The prescription of GLGZD was first recorded in ‘Jin Gui Yao Lue’, a medical book written by Zhongjing Zhang of the Eastern Han Dynasty during the first century (25–220 AD). The formula consists of six crude drugs, including Trichosanthis Radix, Ramulus Cinnamomi, Paeonia lactiflora, Glycyrrhiza, Zingiber officinale Roscoe and Fructus Jujubae at a ratio of 3:3:3:2:3:3. Dried crude drugs were purchased from Tongrentang Chinese Medicine Pharm (Fuzhou, China), a famous and time-honored pharmaceutical brand in the TCM industry in China. They were identified and confirmed by the College of Pharmacology, Fujian University of Traditional Chinese Medicine, Fuzhou, China. The formula was prepared by boiling the herbs in water. After the first decoction (2 h), the suspension was filtered and water was added for the second decoction (1 h). The filtered and mixed suspension from the two decoctions was concentrated under vacuum by using a rotary evaporator to a final concentration of 1.16 g/ml. The samples were then stored at −20°C before use.

The cells were cultured in DMEM/high glucose medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin solution at 37°C under an atmosphere of 5% CO₂ (Thermo Fisher Scientific, Waltham, MA, USA). One day prior to treatment, the culture medium was changed to DMEM/high glucose medium with 0.5% FBS in order to reduce the serum effect. Twenty-four hours after seeding, the medium was renewed with one of the three types of fresh culture medium (medium without glutamate, with 30 mM glutamate, and 30 mM glutamate plus various concentrations of GLGZD), and incubated for 24 h. In a single experiment each treatment was performed in triplicate.

Cells were seeded into 96-well plates at a concentration of 5–6×10⁴ cells/ml. Cell viability was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT assay was performed as follows: 20 μl of 5 mg/ml MTT dissolved in phosphate-buffered saline (PBS) was added to each individual well followed by incubation at 37°C for 4 h. The solution was then removed, and the produced formazan was solubilized in 100 μl dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm using an automated microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Cell viability was expressed as a percentage of the control culture value.

Cells were seeded into 6-well microplates. Following treatment for 24 h, the cells were fixed for 10 min with 4% paraformaldehyde in PBS, washed with PBS, then visualized and photographed under a phase contrast microscope (Leica, Wetzlar, Germany).

The apoptosis of BV-2 cells was determined by flow cytometry on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) with an Annexin V-FITC Apoptosis Detection kit (KeyGen Biotech, Nanjing, China). Staining was performed according to the manufacturer’s instructions. The percentage of cells found in early apoptosis was calculated by counting the number of Annexin V-positive and propidium iodide (PI)-negative cells. The percentage of cells found in late apoptosis was calculated by counting the number of Annexin V-positive and PI-positive cells.

The JC-1 assay kit (KeyGen Biotech) was employed to measure the MMP of BV-2 cells according to the manufacturer’s instructions. Briefly, the cells were seeded into 6-well plates and exposed to glutamate or GLGZD for 24 h. Thereafter, the cells were harvested and resuspended in a mixture of 500 μl culture medium and 500 μl JC-1 staining fluid, and incubated in the dark at 37°C for 20 min. Following two washes with JC-1 staining buffer and incubation in DMEM, the cells were analyzed by flow cytometry. Mitochondria containing red JC-1 aggregates in healthy cells were detectable in the FL-2 channel, and those containing green JC-1 monomers in apoptotic cells were detectable in the FL-1 channel. The values of MMP staining from each sample were expressed as the ratio of red fluorescence intensity over green fluorescence intensity.

We also evaluated the MMP of BV-2 cells in situ. The cells were seeded in a glass-bottom cell sterile culture Petri dish specific for confocal microscopy (diameter of 15 mm, Nest Biotechnology Co., Ltd., Shanghai, China) for 24 h. Following 24 h of treatment with glutamate or GLGZD, the cells were incubated with a mixture of 500 μl JC-1 staining fluid and 500 μl cell culture medium in the dark at 37°C for 30 min. Subsequently, the cells were washed twice with staining buffer preserved in 4°C. Lastly, 1 ml of cell culture medium was added to each specimen and the cells were analyzed using a LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the supplier’s instructions. RNA was quantified by optical density measurements at 260 and 280 nm. Integrity was confirmed by 1% agarose gel electrophoresis. We used 2 μg of RNA in a 20 μl reaction mixture utilizing M-MLV reverse transcriptase (Fermentas, Waltham, MA, USA) according to the supplier’s instructions. The resultant reverse transcription products were stored at −20°C until further use. Mouse polymer, Bcl-2 and Bax primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China) according to the following sequences: Bax sense, 5′-GAGACACCTGA GCTGACCTTG-3′ and antisense, 5′-GAAGTTGCCATCAG CAAACAT-3′; Bcl-2 sense, 5′-ATGTGTGTGGAGAGCGT CAAC-3′ and antisense, 5′-CAGCCAGGAGAAATCAAA CAG-3′; β-actin sense, 5′-GAGACACCTGAGCTGACC TTG-3′ and antisense, 5′-GAGACACCTGACCACCCTG TTGCTGTA-3′. The product size for Bax, Bcl-2 and β-actin sense was 195, 177 and 490 bp, respectively. PCR was carried out with Taq polymerase (Thermo Fisher Scientific Inc., Rockford, IL, USA) according to the supplier’s instructions. The PCR conditions were as follows: 94°C for 4 min, followed by 35 cycles (30 for β-actin) of 1 min denaturation at 94°C, 1 min annealing at 58°C, 1 min polymerization at 72°C, and finally 10 min extension at 72°C. The PCR products were analyzed by 1.5% agarose electrophoresis and visualized using ethidium bromide (0.25 μg/ml) in 0.5X TBE buffer (Tris 40 mM, EDTA 1 mM, boric acid 44 mM) at 80 V (constant voltage). Images of gels were acquired and analyzed by Molecular Imager software (Bio-Rad Laboratories). The density of the PCR bands was expressed as a ratio of the band density divided by that of the housekeeping gene, β-actin.

Cells were harvested and lysed in RIPA buffer containing the protease inhibitor, phenylmethylsulfonyl fluoride (PMSF) (both from Beyotime, Shanghai, China). Cell lysates were collected by centrifugation at 12,000 × g for 10 min at 4°C. Protein concentrations were determined using an enhanced BCA protein assay kit (Beyotime). Equal amounts of protein from each sample (40 μg) were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (12%) and transferred onto PVDF membranes. The membranes were incubated in blocking buffer (non-fat milk) and then incubated overnight at 4°C with rabbit polyclonal antibodies against Bax (CST, 1:1,000, 20 kDa), Bcl-2 (CST, 1:1,000, 26 kDa) or β-actin (1:4,000, 43 kDa) (Beyotime). The membranes were stringently washed and incubated with HRP-conjugated secondary antibodies (Proteintech Group, Chicago, IL, USA), for 1 h at room temperature. After washing, proteins were detected using enhanced chemiluminescence, and images were acquired using a Bio-Image Analysis System (Bio-Rad Laboratories).

Cells were seeded in a glass-bottom cell sterile culture Petri dish specific for confocal microscopy (diameter of 15 mm, Nest Biotechnology Co., Ltd.) for 24 h. Following treatment with glutamate or GLGZD (125, 250, 500 and 1,000 μg/ml) for 24 h, the cells were fixed with 4% formaldehyde in PBS for 30 min at room temperature, rinsed three times in PBS, and incubated in blocking buffer (5% BSA, 10% normal donkey serum, 0.3% Triton X-100 in PBS) for 60 min at room temperature. The cells were then incubated with cleaved caspase-3 antibody (Alexa Fluor 488-conjugated) overnight at 4°C (CST, 1:100). The cells were washed twice with PBS, and incubated with a rhodamine-conjugated phalloidin (F-actin) antibody (cytoskeleton, 1:100) at 37°C for 30 min. Lastly, DAPI (Beyotime, 1:1,000) was added to each specimen for nuclei staining and the cells were analyzed using a LSM 710 laser scanning confocal microscope under a ×40 water objective (Carl Zeiss).

Data are expressed as the means ± standard error of mean (SEM). Data were first analyzed using Portable IBM SPSS Statistics software. A paired-sample t-test was then performed to compare the treated samples, and values of P<0.05 were considered to indicate statistically significant differences.

In our previous study (6), a high-performance liquid chromatography (HPLC) fingerprint was used to control the quality of the GLGZD extract, which revealed that the method we use to prepare GLGZD was efficient and that the product used for this study was pure.

In order to evaluate the effects of GLGZD on glutamate-induced cell death, we first determined the optimal concentration of glutamate which was able to induce BV-2 cell death (Fig. 1A). Increasing concentrations of glutamate were added (15, 20, 25, 30, 35 and 40 mM) and cell survival was measured. The concentration of glutamate that induced approximately 50% cell death, and the appropriate concentration for our experiments, was 30.0 mM.

The glutamate-induced loss of cell viability was markedly attenuated by treatment with GLGZD (Fig. 1B). Following treatment with 30 mM glutamate for 24 h, cells survived for an average of 41.72±6.95% of the control value. Treatment with 125, 250, 500 and 1,000 μg/ml of GLGZD in the presence of 30.0 mM glutamate markedly increased cell viability to 51.37±5.99, 53.24±3.68, 58.02±4.34 and 58.39±4.81%, respectively. These results demonstrated that the glutamate-induced loss of cell viability was partially attenuated by GLGZD in a dose-dependent manner.

In addition to cell viability, we also sought to analyze the morphological characteristics of BV-2 cells cultured in the presence of glutamate with or without GLGZD. BV-2 cells display a characteristic small spherical morphology with more than half of the cells displaying process-bearing sites, often bipolar and tripolar (Fig. 2A). The addition of 30 mM glutamate induced contraction, rounding and even floating of the majority of cells (Fig. 2B). This suggests the involvement of microglial cell apoptosis and necrosis induced by treatment with glutamate. This morphological change was effectively inhibited by 250, 500 and 1,000 μg/ml of GLGZD (Fig. 2D–F).

A quantitative evaluation of apoptosis was then carried out by flow cytometry with an Annexin V/PI test. The apoptotic rate of the cells treated with 30 mM glutamate alone for 24 h markedly increased to 36.46% (Fig. 3). However, treatment with GLGZD reversed this effect. The proportion of apoptotic cells decreased from 36.46% to 24.86, 19.32, 17.11 and 16.06%, when the cells were co-incubated with concentrations of 125, 250, 500 and 1,000 μg/ml of GLGZD, respectively (Fig. 3A–F). A dose-dependent effect was evident, as the highest concentration of GLGZD (1,000 μg/ml) demonstrated the least number of apoptotic cells (Fig. 3G).

Apoptosis is often accompanied by mitochondrial dysfunction, and the decline in MMP is considered as a symbolic event of early cellular apoptosis (14,15). In this study, to investigate the effects of glutamate and GLGZD on mitochondrial function, indicators of mitochondrial activity were monitored using JC-1 staining. The MMP of BV-2 cells treated with glutamate for 24 h was markedly reduced (Fig. 4). The ratio of aggregated JC-1 (FL-2 channel) to monomeric JC-1 (FL-1 channel) was decreased from 79.8 (control group) to 33.7% (glutamate-treated only group) (P<0.01) after 24 h of treatment. When the cells were incubated with 125, 250, 500 and 1,000 μg/ml of GLGZD, the ratio increased from 33.7% to 42.7, 54.5, 78.4 and 79.7%, respectively (Fig. 4A–F). Treatment with GLGZD attenuated the decline in MMP in a dose-dependent manner, as indicated by the increase in red (JC-1 aggregates)/green (JC-1 monomers) ratio (Fig. 4G).

In addition, the BV-2 cells stained with JC-1 exhibited mitochondrial red fluorescence with a little green fluorescence, suggesting that the cells were in a normal polarized state (Fig. 5A). The JC-1 aggregates were dispersed to the monomeric form (green fluorescence) in the glutamate-treated cells (Fig. 5B). However, treatment with GLGZD attenuated the dissipation of the MMP (Fig. 5C–F), corroborating our results from flow cytometry (Fig. 4).

To determine whether GLGZD protects the BV-2 cells from glutamate-induced apoptosis by modulating the Bcl-2 family of proteins, the mRNA and protein levels of Bax and Bcl-2 were estimated using RT-PCR and western blot analysis. We detected an increase in Bax and a decrease in Bcl-2 mRNA levels following exposure to glutamate (Fig. 6A and B). Treatment with GLGZD inhibited the upregulation of Bax and enhanced the upregulation of Bcl-2 slightly at 24 h of glutamate exposure. Thus, GLGZD attenuated the increase in the Bax to Bcl-2 ratio induced by glutamate, a sign of apoptosis inhibition. In addition to the GLGZD modulation of Bax and Bcl-2 mRNA levels, we observed similar results with the protein levels (Fig. 6C and D).

We utilized an antibody specific for the activated form of caspase-3, a caspase that plays an important role in a number of neuronal apoptotic pathways, as an ‘executioner’ of cell death (16). Treatment with glutamate led to the formation of apoptotic nuclei, as assessed by an abundance of green puncta labeled with the antibody to activated caspase-3 (Fig. 7). Cleaved caspase-3 was localized in the nuclei, as it overlapped with apoptotic cell nuclei. Treatment with GLGZD induced the re-localization of caspase-3, out of the nucleus, representing a decrease in caspase-3 expression levels (Fig. 7)