Cell viability was evaluated using cytotoxicity assays. Briefly, PC-12 cells were seeded into 96-well plates with 5,000 cells per well and incubated with drugs or inhibitors at the indicated concentrations for 48 h. The salidroside was added at the concentrations of 12.5, 25, 50, 100 or 200 µM; Aβ_1-42 was added at concentrations of 0.01 to 1 µM; while the inhibitors were added at concentrations of 5 to 20 µM. A volume of 25 µl MTT solution (5 mg/ml) was added to each well and incubated for additional 4 h. Then, dimethyl sulfoxide (DMSO) was added to dissolve the MTT formazan product and the absorbance was determined at 570 nm using a SpectraMax M5 device (Molecular Devices, LLC, Sunnyvale, CA, USA). The relative cell viability rates of the test group were calculated vs. the untreated controls.

The quantity of LDH that is released into the incubation medium when the cell membrane is destroyed was determined for evaluation of Aβ-induced cytotoxicity. A total of 5×10³ PC-12 cells were seeded into 96-well plates. After incubation with 50 µM salidroside for 1 h, followed by incubation with Aβ for additional 24 h, the activity of the LDH that was released into the medium was determined according to the protocol of CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega Corporation, Madison, WI, USA). The fluorescent intensity was determined using a microplate reader (Multiskan™ FC; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The percentage values of the released LDH were normalized to the control group.

PC-002D12 cells were fixed with 3.7% formaldehyde in PBS for 5 min at room temperature and blocked with 5% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) containing 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min at room temperature. The prepared specimens were stained with DAPI (SouthernBiotech, Birmingham, AL, USA) at a concentration of 10 µg/ml at room temperature for 2 min and then observed under a confocal microscope (Nikon Corporation, Tokyo, Japan). Apoptotic cells exhibited nuclear condensation with intensely stained nuclei, while the normal cells did not exhibit nuclear condensation (22).

Cells were seeded in 6-well plates at a density of 5×10⁴ cells/cm². After 24 h of incubation, cells were exposed to 50 µM salidroside for 1 h and to 0.3 µM Aβ for 24 h. Then, some of the cells were incubated with 5 µM fluorescent probe of H2DCF-DA for 20 min and the fluorescence emission was visualized using a fluorescence microscope (Nikon Corporation). The other cells were analysed using the MDA and SOD detection kits (KeyGen Biotech Co., Ltd., Nanjing, China).

PC-12 cells were seeded into a black 96-well plate with 5,000 cells per well, then incubated with 50 µM salidroside for 1 h, followed by incubation with Aβ. Following 24 h, cells were incubated with a fluorescent probe of JC-1 (Beyotime Institute of Biotechnology, Haimen, China) at a concentration of 5 mg/ml for 15 min at 37°C and then washed twice with PBS. The intensity of red and green fluorescence was determined using an Infinite M200 PRO Multimode Microplate (Tecan Group, Ltd., Mannedorf, Switzerland) and a fluorescence microscope (Nikon Corporation). The mitochondrial membrane potential was calculated using the ratio of JC-1 red/green fluorescence intensity, the color changed from red to green when apoptosis occurred and the value was normalized to the control group.

Once PC-12 cells had been incubated with salidroside for 1 h and with Aβ for 24 h, the cells were lysed with lysis buffer of radioimmuno-precipitation assay (RIPA; KeyGen Biotech Co., Ltd.) and centrifuged at 12,500 x g for 5 min at 4°C. Then, the cell lysate was incubated with 2X substrate working solution at room temperature for 30 min in 96-well plates. The activity of caspase-3/7 was determined using the commercially available Caspase-Glo 3/7 Assay (Invitrogen; Thermo Fisher Scientific, Inc.). The fluorescence intensity of each sample was normalized to the protein concentration of the sample. All the percentage values of caspase-3/7 activities were normalized to the control group.

PC-12 cell apoptosis was evaluated with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kit (KeyGen Biotech Co., Ltd.). Upon treatment, the adherent and non-adherent cells were harvested. The cells were then stained with Annexin V-FITC and PI in binding buffer for 15 min. Flow cytometric analysis was performed on a fluorescence-activated cell sorting flow cytometer (BD Biosciences; Franklin Lakes, NJ, USA) and the data were analyzed with Cell Quest software (version 3.4, BD Biosciences).

The cells were lysed with RIPA buffer, the concentration of proteins were determined by Bicinchoninic Acid detection kit (KeyGen Biotech Co., Ltd.). Immunoblots were performed with samples that contained total protein (30 µg), which had been separated by 12% SDS-PAGE gel and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against phosphorylated (p)-ERK1/2 (1:1,000; AF1015; Affinity Biosciences, Cincinnati, OH, USA), total (t)-ERK (1:1,000; AF6240; Affinity Biosciences), p-AKT (1:1,000; AF0016; Affinity Biosciences), t-AKT (1:1,000; AF4718; Affinity Biosciences), B-cell lymphoma (BCL)-2 (1:1,000; AF6139; Affinity Biosciences), BCL2 associated X (Bax; AF0120; 1:1,000; Affinity Biosciences) and GAPDH (1:1,000; AF7021; Affinity Biosciences), followed by incubation with a secondary antibody of goat anti-rabbit IgG-horseradish peroxidase (1:5,000; sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Blots were developed using an enhanced chemiluminescence detection reagent (EMD Millipore). GAPDH was used as an internal loading control.

All experiments were repeated at least in triplicate, all data were evaluated using SPSS v.19.0 (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation. One-way analysis of variance and Bonferroni’s test were used to compare different treatment samples. P<0.05 was considered to indicate a statistically significant difference.

The biological activity of salidroside (Fig. 1A) was assessed by cell viability assays. The cytotoxicity of Aβ_1-42 on PC-12 cells was first examined by MTT assay. As presented in Fig. 1B, exposure of PC-12 cells to Aβ_1-42 (0.001-1 µM) for 24 h induced a dose-dependent decrease in cell viability, thereby demonstrating that Aβ_1-42 could induce toxicity in PC-12 cells. The viability of PC-12 cells was only increased or slightly decreased and the differences compared with the control group were not significant, following treatment with salidroside (12.5-200 µM) for 24 h, thereby indicating that salidroside was not toxic to PC-12 cells under these treatment conditions (Fig. 1C). Compared with the Aβ_1-42 group, pre-treatment with salidroside at concentrations of 25, 50 and 100 µM significantly improved cell viability (P<0.05; Fig. 1D). The multi-combination test results further demonstrated that salidroside protected and rescued PC-12 cells from Aβ_1-42-induced cell death (Fig. 1E).

LDH assay was used for evaluation of the protective activity of salidroside. When pre-treated with 50 µM salidroside for 1 h, PC-12 cells exhibited a significantly reduced Aβ_1-42-induced LDH leakage (from 135-110% relative to the control group; P<0.01; Fig. 2A). Nuclei condensation was detected in PC-12 cells upon exposure to Aβ_1-42 by DAPI staining (Fig. 2B). Pre-treatment with 50 µM salidroside significantly inhibited Aβ_1-42-induced apoptosis compared with Aβ_1-42 alone (from 35-23%; P<0.05 Fig. 2C). To further evaluate the influence of salidroside in response to oxidative stress in PC-12 cells, the cells were treated with Aβ_1-42 for 24 h following being incubated with salidroside for 1 h. Cell apoptosis rates were detected by flow cytometry (Fig. 2D). The apoptosis rate of PC-12 cells increased following 24 h of Aβ_1-42 treatment, while salidroside treatment could protect PC-12 cells from Aβ_1-42-induced apoptosis.

Since the toxicity of Aβ_1-42 may be affected by the generation of ROS, MDA and SOD (23), the inhibitory/promotional effect of salidroside on ROS, MDA, and SOD production was examined. PC-12 cells were pre-treated with or without 50 µM salidroside for 1 h and then treated with Aβ_1-42 for 24 h. Differences in fluorescence intensity were observed among the different groups (Fig. 3A). The results revealed that Aβ_1-42 could effectively induce intracellular ROS generation, whereas salidroside could significantly inhibit the generation of ROS induced by Aβ_1-42 (from 145 to 125% relative to control group; P<0.05; Fig. 3B). While the MDA detection results demonstrated that the MDA levels were significantly increased due to oxidative stress injury but significantly decreased by salidroside pre-treatment (P<0.01; Fig. 3C). By contrast, the SOD activity in salidroside-pretreated cells increased compared with Aβ-treated cells (Fig. 3D).

Previous studies reported that the loss of the mitochondrial membrane potential was involved in the progression of neuron apoptosis caused by Aβ_1-42 during AD (24,25). In the present study, mitochondrial membrane permeability was detected using the JC-1 probe in order to evaluate the anti-apoptotic effects of salidroside. Red and green fluorescence represented high mitochondrial membrane permeability in viable cells and low mitochondrial membrane permeability in apoptotic cells, respectively (Fig. 4A). When incubated with Aβ_1-42 for 24 h, the mitochondrial membrane permeability was significantly depolarized in Aβ_1-42-treated PC-12 cells compared with the control (P<0.01), whereas pre-treatment with salidroside effectively prevented the loss of mitochondrial membrane permeability (Fig. 4B). Treatment of PC-12 cells with 0.3 µM Aβ_1-42 for 24 h triggered a significant increase in caspase-3/7 activity compared with the control (P<0.01), whereas pre-treatment with 50 µM salidroside for 1 h significantly inhibited caspase-3/7 activity (P<0.05; Fig. 4C).

Since the ERK1/2 and AKT signaling pathways are classical apoptosis-associated pathways, the present study evaluated whether the anti-apoptotic effect of salidroside was mediated by the ERK1/2 and AKT signaling pathways. The present study examined the phosphorylated and total expression levels of ERK1/2 and AKT in PC-12 cells treated with salidroside by western blotting. As presented in Fig. 5A-C, the phosphorylation levels of ERK1/2 and AKT gradually increased upon the addition of salidroside in a time and dose-dependent manner.

To confirm the roles of the ERK1/2 and AKT signaling pathways in the inhibitory effect of salidroside on Aβ_1-42-induced apoptosis in PC-12 cells, the specific inhibitors of the ERK1/2 and AKT signaling pathways, PD98059 and LY294002, respectively, were used. The two pathway inhibitors blocked the protective effects of salidroside in cells treated with Aβ_1-42 (Fig. 6A). PC-12 cells were pre-treated with PD98059 or LY294002 (5, 10 and 20 µM) for 30 min and then treated with 50 µM salidroside for 1 h, and the viability of cells was determined by MTT assay 24 h later. The protective effect of salidroside was blocked in the presence of an all concentrations of PD98059 and LY294002 (Fig. 6B and C). Upon staining with DAPI, pyknosis was detected in treated PC-12 cells (Fig. 6D). Pre-treatment with 50 µM salidroside reversed the effect of Aβ_1-42 on PC-12 cells, whereas incubation with PD98059 or LY294002 abolished the protective effect of salidroside (Fig. 6E). The western blot results further demonstrated that upon treatment of cells with Aβ_1-42, the incubation with PD98059 or LY294002 for 24 h inhibited the phosphorylation of ERK1/2 and AKT, respectively. Salidroside reversed the decrease in the phosphorylation levels of ERK1/2 and AKT induced by Aβ_1-42, whereas PD98059 and LY294002 pre-treatment blocked the reversing effects of salidroside (Fig. 6F). The anti Aβ-induced apoptotic effect of salidroside may be mediated by other signaling pathways, such as BCL-2/Bax (Fig. S1); however, further investigation is required.