Biological and Pharmaceutical Bulletin
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Protective Effects of Ginsenoside Rg1 on Astrocytes and Cerebral Ischemic-Reperfusion Mice
Chenghong SunXinqiang LaiXiuyan HuangYaoying Zeng
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2014 Volume 37 Issue 12 Pages 1891-1898

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Abstract

Ginsenoside Rg1 (Rg1), one of the active ingredients in Panax ginseng, has been known to regulate many cellular processes. The purpose of this study was to investigate the protective effects of Rg1 on apoptosis in mouse cultured astrocytes in vitro and a mouse model of cerebral ischemia and reperfusion in vivo. The cell apoptosis was measured by fluorescence microplate reader and xCELLigence system and the Ca2+ overload was recorded by confocal microscopy. The mitochondrial membrane potential and reactive oxygen species (ROS) were determined by flow cytometry. BALB/c mice were subjected to transient middle cerebral artery occlusion (MCAO) and randomly divided into four groups: Sham (sham-operated +0.9% saline), MCAO (MCAO+0.9% saline), Rg1-L (MCAO+Rg1 20 mg/kg) and Rg1-H (MCAO+Rg1 40 mg/kg). Neurological deficit scores, brain water content and infarct volume were evaluated at 24 h after reperfusion. The results showed that Rg1 significantly attenuated H2O2-induced apoptosis in astrocytes. Rg1 efficiently inhibited intracellular Ca2+ overload, loss of mitochondrial membrane potential, and ROS production in astrocytes. In vivo study, it was also observed that Rg1 markedly reduced the neurological deficit scores, brain edema, and infarct volume in the model mice. These results suggest that Rg1 possesses significant neuroprotective effects, which might be related to the prevention of astrocytes from apoptosis.

Stroke is the third leading cause of death and a leading cause of disability worldwide.1) Between 1990 and 2010, the number of stroke-related deaths increased by 26% and disability-adjusted life-years by 19%.2,3) Ischemia and reperfusion is a pathological condition, which is characterized by an initial restriction of blood supply to an organ followed by the subsequent restoration of perfusion and concomitant reoxygenation. A variety of animal models have been developed for modeling ischemic stroke. The middle cerebral artery occlusion (MCAO) model has been utilized extensively, especially in rodents. Ischemic injury encompasses all cell types, including astrocytes. Astrocytes play a fundamental role in the pathogenesis of ischemic neuronal cell death. Efforts targeting the functional integrity of astrocytes may constitute a superior strategy for neuroprotection.4) Several molecular pathways are involved in astrocyte apoptosis, such as Ca2+ overload, oxidative stress, and mitochondrial dysfunction. Therefore, it is very effective and important to protect the central nervous system that preventing astrocytes against cell apoptosis.

Panax ginseng, the fleshy roots of the family Araliaceae plants, has been used as a tonic effect in oriental countries, especially China. With unique triterpenoid saponins, ginsenosides are recognized as the major active ingredient of the herb and up to now more than 150 naturally occurring ginsenosides have been isolated from ginseng, including ginsenoside Rg1 (Rg1).5) As a highly interesting natural saponin compound for drug development, Rg1 has a broad spectrum of established pharmacological functions. Rg1 increased the proliferating ability of neural progenitors to promote neurogenesis.6,7) Rg1 attenuated amyloid β (Aβ)1–40-induced apoptosis in rat cortical neurons via inhibiting the activity of cyclindependent kinase-4 (CDK4), decreasing the phosphorylation of pRB and downregulation the expression of E2F1 mRNA.8) Including Alzheimer’s disease, study of Parkinson’s disease had reported that activation of the insulin-like growth factor-1 (IGF-1) mediated the neuroprotective effects of Rg1 in 6-hydroxydopamine (6-OHDA)-treated rats.9) In addition, Rg1 also shows a neuroprotective effect on brain ischemia by inhibition of Ca2+ influx into primary cultured hippocampal neurons.10) However, the anti-apoptotic effect of Rg1 on the astrocytes remains to be elucidated.

In the current report, we examined the impact of Rg1 treatment in vitro using murine culture astrocytes. MCAO, as a well-characterized model for studies on acute ischemia stroke, was applied to evaluate the effect of Rg1 administration in vivo.

MATERIALS AND METHODS

Animals and Reagents

Rg1 (HPLC purity>98%) was supplied by Sigma-Aldrich (St. Louis, MO, U.S.A.). Lipopolysaccharides (LPS), Ionomycin, H2O2, poly-D-lysine, chloral hydrate and 2,3,5-triphenyltetrazolium chloride (TTC) were obtained from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium, fetal calf serum (FCS), glutamine, β-mercaptoethanol, penicillin and streptomycin were purchased from Gibco Lab (Rockville, MD, U.S.A.). Fluo-4/acetoxymethyl ester (AM), Pluronic F-127, sulforhodamine 101, 4′,6-diamidino-2-phenylindole (DAPI), SYTOX Green, DIOC6(3) and H2DCFDA were obtained from Invitrogen (U.S.A.). Cell Counting Kit-8 was purchased from DOJINDO (Kumamoto, Japan).

Male BALB/c mice (25–30 g) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China). The animal experiments were approved by the Ethics Committee for Experimental Animals at Jinan University and were performed according to the national guidelines for animal welfare.

Cell Isolation and Culture

Primary astrocytes were isolated as described previously with minor modifications.11) Briefly, 1–2 d of newborn mice were decapitated, and the brains were removed and collected in ice-cold phosphate buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The brains were gently passed through nylon meshes of 200 µm pore width. The cell suspension was centrifuged at 4°C for 5 min at 500×g. The cells were resuspended in 10 mL growth medium (DMEM-F12 supplemented with 10% (v/v) FCS, 20 U/mL penicillin and 20 µg/mL streptomycin). Cells were then placed into 75 cm2 tissue culture flasks precoated with poly-D-lysine (20 mg/mL). Cultures were incubated in a CO2 incubator at 37°C in an atmosphere of 5% CO2 in air. The medium was changed after 3 d and then twice a week. On reaching confluence, the cultures were shaken for 3 h at 180 rpm to remove microglia and oligodendrocyte progenitors. The remaining astrocyte monolayers were trypsinized and plated in culture dishes at a density of 3–5×105/mL cells per dish and incubated at 37°C with 5% CO2, humidified to saturation. In these cultures, more than 98% of the cells are positive for sulforhodamine 101 (SR101), an astrocyte-specific marker.12)

Cytotoxicity

Astrocytes were seeded on a 24-well plate and were treated with different concentrations of Rg1 (0, 2.5, 5, 10 and 20 µM) for 48 h. Each concentration should be given 3 repetitions. And then cells were added with 40 µL CCK-8 and incubated for 4 h. The absorbance was then measured at 450 nm using a microplate reader (Bio-Rad, U.S.A.).

Cytotoxicity of Rg1 was also measured by the xCELLigence Cells Analysis System (Roche Inc., Switzerland) which was placed in a humidified incubator maintained at 37°C with 5% CO2. Growth curves were constructed using disposable E-plates with 96 wells containing integral sensor electrode arrays. Briefly, cells were seeded at 5×104/mL in DMEM-F12 medium containing 10% FCS, and each well should be filled with 200 µL medium. The cells were divided randomly into two groups, control group, Rg1 group. Each group should be given 3 repetitions. After 6 h, Rg1 (10 µM) was added to Rg1 group. At the same time, 10 µL normal saline (NS) was added to control group. Cell index was recorded every 5 min.

H2O2-Induced Apoptosis

Astrocytes were seeded on a 24-well plate and were pretreated with different concentrations of Rg1 (0, 2.5, 5 and 10 µM) for 6 h. Each concentration should be given 3 repetitions. And then cells were co-treated with 5 µM H2O2 at 37°C. After being cultured for 48 h, cells were added with 0.5 µM SYTOX Green and incubated at room temperature for 30 min. The absorbance was then measured at 488ex/523em nm using a fluorescence microplate reader (Berthold, Germany). Only the dead cells with compromised plasma membranes could be stained, so the higher mean fluorescence intensity (MFI) meant the higher mortality of cells, on the contrary, the better the cell states.

The effect of Rg1 on H2O2-induced apoptosis was also measured by the xCELLigence Cells Analysis System as the section of cytotoxicity described. The cells were divided randomly into four groups, control group, Rg1 group, H2O2 group, H2O2+Rg1 group. Each group should be given 3 repetitions. After 6 h, Rg1 (10 µM) was added to Rg1 group and H2O2+Rg1 group. At the same time, 10 µL NS was added to control group and H2O2 group. After 12 h of recording, H2O2 group and H2O2+Rg1 group were exposed to 5 µM H2O2, and other groups received NS only.

Intracellular Ca2+ Kinetics

Intracellular Ca2+ kinetics was performed as described previously with minor modifications.13) Briefly, astrocytes were seeded on a petri dish (MatTek, U.S.A.). The Ca2+ indicator Fluo4-AM (2.5 µM) was gently mixed with an equal volume of pluronic F-127. One micro mole SR101, an astrocyte-specific marker, was added simultaneously. Cells were loaded with the mixture and incubated in the dark at 37°C for 30 min. After the incubation, cells were washed by Locke’s buffer (145 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1 mM MgCl2, 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-Na, and 5.6 mM glucose adjusted to pH 7.4 with HCl). The cells were suspended with Locke’s buffer, followed by the addition of 0.5 µM DAPI, a nuclear dye. The cells were pretreated with 10 µM Rg1 or NS for 1960 s and then stimulated by 2.5 µM Ionomycin (Ion). The imaging of time series was performed by LSM 510META confocal microscope (Zeiss, Germany). The regions of interest (ROIs) were analyzed by the Zeiss AIM Image Examiner software.

Analysis of ΔΨm, Reactive Oxygen Species (ROS)

Astrocytes were seeded on a 24-well plate and were pretreated with different concentrations of Rg1 (0, 2.5, 5 and 10 µM) for 6 h. Each concentration should be given 3 repetitions. And then cells were co-treated with 2.5 µM Ionomycin (Ion) or 10 µg/mL LPS at 37°C. After being cultured for 24 h, cells were harvested by a cell scraper and washed two times. The cells were incubated with 20 nM DIOC6(3) or 10 µM H2DCFDA in the dark for 30 min, followed by washing two times. The flow cytometry analysis was then performed using a FACSAria flow cytometer (BD, U.S.A.).

Mouse Model of Cerebral Ischemia and Reperfusion

Middle cerebral artery occlusion (MCAO) surgery was performed as described previously with minor modifications.14) Briefly, mice at the age of 6–8 weeks were anesthetized with chloral hydrate (300 mg/kg, intraperitoneal (i.p.) injection) and the right common carotid artery was exposed at the level of the external and internal carotid artery (ECA and ICA) bifurcation. A 25 mm length of monofilament nylon suture (ϕ 0.20±0.01 mm) was inserted into ECA from the common carotid artery bifurcation and pushed into the ICA for 8–10 mm until a slight resistance was felt, to block the origin of the middle cerebral artery. Afterwards, the skin incision was sutured, and 60 min after MCAO, reperfusion was allowed by withdrawal of the suture thread until the tip cleared the ICA. Animals were then returned to their cages and closely monitored, with body temperature kept at (37±0.5) °C.

Groups and Drug Administration

All mice (n=10 in each group) were randomly divided into four groups, (1) Sham group: animals received sham operation and equal volume of normal saline; (2) MCAO group: animals received MCAO and equal volume of normal saline; (3) Rg1-L group: animals received MCAO and treated with Rg1 at 20 mg/kg; (4) Rg1-H group: animals received MCAO and treated with Rg1 at 40 mg/kg. Rg1 was dissolved in normal saline to prepare concentrations of 20 mg/mL and 40 mg/mL. Different doses of Rg1 or solvent were administered by i.p. at 0.5 h after cerebral ischemia and 12 h after reperfusion.

Neurological Deficit Scores

The neurological deficit scores of four groups mice (n=10 in each group) were determined by an examiner blinded to the experimental groups at 24 h after reperfusion. The deficits were scored with a modified scoring system developed by Longa et al.15) as follows: 0, no neurological deficit; 1, failure to extend right paw fully; 2, circling to right; 3, falling to right; 4, did not walk spontaneously and had depressed levels of consciousness. The neurologic deficit score had been measured for 8 times, and the average value would be obtained from these independent experiments.

Measurement of Brain Water Content

Brain water content was measured using the wet-dry weight method.16) Four groups mice (n=10 in each group) were deep anesthetized with chloral hydrate i.p. at 24 h after reperfusion. The brains were quickly removed and gently blotted with tissue paper to clear small quantities of adsorbent cerebrospinal fluid. Brain samples were immediately weighed on an electronic balance to obtain wet weight. Then they were dried to constant weight in an oven to obtain the dry weight. Brain water content was then calculated with the equation as follows: Brain water content (%)={(wet weight−dry weight)/wet weight}×100%. The brain water content had been measured for 3 times, and the average value would be obtained from three independent experiments.

Measurement of Infarct Volume

To detect the infarction volume, four groups mice (n=10 in each group) were deep anesthetized with chloral hydrate i.p. at 24 h after reperfusion. The brains were quickly removed and cut into four coronal slices of 2-mm thickness, incubated in a 2% solution of with 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 20 min. Infarction volume was measured by a blinded observer using digital imaging (Digital Camera, Olympus MDF-382E) and image analysis software (Adobe Photoshop 5.0). The infarct volume had been measured for 3 times, and the average value would be obtained from three independent experiments.

Statistical Analysis

Data were expressed as mean±S.E.M. Statistical significance was analyzed by one-way ANOVA with Student’s test. Neurological deficit scores were analyzed by Wilcoxon’s rank sum test. Significant difference was considered when p<0.05.

RESULTS

Effect of Rg1 on Cell Viability in Mouse Cultured Astrocytes

First, the effect of Rg1 on the cell viability in astrocytes was assessed using CCK-8 assay. We found that treatment with Rg1 (2.5, 5, 10 µM) for 48 h did not cause any significant viability change from the control level (Fig. 1A). However, the cytotoxicity of Rg1 began to appear while the concentration reached 20 µM (p<0.05).

Fig. 1. Effect of Rg1 on the Cell Viability and Apoptosis of Astrocytes

(A) The cells were co-treated with Rg1 for 48 h, and then measured by CCK-8, * p<0.05 vs. blank control cells. (B) The cells were measured by xCELLigence. (C) The cells were pretreated with Rg1 for 6 h, followed by co-treatment with 100 µM H2O2 for 48 h, and then measured by fluorescence microplate reader with SYTOX Green. (D) The cells were measured by xCELLigence. Data are mean±S.E.M. (n=3). * p<0.05, ** p<0.01 vs. cells treated with H2O2. ##p<0.01 vs. blank control cells.

Next, we used the 10 µM concentration in the real-time xCELLigence assay to examine the toxic effects of Rg1. As shown in Fig. 1B, the cell index (CI) of two groups sharply increased after seeding up to reach their first maximum at 6 h. Thereafter the both CIs slowly decreased to reach a minimum at 10 h to increase again to their second maximum at 48 h, respectively. There was no any significant viability change from the control level, indicating that Rg1 was non-cytotoxic to astrocytes within experimental concentration range.

Rg1 Attenuated H2O2-Induced Apoptosis in Astrocytes

SYTOX Green stain is a high-affinity nucleic acid dye that easily penetrates cells with compromised plasma membranes and yet will not cross the membranes of live cells. As shown in Fig. 1C, 5 µM H2O2 could induce obviously astrocytes apoptosis (p<0.01, vs. control). On the other hand, the pretreatment of Rg1 (2.5, 5, 10 µM) significantly inhibited H2O2-induced cytotoxic effects in a dose-dependent manner.

Similarity with cytotoxicity, the real-time xCELLigence assay was to apply to investigate the effect of Rg1 on astrocytes apoptosis (Fig. 1D). Thereafter the CI of H2O2 group reached a plateau value at 40 h to decrease slowly, where the CI of H2O2+Rg1 group remained increasing (p<0.01, vs. H2O2 group). Namely, it’s very significant that pretreatment of 10 µM Rg1 inhibited H2O2-induced apoptosis in astrocytes.

Rg1 Inhibited Intracellular Ca2+ Overload in Astrocytes

Ionomycin (Ion) greatly raised the level of intracellular calcium ([Ca2+]i), and the ratio of fluorescence intensity (F/F0) of control group (Fig. 2B) and Rg1 group (Fig. 2C) approximately reached their maximum at 2400 s and 3100 s, respectively. Pretreatment of Rg1 had postponed the appearance of maximal [Ca2+]i for 700 s (3100–2400). DAPI was used to stain dead cells since the dye was cell impermeant. As shown in Fig. 2A, the death time of start and end in the control group were 2800 s and 3200 s, respectively. Moreover, both time of the Rg1 group were 3600 s and 3800 s, respectively. Consequently, Rg1 had delayed the death time of start and end for 800 s (3600–2800) and 600 s (3800–3200), respectively, which were almost consistent with 700 s mentioned above.

Fig. 2. The Elevated Intracellular Ca2+ Levels of Astrocytes Treated with Ion Is Inhibited by Rg1

The experiment was described in Materials and Methods. Intensity of fluorescence was collected and calculated at different time intervals for 5 s. (A) is represented for 25 of interest cells per microscope field at each time point in three independent experiments. (B) Control group. (C) Rg1 group. Changes in fluorescence intensity (F) as a function of time are expressed in the form (F/F0), where F0 indicates resting fluorescence intensity. Ion: ionomycin.

Effect of Rg1 on ΔΨm and ROS in Astrocytes

To test whether mitochondrial membrane potential (ΔΨm) and ROS were associated with the protective effect of Rg1 on apoptosis in astrocytes, we stained the cells with DIOC6(3) or H2DCFDA, which measured ΔΨm and ROS, respectively. As shown in Fig. 3A, Ion initiated a hyperlorization of the mitochondrial membrane in astrocytes when compared with control group (p<0.01). Preincubation of Rg1 in the presence of Ion for 6 h blocked ΔΨm loss in a dose-dependent manner. ROS was also produced in mitochondria, whose respiratory chain is a major source. As shown in Fig. 3B, LPS induced the generation of ROS in astrocytes very significantly when compared with control group. Pretreatment of Rg1 in the presence of LPS for 6 h decreased the production of ROS dose-dependently.

Fig. 3. Effect of Rg1 on ΔΨm and ROS in Astrocytes

(A) The cells were pretreated with different concentrations of Rg1 for 6 h, followed by co-treatment with 2.5 µM Ion for 24 h, and then measured by flow cytometry with DIOC6(3). Data are mean±S.E.M. (n=3). * p<0.05, ** p<0.01 vs. cells treated with Ion. ##p<0.01 vs. blank control cells. (B) The cells were pretreated with different concentrations of Rg1 for 6 h, followed by co-treatment with 10 µg/mL LPS for 24 h, and then measured by flow cytometry with H2DCFDA. Data are mean±S.E.M. (n=3). * p<0.05, ** p<0.01 vs. cells treated with LPS. #p<0.05, ##p<0.01, &&p<0.01 vs. blank control cells. Ion: ionomycin.

Neuroprotective Effects of Rg1 against Ischemia and Reperfusion (I/R) in Mice

Neurological deficit scores were evaluated at 24 h after reperfusion. MCAO group got higher scores than Sham group (Fig. 4A). Compared with MCAO group, the neurological deficit scores were significantly reduced in Rg1-H group (p<0.05), but no significant decrease in the score was observed in Rg1-L group. The preliminary results suggested that Rg1 (40 mg/kg) injection significantly reduced body asymmetry in a Longa’s neurological abnormality scores and promoted the functional recovery of mice. Brain water content at 24 h after reperfusion was shown in Fig. 4B. In Sham operated group, water content was 78.38±0.28% and increased to 80.82%±0.31% at 24 h after reperfusion in MCAO group. Compared with MCAO group, water content markedly reduced to 79.27±0.25% (p<0.05) in Rg1-H group, but no statistical significance was observed in Rg1-L group (80.50±0.31%). These data indicated that Rg1 (40 mg/kg) have a significant effect on reducing brain edema. Figure 4C showed typical photographs of TTC-stained sections from solvent and Rg1-treated mice. No infarction was observed in the Sham group. The infarct volume was significantly lessened from 33.89%±2.62% in MCAO group to 30.03%±2.57% in the Rg1-L group and 13.41%±2.53% in Rg1-H group (p<0.01, Fig. 4D). This suggested that the administration of Rg1 (40 mg/kg) exhibited protective effect against ischemia induced cerebral injury.

Fig. 4. Neuroprotective Effects of Rg1 on Cerebral Ischemia and Reperfusion in Mice

BALB/c mice were randomly divided into Sham, MCAO, Rg1-L and Rg1-H groups with 10 mice, respectively. The drug administration and surgery were described in Materials and Methods. (A) The neurological deficit scores result. (B) Brain water content. (C) and (D) were the results of brain infarct volume. (C) Presented one of the representative experiments, the infarct area was unstained, and the normal part was stained in red. (D) was the statistical results. Data are mean±S.E.M. (n=3). * p<0.05, ** p<0.01 vs. MCAO group. ##p<0.01 vs. Sham group.

DISCUSSION

In the present investigation, we demonstrated for the first time the anti-apoptotic effect of Rg1 on cultured astrocytes via inhibiting Ca2+ overload and ΔΨm loss induced by Ion, as well as ROS production stimulated by LPS. In vivo, administration of Rg1 reduced the neurological deficit scores, brain edema, and infarct volume in the I/R mice, which provided an evidence of its neuroprotective activity.

The elevation in intracellular calcium concentration is one of the earliest events during cell apoptosis. The calcium movement is attributed to the depletion from intracellular stores induces the influx of extracellular calcium across the plasma membrane, a mechanism known as the store-operated calcium entry (SOCE).17) In astrocytes, SOCE through calcium channels is one of the main mechanism to increase intracellular Ca2+ concentrations. At higher concentrations (1–10 µM), the ionophore increases the permeability of the plasma membrane, ER and mitochondria to Ca2+ ions.18) In this paper, we found that 2.5 µM Ion greatly raised the level of [Ca2+]i in the cultured astrocytes, pretreatment of Rg1 delayed the appearance of maximal [Ca2+]i and the death time of astrocytes. Moreover, pretreatment of Rg1 also delayed the appearance of maximal mitochondrial calcium when astrocytes were coincubated with Rhod-2, the mitochondrial calcium indicator (data not shown). Therefore, Rg1 reduced calcium overload induced by Ion in the cultured astrocytes, which might be regarded as a potential anti-apoptotic target.

It is well known that an important early apoptotic event is collapse of the mitochondrial membrane potential (ΔΨm), which leads to the opening of mitochondrial permeability transition (MPT) pores, a protein complex that spans both the outer and inner mitochondrial membranes.19) MPT is the critical event for the intrinsic pathway of apoptosis, which marks the frontier between death and life, the “point of no return.”20) Ion increases the permeability of the mitochondrial membrane to Ca2+, which not only raises mitochondrial calcium levels, but also induces ΔΨm reduction. On the other hand, a sustaining elevation of intracellular calcium could induce rapid increases in mitochondrial permeability.21) It has been reported that Rg1 could restore the decreased mitochondrial membrane potential in human neuroblastoma SK-N-SH cells,22) MES23.5 cells,23) and PC12 cells.24) Consistent with these observations, our results suggest that Rg1 significantly blocked ΔΨm loss in the astrocytes.

A variety of studies have demonstrated that reactive oxygen species (ROS) and the resulting oxidative stress play a pivotal role in apoptosis. Previous studies had shown that LPS induced ROS generation, which was mediated in part by direct interaction of TLR4 with Nox4.25) López et al.26) found that ginsenoside Rg1 decreased ROS formation on astrocytes primary culture. Recently, similar results had been demonstrated in cardiomyocytes,27) MES23.5 cells,28) and human umbilical vein endothelial (HUVEC) cells.29) Our present data confirm and extend the observations of others, regarding the anti-apoptotic role of Rg1. The calcium enters the cell through voltage- and agonist-operated channels and also by more unspecific channels, such as those opened by ROS.30) In other words, ROS may stimulate an increase in intracellular calcium concentration. As mentioned above, cell apoptosis will take place as soon as the elevation in intracellular calcium concentration. On the basis of the anti-apoptotic results, we propose the signaling pathways for Rg1 in murine astrocytes, which may be related to Ca2+ overload, oxidative stress, and mitochondrial dysfunction.

To demonstrate the neuroprotective activity of Rg1, we also evaluated its efficacy in the cerebral ischemia and reperfusion mice. The results showed that the administration of Rg1 (40 mg/kg) significantly reduced ischemia damages and reperfusion injury. Previous studies have demonstrated that aquaporin-4 (AQP4) is implicated in the formation and resolution of brain edema, which is one important consequence of cerebral ischemia.3134) Zeng et al.35) found that 10% hypertonic saline exerted anti-edema effects possibly through downregulation of AQP4 expression in the perivascular astrocytes. Hua et al.36) also found that AQP4 played an important role in modulating brain water transport in an astrocytic oxygen-glucose deprivation and reintroduction model. By siRNA knockdown approach, Ding et al.37) showed that downregulation of AQP4 induced glioblastoma cell apoptosis in vitro and in vivo. Moreover, Chu et al.38) found that Granulocyte-colony stimulating factor (G-CSF) led to neurological functional improvement in mice by associating with reduction of brain edema and neuronal death and apoptosis and statistical analysis suggested AQP4 was required for these effects. Recently, Rg1 was found to improve neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of AQP4 expression.39) With the results of this study, we considered that Rg1 markedly reduced the brain water contents of MCAO mice, which might be related to the suppression of astrocytes apoptosis by Rg1. This preliminary study needs further research to confirm whether AQP4 is involved in astrocytes apoptosis and brain edema.

In summary, the present study demonstrated that Rg1 possesses anti-apoptotic effects on astrocytes, which might be association with the inhibition of Ca2+ overload, ΔΨm loss, and ROS production. Rg1 also exerts neuroprotective effects on cerebral ischemia and reperfusion mice, which might be related to prevent astrocytes against apoptosis. These findings provide further evidence for the pharmacological specificity of Rg1.

Acknowledgment

This study was supported by Science and Technology Development Program of Guangdong Province (No. 2010A080407005).

REFERENCES
 
© 2014 The Pharmaceutical Society of Japan
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