Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms

Journal of Ethnopharmacology 159 (2015) 102–112

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Research Paper

Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms Yumao Jiang, Zongyang Li, Yamin Liu, Xinmin Liu, Qi Chang, Yonghong Liao, Ruile Pan n Institute of Medicinal Plant Development, Chinese Academy of Medical Science, Peking Union Medical College, Beijing 100193, China

art ic l e i nf o

a b s t r a c t

Article history: Received 10 August 2014 Received in revised form 28 October 2014 Accepted 29 October 2014 Available online 15 November 2014

Ethnopharmacological relevance: The root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant which has been employed as a panacea for more than 2,000 years in China. It has the actions of invigorating primordial qi, recovering pulse and desertion, engendering liquid, and calming spirit. The water extract of Panax ginseng (WEG) has been used to treat kinds of central nervous system disorders, such as depression, insomnia, Alzheimer's disease and Parkinson's disease. Our previous work has demonstrated that WEG possessed antidepressant-like activities in both acute and chronic stress models of depression. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of WEG on corticosteroneinduced apoptosis. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of WEG. Materials and methods: The PC12 cells were treated with 250 μmol/L corticosterone in the absence or presence of WEG for 24 h, then 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) detection, Hoechst33342 staining and TUNEL staining were investigated to confirm the neuroprotection of WEG. Then, mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca2 þ ([Ca2 þ ]i), reactive oxygen species (ROS) concentration, and the expression level of glucocorticoid receptor (GR), heat shock protein 90 (Hsp90), histone deactylase 6 (HDAC6), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible protein 153 (GADD153), X-box DNA-binding protein-1 (XBP-1), caspase-12, cytochrome C, inhibitor of caspase-activated deoxyribonuclease (ICAD), caspase-3 and caspase-9 were assessed by Western Blot analysis to understand the molecule mechanisms of neuroprotection of WEG. Results: WEG partly reversed corticosterone-induced damage in PC12 cells, which increased cell viability, decreased LDH release, and attenuated corticosterone-induced apoptosis as compared with the corticosterone-treated group. Mechanistically, compared with the corticosterone-treated group, WEG strongly attenuated [Ca2 þ ]i overload and ROS level, and restored mitochondrial function, including mPTP and MMP. Furthermore, WEG strongly up-regulated the expression of GR and HDAC6, and down-regulated the expression of Hsp90, cytochrome C, ICAD, caspase-3, caspase-9 as well as endoplasmic reticulum (ER) stress-related proteins, such as GADD153, GRP78, XBP-1, and caspase-12. Conclusion: WEG possessed neuroprotection against corticosterone-induced damage in PC12 cells, and the underlying molecule mechanisms was depended on the intervening of HDAC6 and HSP90 of the GR-related function proteins, and subsequent restoration of ER and mitochondria functions. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Panax ginseng C.A. Meyer Mitochondria stress Endoplasmic reticulum stress Glucocorticoid receptor Heat shock protein 90 Histone deactylase 6

1. Introduction Depression, especially major depressive disorder, which is a tragedy for patients and families, has been considered as a pathologic change of the physiological adaptation of the brain (Leonard, 2010). Exposure n Correspondence to: Institute of Medicinal Plant, Peking Union Medical College, Chinese Academy of Medical Sciences. No. 151, North Road Malianwa, Haidian District, Beijing 100193, China. Tel.: (86)-10–57833275; fax: þ 86 10 57833299. E-mail address: [email protected] (R. Pan).

http://dx.doi.org/10.1016/j.jep.2014.10.062 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

to environmental and work-related stress has been viewed as the leading cause of this change, since during the repeated bouts of severe aggression; physiological adaptation failed to maintain the response to the negative effects of the stressor and sustained homoeostasis (Yoon et al., 2011; Hammen et al., 2009; Holsen et al., 2011). Among these changes, the most significant counteraction is the hyperactivity and impaired feedback of the hypothalamic –pituitary–adrenal axis, and induces overproduction of glucocorticoids (Rubin et al., 1987; Murray et al., 2008). Correspondingly, there has been a close relationship between the elevated circulating glucocorticoids which induced by

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long-term excessive stress and the impairment of neurons in the dentate gyrus of the hippocampus (Sapolsky, 2001; 1985). In addition, previous research has reported that exogenous administration of high dose of corticosterone induced changes in behavior, neurochemistry and brain anatomy (Crupi et al., 2011). Furthermore, the reduced hippocampal volumes and decreased hippocampal cells were observed in major depression patients by magnetic resonance imaging study and postmortem analyses (Saylam, et al., 2006; Stockmeier, et al., 2004). These interrelated results establish a link between hippocampal damage and the subsequent formation of depressive symptoms (Sapolsky et al., 1984). The neuron injury in hippocamp by chronic corticosterone treatment would be reversed by antidepressants (Hellsten et al., 2002). Therefore, it is speculated that the protection against neuron damage induced by high concentration of corticosterone might be a potential mechanisms of antidepressant agents. The root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant employed as a panacea due to its tonic functions for more than 2,000 years. As a traditional Chinese medicine (TCM), ginseng is often given orally as water decoction, and the clinical indications involved in the treatment of antidepressant and antianxiety (Kennedy and Scholey, 2003). It has been proved that ginseng showed anti-stress effects (Lee, et al., 2006; Pannacci, et al., 2006; Ramesh, et al., 2012) and improved mood and anxiety adjustment for postmenopausal women (Tode, et al., 1999; Wiklund et al., 1999). Our previous work also demonstrated that the extract from Panax ginseng possessed antidepressant-like activities in both forced swimming test and chronic stress models of depression (Dang, et al., 2009). However, the antidepressant mechanism of Panax ginseng is still not clear. As a lot of studies have proved that ginsenosides display the central nervous system protective effects, which increase neuronal survival, promote neurite growth, and rescue neurons from apoptosis (Yan et al., 2013; Wu et al., 2012). Therefore, there is a reason to believe that neuroprotective effect might be contributing to the in vivo antidepressant–like effect. In the recent years, the use of exogenously co-incubated corticosterone with rat pheochromocytoma (PC12) cells has been extensively adopted as an in vitro model to study the impairment of neuron and a serious depression-like syndrome with programmed cell death during the chronic stress (Li, et al., 2013; Zhang, et al., 2010). In present study, PC12 cell line was employed to study the neuroprotective effect and the potential mechanisms of WEG. Our result suggested that cytoprotection of WEG may be through suppression the mitochondria-dependent and ER stress-dependent cell death pathways in the organelle level as well as the remediation of GR related pathway in molecule level.

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through its microscopic and macroscopic characteristics by Professor Bengang Zhang of the Institute of Medicinal Plant, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, where the voucher specimens (No. 20120905) have been deposited in Herbarium of the institute. The powdered ginseng root (100.0 g) was extracted with boiling water for two times (each time for 30 min), the filtrate was combined and evaporated in vacuo to get WEG (20.5 g). The ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd in WEG were analyzed on a Waters ACQUITY UPLC (ELSD) system. A ACQUITY UPLC BEH C18 (50 mm  2.1 mm ID, 1.7 μm) was used with an injection volume of 2 μl for the UPLC separation. The mobile phases consisted of (A) acetonitrile and (B) H2O at a flow rate of 0.3 ml/min. The gradient elution was used as follows: 0-3 min, 19% A; 3-4 min, 19%-21% A; 4-5 min, 21%-26% A; 5-9 min, 26%27% A; 9-12 min, 27%-32% A; 12-15 min, 32%-43% A;1518 min, 43%-60% A; 18-20 min, 100% A. UV absorption was measured at 210 nm. All solutions were filtered through a 0.22 μm filter prior to detection. The gensenosides were identified by comparison of the retention time with authentic standard and the amount of each ginsenoside was quantified by the external standard method. The contents of Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd were 15.5, 15.2, 5.4, 0.75, 16.36, 5.21, 4.45, 0.5, 2.04 mg/g, respectively. 2.3. Cell culture and treatment

2. Materials and methods

PC12 cells were purchased from cell resource center of Chinese academy of medical science (Beijing, China). The cells were maintained in DMEM medium supplemented with penicillin (100 unit/ml), streptomycin (100 μg/ml), 10% fetal bovine serum and 5% horse serum in humidified atmosphere of 5% CO2 and 95% air at 37 1C. Firstly, the appropriate damage concentration of corticosterone was selected on the basis of our previous study (Li et al., 2013). In brief, different concentrations of corticosterone (50, 150, 250, 350 and 450 mmol/L) were incubated with PC12 cells for 24 h, and the cell viability was determined by MTT. When treated with 250 mmol/L corticosterone for 24 h, the cell viability decreased to approximately 50%, which induced cell injury without inducing cell death, and was used in subsequent experiments. To research the neuroprotective effect of WEG, the cells were divided into eight equal groups: non-treated control, 250 μmol/L corticosterone and 250 μmol/L corticosterone plus WEG (6.25, 12.5, 25, 50, 100 and 200 μg/ml) in the experiments. Experiments were executed for 24 h after the cells were seeded. WEG were applied for 24 h prior to the treatment with corticosterone, and then the cells were co-incubated with corticosterone and WEG for another 24 h.

2.1. Materials

2.4. Determination of cell viability

Fetal bovine serum, horse serum, penicillin and streptomycin were obtained from Gibco (Grand Island, NY, USA). Dulbecco's Modified Eagle Medium (DMEM) and Corticosterone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies for cytochrome C (1∶100), ICAD (1∶500), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1∶200), β-actin (1∶200), caspase-9 (1∶200), caspase-3 (1∶200), GRP78 (1∶200), GADD153 (1∶200), XBP-1 (1∶200), GR (1∶200), HDAC6 (1∶200), HSP90 (1∶200) and caspase-12 (1∶200) and the second antibodies labeled with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (1∶1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents are of analytical grade.

Cell viability was determined by MTT colorimetric assay. At the end of treatment, the medium was taken out, and then fresh medium containing 0.5 mg/ml MTT was added to each well. The mixture was incubated for 4 h at 37 1C. Then the culture medium was replaced with equal volume of DMSO to dissolve formazan crystals. After shaking at room temperature for 10 min, absorbance of each well was determined at 570 nm by using the microplate reader (TECAN, Sunrise, Austria). Cell viability was showed as a percentage of non-treated control.

2.2. Preparation of WEG Ginseng roots were obtained from Fusong county, Jilin province, China, on September, 2012. The plant was authenticated

2.5. Lactate dehydrogenase (LDH) release assay LDH is a soluble cytosolic enzyme presented in most eukaryotic cells, it released into the culture medium upon cell death because of the damage of plasma membrane. The increase of LDH activity in the culture medium is proportional to the number of lysed cells. At the end of the drug treatment, the supernatant was collected, and

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the amount of LDH release was determined by using an assay kit according to the manufacturer's protocol. Briefly, the supernatant and cell lysates were transferred to 96-well plates and incubated

with 1 mg/ml NADH in pyruvate substrate solution for 15 min at 37 1C. After additional incubation for 15 min with 2,4- dinitrophenylhydrazine at 37 1C, the reaction was stopped by adding 0.4 mol/L NaOH. The changes in absorbance were determined at 440 nm by using a spectrophotometric microplate reader (TECAN, Sunrise, Austria). LDH leakage was expressed as the percentage (%) of the total LDH activity (LDH in the supernatantþLDH in the cell lysate), according to the equation: %LDH released¼ (LDH activity in the medium/total LDH activity)  100%. 2.6. Assessment with Hoechst 33342 staining Hoechst 33342 staining distinguishing apoptotic from normal cells based on nuclear chromatin condensation and fragmentation was used for the qualitative and quantitative analyses of the apoptotic cells. PC12 cells were cultured in 6-well plates for 24 h. After treatment, the cells were incubated with 5 μg/ml Hoechst 33342 for 15 min, washed twice with phosphatebuffered saline (PBS), and then visualized by inverted fluorescence microscopy (Leica, Germany). The apoptotic nuclei were counted in at least 200 cells from five nonoverlapping fields in all treatment, and expressed as a percentage of the total number of nuclei counted. 2.7. TUNEL staining

Fig. 1. Effect of WEG at different concentrations on the cell viability and LDH leakage in corticosterone-treated PC12 cells y (A) The cell viability by MTT assay; (B) The LDH leakage assay. The results are expressed as mean7SD (n¼ 4). ##po0.01 as compared with control group; nnpo0.01 as compared with the corticosterone group.

The DNA fragmentation of the apoptotic PC12 cells was detected by using the terminal deoxynucleotidyl transferasemediated biotinylated UTP nick end labeling TUNEL kit (Genmedscientificsinc, USA). The cells were cultured on cover slips for 24 h.

Fig. 2. Effects of WEG on the cell survival in corticosterone-treated PC12 cells by Hoechst 33342 staining method. (A) Representative images by Hoechst 33342 staining, arrowheads in the pictures indicate the nuclei of the apoptotic cells (Hoechst-positive cells). (B) The apoptosis rate was determined by calculating the percentage of Hoechstpositive cells over the total number of cells. The values are mean 7 SD (n¼ 4). ##p o0.01 as compared with the control group; nnp o 0.01 as compared with corticosterone group. Cort: corticosterone.

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At the end of the drug treatment, the cells were fixed by incubation in a 10% neutral buffered formalin solution for 30 min at room temperature. Then the cells were incubated with a methanol solution containing 0.3% H2O2 for 30 min at room temperature, and then did with a permeabilizing solution (0.1% sodium citrate and 0.1% Triton X-100) for 2 min at 4 1C. The cells were incubated with the TUNEL reaction mixture for 60 min at 37 1C and visualized by using an inverted fluorescence microscopy (Leica, Germany). TUNEL-positive nuclei were counted in five randomly selected fields per cover slip, and then converted to percentage by comparing TUNEL-positive counts with the total cell nuclei as determined by Hoechst 33342 counterstaining. 2.8. Measurement of intracellular reactive oxygen species (ROS) level ROS level was measured by using DCFH-DA method (Yokozawa, et al., 2007). DCFH-DA is a non-fluorescent compound, and it can be enzymatically converted to highly fluorescent compound, DCF, in the presence of ROS. In brief, after the treatment, PC12 cells were washed with D-Hanks and incubated with DCFH-DA at a final concentration of 10 μmol/L for 30 min at 37 1C in darkness. The fluorescence intensity was measured in the microplate reader (SpectraFluor; Tecan, Sunrise, Austria) at an excitation wavelength of 485 nm and an emission wavelength of 538 nm after the cells were washed three times with D-Hanks solution to remove the extracellular DCFH-DA. The level of intracellular ROS was showed as a percentage of non-treated control. 2.9. Measurement of intracellular Ca2 þ concentration The concentration of [Ca2 þ ]i was determined as described previously elsewhere (Zhu et al., 2006). In brief, PC12 cells were plated on 24-well plates at a density of 1  105cells/ml. At the end of the treatment, the cells were collected and incubated with cultured medium containing 5 μmol/L Fura-2/AM at 37 1C for 45 min. Subsequently, the cells were washed and resuspended with cold balanced salt solution buffer containing 0.2% bovine serum albumin. The cells were incubated at 37 1C for another 5 min just before measurement. The concentration of [Ca2 þ ]i was determined by alternating excitation wavelengths of between 340 and 380 nm with emission at 510 nm, using a fluorescence spectrophotometer (SpectraFluor; Tecan, Sunrise, Austria). 2.10. Measurement of mitochondrial permeability transition pore (mPTP) opening The opening of mPTP in corticosterone-treated PC12 cells was determined by using the Calcein-cobalt quenching method. The cells were seeded at a density of 1  105 cells/ml in 6-well plates. After the treatments, the cells were loaded with calcein dye and in the presence of cobalt chloride (1 mmol/L CoCl2) for 30 min at 37 1C. Pictures were obtained by using an inverted fluorescence microscopy (Leica, Germany) with excitation and emission wavelengths of 488 and 505 nm, respectively. 2.11. Measurement of mitochondrial membrane potential (MMP) 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide (JC- 1, Invitrogen, Eugene, OR, USA) was used to determine the changes on MMP in corticosterone-treated PC12 cells. In brief, the cells were cultured in complete medium at a density of 1  105 cells/ml in 6-well plates, and then incubated with JC-1 (2 mmol/L final concentration) for 30 min in the darkness. After incubation, the cells were washed twice with PBS and visualized by using an inverted fluorescence microscopy (Leica, Germany). Monomeric JC-1 green fluorescence emission and aggregate JC-1 red

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fluorescence emission were measured on a microplate reader (SpectraFluor; Tecan, Sunrise, Austria). The MMP of PC12 cells in all treatment groups was calculated as the ratio of red to green fluorescence. 2.12. Western blot analysis At the end of treatments, PC12 cells were collected, washed once with PBS, and then lysed with a cell lysis buffer containing 1% phenylmethylsulfonyl fluoride. The whole cell lysates were centrifuged at 14,000 rcf for 20 min at 4 1C and after that, the supernatants were collected. The cytosolic fractions were obtained by differential centrifugation described by previous study (Sugawara, et al., 2002) for the analysis of cytochrome C release. The concentration of protein was determined by bicinchoninic acid protein assay. Equal amounts of protein (30 μg) were separated by electrophoresis on appropriate concentration of sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes. These membranes were incubated with 5% (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 90 min to block nonspecific binding sites. Then, they were incubated overnight at 4 1C with the primary antibodies. After washing three times with TBST, the membranes were incubated for 1 h at room temperature with the secondary antibodies. After rewashing four times with TBST, the bands were developed by enhanced Chemiluminescence. 2.13. Statistical analysis The results are presented as means 7 standard deviation (SD). Every statistical analysis was performed with one-way ANOVA followed by a Dunnett's test using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Differences were accepted as statistically significant at p o0.05. All the experiments were performed for a minimum of three times.

3. Results 3.1. Effect of WEG on corticosterone-induced apoptosis in PC12 cells by MTT According to the results of MTT assay (Fig. 1A), the viability of PC12 cells was measured with the absorbance of formazan detected in each well, when the cells were exposed to corticosterone (250 μmol/L) for 24 h, the cell viability was significantly decreased as compared with the control group (p o0.01), and the survival rate was 47.41% of non-treated control. When the cells were pretreated with WEG at different concentrations (12.5, 25, 50, 100 and 200 μg/ml) for 24 h, in the presence of 250 μmol/L corticosterone for 24 h, the cell viability were increased in a dosedependent manner, and revealed significant difference as compared with the corticosterone-treated group. The result displayed that WEG (100 μg/ml and 200 μg/ml) possessed the best neuroprotective effects (p o0.01). In order to do the further research of the protective effect and the possible mechanism of WEG against corticosterone, 12.5, 25, 50 and 100 mg/ml were chosen to do the experiments of LDH release, intracellular calcium and intracellular ROS level determination, while 100 mg/ml was selected in investigation of the other experiments. 3.2. Effect of WEG on corticosterone-induced LDH leakage in PC12 cells As shown in Fig. 1B, after treatment with 250 μmol/L corticosterone for 24 h in PC12 cells, the LDH leakage was obviously

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increased as compared with the control group (p o0.01), and the percentage of LDH leakage in corticosterone group was 497.39% while 100% in control group. Pretreatment of the cells with various concentrations of WEG (12.5, 25, 50 and 100 μg/ml) in the presence of 250 μmol/L corticosterone for 24 h could lead to the decrease of LDH leakage significantly (po 0.01, p o0.01, p o0.01 and p o0.01, respectively), and the percentage of LDH leakage was 345.10%, 318.30%, 307.19% and 273.86%, respectively.

3.3. Effect of WEG on corticosterone-induced apoptosis in PC12 cells by Hoechst 33342 staining PC12 cells treated with 250 μmol /L corticosterone for 24 h showed typical characteristics of apoptosis, including the condensation of chromatin, the shrinkage of nuclei using Hoechst 33342 staining as shown in Fig. 2A. The amount of apoptotic nuclei was markedly increased, and the apoptosis rate increased to 47.40%

Fig. 3. Effect of WEG on the cell survival in corticosterone-treated PC12 cells by TUNEL staining. (A) Representative images of TUNEL-positive cells (green, top row), Hoechst counterstaining (blue, middle row) and merging (bottom row) (B) Quantification of TUNEL staining. The histogram shows the relative proportion of TUNEL-positive cells in different treatment groups. The values are mean7SD (n¼ 4). ##po0.01 as compared with the control group; nnpo0.01 as compared with the corticosterone group. Cort: corticosterone.

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relative to the control group (100%, p o0.01). However, pretreatment with WEG (100 μg/ml) in the presence of corticosterone (250 μmol/L), the number of apoptosis cells was obviously decreased, and the apoptosis rate reduced to 19.81% relative to the control group (100%, p o0.01) (Fig. 2B). 3.4. Effect of WEG on internucleosomal DNA fragmentation in corticosterone -induced PC12 cells

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3.7. Effect of WEG on mitochondrial apoptotic pathway in corticosterone-induced PC12 cells 3.7.1. Effect of WEG on the opening of mPTP in corticosteroneinduced PC12 cells The influence of WEG on corticosterone-induced mPTP opening was assayed by using the calcein-cobalt quenching method.

To further investigate the effect of WEG on corticosterone-induced apoptosis in PC12 cells, TUNEL staining was carried out. As shown in Fig. 3A, sparse numbers of TUNEL-positive cells exhibited internucleosomal DNA fragmentation were found in normal cells. However, a large number of TUNEL-labeled cells (Green) was observed in corticosterone-treated cells. Pretreatment with WEG during the incubation with corticosterone for 24 h, TUNEL-positive cells were markedly reduced. The quantification of TUNEL staining revealed that treatment with corticosterone yielded 51.07% of TUNEL-positive cells as compared with the total cells (Fig. 3B). However, the cells pretreated with WEG, observably decreased the ratio of TUNEL-labeled cells to 24.35% as compared with corticosterone-treated group. 3.5. Effect of WEG on corticosterone-induced intracellular Ca2 þ concentration in PC12 cells The intracellular Ca2 þ concentration was measured by Fura-2/AM fluorescence -labeled assay, and the ratio of fluorescence intensities was determined to represent the intracellular Ca2 þ concentration in PC12 cells. After the treatment of the cells with 250 μmol/L corticosterone for 24 h, the ratio of fluorescence intensity of [Ca2 þ ]i was markedly increased compared with the control group. Although in WEG (12.5, 25, 50 and 100 μg/ml) groups, the corticosteroneinduced [Ca2 þ ]i overloading was attenuated (Fig. 4). The result indicated that WEG can observably decrease the intracellular Ca2 þ concentration induced by corticosterone in the cells.

Fig. 4. Effect of WEG on the concentration of [Ca2 þ ]i in corticosterone-treated PC12 cells. The values given are the mean 7 SD (n¼4); ##p o0.01 as compared with the control group; **po 0.01 as compared with the corticosterone group.

3.6. Effect of WEG on corticosterone-induced ROS in PC12 cells As shown in Fig. 5, after exposed to 250 μmol/L corticosterone for 24 h, the intracellular ROS level of the PC12 cells markedly increased to 194.40% relative to the control value (100%, p o0.01), which suggests that corticosterone may induce oxidative stress. When the cells were incubated with different concentrations of WEG (12.5, 25, 50 and 100 μg/ml) in the presence of 250 μmol/L corticosterone for 24 h, the intracellular ROS levels significantly decreased to 167.01%, 154.90%, 145.90% and 123.48% of the control value (100%, p o0.01), respectively.

Fig. 5. Effect of WEG on corticosterone-induced ROS in PC12 cells. DCF fluorescence reflects ROS level. The values given are the mean7SD (n¼4). ##po0.01 as compared with the control group; nnpo0.01 as compared with the corticosterone group.

Fig. 6. Effect of WEG on the opening of mPTP in corticosterone-treated PC12 cells. Green fluorescence is representative of healthy mitochondria fluorescence. Treatment with corticosterone caused a decrease in green fluorescence compared to control; pretreatment with WEG in the presence of corticosterone results in intensity of green fluorescence. Cort: corticosterone.

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Calcein could selectively gather in the mitochondria and illustrated the green fluorescence in normal PC12 cells. However, when mPTP opened unusually, the profound release of calcein to the cytosol would be quenched by cobalt. As shown in Fig. 6, treatment with corticosterone (250 μmol/L) caused a significant decrease in green fluorescence relative to the control group, which is consistent with mPTP opening. A comparatively intensive green fluorescence was observed on WEG (100 μg/ml)-pretreated group

in the presence of corticosterone. The result reflected that WEG significantly induced the closure of the mPTP. 3.7.2. Effect of WEG on corticosterone-induced MMP in PC12 cells The effect of WEG on corticosterone-induced MMP was assayed by JC-1 staining. JC-1 manifests potential-dependent accumulation, and it accumulates and forms dimeric J-aggregates giving off a bright red fluorescence in the mitochondria in normal cells.

Fig. 7. Effects of WEG on MMP in corticosterone-treated PC12 cells. (A) Representative images of PC12 cell stained with the MMP-sensitive dye JC-1. Red fluorescence is from JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, while green fluorescence is emitted by cytosolic JC-1 monomers and indicates MMP dissipation. Merged images indicate the co-localization of JC-1 aggregates and monomers. Images shown are representative of three independent experiments. (B) The MMP of cells in each group was calculated as the fluorescence ratio of red to green. The MMP was analyzed using a fluorescence microplate reader after JC-1 staining. The results are expressed as a percentage of control. ##po0.01 versus control group, nnpo0.01 versus groups treated with cortisone. Results were expressed as mean7SD (n¼4). Cort: corticosterone.

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Fig. 8. Effects of WEG on the expression of cytochrome C, caspase-9, caspase-3 and ICAD in corticosterone-treated PC12 cells by western blot analysis. All experiments included control, corticosterone and corticosterone plus WEG groups, and used GAPDH as the loading control. The data are presented as the means7 SD (n ¼ 3). Cort: corticosterone.

Fig. 9. Effects of WEG on the expression of GRP78, GADD-153, XBP-1 and caspase-12 in corticosterone-treated PC12 cells by western blot analysis. All experiments included control, corticosterone and corticosterone plus WEG groups, and used GAPDH and β-actin as the loading control. The data are presented as the means 7 SD (n¼ 3). Cort: corticosterone.

3.9. Effect of WEG on the GR-related pathway in corticosteronetreated PC12 cells

Fig. 10. Effects of WEG on the expression of GR in corticosterone-treated PC12 cells by western blot analysis. All experiments included control, corticosterone and corticosterone plus WEG groups, and used GAPDH as the loading control. The data are presented as the means 7SD (n ¼ 3). Cort: corticosterone.

However, when the potential is destroyed, the dye cannot access the cytomembrane and remain in the cytoplasm in monomeric form giving off a bright green fluorescence. Therefore, mitochondrial depolarization is expressed by a decrease in the red/green fluorescence intensity ratio. As shown in (Fig. 7A), PC12 cells treated with corticosterone (250 μmol/L) represented a marked decrease in the red/green fluorescence ratio as compared with the control group (po0.01). In contrast, WEG (100 μg/ml) pretreatment reduced the effect of corticosterone on the red/green fluorescence intensity ratio (Fig. 7B). Consequently, the result showed the restoration of WEG on the MMP in corticosterone-induced PC12 cells. 3.7.3. Effect of WEG on cytochrome C, caspase-9, caspase-3 and ICAD activation in corticosterone-induced PC12 cells In order to further discuss the mitochondrial apoptotic pathway in the neuroprotective activity of WEG against corticosteroneinduced PC12 cells, the activation of cytochrome C, Caspase-9, Caspase-3 and ICAD was detected. As shown in Fig. 8, the expressions of the four proteins were markedly increased in the corticosterone (250 μmol/L)-treated group as compared with the control. However, their activities were significantly decreased on WEG (100 μg/ml) pretreatment groups. 3.8. Effect of WEG on ER stress activation in corticosterone-induced PC12 cells In order to explore whether corticosterone-induced apoptosis in PC12 cell is related to ER stress, the activation of ER biomarkers, GRP78, GADD-153 and XBP-1, caspase-12 was analyzed by western blot. As shown in Fig. 9, the expression of GRP78, GADD-153, XBP-1 and caspase-12 significantly increased in PC12 cells after 250 μmol/L corticosterone treatment. However, the up-regulation of these biomarkers was attenuated by pretreatment with WEG (100 μg/ml).

3.9.1. Effect of WEG on GR expression in corticosterone-treated PC12 cells As shown in Fig. 10, the expression of GR was up-regulated in the corticosterone -treated group. However, pretreatment with WEG (100 μg/ml) attenuated this change. Meanwhile, the intensity of GR expression in WEG-treated group was similar to that in the control group. The result indicated that WEG can inhibit the up-regulation of the expression of GR induced by corticosterone in PC12 cells, but have no significant effect on GR.

3.9.2. Effect of WEG on HDAC6 and Hsp90 expression in corticosterone-treated PC12 cells The activations of Hsp90 and HDAC6 were analysed with western blot. As shown in Fig. 11, the expression of HDAC6 was significantly up-regulated in the corticosterone-treated PC12 cells; however, the expression of Hsp90 was decreased compared to that of the control group. Moreover, these changes attenuated by pretreatment with WEG (100 μg/ml).

4. Discussion Ginseng is a famous and valuable herb in TCM, and its water extract has been used to treat various central nervous system disorders, such as depression, insomnia, Alzheimer's disease, Parkinson's disease and cerebral ischemia (Niederhofer, 2009; Einat, 2007). As neuroprotection is defined as a therapeutic intervention that prevents the death of vulnerable neurons, slows down disease progression and delays transition from the preclinical to the clinical stage (Djaldetti et al., 2003), the effect of ginseng on central nervous system disorders is likely to be through neuroprotection (Wu et al., 2012). Accordingly, the neurotrophic and neuroprotective effects of ginseng in cells and animals have recently been reviewed (Chen et al., 2014). Among the reported neuroprotective effects of ginseng in cell lines included neurotoxicity induced by 6-OHDA, β-amyloid and 1-methyl-4phenylpyridinium ion (Wei et al., 2008; Ge et al., 2010; Hu et al., 2011). The present study demonstrated that WEG possessed the protective activity against corticosterone-induced damage in PC12 cells, which was confirmed by MTT assay, LDH detection, Hoechst33342 staining and TUNEL staining. In order to gain better understanding of WEG protective effect, we investigated the GR-related, the ER-stress and mitochondria-dependent cell death pathways.

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Fig. 11. Effects of WEG on the expression of Hsp90 and HDAC6 in corticosteronetreated PC12 cells by western blot analysis. All experiments included control, corticosterone and corticosterone plus WEG groups, and used β-actin as the loading control. The data are presented as the means 7SD (n¼ 3). Cort: corticosterone.

It is clear that GR mediates the biological effects of glucocorticoid by acting as a transcription factor which has been well characterized as trans-activation during the cell progression (Hardy et al., 2005; Yemelyanov et al., 2007). Upon binding to the ligand which is corticosterone in the PC12 cells, GR becomes activated and translocates into the nucleus where it controls specific transcriptional programs and modulates a variety of gene expression associated physiological responses (De Bosscher et al., 2003; U, M., Shen et al., 2004). So, we took the investigation of the intervention of WEG which might disturb the corticosterone-induced activation of GR. The results showed that WEG attenuated the reinforcement of the GR expression which induced by corticosterone, and showed no significant influence on PC12 cells, which revealed that WEG is not like the saikosaponin D, which is an agonist of GR by our previous study (Li et al., 2014). These results suggested the WEG might impact the GR on the upstream site of the ligand binding. With the exception of ligand binding, the formation and activization of GR would experience a form of precursor complexus, and the biochemical reconstitution of GR heterocomplex indicates the prerequisite role for the molecular chaperone Hsp90-dependent maturation and modification of GR complex (Cohen et al., 2004; Cadepond et al., 1991; Richter and Buchner, 2001). It is important that GR is regularly combined with Hsp90, which reinforces the affinity of the GR heterocomplex to ligand binding (Banerji, 2009; Pratt and Dittmar, 1998). The study of invertible protein acetylation has been characteristically linked to mechanistic insight into the basic steps of Hsp90dependent complex (Kovacs et al., 2005; Murphy et al., 2005). HDAC6 has been revealed a comprehensive range of biological activity that involves protein acetylation in adjustment and control of microtubules, hydrophilia of the enzyme, and the processing of misfolded protein aggregates (Fazi et al., 2009; Lee et al., 2012). These observations implied that HDAC6 might play a crucially regulatory role in Hsp90-based decoration and formation of the GR heterocomplex (Kovacs et al., 2005). In the present study, we investigated the expression of HDAC6 and Hsp90 under the corticosterone-damaged condition, and the results clearly demonstrated WEG reversed the overexpression of HDAC6 and the attenuation of detached Hsp90. Consequently, WEG influenced the formation of the GR heterocomplex and down-regulated the expression of GR. As the vital organelles, ER and mitochondria, are generally viewed as important organelles being capable of transforming and optimizing their statement and function in response to adapting cellular circumstance (Kleizen and Braakman, 2004). Therefore, we shift the research emphasis to the ER-stress and mitochondriadependent cell death pathway for the sake of illuminating whether the interaction of WEG on GR-related pathway spread to the downstream intracellular organelles. ER is an important organelle for protein biosynthesis, folding, assembly, and modification, and might act as the site where apoptotic cascades are generated and integrated to trigger the death response. Several mechanisms by which apoptotic signals are generated at the ER include: (1) up-regulated Ca2þ release from ER to cytoplasm and mitochondria; (2) cleavage and activation of procaspase-12; (3) increased GR occupancy could promote the expression of CHOP/

GADD-153CHOP. In addition, the process of acetylation might induce the stress-dependent unfolded protein response in ER (Zismanov et al., 2013), and can be regulated and influenced by the HDAC6. Moreover, CHOP/GADD-153 could mediate the dissociation of GRP78 from ER transmembrane receptor PRK-like ER kinase, inositol-requiring enzyme 1, leading to further ER stress (Bertolotti et al., 2000, Shen et al., 2002). Upon the immoderate ER stress, the basic leucine zipper family transcription factor (XBP-1) would be reinforced, and the enrichment of XBP-1 expression affects the cell fate between cytoprotective and proapoptotic outcomes (Lee et al., 2003).To determine whether the neuroprotection of WEG on corticosterone-induced apoptosis in PC12 cells is depended on the ER-stress, the expression of CHOP/GADD-153, GRP78, XBP-1, Caspase-12, the intracellular Ca2þ concentration was measured. Our data suggested that pretreatment with WEG significantly suppressed the over-expression of GADD153, GRP78, XBP-1 and caspase-12 and the maladjustment of intracellular Ca2 þ induced by corticosterone. Mitochondria used to be regarded as the cell's power station and an isolated organelle, however, this concept has been gradually updated with the acknowledgement that the function within a highly dynamic integrated intracellular membrane network is ceaselessly remodeled by both fusion and fission events (Chan et al., 2006; Deniaud et al., 2008). Two organelles interact both physiologically and functionally with 20% of the mitochondrial surface in direct contact with the ER (Kornmann et al., 2009), and one of the most important events of their interaction is Ca2 þ signaling between the two organelles. The overbalance of Ca2 þ released from the ER would lead to depolarization of the mitochondria inner membrane, and also induced cytochrome C release, as well as the activation of the ROS and caspase-regulated apoptosis pathway (Baumgartner et al., 2009; Malli et al., 2005; Boitier et al., 1999). In another way, ROS can also mediate ER stress (Shen et al., 2014). Therefore, we analyzed the expression of Caspase-3, Caspase-9, cytochrome C and ICAD, and investigated the opening of mPTP and disrupted MMP and ROS. Our data demonstrated that pretreatment with WEG significantly suppressed the over-expression of Caspase-3, Caspase-9, cytochrome C and ICAD, and partly reversed the maladjusted intracellular ROS concentration, abnormally opening of mPTP and the depolarization of MMP, which induced by corticosterone. These findings proved that WEG ameliorated corticosterone-induced the mitochondria dysfunction. From the above studies, our results demonstrated the obviously neuroprotection of WEG against the impairment induced by corticosterone in vitro. This neuroprotection is probably associated with inhibition of mitochondrial-dependent and ER stress-dependent cell death pathways in the organelle level as well as the remediation of GR related pathway in molecular level. Moreover, our findings supported the protection of WEG against corticosterone was depended on the deacetylation and chaperone complexes intervening, and subsequent restoration of ER and mitochondria function. Although more detailed molecular mechanism studies are necessary to fully clarify the neuroprotection of WEG, these results should encourage further in vivo studies.

Acknowledgments This work was financially supported by International Science and Technology Cooperation of China (2011DFA32730 and 1108) and National Science and Technology major projects (2012ZX09301002-001).

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