide

LDH:lactate dehydrogenase

ROS:reactive oxygen species

GSH:reduced glutathione

GSSG:oxidized glutathione

TBARS:thiobarbituric acid-reactive substance

DCFH-DA:6-carboxy-2′,7′-dichloroflurescin diacetate

Vit E:vitamin E

Introduction

Glutamate is thought to be involved in the pathogenesis of a number of different neurodegenerative diseases [1]. There are two forms of glutamate toxicity: receptor initiated excitotoxicity [2] and non-receptor-mediated oxidative glutamate toxicity [3]. Oxidative glutamate toxicity is initiated by high concentrations of extracellular glutamate that prevent cystine uptake into the cells, followed by the depletion of intracellular cysteine and the loss of GSH. With a diminishing supply of GSH, there is an accumulation of excessive amounts of ROS and ultimately cell death. Since oxidative glutamate toxicity is not only distinct from both classical apoptosis and necrosis but also dependent upon oxidative stress and ROS production, it is termed oxytosis [4].

Scutellarin (Fig. [1]), a known flavone 7-O-glucuronide, is a primary active ingredient in breviscapine. Breviscapine is a mixture extracted from the Chinese herb, Erigeron breviscapus (Vant.) Hand.-Mazz. and contains scutellarin, 4′-hydroxybaicalein 7-O-β-D-glucopyranosiduronic acid methyl ester, baicalein 7-O-β-D-glucopyranoside, etc. [5]. It has been proved that breviscapine is effective for ischemic cerebrovascular diseases. In vivo, breviscapine and its preparation can protect against cerebral ischemia-reperfusion injury by many pathways of action [6]. In China breviscapine injections are extensively used for the treatment of ischemic cerebrovascular diseases. In the course of screening compounds of breviscapine, we found that scutellarin showed a stronger antioxidative activity. However, there is insufficient information on the neuroprotection of scutellarin, as a major component of breviscapine, especially at the cellular level. In this study, we chose PC12 cells to investigate the effects of scutellarin on glutamate-induced oxidative cytotoxicity.

Materials and Methods

Materials

The plants of Erigeron breviscapus (Vant.) Hand.-Mazz were collected in May 2001 in Qiubei County, Yunnan Province of China. A voucher specimen (0 105 169), authenticated by Professor J.-Q. Zhou, was deposited in the herbarium of Anhui College of Traditional Chinese Medicine, Hefei, P. R. of China. The isolation of scutellarin (Fig. [1]) was performed as described by Zhang et al. [7]. Its purity, determined by HPLC method, was 98.06 %. 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Fluka, and Dulbeco’s Modified Eagles Medium (DMEM) from Gibco. 6-Carboxy-2′,7′-dichlorofluorescin diacetate (DCFH-DA), thiobarbituric acid-reactive substances (TBARS), tetraethoxypropane, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and bovine serum albumin (BSA) were purchased from Sigma. D-α-Tocopherol succinate (vitamin E) was purchased from BASF Vitamins Co. Ltd. All other chemicals were of analytical grade.

Cell culture and treatment

The PC12 cell line was obtained from Shanghai Institute of Cell Biology and maintained at 37 °C in a humidified atmosphere containing 5 % CO₂ in high glucose DMEM supplemented with 15 % (V/V) heat-inactivated fetal calf serum, 100 kU/L penicillin and 100 mg/L streptomycin. Cells were plated at 5,000 cells/cm² on poly-L-lysine coated (10 μg/mL) 24-well plates (0.5 mL/well) for assays of LDH, GSH, GSSG, GSSG reductase activity, and lipid peroxidation products, and on 96-well plates (100 μL/well) for cell survival and ROS assay. Scutellarin or vitamin E was added to the culture 0.5 h prior to glutamate addition for all assays. Scutellarin and vitamin E test samples were made by diluting 1000-fold concentrated solutions prepared in 100 % ethanol; 0.1 % (V/V) ethanol had no protective or toxic effect by itself. Control cultures were performed in the presence of ethanol, under the same culture conditions.

MTT assay for cell survival

Cell survival was evaluated by the MTT reduction assay [8]. In brief, The confluent monolayers of PC12 cells were treated with different concentrations of scutellarin (0.1 - 10 μM) or 10 μM vitamin E plus glutamate for 24 h. Cell culture medium in each well was aspirated and replaced with fresh DMEM containing 0.5 mg/mL MTT. After 4 h incubation at 37 °C, 100 μL of solubilization solution (50 % dimethylformamide and 20 % SDS, pH 4.8) were then added to each individual well. The mixtures were kept overnight at 37 °C and then the amount of MTT formazan was quantified by determining the absorbance at 570 nm using an ELISA plate reader (Hua Dong Electronic Co, Nanjing, China). Background correction was performed with extracts of cells not treated with MTT.

Assay of LDH leakage

To supplement the MTT assay, we used a commercially available detection kit to measure LDH activity released from PC12 cells. After cells were exposed to 10 mM glutamate in the presence of scutellarin or vitamin E for 24 h, total culture media were collected and centrifuged to remove contaminating cells and cellular debris. The volume of media was then measured. 50 μL of each sample were transferred to a 10-mL test tube. LDH assay reagents were added to each tube according to the protocol (provided by Nanjing Jiancheng Bioengineering Institute) [9] and the absorbance of samples was measured at 440 nm. To assay LDH inside the cells, a hypotonic solution containing 15 mM Tris, at pH 7.4, was added to each well, and cells were incubated for 30 min with gentle shaking. LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the medium + LDH in the cells), according to the equation %LDH released = (LDH activity in the medium/total LDH activity) × 100.

Measurement of intracellular ROS accumulation

The cellular ROS was quantified as described by Hong et al. [10]. Briefly, the cells on poly-L-lysine coated 96-well plate were incubated with 100 μM DCFH-DA in the loading medium in 5 %CO₂/95 % air at 37 °C for 30 min. After DCFH-DA was removed, the cells were washed once with DMEM and incubated for 30 min in DMEM containing different concentrations of scutellarin (0.1 - 10 μM) or vitamin E (10 μM). Then 10 mM glutamate was added. After 30 min incubation, the fluorescence from each well was quantified using 1420 Victor² V (Perkin Elmer Life Science, USA). The percentage increase in fluorescence per well was calculated by the formula [(Ft₃₀ - Ft₀)/Ft₀ × 100], where Ft₀ refers to the fluorescence at time 0 min and Ft₃₀ is the fluorescence at time 30 min in the presence of glutamate.

Thiobarbituric acid-reactive substance (TBARS) assay

After treatment with pharmacological agents, the amounts of lipid peroxide were quantified using TBARS [11]. Briefly, the cell homogenate (prepared in 0.5 mL PBS with 1 % SDS from 4 × 10⁶ cells/pellet) was mixed with 3 mL of 1 % phosphoric acid, 1 mL of 0.67 % thiobarbituric acid and 0.04 % BHT in glass test tubes, and the mixtures were incubated in a boiling water bath for 60 min. Marbles were placed on the tops of tubes during the incubation period to avoid excessive loss of reaction mixture. After cooling the tubes in ice, 1.5 mL of n-butanol were added and the reaction mixture was centrifuged at 1000 g for 10 min. The absorbance of the supernatant was read at 535 nm. The concentrations of TBARS were calculated using tetraethoxypropane as a reference standard.

Protein concentrations in cell homogenates were determined using the Coomassie blue method [12]. BSA was used as a reference standard. The quantities of TBARS were expressed in terms of amount (nmol) per mg protein and converted to percentage of control values to compensate for variations of absolute weights of lipid oxides among the different experiments.

Glutathione assay

The GSH content was measured using a method described by Cooper et al. with minor modification [13]. Briefly, cells were incubated for 24 h in the presence of scutellarin or vitamin E plus glutamate and then washed with cold PBS once and lysed with 2.5 % sulfosalicyclic acid, quickly frozen at -70 °C and thawed to ensure cell lysis. Cell lysates were centrifuged at 10,000 g for 10 min and 100 μL of supernatant were added to 10 μL H₂O. After 5 min incubation, 20 μL of 3 M K₃PO₄ ( pH 13.5 ) were added for 10 min, followed by 50 μL of 10 % sulfosalicyclic acid. GSH levels were assayed by adding 100 μL of a reaction buffer containing 100 mM sodium phosphate, 1 mM EDTA, 0.5 mM 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), 0.3 mM NADPH, and 2.8 units/mL GSH reductase. To measure oxidized GSH (GSSG), 100 μL of supernatant were added to 900 μL of 11 mM N-ethylmaleimide in 100 mM potassium phosphate buffer, containing 5 mM EDTA, pH 7.5. After 20 min incubation at room temperature, samples were assayed by the DTNB method. The reactions were monitored by the changes in absorbance at 412 nm with a microplate reader. The intracellular GSH and GSSG contents calculated from a standard curve and normalized to total cell protein. The protein content was determined using the Coomassie blue method [12]. Results were expressed in nmol GSH or GSSG/mg protein.

Estimation of GSSG reductase activity

GSSG reductase activity was assessed by monitoring the activity of the cytoplasmic enzyme spectrophotometrically, according to manufacturer’s protocol. The rate of conversion of NADPH was followed at 340 nm. The results were expressed as catalytic activity concentration (C) following the equation C (U/mg) = (ΔA)/(ε × d × Δt × v × mg) where ΔA/Δt refers to the rate of absorbance change, ε refers to the linear millimolar absorption coefficient of NADPH (0.631 × mmol^-1 × mm^-1), d is the light path (10 mm), v is the sample volume and mg is the sample protein content expressed in mg.

Cells were incubated with glutamate plus scutellarin or vitamin E for 24 h. The medium was then discarded and the cells were incubated in a hypotonic solution containing 15 mM Tris, at pH 7.4. The enzymatic assay was performed on 100 μL of centrifugation supernatants (14,000 × g, 5 min), and pellets, resuspended in lysis buffer, were used for protein determination [12]. Results for GSSG reductase activity were expressed as U/mg protein.

Data analysis

Throughout the test, data were expressed as means ± SD of n experiments. Statistical significance analysis was determined using Student’s t-test.

Results

Glutamate exposure produced severe inhibition of MTT reduction in PC12 cells without treatment of scutellarin (Table [1]). When cells were preincubated with scutellarin (0.1 - 10 μM) or vitamin E (10 μM), glutamate-induced cell toxicity was significantly attenuated. The protective effect of scutellarin was dose-dependent and maximal at 100 μM (0.97 ± 0.05). Scutellarin showed no cell protection at a dose of 0.01 μM (data not shown). The viability of the cell culture was not modified when control groups were pretreated with 10 μM scutellarin as compared to the vehicle-treated control group (102 ± 3 vs. 100 ± 1 for MTT), indicating that the protective effect of scutellarin is not attributable to an effect on cell division and/or neurite outgrowth. To further investigate the protective effects of scutellarin, the LDH leakage, another non-morphological cell toxicity was assessed. As shown in Table [1], a significant increase in LDH release reflecting cytotoxicity was observed after 24 h exposure to 10 mM glutamate. The preincubation of cells with 0.1, 1, and 10 μM scutellarin, or 10 μM vitamin E, significantly attenuated this increase in LDH release. The pattern of protective effect of scutellarin on glutamate-induced cytotoxicity determined by LDH assay was similar to that determined by the MTT assay.

Glutamate induced about twice the DCF fluorescence at a concentration of 10 mM as compared with the control (21.26 ± 2.27 % vs. 11.25 ± 1.21). Cultures pretreated with 0.1, 1, 10 μM scutellarin or 10 μM vitamin E showed significantly reduced intensity of DCF labeled cells when compared to glutamate-treated cultures (Table [2]). The resultant oxidative stress assay showed that glutamate exposure produced a significant increase in lipid peroxidation, representing an 85 % increase above untreated control levels. Pretreatment with 0.1, 1, 10 μM scutellarin and 10 μM vitamin E significantly alleviated glutamate-induced lipid peroxide production (Table [2]).

Exposure of cells to 10 mM glutamate for 24 h significantly reduced intracellular GSH levels, while intracellular GSSG levels were markedly increased. Resulting decrease of GSH/GSSG ratio was shown (Table [3]). Treatment of the cells with scutellarin or vitamin E completely failed to protect the cells against the glutamate-induced depletion of GSH levels (p > 0.05). This treatment decreased the production of GSSG in PC12 cells, and subsequently increased the GSG/GSSG ratio. Glutamate exposure down-regulated GSSG reductase activity (p < 0.01, 20.01 ± 2.88 U/mg protein after incubation with 10 mM glutamate in comparison with 30.01 ± 3.36 U/mg protein in control cells). Pretreatment of scutellarin or vitamin E protected cells against glutamate-induced reduction of the enzymatic activity of GSSG reductase (Fig. [2]). (The activities in cells incubated with 10 mM glutamate were 25.13 ± 2.56, 25.55 ± 3.31 and 28.12 ± 4.26 U/mg protein in the presence of 0.1, 1, and 10 μM scutellarin, respectively, and 27.84 ± 2.81 U/mg protein in the presence of 10 μM vitamin E).

Discussion

Our studies have revealed that high concentrations of extracellular glutamate exposure induced oxidative glutamate toxicity (oxytosis), characterized by a number of potentially detrimental changes in intracellular ROS, lipids, GSH, GSSG and GSSG reductase activity in PC12 cells, and that treatment with the antioxidant scutellarin can inhibit intracellular accumulations of ROS and products of lipid peroxidation, up-regulate GSSG reductase activity, improve cell redox state as indicated by the GSH/GSSG ratio, and significantly attenuate glutamate-induced cytotoxicity as evaluated by the MTT and LDH assays. Scutellarin failed to protect against the glutamate-induced depletion in GSH levels, but it could compensate for GSH loss in protecting cells from this challenge, indicating that GSH depletion is not sufficient in itself to cause cell death. It is a central finding of our study that both scutellarin and vitamin E can protect GSH-depleted cells from death.

GSSG reductase is the enzyme responsible for the conversion of GSSG into GSH using reducing equivalents of NADPH [3]. Up-regulation of the enzyme in the presence of scutellarin involved in GSH metabolism is an important mechanism of protection against oxytosis. The reduction of intracellular GSSG by scutellarin may contribute to compensation for GSH and up-regulation of GSSG reductase activity. How scutellarin up-regulates GSSG reductase activity remains to be investigated. This may be associated with the antioxidant response element contained in its gene [14]. The reduced GSSG levels resulted in an increase in the GSH/GSSG ratio and the cellular redox state was improved to enhance resistance to oxidative stress insult in the presence of scutellarin.

In the present study, we used vitamin E as a positive control instead of ascorbate. Ascorbate in culture medium is rapidly oxidized to dehydroascorbate, which is transported into cells by the glucose transporter (GLUT), and generates an oxidative stress which triggers apoptosis [15]. Vitamin E, a classical antioxidant, has been considered to have protective effects on glutamate cytotoxicity in vitro [16]. The comparison of the two antioxidants with different structures indicated that scutellarin, presumably having high catalytic activities or greater membrane permeability, exhibited a higher potency against oxidative stress induced by extracellular glutamate than vitamin E. One possible mechanism underlying the effectiveness of scutellarin against oxidative stress induced by glutamate involves its polyphenolic structure since it is known that plant-derived polyphenolic compounds are potent antioxidants and free radical scavengers [17], whereas the protective action of vitamin E may result primarily from its ability to donate hydrogen to radicals, usually peroxyl and alkoxyl radicals, thus preventing the propagation of lipid peroxidation [16].

In summary, scutellarin possesses potent antioxidative properties and protects cells from oxidative glutamate toxicity, which suggests that it may slow the clinical progression of the diseases in which oxidative stress and excessive glutamate release (or deficient uptake) seem to be involved.

Acknowledgements

The authors are grateful to Professor Wen-Cai Ye and Professor Gwen Crotts for their valuable comments on this manuscript.

References

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Guo-Qing Liu

Department of Pharmacology

China Pharmaceutical University

24 Tong Jia Xiang

Nanjing 210009

The People’s Republic of China

Fax: +86-25-3271340

Email: haohongchina@hotmail.com

References

• 1 Coyle J T, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders.  Science. 1993;  262 689-95
  CrossrefPubMedSearch in Google Scholar
• 2 Choi D W. Glutamate neurotoxicity and diseases of the nervous system.  Neuron. 1988;  1 623-34
  CrossrefPubMedSearch in Google Scholar
• 3 Murphy T H, Miyamoto M, Sastre A, Schnaar R L, Coyle J T. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress.  Neuron. 1989;  2 1547-58
  CrossrefPubMedSearch in Google Scholar
• 4 Tan S, Schubert D, and Maher P. Oxytosis: A novel form of programmed cell death.  Current Top Medical Chemistry. 2001;  1 497-506
  PubMedSearch in Google Scholar
• 5 Rao Y, Wei H Z, Wang Y M, Luo G A. Analysis and determination of scutellarin in preparation using electrophoresis.  Chinese Traditional Patent Medicine. 2002;  24 584-6
  PubMedSearch in Google Scholar
• 6 Shuai J, Dong W W. Experimental research of PKC inhibitor, Erigeron breviscapus on the ischemic/reperfusional brain injury.  Chinese Pharmacological Bulletin. 1998;  14 75-7
  PubMedSearch in Google Scholar
• 7 Zhang W D, Chen W S, Wang Y H, Yang G J, Kong D Y, Li H T. Studies on the flavone glycosides from the extract of Erigeron breviscapus .  Chinese Traditional and Herbal Drugs. 2000;  31 565-68
  PubMedSearch in Google Scholar
• 8 Hansen M B, Nielsen S E, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.  Journal of Immunological Methods. 1989;  119 203-10
  CrossrefPubMedSearch in Google Scholar
• 9 Zhou L J, Song W, Zhu X Z, Chen Z L, Yin M L, Cheng X F. Protective effects of bilobalide on amyloid beta-peptide 25 - 35-induced PC12 cell cytotoxicity.  Acta Pharmacological Sinica. 2000;  21 75-9
  PubMedSearch in Google Scholar
• 10 Hong W, Joseph J A. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader.  Free Radical Biology & Medicine. 1999;  27 612-16
  CrossrefPubMedSearch in Google Scholar
• 11 Uchiyama M, Mihara M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test.  Analytical Biochemistry. 1978;  86 271-8
  PubMedSearch in Google Scholar
• 12 Sedmak J J, Grossero S E. A rapid sensitive and versatile assay for protein using Coomassie blue G 250.  Annals of Biochemistry. 1977;  79 544-52
  PubMedSearch in Google Scholar
• 13 Cooper A J, WA P ulsinelli, TE D uffy. Glutathione and ascorbate during ischemia and postischemic reperfusion in rat brain.  Journal of Neurochemistry. 1980;  35 1242-5
  PubMedSearch in Google Scholar
• 14 Duffy S, So A, Murphy T H. Activation of endogenous antioxidant defenses in neuronal cell prevents free radical-mediated damage.  Journal of Neurochemistry. 1998;  71 69-77
  PubMedSearch in Google Scholar
• 15 Song J H, Shin S H, Wang W, Ross G M. Involvement of oxidative stress in ascorbate-induced proapoptotic death of PC12 cells.  Experimental Neurology. 2001;  169 425-37
  CrossrefPubMedSearch in Google Scholar
• 16 Pereira C M, Oliveira C R. Glutamate toxicity on a PC12 cell line involves glutathione (GSH) depletion and oxidative stress.  Free Radical Biology & Medicine. 1997;  23 637-47
  CrossrefPubMedSearch in Google Scholar
• 17 Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms.  Free Radical Biology & Medicine. 2001;  30 433-46
  CrossrefPubMedSearch in Google Scholar

Guo-Qing Liu

Department of Pharmacology

China Pharmaceutical University

24 Tong Jia Xiang

Nanjing 210009

The People’s Republic of China

Fax: +86-25-3271340

Email: haohongchina@hotmail.com