Inhibition of NMDA Receptors Underlies the ... - Semantic Scholar

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

00722

The American Journal of Chinese Medicine, Vol. 37, No. 4, 759–770 © 2009 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine

Inhibition of NMDA Receptors Underlies the Neuroprotective Effect of Ginsenoside Rb3 Liang-Liang Peng,∗ Hong-Mei Shen,∗ Zheng-Lin Jiang,∗ Xia Li,∗ Guo-Hua Wang,∗ Yun-Feng Zhang† and Kai-Fu Ke† ∗Department of Neuropharmacology, Institute of Nautical Medicine †Department of Neurology, Affiliated Hospital

Nantong University, Nantong, Jiangsu 226001, China

Abstract: In order to investigate the mechanisms underlying the neuroprotective effect of ginsenoside Rb3 , rat hippocampal neurons were primarily cultured, and exposed to 1 mM N-methyl-D-aspartate (NMDA), cell viability and lactate dehydrogenase leakage were measured. Ca2+ influx was determined by calcium imaging with a laser confocal microscopy. The influences of ginsenoside Rb3 on these variables were examined. Patch-clamp technique was used to observe the effects of ginsenoside Rb3 on NMDA-evoked current. The results show that treatment of Rb3 raised the neuronal viability, reduced the leakage of lactate dehydrogenase, and inhibited NMDA-elicited Ca2+ influx in a dose-dependent manner. In the presence of Rb3 , NMDA-evoked peak current was inhibited, and Ca2+ -induced desensitization of NMDA current was facilitated. It is suggested that ginsenoside Rb3 could exert a neuroprotective role on hippocampal neurons, a role which was partly mediated by the facilitation of Ca2+ -dependent deactivation of NMDA receptors, and the resultant reduction of intracellular free Ca2+ level. Keywords: Ginsenoside Rb3 ; NMDA Receptors; Excitotoxicity; Neuroprotection.

Introduction Treatment of ischemic cerebral stroke remains unsatisfactory because of the complexity of the pathophysiological processes of brain injury (Labiche and Grotta, 2004; Sugawara et al., 2004; Lo et al., 2005; Segura et al., 2008) and the difficulties of neuronal regeneration (Popa-Wagner et al., 2007; Hess and Borlongan, 2008). Almost no neuroprotective agents with affirmative curative efficacy could be used clinically to alleviate neuronal damage (Cheng et al., 2004; Green, 2008; Segura et al., 2008). However, the recent success of a Correspondence to: Dr. Zheng-Lin Jiang, Department of Neuropharmacology, Institute of Nautical Medicine, Nantong University, 19 Qixiu Road, Chongchuan District, Nantong, Jiangsu, 226001 China. Tel: (+86) 513-85051796, Fax: (+86) 513-8505-1796, E-mail: [email protected]

759

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

760

00722

L.-L. PENG et al.

“Fast-Mg2+ ” therapy in the treatment of ischemic stroke (Saver et al., 2004) brings us hope in the search of new neuroprotective agents. Ginsenosides are generally considered as the principal bioactive ingredients of Panax ginseng C.A. Meyer (Araliaceae). Over 30 different ginsenosides have been identified and classified into three subclasses: panaxadiol, panaxatriol, and oleanolic acid type ginsenosides (Attele et al., 1999). Ginsenoside Rb3 belongs to the panaxadiol ginsenosides family. There is growing evidence suggesting that total saponins and several ginsenosides have neuroprotective and/or neurotrophic effects on neurons (Ji et al., 2005; Liao et al., 2002; Rudakewich et al., 2001; Tian et al., 2005). Our study has indicated that ginsenosides Rb1 , Rb3 , Rg1 , Rh2 , and F11 could protect rat hippocampal neurons and alleviate neuronal damage induced by hypoxic/ischemic insults (Jiang and Jiang, 2003; Shen et al., 2006a; 2006b; Jiang et al., 2007a). Ginsenoside Rb3 is one of the main active ingredients of ginseng; the mechanisms of its neuroprotective effect are unclear. Excitotoxicity of glutamate is an important process in the pathophysiological response cascades leading to hypoxic/ischemic brain injury, in which activation of N-methyl-Daspartate (NMDA) receptors (NMDAR) and especially calcium overload are involved (White et al., 2000; Berliocchi et al., 2005). Several reports have recently shown that some ginsenosides could modulate the function of NMDAR (Kim et al., 2002; 2004; Kim and Rhim, 2004; Lee et al., 2006). However, it is not identified that ginsenoside Rb3 could modulate the function of NMDAR and thereby contribute to its neuroprotective effect. In the present study, we observed the neuroprotective effect of ginsenoside Rb3 in cultured rat hippocampal neurons which were exposed to a high concentration of NMDA, and its inhibitory influence on NMDA receptors. Materials and Methods Animals and Chemicals Sprague-Dawley rats were obtained from the Experimental Animal Center of Nantong University, Nantong, China. All procedures in this study were in accordance with the institutional guidelines, which comply with international rules and policies. Ginsenoside Rb3 , extracted from the stem and leaf of Jilin Panax ginseng, was purchased from the Department of Organic Chemistry, Medical School of Jilin University, Changchun, China, and the purity (HPLC analysis) was > 95% (Lot # 20060718). Common inorganic salts including NaCl, KCl, CaCl2 , MgCl2 , and NaOH were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), dimethylformamide, sucrose, glucose from Xilong Chemical Co. Ltd. (Guangzhou, China), dimethylsulfoxide (DMSO) from Shenhe Chemical Reagent Co. Ltd. (Shanghai, China). The chemicals for neuronal culture were products of Invitrogen Corporation (Carlsbad, USA). Acetoxymethyl-ester form of fluo-3 (Fluo-3/AM), AP-4, CH3 O3 SCs, CsOH, cytosine arabinoside, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT), EGTA, glycine, HEPES, Mg-ATP, MK-801, Na-GTP, NMDA, sodium dodecylsulphate, and other chemicals except those indicated elsewhere were purchased from Sigma-Aldrich Corporation (Saint Louis, USA).

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

00722

INHIBITON OF RB3 ON NMDA RECEPTORS

761

Cell Culture Briefly, the hippocampi isolated from one-day-old newborn rats were cut into pieces, and digested with 0.125% trypsin at 37◦ C for 15 min. The digested brain tissue pieces were mildly triturated in a culture dish using heat-polished pipettes to dissociate hippocampal cells. The dissociated cells were then plated on to a 96-well plate or a coverslip (1 × 1 cm) coated with poly-L-lysine overnight. Cells were grown in the plating medium with 85% high glucose Dulbecco’s Minimum Essential Medium (DMEM, 12100-046), 15% fetal bovine serum (Hyclone, Logan, USA), and 106 U/L penicillin-streptomycin (Merro Pharmaceutical Factory, Dalian, China). Six hours later, the plating medium was changed to the feeding medium with 98% neurobasal medium (21103-049), 2% B27 (17504-044), 2 mM glutamine, and 106 U/L penicillin-streptomycin. The day of plating was counted as day-in-vitro (DIV) 0. On DIV 3, cytosine arabinoside (10 µM) was added to prevent proliferation of glial cells. The feeding medium was changed every 3 days. Cultures were maintained in a 5% CO2 incubator at 37◦ C. The cultured neurons on DIV 8 were used for the subsequent experiments. Excitotoxicity Induced by NMDA Exposure Cultured hippocampal neurons were rinsed twice with extracellular solution containing (in mM): NaCl 145, KCl 3, HEPES 10, CaCl2 •2H2 O 3, MgCl2 •6H2 O 2, and glucose 8. The neurons were then incubated at 37◦ C in DMEM containing NMDA (1 mM) for different durations. After exposure of NMDA, the neurons were washed twice with extracellular solution, and subsequently fed with high glucose DMEM and returned to the normal incubator for a 24-hours recovery. The neurons for the control underwent the same procedure without the exposure of NMDA. Measurements of Cell Damage Cell viability was assessed by MTT method. 24 hours after glutamate treatment, the culture medium was removed from the 96-well plate, and the neurons were rinsed twice with extracellular solution. MTT solution (25 µl) was added into each well (final concentration 0.1%). Following incubation at 37◦ C for 4 hours, 100 µl sodium dodecylsulphate solution of 20% (dissolved in dimethylformamide) was mixed into each well to dissolve the resultant dark blue crystal for 20 hours. Absorbance of each well was measured at a wavelength of 570 nm (OD 570) with the Universal Microplate Reader (ELx 800, BioTek Instruments, Inc., Winooski, USA). Neuronal damage was evaluated by measurement of lactate dehydrogenase (LDH) leaked into the culture medium. LDH activity was measured followed by the protocol of the LDH kit (Jiancheng Bioengineering Institute, Nanjing, China). Twenty hours after glutamate treatment, 0.4 ml of culture medium was mixed with 1.3 ml nicotinamide adenine dinucleotide (NADH) solution and 1.3 ml sodium pyruvate solution. Both agents were dissolved in potassium phosphate buffer (100 mM K2 HPO4 , adjusted to pH 7.5 with KH2 PO4 ). The mixed solution was immediately assayed with a spectrophotometer (UV-2450, Shimadzu Corporation, Kyoto, Japan) by monitoring the conversion of NADH to NAD at 340 nm at 37◦C,

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

762

00722

L.-L. PENG et al.

coupled with the reduction of pyruvate to lactate. LDH activity is expressed as units/ml, with one unit of activity representing the amount of LDH that causes a decrease of 0.001 absorbance unit of NADH per minute in the presence of sodium pyruvate. Intracellular Free Ca2+ Imaging For intracellular free Ca2+ imaging, Fluo-3/AM was used as a fluorescent Ca2+ indicator. Fluo-3/AM was dissolved in DMSO with a final work concentration of 0.1% DMSO. The cultured hippocampal neurons were loaded with 5 µM Fluo-3/AM for 45 min at 37◦C in extracellular solution. After being washed three times with extracellular solution, the neurons were incubated at 37◦ C for another 30 min to complete the deesterification of Fluo-3/AM. Intensity of fluorescence with the excitation wavelength at 485 nm and emission wavelength at 525 nm was recorded every 30 sec for 5 min by using a laser scanning confocal microscope (TCS SP2, Leica Microsystems, Heidelberg, Germany). All image data were collected and analyzed with the Leica control software of the microscope. The increase of intracellular free Ca2+ was determined according to the following equation: Ca2+ influx (%) = (F525 Fbase,525 )/Fbase,525 × 100, where F525 is the fluorescence intensity measured after each treatment, and Fbase,525 the basal fluorescence intensity. Recording of NMDA-Activated Currents NMDA current (INMDA) was recorded via a pipette filled with a pipette solution containing (in mM): CH3 O3 SCs 135, NaCl 8, HEPES 10, EGTA 0.5, Mg-ATP 4, and Na-GTP 0.3. The pH was adjusted to 7.25 with 1 M CsOH, and the osmolarity to 285 mOsm with sucrose. Conventional whole-cell patch-clamp technique was adopted with the neurons in a bath solution containing (in mM): NaCl 150, KCl 3, HEPES 10, CaCl2 •2H2 O 3, glucose 8 (pH 7.4 adjusted with 1 M NaOH, and osmolarity 300–310 mOsm adjusted with sucrose). Amplifier MultiClamp 700A, AD converter Digidata 1320A and software pCLAMP 8.0 (Axon Instruments, Forster City, USA) were used. When filled with the solution, the pipette had a resistance of 5–10 M. The junction potential between the pipette and the bath solution was −9.9 mV. This value was calculated using the junction potential calculation system of Clampex 8.0, pCLAMP 8.0, and nulled just before forming giga-seal. In most experiments, series resistance (Rs) before compensation was 10–20 M. Routinely, 70–80% of the Rs were compensated. Only neurons with Rs less than 20 M were selected for further tests. Membrane potential was held at −70 mV during the recording of INMDA. In order to record INMDA, NMDA was applied to each cultured neuron via a perfusion system MPS-2 (World Precision Instruments, Sarasota, USA). The tip (250 µm diameter) of a drug application pipette was usually placed 100 µm away from the cell recorded. The bath was continuously perfused with the bath solution throughout all experiments. All current responses were elicited in this solution except those indicated elsewhere and at an ambient temperature of 23–25◦C. The desensitization degree of NMDA currents was determined by the following equation: Id = 1– Iss /I p , where I p indicates the peak current, and Iss the steady state current of INMDA (Fig. 1).

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

00722


763

Figure 1. NMDA current (INMDA ) recorded with whole-cell mode in a cultured hippocampal neuron.

Drug Application Ginsenoside Rb3 , AP-4, MK-801 and glycine were dissolved in the culture medium, extracellular solution or bath solution as needed, and administered simultaneously with NMDA. Data Analysis The softwares Clampfit 8.0 (Axon Instruments, Forster City, USA) and SigmaPlot 2001 (Jandel Scientific, Costa Madre, USA) were applied for data analysis and plotting in the present study. All data are presented as mean ± SE. Student’s t-test was used for statistical analysis of data between two groups. Data of multiple groups were analyzed with oneway ANOVA and post hoc Newman-Keuls’ test. Differences were considered statistically significant at a level of p < 0.05. Results Rb3 Inhibition of NMDA-Induced Excitotoxicity When the cultured hippocampal neurons were exposed to 1 mM NMDA with increasing duration (from 15 min to 120 min), LDH leakage was enhanced in a time-dependent manner (Fig. 2A, p < 0.05). After exposure to NMDA for 60 min, LDH leakage was increased significantly from 22.9 ± 7.7 U/ml/min of the control to 63.9 ± 1.4 U/ml/min (Fig. 2A, p < 0.05). In the presence of ginsenoside Rb3 , however, the leakage of LDH was reduced as a function of the concentration of ginsenoside Rb3 . (Fig. 2B, p < 0.05 or 0.01). Similarly, ginsenoside Rb3 dose-dependently inhibited the reduction of cell viability induced by a 60min exposure to 1 mM NMDA (Fig. 2C, p < 0.05 or 0.01). However, 30 µM ginsenoside Rb3 did not further inhibit the leakage of LDH or elevate the cell viability of hippocampal neurons after NMDA exposure (Figs. 2B and 2C).

July 8, 2009 12:52 WSPC

764

WS-AJCM SPI-J000

00722

L.-L. PENG et al.

Inhibition of Rb3 on NMDA-Elicited Ca2+ Influx After a period of control records, 1 mM NMDA was added and an abundant Ca2+ influx in the cultured hippocampal neurons was elicited (Fig. 3B). This elevation of intracellular free Ca2+ concentration was dose-dependently blocked by AP-4, an NMDA receptor antagonist, and MK-801, an open channel blocker (Fig. 3E, p < 0.05 or 0.01). With a simultaneous application of 1 nM to 3 µM ginsenoside Rb3, the NMDA-elicited Ca2+ influx was reduced dose-dependently (Fig. 3E, p < 0.05 or 0.01). Compared to the control, an increase to 130 ± 0.9% in the NMDA-elicited of intracellular free Ca2+ was decreased to 10.3 ± 0.6%

(A)

(B) Figure 2. Inhibition effect of ginsenoside Rb3 on NMDA-induced cell damage. (A) Mean values of lactate dehydrogenase (LDH) leakage in cultured hippocampal neurons (n = 8). NMDA (1 mM) exposure with indicated durations produced a time-dependent increase of LDH leakage. ∗p < 0.05 and ∗∗p < 0.01, vs. control. (B) LDH leakage after use of Rb3 (n = 8). An addition of ginsenoside Rb3 at indicated concentrations inhibited LDH leakage induced by a 60-min exposure of NMDA. (C) Cell viability of cultured hippocampal neurons after a 60-min exposure of NMDA (n = 10). In the presence of ginsenoside Rb3 at indicated concentrations, neuronal viability was elevated in a dose-dependent manner. ∗p < 0.05 and ∗∗p < 0.01, vs. NMDA only. # , p < 0.01, vs. that without NMDA.

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

00722


765

(C) Figure 2. (Continued)

after the administration of 3 µM ginsenoside Rb3 (Fig. 3E, p < 0.01). When the extracellular Ca2+ level was elevated, the NMDA-elicited Ca2+ influx was increased in a dose-dependent manner (Fig. 3F), however, ginsenoside Rb3 (0.01 µM) significantly inhibited this dosedependent increase when the extracellular Ca2+ concentration at1 and 3 mM (Fig. 3F, p < 0.05 or 0.01).

(A)

(B)

(C)

(D)

Figure 3. Inhibition of ginsenoside Rb3 on NMDA-elicited Ca2+ influx. (A)–(D) Examples of Ca2+ image in cultured hippocampal neurons before and after addition of 1 mM NMDA. Scale bar, 60 µm for four images. (A) Control for B. (B) Increase of Ca2+ influx after NMDA addition. (C) Control for D. (D) Inhibition of Ca2+ influx after 1 µM Rb3 addition. (E) Mean values of increased intracellular Ca2+ levels ([Ca2+ ]i ) after indicated treatments (n = 20). Similar to NMDAR antagonists AP-4 and MK-801, ginsenoside Rb3 reduced NMDA-elicited Ca2+ influx as a function of concentration. ∗p < 0.05 and ∗∗p < 0.01, vs. NMDA alone. (F) Elevation of NMDAinduced Ca2+ influx by increasing extracellular Ca2+ concentration ([Ca2+ ]o ) and inhibited by ginsenoside Rb3 (n = 20). ∗p < 0.05 and ∗∗p < 0.01, vs. that without Rb3 .

July 8, 2009 12:52 WSPC

766

WS-AJCM SPI-J000

00722

L.-L. PENG et al.

(E)

(F) Figure 3. (Continued)

Influence of Rb3 on NMDA-Elicited Current and Ca2+ -Dependent Deactivation NMDA elicited current response from 85% of the cultured hippocampal neurons (n = 200) in a concentration-dependent manner. An addition of 10 µM glycine remarkably increased INMDA, and shifted the dose-response curve of INMDA to the left. Meanwhile, as compared to the control, the desensitization degree (Id ) was decreased for almost all current responses elicited by any concentration of NMDA in the presence of 10 µM glycine (data not shown). Ginsenoside Rb3 itself did not evoke any current response in the cultured hippocampal neurons, but reduced NMDA-elicited peak current to different extents and facilitated the desensitization course at the same time (Fig. 4A). The glycine-independent desensitization, INMDA was measured in 1 µM glycine and 3 mM extracellular Ca2+ . For measurement of the glycine-dependent desensitization, INMDA was

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

00722


767

(A)

(B)

(C) Figure 4. Effects of ginsenoside Rb3 on the current response elicited by 300 µM NMDA. (A) Examples of INMDA after use of Rb3 . Ginsenoside Rb3 at indicated concentrations decreased the peak response and facilitated the desensitization course of INMDA . (B) Examples of Ca2+ -dependent desensitization of INMDA and the effect of ginsenoside Rb3 . (C) Mean values of Ca2+ -dependent desensitization (Id ) of INMDA with or without use of ginsenoside Rb3 (n = 6 for each group). ∗p < 0.05 and ∗∗p < 0.01, vs. that without Rb3 .

July 8, 2009 12:52 WSPC

768

WS-AJCM SPI-J000

00722

L.-L. PENG et al.

elicited with maintained 3 mM Ca2+ , and glycine was applied increasingly from 0.01 µM to 10 µM. Ca2+ -dependent deactivation was determined when INMDA was recorded with the concentration of glycine maintained at 1 µM and the extracellular Ca2+ level was increased from 0.03 mM to 3 mM. Unfortunately, ginsenoside Rb3 at 0.01–30 µM did not exert constant influence on both the glycine-dependent and glycine-independent desensitizations of INMDA (data not shown). However, ginsenoside Rb3 facilitated the Ca2+ -dependent deactivation of INMDA , i.e., as shown in Fig. 4B, the peak response was reduced and the desensitization degree (Id ) was increased. At the extracellular Ca2+ concentration at 1 and 3 mM, Id was significantly elevated by ginsenoside Rb3 from 0.45 ± 0.06 and 0.50 ± 0.07 to 0.77 ± 0.13 and 0.98 ± 0.07 respectively (Fig. 4C, p < 0.01). Discussion The present study proved for the first time that ginsenoside Rb3 could protect cultured rat hippocampal neurons from NMDA-induced excitotoxicity. We found that Rb3 in a dosedependent manner reduced LDH leakage from neurons and raised neuronal viability after NMDA exposure. Meanwhile, ginsenoside Rb3 inhibited NMDAR-mediated Ca2+ influx dose-dependently. It is suggested that ginsenoside Rb3 could exert a neuroprotective effect on neurons and an inhibitory influence of Rb3 on NMDA receptors is attributable. The neuroprotective effect of ginsenoside Rb3 that we observed in the present study is consistent with several studies we had reported in Chinese journals (Fan et al., 2006; Shen et al., 2006a; 2006b; Jiang et al., 2007a). We have found protective effects of ginsenoside Rb3 on oxygen/glucose deprived hippocampal slices (Shen et al., 2006b; Jiang et al., 2007a), glutamate-exposed hippocampal neurons (Shen et al., 2006a), hypoxia-treated hippocampal (Shen et al., 2006b) or cortical neurons (Li et al., 2004), and rat brain with middle cerebral artery occlusion (Fan et al., 2006). Rb3 in these studies reduced the release of LDH and the expression of iNOS and caspase-3, elevated neuronal viability, and promoted the recovery of neuronal function. In other studies, our results have shown that ginsenoside Rb3 could raise the number of GABA-positive hippocampal neurons in rat after hypoxic exposure (Shen et al., 2005) and inhibit the increase of persistent sodium current in cultured hippocampal neurons after oxygen/glucose deprivation (Jiang et al., 2007b). This neuroprotective effect of ginsenoside Rb3 presented here and reported in our previous studies leads us to add a new member into the ginsenosides family which possesses neuroprotective efficacy. For further research on the mechanism of the neuroprotective effect of ginsenoside Rb3 , we observed the influence of Rb3 on the current responses elicited by NMDA in the cultured hippocampal neurons. The results showed that Rb3 facilitated the Ca2+ -dependent deactivation of NMDAR. Glutamate excitotoxicity is an important initial process in the pathophysiological response cascades leading to hypoxic/ischemic brain injury, in which activation of NMDAR and especially calcium overload are centrally involved (Berliocchiet al., 2005; White et al., 2000). Therefore, the inhibitory influence of ginsenoside Rb3 on NMDAR activity that we observed, which induces a reduction in the intracellular free Ca2+ level while NMDAR is over-activated may partly underlie the neuroprotective effect of Rb3 .

July 8, 2009 12:52 WSPC

WS-AJCM SPI-J000

00722


769

This inhibitory effect of Rb3 on NMDAR has not been reported until now. However, Kim et al. (2002) have observed an inhibitory effect of total saponins and 10 ginsenosides on NMDAR, i.e., at a concentration of 10 µM, ginsenosides Rb2 , Rd, Re, Rf, and Rg2 had little or no significant effect while Rb1 , Rc, Rg3 , Rh1 , and Rh2 produced a notable inhibition on NMDA-induced [Ca2+ ]i increase, and among these five effective ginsenosides, Rg3 produced the highest inhibitory effect. Moreover, they found that Rg3 reduced NMDAinduced current remarkably. However, they did not investigate the effect of ginsenoside Rb3 . Their further study (Kim et al., 2004) has revealed that ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in cultured rat hippocampal neurons. Lee et al. (2006) have reported that 20(S)-ginsenoside Rh2 inhibits NMDAR via an interaction with polyamine-binding sites in cultured rat hippocampal neurons. Our recent study has indicated that ginsenoside Rg1 protects neurons from hypoxic-ischemic injury partly by inhibiting Ca2+ influx through NMDA receptors (Zhang et al., 2008). The present study extends the research concerning the inhibitory effect of ginsenosides on NNDAR activity. In conclusion, ginsenoside Rb3 exerted a neuroprotective role on cultured rat hippocampal neurons subjected to excitotoxicity of NMDA. This effect was partly mediated through the facilitation of Ca2+ -dependent deactivation of NMDA receptors, and the resultant reduction of intracellular free Ca2+ level. Acknowledgments This study was supported by grants from the Administration of Science and Technology of Jiangsu (Project No. BK2003036) and from the Administration of Education of Jiangsu (Projects No. 03KJD310185, KJS02052 and 05KJD350159), Jiangsu Province, China. References Attele, A.S., J.A. Wu and C.S. Yuan. Ginseng pharmacology: multiple constituents and multiple actions. Biochem. Pharmacol. 58: 1685–1693, 1999. Berliocchi, L., D. Bano and P. Nicotera. Ca2+ signals and death programmes in neurons. Philos. Trans. R. Soc. Lond. B 360: 2255–2258, 2005. Cheng, Y.D., L. Al-Khoury and J.A. Zivin. Neuroprotection for ischemic stroke: two decades of success and failure. NeuroRx. 1: 36–45, 2004. Fan, X.J., K.F. Ke, Z.L. Jiang and G.H. Wang. Neuroprotective effect of ginsenosides on focal cerebral ischemia/reperfusion in rats. Natl. Med. J. China 86: 2071–2074, 2006. Green, A.R. Pharmacological approaches to acute ischaemic stroke: reperfusion certainly, neuroprotection possibly. Br. J. Pharmacol. 153(Suppl. 1): S325–338, 2008. Hess, D.C. and C.V. Borlongan. Stem cells and neurological diseases. Cell Prolif. 41(Suppl. 1): 94–114, 2008. Ji, Y.C., Y.B. Kim, S.W. Park, S.N. Hwang, B.K. Min, H.J. Hong, J.T. Kwon and J.S. Suk. Neuroprotective effect of ginseng total saponins in experimental traumatic brain injury. J. Korean Med. Sci. 20: 291–296, 2005. Jiang, S. and Z.L. Jiang. Protective effect of ginsenoside Rb1 on ischemic brain injury in rat. J. Apoplexy Nerv. Dis. 20: 415–417, 2003. Jiang, S., Z.L. Jiang and Y.M. Zeng. The protective effect of nine ginseng saponin monomers on ischemic brain injury in rat. Pharmacol. Clin. Chin. Mat. Med. 23: 19–21, 2007a.

July 8, 2009 12:52 WSPC

770

WS-AJCM SPI-J000

00722

L.-L. PENG et al.

Jiang, W.W., Z.L. Jiang and K.F. Ke. The effect of ginsenoside Rb3 on the persistent sodium current in ischemic and normal neurons. Pharm. Clin. Res. 15: 444–448, 2007b. Kim, S., K. Ahn, T.H. Oh, S.Y. Nah and H. Rhim. Inhibitory effect of ginsenosides on NMDA receptormediated signals in rat hippocampal neurons. Biochem. Biophys. Res. Commun. 296: 247–254, 2002. Kim, S., T. Kim, K. Ahn, W.K. Park, S.Y. Nah and H. Rhim. Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons. Biochem. Biophys. Res. Commun. 323: 416–424, 2004. Kim, S. and H. Rhim. Ginsenosides inhibit NMDA receptor-mediated epileptic discharges in cultured hippocampal neurons. Arch. Pharm. Res. 27: 524–530, 2004. Labiche, L.A. and J.C. Grotta. Clinical trials for cytoprotection in stroke. NeuroRx. 1: 46–70, 2004. Lee, E., S. Kim, K.C. Chung, M.K. Choo, D.H. Kim, G. Nam and H. Rhim. 20(S)-ginsenoside Rh2 , a newly identified active ingredient of ginseng, inhibits NMDA receptors in cultured rat hippocampal neurons. Eur. J. Pharmacol. 536: 69–77, 2006. Li, A.H., K.F. Ke, X.M. Wu and S.Y. Bao. Effects of ginsenosides Rb1 , Rb3 , Rg1 against ischemic injury of cultured cortical neurons and its mechanism. J. Apoplexy Nerv. Dis. 21: 231–233, 2004. Liao, B., H. Newmark and R. Zhou. Neuroprotective effects of ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp. Neurol. 173: 224–234, 2002. Lo, E.H., M.A. Moskowitz and T.P. Jacobs. Exciting, radical, suicidal: how brain cells die after stroke. Stroke 36: 189–192, 2005. Popa-Wagner, A., S.T. Carmichael, Z. Kokaia, C. Kessler and L.C. Walker. The response of the aged brain to stroke: too much, too soon? Curr. Neurovasc. Res. 4: 216–227, 2007. Rudakewich, M., F. Ba and C.G. Benishin. Neurotrophic and neuroprotective actions of ginsenosides Rb1 and Rg1 . Planta Med. 67: 533–537, 2001. Saver, J.L., C. Kidwell, M. Eckstein and S. Starkman. Prehospital neuroprotective therapy for acute stroke: results of the field administration of stroke therapy–magnesium (FAST–MAG) pilot trial. Stroke 35: e106–e108, 2004. Segura, T., S. Calleja and J. Jordan. Recommendations and treatment strategies for the management of acute ischemic stroke. Expert Opin. Pharmacother. 9: 1071–1085, 2008. Shen, H.M., Z.L. Jiang and X.S. Gu. Effects and mechanisms of ginsenoside Rb3 on glutamate excitotoxic injury in cultured neurons of rat hippocampus. Chin. J. Appl. Physiol. 22: 31–34, 2006a. Shen, H.M., Z.J. Zhang, S. Jiang and Z.L. Jiang. Protective effects of ginsenoside Rb3 on hypoxic/ischemic brain injury and involved mechanisms. Chin. J. Appl. Physiol. 22: 302–306, 2006b. Shen, H.M., Z.J. Zhang and Z.L. Jiang. Effects of ginsenoside Rb3 on GABA in brain tissue in rats with injury of hypoxia. J. Clin. Neurol. 18: 455-456, 2005. Sugawara, T., M. Fujimura, N. Noshita, G.W. Kim, A. Saito, T. Hayashi, P. Narasimhan, C.M. Maier and P.H. Chan. Neuronal death/survival signaling pathways in cerebral ischemia. NeuroRx. 1: 17-25, 2004. Tian, J., F. Fu, M. Geng, Y. Jiang, J. Yang, W. Jiang, C. Wang and K. Liu. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebral ischemia in rats. Neurosci. Lett. 374: 92-97, 2005. White, B.C., J.M. Sullivan, D.J. DeGracia, B.J. O’Neil, R.W. Neumar, L.I. Grossman, J.A. Rafols and G.S. Krause. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J. Neurol. Sci. 179: 1-33, 2000. Zhang, Y.F., X.J. Fan, X. Li, L.L. Peng, G.H. Wang, K.F. Ke and Z.L. Jiang. Ginsenoside Rg1 protects neurons from hypoxic–ischemic injury possibly by inhibiting Ca2+ influx through NMDA receptors and L-type voltage-dependent Ca2+ channels. Eur. J. Pharmacol. 586: 90-99, 2008.