Ginsenoside Rg1 protects primary cultured rat hippocampal neurons from cell apoptosis induced by β-amyloid protein

Abstract

Objectives: Estrogen is known to prominently benefit neuronal syndromes and neurodegenerative diseases. Ginsenoside Rg1, an active ingredient found in a Chinese plant, ginseng root, was previously demonstrated to exert estrogen-like activity. This study was performed to assess the neuroprotective effect of ginsenoside Rg1 against apoptosis induced by β-amyloid protein 25–35 (Aβ_25–35) in primary cultured rat hippocampal neuronal cells as well as in the underlying mechanisms.

Methods: We first measured cell viability and lactate dehydrogenase (LDH) release from primary cultured rat hippocampal neurons. After that, the inhibition effects of ginsenoside Rg1 on neuronal cell apoptosis were evaluated with flow cytometric analysis. Furthermore, western blot analysis was used for detecting the expression of apoptosis-related proteins Bcl-2, Bax, and active caspase 3.

Results: The results show that ginsenoside Rg1 could increase neuronal viability and reduce LDH release; rescue cell apoptosis induced by Aβ_25–35; decrease the expression of caspase 3, increase the ratio of Bcl-2/Bax at the protein levels compared with the cells only treated with Aβ_25–35.

Conclusions: Taken together, our results indicate that the apoptosis induced by Aβ_25–35 could be reversed by ginsenoside Rg1. Furthermore, this neuroprotective effect is probably mediated by up-regulating the ratio of Bcl-2/Bax that activates caspase 3.

Keywords::

• Alzheimer’s disease
• β-amyloid protein
• ginsenoside Rg1
• neuroprotection

Introduction

Pathologically, Alzheimer’s disease (AD) is manifested by selective oxidative stress-induced neuronal cell death, the deposition of β-amyloid (Aβ) peptide into senile plaques in the extracellular space, and the formation of neurofibrillary tangles inside the neuron. Neuronal apoptosis induced by Aβ was proved to be closely associated with AD (Kawasumi et al., 2002; Pereira et al., 2004). Different therapeutic efforts are targeted at blocking Aβ aggregation, Aβ production, Aβ-induced toxicity, and oxidative stress (Zhu et al., 2004; Irie et al., 2005; Tanzi & Bertram, 2005). Estrogen is shown to decrease the production of Aβ and to be effective against Aβ-induced toxicity in neurons (Green et al., 1996; Xu et al., 1998; Goodenough et al., 2003). However, we have to recognize the side effects of estrogen replacement therapy (ERT). It probably increases proliferative and oncogenic activities on non-neuronal cells such as cells in the breast and endometrium (Beresford et al., 1997; Collaborative Group on Hormonal Factors in Breast Cancer, 1997).

Accordingly, growing attention has been paid to phytoestrogens for their potential therapeutic use in a range of hormone-dependent diseases. They have fewer side effects and have been proven to possess neuroprotective activity (Mackey & Eden, 1998; Kim et al., 2005). Panax ginseng C.A. Meyer (Araliaceae) is an important Chinese herbal medicine that has been widely used in restoring harmony and improving memory for thousands of years in China, and recently it is being widely studied in the west. Ginsenosides were reported to be the most abundant and active compounds contributing to the pharmacological actions of ginseng (Huang & Williams, 1999). As one of nearly 40 different ginsenosides that have been identified from ginseng, ginsenoside Rg1 has been attracted considerable attention. In recent years, ginsenoside Rg1 has been termed as a phytoestrogen as it exhibits estrogenic properties (Chan et al., 2002; Leung et al., 2007; Gao et al., 2009). However, the molecular mechanisms underlying the neuroprotective effect of ginsenoside Rg1 have not been fully studied. Our experiments were performed to explore whether ginsenoside Rg1 could protect primary cultured rat neuronal cells against insult of Aβ_25–35. Furthermore, in our experiments, it was demonstrated that ginsenoside Rg1 regulates the expression of Bcl-2, bax, and caspase 3 and thus inhibit the neuronal cell apoptosis induced by Aβ.

Materials and methods

General chemicals

Ginsenoside Rg1 was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of ginsenoside Rg1 for experiments was >99%. Aβ_25–35, 17β-estradiol (E2), dimethyl sulfoxide (DMSO), arabinosylcytosine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and poly-d-lysine were purchased from Sigma-Aldrich (St. Louis, MO).

Primary antibodies of Bcl-2, Bax, and active form of caspase 3 were purchased from Cell Signaling Technology (San Francisco, CA). Dulbecco’s modified Eagle’s medium/F12 medium (DMEM/F12) and trypsin were purchased from Gibco (Burlington, Ontario, Canada). Fetal bovine serum was obtained from Sijiqing (Hangzhou, China).

Primary neuronal cell cultures

Primary cultured hippocampal neurons were prepared from embryonic brains of Wistar rats at Days 16–18 of gestation as previously described (Bastianetto et al., 1999; Xie et al., 2000). After removal of meninges in DMEM/F12 medium, hippocampi were cut into small pieces of 1 mm³ and treated with 0.125% trypsin for 15 min at 37°C. The reaction was stopped using DMEM/F12 medium supplemented with 10% fetal bovine serum. The pellet was dissociated by repeated passage through a series of fire-polished constricted Pasteur pipettes and passed through a nylon mesh. Finally, cells were plated on polyethylenimine-coated (0.1 mg/mL) 6-well, 12-well, 24-well plates or culture flasks with 90% DMEM/F12 medium, 10% fetal bovine serum, 10 U/mL penicillin and 10 U/mL streptomycin at a density of 1 × 10⁵ cells/mL. The cultures were placed in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The cells were exposed to arabinosylcytosine (5 μg/mL) for 24 h at Day 3 to suppress the proliferation of glial cells. Cells were used for experiments after 10 days in vitro.

Exposure to neuronal cells

Ginsenoside Rg1 was dissolved in sterile distilled water at a concentration of 0.4 mg/mL as a stock solution. Aβ_25–35 at a concentration of 1 mg/mL was dissolved in DMEM/F12 medium and incubated for 7 days at 37°C prior to use. E2 was dissolved at 10 mM in ethanol and then diluted into DMEM/F12 media to a concentration of 10 μM.

Neuronal cells were treated with Aβ_25–35 and either ginsenoside Rg1 or E2 after refreshing the medium at Day 7. The experiments were performed 72 h later.

Analysis of cell injury

Cell viability was determined by the MTT method. Cells (5 × 10⁴ cells/well) were seeded in 96-well plates. In brief, after various treatments, cells were incubated with MTT solution (25 μL, 5 mg/mL). Four hours later, cells were exposed to DMSO by shaking for 10 min. Absorbance of each well was measured at a wavelength of 570 nm with a universal microplate reader (ELx 800, BioTek Instruments, Winooski, VT). The optical density of untreated controls was set at 100%.

Cell viability was also evaluated by measurement of lactate dehydrogenase (LDH) (Biovision, USA) released into the medium. LDH activity was determined according to the manufacturer’s instructions. Colorimetric absorbance was measured at 570 nm with a microplate reader (ELx800).

Assays were repeated in three independent experiments, each performed in quintuplicate.

Flow cytometric analysis of Annexin V/PI staining

Cell apoptosis was investigated by staining cells with both Annexin V and PI (Biovision, Mountain View, CA). The 1–5 × 10⁵ cells were collected by centrifugation after various treatments. Then cells were incubated with Annexin V and PI according to the manufacturer’s instructions for 5 min at room temperature in the dark. Apoptotic cells can be recognized and distinguished from necrotic cells using flow cytometric analysis (FACSCalibur, Franklin Lakes, NJ). The percentage of apoptotic cells was determined from a DNA histogram as the ratio of the hypo-diploid cell population (the sub-G1 peak) to the total number of cells.

Protein expression detection by western blot analysis

The protein expression of Bcl-2, Bax, and active caspase 3 was determined by western blot analysis. Cells were collected on ice in lysis buffer containing Tris–HCl (pH 6.8, 125 mM), 2% sodium dodecyl sulfate (SDS), 10% glycerol, and 2-mercaptoethanol (pH 6.8, 200 mM). Then SDS-polyacrylamide gel electrophoresis was performed and the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was washed in Tris-buffered saline and blocked for 2 h in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS/T) at room temperature. The membrane was incubated in 5% milk with primary antibodies (1:1000) overnight at 4°C. After that, the membrane was washed three times for 5 min each with 10 mL TBS/T and incubated with secondary anti-rabbit HRP antibody (1:3000) with gentle agitation for 1.5 h at room temperature. The bands were visualized by the enhanced chemiluminescence (ECL) method and exposed to an X-ray film. The intensity of the bands was quantified using a densitometer. For quantification, densities were normalized by corresponding blots for actin.

Statistical analysis

All data are expressed as mean ± SD. Statistical comparisons were performed by one-way analysis of variance (ANOVA) and the least significant difference (LSD) t-test. A P-value of <0.05 was considered statistically significant.

Results

Establishment of Aβ_25–35-insulted primary culture neuronal cell model

The aggregation of Aβ and Aβ_25–35 are considered to be critical events that induce neuronal apoptosis (Pike et al., 1995). Therefore, in this study we used Aβ_25–35 to induce neuronal toxicity. Various concentrations of Aβ_25–35 were added into the culture medium to determine a suitable concentration for the model. Cell viability was assessed by MTT method and LDH release assessment. As shown in Figure 1, the toxicity of Aβ_25–35 to neuronal cells was in a dose-dependent manner. The results showed decreased levels of MTT reduction by 34.7% after treatment with 5 μM Aβ_25–35 and by 30% after treatment with 10 μM Aβ_25–35 for 3 days at 37°C (Figure 1A); LDH release assessment obtained the same results (Figure 1B). Accordingly, we eventually chose a 3-day treatment of 5 μM Aβ_25–35 for further experiments.

Figure 1.  Effect of Aβ_25–35 on the viability of primary cultured neuronal cells. The 7-day-old cells were treated with various concentrations of Aβ_25–35 (0, 2.5, 5, 7.5, 10 μm) for 3 days and then measured. (A) Cell viability and (B) LDH release. Data represent means ± SD, n = 3.

Ginsenoside Rg1 inhibits the cytotoxicity of Aβ_25–35

The neuroprotective effects of ginsenoside Rg1 in preventing Aβ_25–35-induced cytotoxicity were determined. Neuronal cells underwent various treatments for 3 days before the viability test. As illustrated in Figure 2, after treatment with Aβ_25–35, the cell viability was decreased remarkably to 65.3 ± 5.5% and LDH release was increased to 293.7 ± 19.3%. The cell viability was decreased and LDH release was inhibited in the ginsenoside Rg1 group and E2 group compared with the model group. Ginsenoside Rg1 groups produced dose-dependent effects on LDH release.

Figure 2.  Ginsenoside Rg1 inhibits Aβ_25–35-induced neurotoxicity of primary cultured neuronal cells. Primary cultured neuronal cells were treated with E2, various concentrations of ginsenoside Rg1 and Aβ_25–35 of 5 μM for 3 days. (A) Cell viability and (B) LDH release. Data represent means ± SD, n = 3. *P < 0.01 versus the control group, **P < 0.05 versus the model group, and #P < 0.01 versus the model group.

Ginsenoside Rg1 protects apoptosis induced by Aβ_25–35

To determine the cell apoptosis after various treatments, Annexin V and PI were used to stain cells and were then distinguished by flow cytometric analysis. As seen in Figure 3, the early apoptotic cells are presented in the lower right quadrant of the fluorescence-activated cell-sorting histogram (Annexin V FITC⁺/PI⁻). The late apoptotic cells are presented in the upper right quadrant (Annexin V FITC⁺/PI⁺). As seen in Figure 4, various concentrations of ginsenoside Rg1 significantly reduced the numbers of early apoptotic cells to 11.5 ± 1.32% (10⁻⁸ M), 7.12 ± 1.16% (10−⁷ M), 6.88 ± 2.26% (10⁻⁶ M) compared with 18.19 ± 0.77% of the model group. The numbers of early apoptotic cells were reduced to 6.56 ± 1.91% in the E2 group. These results suggest that both ginsenoside Rg1 and E2 could rescue apoptosis induced by Aβ_25–35.

Figure 3.  Detection of Ginsenoside Rg1 protecting Aβ_25–35-induced apoptosis by flow cytometry analysis of Annexin V/PI staining. The early apoptotic cells are presented in the lower right quadrant (Annexin V FITC⁺/PI⁻). The late apoptotic cells are presented in the upper right quadrant (Annexin V FITC⁺/PI⁺). Viable cells are presented in the lower left quadrant (Annexin V FITC⁻/PI⁻). Necrotic cells are shown in the upper left quadrants (Annexin V FITC⁻/PI+). (A) Control, (B) model, (C) Rg1 10⁻⁸ M, (D) Rg1 10⁻⁷ M, (E) Rg1 10⁻⁶ M, and (F) E2 10⁻⁸ M.

Figure 4.  The percentage of apoptotic cells after Annexin V/PI staining. Data represent means ± SD, n = 3. *P <0.01 versus the control group and **P < 0.05 versus the model group.

Expression of Bcl-2, Bax, and active caspase 3 protein

As shown in Figures 5 and 6A, levels of Bcl-2 protein were up-regulated after being treated with ginsenoside Rg1 and E2, and levels of Bax protein were down-regulated (Figures 5 and 6B). It resulted in an up-regulation of the Bcl-2/Bax ratio (Figures 5 and 6C). As illustrated in Figures 5 and 6D, ginsenoside Rg1 down-regulated the activation of caspase 3 similar to E2.

Figure 5.  Protein levels of Bcl-2, Bax, Bcl-2/Bax, and active caspase 3 were changed by ginsenoside Rg1.

Figure 6.  The ratio of protein expressions. (A) Bcl-2/actin, (B) Bax/actin, (C) Bcl-2/Bax, and (D) active caspase 3/actin. Data represent means ± SD. n = 3. *P < 0.01 versus control group, **P < 0.05 versus the model group, and #P <0.01 versus the model group.

Discussion

A large body of evidence suggests that estrogen plays a profound neuroprotective role in Alzheimer’s and Parkinson’s diseases (Behl, 2002). In recent years, because of the side effects of estrogen in clinical evidence, ginseng has been widely studied as it was shown to possess estrogen-like activity. It was found that ginsenoside Rg1 pretreatment reduced inducible nitric oxide (NO) synthase protein level and NO production (Chen et al., 2003). Gao et al. (2009) have reported that ginsenoside Rg1 may attenuate 6-OHDA-induced apoptosis and its action might involve the activation of IGF-IR signaling pathway and ER signaling pathway. Leung et al. (2007) have reported that ginsenoside Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of primary cultured nigral neurons against rotenone toxicity. However, its molecular mechanisms implicated in the protective actions are not fully understood. In the present study, we attempt to study the mechanism of the neuroprotective effect of ginsenoside Rg1, one of the most abundant ginsenosides found in ginseng.

Our results show that ginsenoside Rg1 could protect neurons from damage induced by Aβ_25–35 insults similar to E2. Additionally, ginsenoside Rg1 increased neuronal viability and reduced LDH release in cultured neuronal cells. We found that after being treated with Aβ_25–35 for 3 days, cell viability had dramatically decreased and that ginsenoside Rg1 could reverse this. Moreover, in Annexin V/PI staining tests, with ginsenoside Rg1 treatment, the neuronal cells appeared less apoptotic than those treated with only Aβ_25–35. All these findings demonstrated that ginsenoside Rg1 could protect cells against Aβ_25–35-induced toxicity in primary cultured neurons.

Caspases (cysteine aspartate-specific proteases) are a family of intracellular proteins involved in the initiation and execution of apoptosis (Ashe & Berry, 2003). Apoptosis is promoted by proteolysis that is largely due to the activity of caspases (Miller, 1997; Takai et al., 1998). Caspases are synthesized as inactive proenzymes in the cytoplasm and are activated by cleavage of specified aspartate residues, thus initiating a cascade of proteolytic cleavage leading to activation of downstream caspases with cellular substrates, such as poly(ADP-ribose) polymerase (PARP) and lamins (Jellinger, 2000). To date, >14 mammalian caspases have been identified (Thornberry & Lazebnik, 1998). Caspase 3 is a key executor caspase (Kim et al., 1997); its activation is considered specific for the apoptotic process and defines an irreversible stage in the cell-death cascade (Jellinger, 2000). In our experiment, ginsenoside Rg1 and E2 decreased the expression of active caspase 3 at the protein levels compared with the cells treated with Aβ_25–35. This indicated that the activation of caspase 3 plays an important role in the neuroprotective effect of both ginsenoside Rg1 and E2.

It was found that in apoptosis triggered by many stimuli, mitochondria play a pivotal role in coordinating caspase activation through the release of cytochrome c (Solange & Martinou, 2000). Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria, as well as a gene with clear anti-apoptotic properties in neurodegenerative conditions (Yang et al., 1997; Nuydens et al., 2000). Thus, one possible role of Bcl-2 in the prevention of apoptosis is to block cytochrome c release from mitochondria (Yang et al., 1997). Currently, 15 Bcl-2 family proteins have been identified in mammals (Gross et al., 1999). This family includes proteins that can promote either cell survival, such as Bcl-2, Bcl-XL, Mcl-1, A1, Bcl-W, or cell death like Bax, Bak, Bcl-XS, Bok (Adams & Cory, 1998; Chao & Korsmeyer, 1998). Sato et al. (1994) suggested that Bax is a cell-death effector whose activity is neutralized by binding of Bcl-2. The ratio between these two subsets helps determine, in part, the susceptibility of cells to a death signal (Oltvai et al., 1993). Data from the study demonstrated an increase of Bcl-2, decrease of Bax, as well as a dramatic up-regulation of the Bcl-2/Bax ratio in the ginsenoside Rg1 group and E2 group neuron cells. These results are in line with our other results described earlier. This indicated that ginsenoside Rg1 and E2 protect the neuron cells from apoptosis induced by Aβ_25–35 insults, probably because it could increase the ratio of Bcl-2/Bax that down-regulates the activation of caspase 3 and finally inhibits neuronal cell apoptosis. Moreover, further studies are required to determine whether estrogen receptors are involved in ginsenoside Rg1-related neuroprotection, or whether there are other signal transduction pathways involved.

Conclusion

In conclusion, ginsenoside Rg1 exerted a neuroprotective role on the apoptosis neurons induced by Aβ_25–35 similar to E2. However, there are fewer side effects of using ginsenoside Rg1 compared with E2. This proves that more attention should be paid to clarify the further mechanisms of ginsenoside Rg1, which will help us to discover more potent neuroprotective candidates.

Declaration of interest

This work was supported by the Health Department of Shandong Province, Jinan, China. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.