A neuronal suspension was prepared from the pregnant rats, as outlined previously (11). Briefly, the pregnant rats were anesthetized with 1.5 ml 10% chloral hydrate (Sangon Biotech Co., Ltd., Shanghai, China) and then fixed on the animal operating table. Their abdominal skin was disinfected with a 75% alcohol gauze and laparotomy was performed. The fetal rats were carefully removed from the uterus and their brain was removed following removal of the scalp and skull. The brain tissues were placed in cold D-Hank's solution (Gibco; Thermo Fisher Scientific, Inc.) containing 4.5% glucose in a petri dish. Subsequently, the fetal rats meninges and blood vessels were removed under a Leica S6 E stereomicroscope (Leica Microsystems, Wetzlar, Germany), and the brain cortex tissue was isolated and cut into 1×1×1 mm tissue sections. The sections were then placed into a 0.25% pancreatic enzyme EDTA solution at 37°C for 20 min, followed by termination of digestion by addition of 5 ml Dulbecco's Modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% horse serum and 10% fetal bovine serum for 5 min. The cells were isolated from the sections by a mechanical method using a Pasteur pipette, and passed through a 200 mesh stainless steel sieve. Next, the cells were counted and adjusted to a concentration of 5×10⁶/ml using DMEM.

Subsequently, 0.1 ml cells were seeded at a density of 5×10⁵ cells/ml into 96-well culture plates coated with polylysine, and were subsequently stored in a 5% CO₂ incubator (Thermo Fisher Scientific, Inc.) at 37°C with saturated humidity. After 4 h, Gibco Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.), supplemented with 10% equine serum, was removed from the plates, and the cell cultures were incubated with 2.0% B-27 neurobasal medium for 4 days. Subsequently, the cells were incubated with RDPS (0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.0, 4.0 or 8.0 g/l) for 48 h, after which the culture medium (0.1 ml) was removed and added to 96-well plates containing 0.5% MTT for 4 h. Following removal of the culture media, 0.15 ml dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to the wells, and the plates were agitated for 10 min. Absorbance [optical density (OD)] values were measured at 490 nm using a microplate reader (EL×800™; BioTek Instruments, Inc., Winooski, VT, USA).

Cytotoxicity and MTT analyses were conducted as outlined previously (11). Briefly, the neurons were treated with RDPS (0.025, 0.05, 0.10, 0.25, 0.50, 1.0 or 2.0 g/l) for 4 h, after which the 96-well plates were stored for 12 h in a hypoxia incubator (YQX-II Anaerobic Incubator; Shanghai Hengyue Medical Instruments Co., Ltd, Shanghai, China), containing 85% nitrogen, 10% hydrogen and 5% CO₂, at 37°C. Subsequently, the plates were transferred to a 5% CO₂ reoxygenation incubator for 24 h, after which the culture media (0.1 ml) was removed and added to 96-well plates containing 0.5% MTT for 4 h. Subsequently, the culture media was removed, 0.15 ml DMSO was added to the wells, the plates were agitated for 10 min, and the OD values were measured at 490 nm using a microplate reader.

The harvested rat neuronal cells were divided into five groups, as follows: i) Normal control group (C), in which the neurons (5×10⁵ cells/ml) were cultured in an incubator at 37°C, containing 5% CO₂ and saturated humidity for 6 days; ii) apoptosis model group (A), in which neurons (5×10⁵ cells/ml) were cultured in an incubator at 37°C, containing 5% CO₂ and saturated humidity for 4 days, after which the neurons were placed in a hypoxia incubator for 12 h, followed by transfer to a 5% CO₂ reoxygenation incubator for 24 h; iii) 0.025 g/l RDPS-treated group (RDPS1); iv) 0.05 g/l RDPS-treated group (RDPS2); v) 0.1 g/l RDPS-treated group (RDPS3); and vi) 0.25 g/l RDPS-treated group (RDPS4). Generation of the RDPS-treated groups involved culturing the rat neurons (5×10⁵ cells/ml) in an incubator containing 5% CO₂ at 37°C, with saturated humidity for 4 days, after which the cells were incubated with the appropriate concentration of RDPS for 4 h. Subsequently, the neurons were cultured under hypoxic conditions for 12 h, and then incubated for 24 h in a 5% CO₂ reoxygenation incubator.

Hoechst 33342 fluorescence staining was conducted as outlined previously (11). The apoptotic neurons were observed using fluorescence microscopy. Briefly, the neurons (>200) were counted randomly under a high power microscope (DMI 3000; Leica Microsystems GmbH, Wetzlar, Germany) and the apoptotic rate was calculated as follows: Apoptotic rate (%) = (number of apoptotic neurons/total number of neurons) × 100%.

The neurons were digested using 0.02% ethylenediaminetetraacetic acid (EDTA) and 0.125% pancreatin solution (Sigma-Aldrich), and the resulting neuronal cell suspension was centrifuged for 5 min at 300 × g, after which the supernatant was removed. The neurons were washed twice with phosphate-buffered saline (PBS), followed by centrifugation for an additional 5 min (300 × g). The cells (1–5×10⁵ cells/ml) were suspended in 500 µl binding buffer, after which 5 µl Annexin-FITC and 5 µl PI was added, with agitation. The cells were incubated at room temperature in the dark for 10 min, followed by centrifugation for 5 min at 300 × g. Subsequently, the labeling liquid was removed and the cells were washed once with incubation buffer. The cells were analyzed in a flow cytometer (Coulter Epics xL; Beckman Coulter Inc., Brea, CA, USA) with argon ion laser-excited fluorescence at 488 nm. Flowjo 7.6 software was used to analyze the results of the flow cytometric analysis (Tree Star Inc., Ashland, OR, USA).

Rhodamine 123 staining was performed according to the manufacturer's protocol. Briefly, hypoxia/reoxygenation cultured neurons were digested using 0.02% EDTA and 0.125% pancreatin solution, and the resulting neuronal cell suspension was centrifuged for 5 min at 300 × g, after which the supernatant was removed. The neurons were washed with PBS three times and Rhodamine 123 dye (final concentration, 0.005 g/l) was added. Following incubation for 20 min, the neurons were washed three times with PBS and incubated for 60 min. Subsequently, the neurons were collected and analyzed in a flow cytometer with argon ion laser-excited fluorescence at 488 nm. Flowjo 7.6 software was used to analyze the fluorescence intensity.

The PCR assay (MyCycler™ Thermal Cycler; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was performed according to the manufacturer's protocol. Briefly, total mRNA extraction was performed as follows: TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) extraction for 5 min, followed by chloroform treatment for 2 min, centrifugation at 12,000 × g for 15 min, isopropyl alcohol treatment for 20 min, followed by further centrifugation at 12,000 × g for 10 min. The supernatant was removed and 75% ethanol precipitation was performed, followed by centrifugation at 7,500 × g for 5 min, supernatant removal and air drying. Diethylpyrocarbonate-treated water was then added, in order to dissolve the mRNA, at 65°C for 10–15 min. The OD value of the RNA was measured at 260 nm using a SmartSpec Plus ultraviolet spectrophotometer (Bio-Rad Laboratories, Inc.).

The RNA OD value was used to calculate the concentration of RNA, as follows: RNA concentration (mg/ml) = 40 × OD₂₆₀ value × dilution ratio/1,000. Reverse transcription of mRNA into cDNA was performed as follows: Total mRNA (3 µl), 1 µl random primer (0.1 µg/ml), 10 µl 2X TS Reaction mix, 1 µl TransScript™ RT/RI Enzyme mix and 5 µl ribonuclease-free water, was mixed and incubated at 25°C for 10 min, 42°C for 30 min and 85°C for 5 min.

The β-actin, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) genes were amplified according to the following protocol: The cDNA (3 µl), 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 25 µl 2X TransTap™ HiFi PCR SuperMix II and 20 µl double distilled H₂O, were mixed and subjected to 32 PCR cycles (94°C for 5 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and 72°C for 10 min) for β-actin and caspase-3, and 32 PCR cycles (94°C for 5 min, 94°C 30 sec, and 58°C for 30 sec, 72°C 1 min and 72°C for 10 min) for Bax and Bcl-2. Agarose gel electrophoresis was performed using 5 µl of the PCR products at 120 V for 45 min. The gray-scale value of each DNA band was measured using Quantity One software (Bio-Rad Laboratories, Inc.). The levels of gene expression were quantified by calculating the ratio of the OD values of the respective gene:OD value of the internal control.

The gene primer sequences were as follows: β-actin (432bp) forward, 5′-TCA GGT CAT CAC TAT CGG CAA T-3′ and reverse, 5′-AAA GAA AGG GTG TAA AAC GCA −3′; caspase-3 (159bp) forward, 5′-GCA TGC CAT ATC ATC GTC AG-3′ and reverse, 5′-GGA CCT GTG GAC CTG AAA AA-3′; Bax (173 bp) forward, 5′-GAT CAG CTC GGG CAC TTT AG-3′ and reverse, 5′-TGC AGA GGA TGA TTG CTG AC-3′; and Bcl-2 (223bp) forward, 5′-ATG CCG GTT CAG GTA CTC AG-3′ and reverse, 5′-CGA CTT TGC AGA GAT GTC CA-3′.

Immunocytochemical staining was performed, as outlined previously (11). Briefly, the cells were incubated with rabbit anti-rat Bcl-2 (dilution, 1:200; cat. no. D2010), rabbit anti-rat Bax (dilution, 1:200; cat. no. 12910), or rabbit anti-caspase-3 (dilution, 1:400; cat. no. E1410) polyclonal primary antibodies, followed by incubation with a goat anti-rabbit immunoglobulin G secondary antibody (cat. no. 202012; all antibodies were purchased from Zhongshan Golden Bridge Biotechnology, Beijing, China). The cells that were brown in appearance under a light microscope (BX43; Olympus Corp., Tokyo, Japan) were designated positive, whereas unstained or ‘buffy’ cells were considered negative. A total of 200 cells were randomly counted in order to calculate the positive rate, as follows: Positive rate(%) = (the number of positive cells/total number of cells) × 100%.

Data are presented as the mean ± standard deviation. Experimental data that conformed to a normal distribution and homogeneity of variance were analyzed using a one-way analysis of variance, and post hoc tests were used for comparison between two groups. Experimental data that did not fit a normal distribution or homogeneity of variance were analyzed using a non-parametric test. P<0.05 was considered to indicate a statistically significant difference. For statistical analysis, the SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA) was used.