Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells

Journal of Ethnopharmacology 111 (2007) 458–463

Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells Na Li a , Bin Liu b , Dean E. Dluzen b , Yi Jin a,∗ a

b

Department of Physiology, Medical College of Qingdao University, Qingdao 266071, PR China Department of Anatomy, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272, USA Received 3 June 2006; received in revised form 21 November 2006; accepted 8 December 2006 Available online 20 December 2006

Abstract We investigated the effect of ginsenoside Rg2 on neurotoxic activities induced by glutamate in PC12 cells. The cells were incubated with glutamate (1 mmol/L), glutamate and ginsenoside Rg2 (0.05, 0.1, 0.2 mmol/L) or nimodipine (5 ␮mol/L) for 24 h. The cellular viability was assessed by MTT assay. The lipid peroxidation products malondialdehyde (MDA) and nitrogen oxide (NO) were measured by a spectrophotometric method. Fura2/AM, as a cell permeable fluorescent probe for Ca2+ , was used to detect intracellular Ca2+ concentration ([Ca2+ ]i ) using a monespectrofluorometer. Immunocytochemical techniques were employed to check the protein expression levels of calpain II, caspase-3 and ␤-amyloid (A␤)1–40 in PC12 cells. The results showed that glutamate decreased the cell viability, increased [Ca2+ ]i , lipid peroxidation (the excessive production of MDA, NO) and the protein expression levels of calpain II, caspase-3 and A␤1–40 in PC12 cells. Ginsenoside Rg2 significantly attenuated glutamate-induced neurotoxic effects upon these parameters at all doses tested. Our study suggests that ginsenoside Rg2 has a neuroprotective effect against glutamateinduced neurotoxicity through mechanisms related to anti-oxidation and anti-apoptosis. In addition, the inhibitory effect of ginsenoside Rg2 against the formation of A␤1–40 suggests that ginsenoside Rg2 may also represent a potential treatment strategy for Alzheimer’s disease. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Panax ginseng; Neuroprotection; Excitotoxicity; Alzheimer’s disease

1. Introduction The root of Panax ginseng is a popular herbal medicine for the treatment of weakness and fatigue and has been used for several thousand years in Asia. Clinical studies demonstrated that Panax ginseng may improve psychological functions, immune functions and conditions associated with diabetes (Kiefer and Pantuso, 2003). The main active components of Panax ginseng are ginsenosides, which have been shown to have a variety of beneficial effects, including anti-inflammatory, antioxidant, and anticancer effects. In addition, several ginsenosides have also been demonstrated to exert a neuroprotective effect in primary cultured neurons or neuronal models in vitro. For example, ginsenosides Rb1 (Liu and Zhang, 1995; Kim et al., 1998) and Rg3 (Kim et al., 1998) can significantly attenuate glutamate-induced

∗ Corresponding author at: Department of Physiology, Medical College of Qingdao University, 38 Dengzhou Road, Qingdao 266021, PR China. Tel.: +86 532 82991203; fax: +86 532 83801449. E-mail address: jin [email protected] (Y. Jin).

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.12.015

neurotoxicity in cultured rat cortical or hippocampal cells. Ginsenoside Re prevents PC12 cells from cytotoxicity induced by serum-free medium and beta-amyloid peptide (Ji et al., 2006). Ginsenoside Rg2 represents another active component of Panax ginseng, which has been shown to block calcium channels and displays anti-free-radical activity (Jiang et al., 1996). There are few studies which have focused upon the effects of ginsenoside Rg2 in the central nervous system. Glutamate is one of the principal excitatory neurotransmitters in the brain, and its interactions with specific neurocyte membrane receptors are responsible for many neurological functions, including cognition, memory, movement, and sensation (Gasic and Hollmann, 1992). Excitatory neurotransmitters also play an important role in the developmental plasticity of synaptic connections in the nervous system (Lipton and Kater, 1989). However, in a variety of pathologic conditions, including stroke and various neurodegenerative disorders, such as Alzheimer’s disease (AD), excessive activation of glutamate receptors may mediate neuronal injury or death. This form of injury appears to be predominantly induced by excessive influx of calcium into neurons through ionic channels triggered by activation of gluta-

N. Li et al. / Journal of Ethnopharmacology 111 (2007) 458–463

mate receptors (Choi, 1985). In addition, glutamate is involved in the formation of pathological hallmarks of AD including senile plaques (SP) and neurofibrillary tangles (NFTs). However, the mechanisms involved with these effects are not very clear. A chief component of SP is amyloid-beta (A␤), which can be generated from the amyloid-beta precursor protein (APP) directly and efficiently cleaved by caspase-3 during apoptosis (Gervais et al., 1999). The elevated A␤ peptide formation also represents one of the pathological characteristics of AD. In the present study, we examined whether ginsenoside Rg2 could exert neuroprotective effects against glutamate-induced neurotoxicity in PC12 cells, which is a widely used in vitro model of neuronal function. In specific, a series of experiments were performed in which we investigated: (1) the possible mechanisms related with oxidative stress and apoptosis, (2) whether ginsenoside Rg2 could affect of formation of A␤, which is induced by glutamate, and (3) the interaction between A␤ and caspase-3 following treatment of the PC12 cells.

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and Fura-2/AM were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies (calpain, caspase-3 and A␤(1–40)), SABC kit and DAB were purchased from Boster Biological Technology Ltd. (Wuhan, China). 2.2. Cell culture and treatments

2. Materials and methods

Rat pheochromocytoma cells (PC12 cells, a gift from Institute of Neuroscience, Chinese Academy of Sciences) were maintained in Dubecco’s modified Eagle’s medium (DMEM) containing 5% fetal calf serum, 10% horse serum, 100 U/mL penicillin, and 100 ␮g/mL streptomycin in a CO2 incubator at 37 ◦ C. Prior to the treatments, PC12 cells were rinsed by DMEM for three times (5 min each), then covered with serum-free DMEM in which glutamate (1 mmol/L) or glutamate (1 mmol/L) with ginsenoside Rg2 (0.05, 0.1, 0.2 mmol/L) or nimodipine (5 ␮mol/L) were added. PC12 cells were subsequently tested for viability as assessed with assays associated with MTT, malondialdehyde (MDA), nitrogen oxide (NO) and intracellular-free Ca2+ ([Ca2+ ]i ) after incubation in a CO2 incubator for 24 h.

2.1. Materials

2.3. Cell viability assay

Fig. 1 shows the chemical structure of ginsenosides Rg2 , which was provided by Jilin Academy of Traditional Chinese Medicine and Materia Medica (China). 3-[4,5-Dimethyllthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT), Triton X-100, ethyleneglycol bis(2-aminoethylether) tetraacetic acid (EGTA)

PC12 cells were inoculated in a 96-well microplate (105 cells/well in 100 ␮L medium) for 24 h. After washing with PBS the PC12 cells were incubated with glutamate (1 mmol/L), or glutamate (1 mmol/L) with ginsenosides Rg2 (0.05, 0.1, 0.2 mmol/L) or Nimodipine (5 ␮mol/L) for 24 h, respectively, at which time MTT (10 ␮L, 0.5 g/L) was added to each culture well. After incubation at 37 ◦ C for an additional 4 h, the formazan crystals were dissolved by addition of 150 ␮L dimethyl sulfoxide (DMSO), and the plates were shaken vigorously to ensure complete solubilization. Formazan absorbance was assessed at 570 nm by a 550 BIO-RAD microplate reader. 2.4. Measurement of lipid peroxidation MDA, a terminal product of lipid peoxidation, was measured to estimate the content of lipid peoxidation in PC12 cells. MDA concentration in cell homogenates was determined with commercial kits purchased from Jiancheng Bioengineering Institute (Nanjing, China), using the thiobarbituric acid method. The assay was based on the conjugation ability of MDA with thiobarbituric acid, to form a red product which has maximum absorbance at 532 nm. NO was also determined with commercial kits purchased from Jiancheng Bioengineering Institute (Nanjing, China), using the nitrate reductase method. Since the chemical character of NO is very active, being rapidly converted to NO− 2 and NO3 − in vivo, the nitrate reductase can reduce NO3 − to NO2 − , producing a yellow product which has maximum absorbance at 550 nm when reacted with developer. 2.5. Measurement of intracellular calcium concentration ([Ca2+ ]i )

Fig. 1. Chemical structure of ginsenoside Rg2 (molecular formula: C42 H72 O13 ; molecular weight: 748).

After the treatments as described above, PC12 cells were collected and prepared to generate a 2 mL cell suspension for

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every sample. Fura-2/AM (final concentration 5 ␮mol/L) was added to the cell suspension. The suspension was shaken at 37 ◦ C for 50 min, and then centrifuged twice at 1000 rpm for 5 min. The cells were re-suspended in HEPES buffer solution, containing NaCl 132, KCl 3, glucose10, HEPES 10 and CaCl2 1 mmol/L, pH 7.4. A 930-spectrophotofluorometer (Third Analytical Apparatus Station, Shanghai) was used for fluorescence determination. The condition of measurement was as follows: alternate: λ time scan: EX: 340 and 380 nm, EM: 500 nm; bandwidth: 10 nm, the excitation spectrum at 340 to 380 nm at 37 ◦ C. After Fura-2/AM loading, the peak of excitation spectrum in samples was tested from 380 to 340 nm. When Triton X-100 (final concentration of 0.1%) was added to lyse the plasmatic membranes, the spectrum peak in 340 nm increased. By addition of EGTA (final concentration 5 mmol/L, pH >8.5), the spectrum of Fura-2 released a peak at 380 nm. Intracellular calcium concentrations were calculated according to the method of Grynkiewicz et al. (1985). 2.6. Immunoperoxidase cell staining PC12 cells samples for immunocytochemistry were plated on sterile polylysine coated glass coverslips. Twelve hours later, PC12 cells were treated for 24 h as described above. Then PC12 cells were washed briefly with PBS (pH 7.4), fixed with 4% polymerisatum for 30 min and then washed with PBS for 5 min. Subsequently, specimens were incubated for 10 min in 1–2 drops (∼50 ␮L) of HRP inhibitor (3% H2 O2 ) and 15 min in 1–2 drops of serum block. Then 1–2 drops of pre-diluted primary antibody (rabbit anti-calpain II polyclonal antibody, 1:200, rabbit anti-caspase-3 polyclonal antibody, 1:500 and rabbit anti-A␤ polyclonal antibody, 1:500) were immediately added to the specimens and incubated overnight at 4 ◦ C. The specimens were further incubated for 10 min with 1–2 drops of biotinylated secondary antibody and for 10 min with 1–2 drops of HRP-sterptavidin complex (SABC). Two to three drops of HRP substrate mixture (DAB) were added, and the whole reaction was developed for 30 s to 10 min until the desired stain intensity was obtained. Control groups were included to determine the specificity of the immunostaining. The staining of calpain II, caspase-3 and A␤ was observed and imaged with an Olympus BX50-camera (Japan). 2.7. Statistical analysis Data were expressed as the mean ± S.D. Statistical differences were evaluated by using one-way ANOVA. The level for a statistically significant difference was set at p < 0.05. 3. Results 3.1. Neuroprotective effects of ginsenoside Rg2 against glutamate-induced neuronal viability Viability of the PC12 cells was tested by MTT assay. The optical densities (OD) of the cells were decreased by incubation with glutamate (1 mmol/L) for 24 h. However, these

Fig. 2. Ginsenoside Rg2 and nimodipine diminished glutamate-induced reductions in OD values. N = 6/group. Data represent mean ± S.D. a p < 0.01 vs. control group; b p < 0.01 vs. glutamate group.

glutamate-induced reductions in OD values could be diminished by treatment with all concentrations of ginsenoside Rg2 (0.05–0.2 mmol/L) and in response to nimodipine (0.05 ␮mol/L) when administered simultaneously with the glutamate (Fig. 2). 3.2. Ginsenoside Rg2 attenuates glutamate-induced lipid peroxidation increase When PC12 cells were exposed to glutamate (1 mmol/L) for 24 h, an increase in lipid peroxidation level, as indicated by the excessive formation of MDA and NO in PC12 cells, was observed to 200–250% of control values (Table 1). Coincubation with ginsenoside Rg2 at all concentrations tested and nimodipine significantly decreased lipid peroxidation (a decrease in the formation of MDA and NO) as compared with levels observed with the glutamate group. 3.3. Effect of ginsenoside Rg2 against Ca2+ increases induced by glutamate Exposure to glutamate (1 mmol/L) for 24 h resulted in a significant increase in the level of intracellular Ca2+ compared with the control. These increases in intracellular-free Ca2+ level were clearly inhibited in the presence of all concentrations of ginsenoside Rg2 (0.05, 0.1, 0.2 mmol/L) and nimodipine (5 ␮mol/L) (Fig. 3). Table 1 Effect of ginsenoside Rg2 on MDA and NO formation induced by glutamate Drug (mmol/L)

NO (␮mol/L)

Control Glutamate (1) Nimodipine (0.005) + glutamate (1) Ginsenoside Rg2 (0.05) + glutamate (1) Ginsenoside Rg2 (0.1) + glutamate (1) Ginsenoside Rg2 (0.2) + glutamate (1)

32.27 65.87 42.41 48.89 46.50 38.66

± ± ± ± ± ±

8.69 5.84a 8.16b 7.32b 7.66b 6.70b

MDA (nmol/mL) 0.27 0.63 0.36 0.30 0.33 0.30

± ± ± ± ± ±

0.13 0.13a 0.13b 0.17c 0.20c 0.15b

N = 5. Data represent mean ± S.D. a p < 0.01 vs. control; b p < 0.01, c p < 0.05 vs. glutamate group.

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A␤ were decreased when ginsenoside Rg2 (0.2 mmol/L) was co-incubated with glutamate for 24 h (Fig. 4). 4. Discussion

Fig. 3. Ginsenoside Rg2 and nimodipine inhibited the enhancement of intracellular-free Ca2+ level induced by glutamate. N = 5/group. Data represent mean ± S.D. a p < 0.01 vs. control group; b p < 0.01 vs. glutamate group.

3.4. Effects of ginsenoside Rg2 on the expression of proteins affected by glutamate Immunocytochemistry was used to determine the protein expression levels of calpain II, caspase-3 and A␤1–40. The protein expression levels of calpain II, caspase-3 and A␤ were all increased in the presence of glutamate (1 mmol/L). In contrast, the protein expression levels of calpain II, caspase-3 and

Our data provide strong evidence for the protective effects of ginsenoside Rg2 against glutamate-induced injury to PC12 cells. Glutamate is a major excitatory neurotransmitter working at a variety of excitatory synapses in the nervous system. It plays important roles in cellular process underlying synaptic plasticity, neuronal development and excitation via the activation of glutamate receptors (Bleich et al., 2003; Conn, 2003). High concentrations of glutamate have been shown to induce neuronal damage and cell loss in vitro in studies using cultured neurons (Choi, 1987; Ankarcrona et al., 1995). Analogous responses have been also observed in acute degenerative diseases such as strokes and traumatic brain injury, or chronic degenerative diseases including Parkinson’s disease, Alzheimer’s disease and multiple sclerosis (Koh et al., 1990; Turski et al., 1991; Smith et al., 2000). In the present study, we observed that ginsenoside Rg2 could effectively protect PC12 cells from the insult of glutamate as indicated by inhibiting reductions of MTT induced by glutamate. The glutamate-induced Ca2+ overload hypothesis has been widely accepted as the mechanism of neuronal injury in glutamate-induced excitotoxicity (Choi and Rothman, 1990; Kristian and Siesjo, 1996; Nishizawa, 2001). Calcium overload could elicit a series of neuronal injury events, resulting

Fig. 4. Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity as indicated by protein expression levels of calpain II, caspase-3 and A␤. Representative photomicrographs of PC12 cells stained with antibodies to calpain II, caspase-3 and A␤ (400× magnifications) are shown. Cells received no treatment (Control, A), glutamate (B) or ginsenoside Rg2 and glutamate (C) treatments. Antibody detection was accomplished using a diaminobenzidine colorimetric method.

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in the over stimulation of proteolytic enzymes, lipid peroxidation, free-radical formation (superoxide and nitric oxide), or immediate-early gene expression (Braughler and Hall, 1989; Dawson et al., 1991; Chen et al., 1992; Lipton et al., 1993; Smeyne et al., 1993), thereby contributing to the excitotoxic process. A previous study reported that ginsenoside Rg2 was capable of blocking calcium channels and exerting anti-free radical actions when tested in cultured myocardiocytes (Jiang et al., 1996). Our current results now show that ginsenoside Rg2 can significantly decrease the elevated level of intracellular Ca2+ and reduce the lipid peroxidation (the excessive production of MDA and NO) induced by glutamate in neuronal cells. In differentiated PC12 cells, patch-clamp analysis showed that NMDA, applied extracellularly, induced an inward current and an elevation in intracellular-free Ca2+ , which was abolished by the NMDA receptor antagonist, MK-801. These results indicate that differentiated PC12 cells express functional NMDA receptors (Casado et al., 1996; Kobayashi and Millhorn, 2000). However, although MK-801 is capable of blocking NMDA receptor and inhibiting excess glutamate release, it can also induce dose-dependent impairments of learning and memory through blocking NMDA glutamate receptor (Benvenga and Spaulding, 1988; Butelman, 1989). Nimodipine, a L-type calcium channel blocker, which is used clinically, was selected as a positive control due to the diminished side-effects and its ability to serve as a positive control for observing the effects of glutamate and ginsenoside Rg2 on intracellular calcium levels. Whether the reductions in intracellular-free Ca2+ that we observed to ginsenoside Rg2 also involve interactions with the NMDA receptor remains to be determined. The elevation of intracellular-free Ca2+ level also mediates the activation of Ca2+ -dependent enzymes, such as calpains (calcium-dependent, neutral proteases, active in the cells responding to signals inducing a rise of cytoplasmic Ca2+ ) which are proposed to participate in the turnover of cytoskeletal proteins and regulation of kinases, transcription factors, and receptors (Tremper-Wells et al., 2002; Scholzke et al., 2003). Calpains have mainly been implicated in excitotoxic neuronal injury and necrosis. In addition, calpain II (requiring mM Ca2+ ) had previously been implicated in the regulation of apoptosis of some cell types by interaction with caspase-3, which has been identified as a key executor of apoptosis; whereas calpain II has mainly been implicated in excitotoxic neuronal injury (Blomgren et al., 2001). In this study, we investigated the protein expression of calpain II and caspase-3 in PC12 cells using immunocytochemistry methods. The results showed that the protein expression of both calpain II and caspase-3 increased by glutamate treatment for 24 h were inhibited by ginsenoside Rg2 . Such data suggest that ginsenoside Rg2 may have an antiapoptotic property which could serve as one of the possible mechanism underlying its cellular protection. Caspase-3 is not only involved in the execution of cell apoptosis, but also in proteolytic cleavage of Alzheimer’s APP and amyloidogenic A␤ peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury.

Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of AD brains and co-localization of its APP cleavage product with A␤ in senile plaques. Caspase-3 thus appears to play a dual role in proteolytic processing of APP and the resulting propensity for A␤ peptide formation, as well as in the ultimate apoptotic death of neurons in AD (Gervais et al., 1999). To investigate whether glutamate can induce the formation of A␤ by activating caspase3, we measured the expression levels of A␤ in PC12 cells. The results showed a strong positive reaction of A␤ in PC12 cells treated by glutamate for 24 h, whereas weak positive reactions were found in ginsenoside Rg2 -treated and control groups. Our data support the theory of glutamate inducing the formation of A␤ (Louzada et al., 2001), and, in this way can be involved in the pathomechanism of AD. In conclusion, ginsenoside Rg2 can efficiently protect PC12 cells against glutamate-induced neuronal injury and formation of A␤. The protective effects are mediated by inhibiting excessive Ca2+ influx triggered by glutamate, reducing the lipid peroxidation and down-regulating the expression of pro-apoptotic factor calpain II and caspase-3, to further block the proteolytic cleavage of Alzheimer’s APP and amyloidogenic A␤ peptide formation. It should be noted that several other ginseng saponins including Re, Rb1, Rg1 and Rg3 can also exert neuroprotective effects against the neurotoxicity induced by glutamate (Kim et al., 1998; Liao et al., 2002), ␤-amyloid peptide (Rudakewich et al., 2001; Ji et al., 2006), serum-free medium (Ji et al., 2006), kainic acid (Lee et al., 2002; Liao et al., 2002) and MPTP (Rudakewich et al., 2001). Ginsenosides appear to be a promising agent not only for the treatment of Alzheimers’ disease (Chen et al., 2006; Ji et al., 2006), but also spinal cord injuries (Liao et al., 2002) and cerebral stroke (Zhang et al., 2006). Additional experimental and clinical studies will be required to assess the potential for clinical application of ginseng or its saponins. Acknowledgements We thank Prof. Longyun Li from Jilin Academy of Traditional Chinese Medicine and Materia Medica, China for gifting ginsenoside Rg2 . This work was supported by Ministry of Science and Technology of PR China. References Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., Nicotera, P., 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961–973. Benvenga, M.J., Spaulding, T.C., 1988. Amnesic effect of the novel anticonvulsant MK-801. Pharmacology Biochemistry and Behavior 30, 205–207. Bleich, S., Romer, K., Wiltfang, J., Kornhuber, J., 2003. Glutamate and the glutamate receptor system: a target for drug action. International Journal of Geriatric Psychiatry 18, S33–S40. Blomgren, K., Zhu, C., Wang, X., Karlsson, J.O., Leverin, A.L., Bahr, B.A., Mallard, C., Hagberg, H., 2001. Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of “pathological apoptosis”? Journal of Biological Chemistry 276, 10191–10198. Braughler, J.M., Hall, E.D., 1989. Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Radical Biology and Medicine 6, 289–301.

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