Trypsin and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); 6-well and 96-well cell culture plates were purchased from Costar (Corning Incorporated, Corning, NY, USA). Anti-S100β (S100β; cat. no. BA120; 1:200) antibody and the 3,3-diaminobenzidine tetrahydrochloride (DAB) kit were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China), Dulbecco's modified Eagle's medium/F-12 supplement (DMEM/F-12), fetal bovine serum (FBS) and 3–(4,5)-dimethylthiahiazol(-z-y1)-3,5-di-phenyltetrazo-lium-bromide (MTT) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Dimethyl sulfoxide (DMSO), Hoechst 33258 and proteinase K were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Multiskan GO Microplate Spectrophotometer was obtained from Thermo Fisher Scientific, Inc. Other reagents and instruments used in the present study were purchased from the following companies: Hematoxylin-eosin (HE) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); RNeasy RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China); reverse transcription (RT) kit (Fermentas; Thermo Fisher Scientific, Inc.); Fast-Start Universal SYBR Green Master Mix (Roche Diagnostics GmbH, Mannheim, Germany); quantitative polymerase chain reaction (qPCR) detection system (RealPlex4; Eppendorf, Hauppauge, NY, USA); LIVE/DEAD viability assay kit (Invitrogen; Thermo Fisher Scientific, Inc.); laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan); and upright microscope (Olympus Corporation, Tokyo, Japan).

The RSC96 cell line consists of spontaneously immortalized rat Schwann cells, which are derived from the long-term culture of rat primary Schwann cells. RSC96 cells were purchased from the China Center for Type Culture Collection (Wuhan, China), and were cultured in DMEM/F-12 supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics in a humidified atmosphere containing 5% CO₂ and 95% air at 37°C. The culture medium was replaced every 3 days after plating. RSC96 cells were passaged with 0.25% trypsin when cell confluence reached 80–90%. Confluent RSC96 cells were subsequently treated at the indicated times with the indicated concentrations of Andro.

Andro was purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Prior to experimentation, Andro was dissolved in DMSO in order to generate a 100 mM stock solution, and was stored at −4°C. The Andro stock solution was diluted with cultured medium to provided various concentrations and added to the cell culture for subsequent experiments. Prior to use, the culture medium contained 1.5625, 3.125 and 6.25 µM Andro was filtered using 0.22 µm filters for sterilization.

Cell viability was estimated using a colorimetric assay based on the conversion of MTT into a blue formazan product. The cells were plated at 800 cells/well in 96-well cell culture plates and were pretreated with various concentrations of Andro (0–50 µM) for 3 days in a 5% CO₂ humidified incubator at 37°C. MTT (5 mg/ml) was then added to each well and the plates were incubated in the dark at 37°C for 4 h. Subsequently, culture medium was removed and the cells were treated with 150 µl DMSO to dissolve the formazan product. The cells were incubated in DMSO with agitation for 10 min. Optical density of each sample was measured using a Multiskan GO Microplate Spectrophotometer at 570 nm. Five individual cultures were used for each test. The experiments were carried out in quintuplicate.

Based on the results of the cytotoxicity assay, three doses of Andro, which exhibited a positive effect, were selected (1.5625, 3.125 and 6.25 µM), alongside a control group (0 µM Andro) for cell proliferation analysis. RSC96 cells in the various groups were cultured for 2, 4 and 6 days in a 5% CO₂ humidified incubator at 37°C prior to subsequent experiments. Cells were digested with 0.25% trypsin and were resuspended in phosphate-buffered saline (PBS) containing 60 µg/ml proteinase K for 6 h at 60°C. After dyeing with Hoechst 33258, cell proliferation was determined by detecting DNA production using an ultraviolet spectrofluorometer; calf thymus DNA was used as a standard. The excitation wavelength was 346 nm and the emission wavelength was 460 nm. The experiments were carried out in quintuplicate.

Cells were cultured for 2, 4 and 6 days, and were fixed in 4% paraformaldehyde for 40 min at room temperature for subsequent HE staining. Cells were incubated with a nuclear dye for 3 min, followed by a 10 sec incubation with HE. Subsequently, the cells were rinsed with PBS, naturally dried and sealed with neutral gum. Cells were then examined, and images were captured under an upright microscope.

Cell viability was determined using the LIVE/DEAD viability assay kit. Briefly, cells on coverslips were rinsed quickly with PBS (0.01 mol/l, pH 7.4) to remove the medium. Subsequently, 1 µM calcein-acetoxym-ethyl (calcein-AM) and 1 µM propidium iodide (PI) were added to the cell cultures and were incubated in the dark for 5 min at 37°C. Images were captured using a laser scanning confocal microscope.

S100β protein expression was detected by immunohistochemical staining using anti-S100 (S100β), according to the manufacturer's protocol. Briefly, cells on coverslips were rinsed quickly with PBS (0.01 mol/l, pH 7.4) to remove the medium. Subsequently, the cells were fixed in 4% paraformaldehyde at room temperature for 40 min. After washing three times with PBS and permeabilizing with 3% Triton X-100 for 5 min, cells were incubated with 3% H₂O₂ for 10 min at room temperature, in order to suppress endogenous peroxidase activity. The cells were then treated with goat serum for 10 min at room temperature to block nonspecific staining. Subsequently, the cells were incubated with rat monoclonal anti-S100 antibody (S100β; 1:150 dilution) overnight at 4°C in a humidified chamber. After washing three times with PBS, secondary antibodies (cat. no. SP-9000; 1:50; OriGene Technologies, Inc., Beijing, China) and biotin-labeled horseradish peroxidase (OriGene Technologies, Inc.) were successively added for 15 and 10 min at room temperature. The chromogenic reaction of S100 was visualized using a DAB kit, and the slides were counterstained with hematoxylin. Finally, cells were gradually dehydrated, sealed with neutral gum, observed, and images were captured under an upright microscope.

To further explore the effects of Andro on the expression of Schwann cell-specific genes, BDNF, GDNF and CNTF mRNA expression was analyzed by RT-qPCR. Total RNA was extracted from RSC96 cells using an RNeasy RNA extraction kit, according to the manufacturer's protocol. Reverse transcription of RNA was performed at 25°C for 5 min, 42°C 60 min and then 72°C for 5 min using a reverse transcription kit (Fermentas; Thermo Fisher Scientific, Inc.). The RT-qPCR reactions were performed using a qPCR detection system with a FastStart Universal SYBR Green Master Mix under the following conditions: 10 min at 95°C, 15 sec at 95°C and 1 min at 60°C for 35 cycles. The primer sequences (BGI, Shenzhen, China) for BDNF, GDNF, CNTF and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; internal control) are listed in Table I. The melting curve data were collected to verify PCR specificity. Each gene was analyzed in triplicate to diminish operation errors. Relative gene expression levels were calculated using the 2^−ΔΔCq method (28), and were normalized to GAPDH gene expression. Each gene was analyzed in quintuplicate to reduce randomization error.

Statistical analyses were conducted using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance followed by Dunnett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

The present study examined the cytotoxicity of various concentrations of Andro on RSC96 cells using the MTT assay. Cells were treated with increasing concentrations of Andro (0–50 µM). As shown in Fig. 2, compared with the control group (0 µM), treatment with Andro between 0.78 and 12.5 µM exhibited low cytotoxicity. In addition, 0.78–12.5 µM Andro significantly accelerated cell growth (P<0.05) with the most obvious effect being observed when used at 3.125 µM (P<0.05). However, Andro exhibited a suppressive effect on RSC96 cells in vitro when used between 12.5 and 50 µM, as compared with the control group.

As presented in Fig. 3, RSC96 cells treated with 1.5625, 3.125 and 6.25 µM Andro exhibited increased proliferation compared with the control group (0 µM Andro). Proliferation was determined according to DNA content (P<0.05), which was markedly higher in the Andro groups compared with in the control group after the same culture period. Among the three concentrations, 3.125 µM Andro exhibited the strongest effect on cell growth at all time points.

HE staining was conducted using an upright microscope to assess the morphology of RSC96 cells. The images indicated that the Andro groups exhibited increased cell growth compared with the control group at the same time point (Fig. 4). There were no marked differences in Schwann cell morphology between the groups after 6 days of culture. Compared with the control group, RSC96 cells in the presence of Andro grew better and had a distinctive proliferative tendency that gradually increased with time. In addition, when used at 3.125 µM, Andro was able to enhance the proliferation of RSC96 cells compared with the other two concentrations in vitro.

As presented in Fig. 5 viable cells and dead cells were stained with calcein-AM/PI. The results demonstrated that Andro exerted positive effects on survival. Images of calcein-AM/PI staining demonstrated that the survival of cells in the Andro groups was increased compared with in the control group. Consistent with the results of a cell proliferation assay (Fig. 4), more viable cells than dead cells were detected in the Andro groups, thus implying that Andro was able to better support cell growth compared with the control group. Among the Andro groups, treatment with 3.125 µM exhibited the best effects, as evidenced by an increase in the number of viable cells.

The present study detected Schwann cell-specific protein S100β expression using immunohistochemical staining (Fig. 6). Positive S100β staining was increased in the Andro groups compared with the control group at the same time points. Among the three doses of Andro tested, 3.125 µM was superior compared with the others in terms of phenotypic maintenance of Schwann cells.

The mRNA expression levels of RSC96 cell-specific genes were determined by RT-qPCR analysis. Nerve growth factor (NGF) and several neurotrophic factors, including BDNF, GDNF and CNTF, have key roles in Schwann cells and the regeneration of peripheral nerves. The mRNA expression levels of BDNF, GDNF and CNTF were significantly increased in the Andro-treated groups compared with the control group (Fig. 7) except for BDNF levels at 6.25 µM concentratio. Furthermore, among all of the groups, 3.125 µM Andro exhibited the best effect on upregulation of BDNF, GDNF and CNTF.