The MC3T3-E1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were cultured at 37°C in a humidified atmosphere of 5% CO₂. Then, the cells (~2×10⁵) were treated with dioscin (0.1, 0.5, 1, 5, 10 and 25 µg/ml; HY-N0124; Medchemexpress, Monmouth Junction, NJ, USA) for 24, 48 and 72 h. CAPE (1 µM; S7414; Selleck Chemicals, Houston, TX, USA) was used to inhibit p-NF-κB-P65.

Cell proliferation was measured using a 3-dimethylthiazol-2-y-(4,5)-2, 5-diphenyltetrazolium bromide (MTT) assay. The MC3T3-E1 cells were seeded in 96-well culture plates at a density of 5×10³ per well. The medium was removed and the cells were treated with dioscin (0.1, 0.5, 1, 5, 10 and 25 µg/ml) for 24, 48 and 72 h. Subsequently, MTT (10 µl per well; 5 mg/ml) solution was added to each well and incubated at 37°C for 4 h. Finally, the optical density was determined at 570 nm using an ELISA plate reader (Model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total proteins were extracted from the MC3T3-E1 cells in RIPA buffer with 0.5% sodium dodecyl sulfate (SDS) and 3% proteinase inhibitor cocktail (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min on ice. The concentration of protein was determined using a BCA protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). For western blot analysis, the proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Following blocking with 5% bovine serum albumin (A-3912; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h, the membranes were incubated at 4°C overnight with the following polyclonal antibodies: ASPP2 (ab36004; 1:1,000; Abcam Biotechnology, Cambridge, UK), phosphorylated (p)-NF-κβ p65 (sc-166748; 1:1,000; Santa Cruz, CA, USA), alkaline phosphatase (ALP; ab133602; 1:10,000; Abcam), microtubule-associated protein 1 light chain 3β (LC3B; 2775; 1:800; Cell Signaling Technology, Inc, Shanghai, China), bone gla protein (BGP; sc-74,495; 1:400; Santa Cruz) and GAPDH (sc-365062; 1:500; Santa Cruz). The membranes were washed in PBS 3 times for 10 mins and then incubated with horse radish peroxidase-conjugated secondary antibodies that against mouse IgG (A9309; Sigma-Aldrich; Merck KGaA) or rabbit IgG (ab97051; Abcam) with 1:2,000 dilution for 2 h at room temperature. The intensities of the bands were quantified using an image analysis system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA).

The cells were seeded on polylysine-coated glass slides (Sigma-Aldrich; Merck Millipore) in 6-well plates with a density of 1–5×10⁵/ml, fixed in 4% paraformaldehyde for 15 min, washed with PBS and permeabilized with 0.1% Triton-X-100 (Sigma-Aldrich; Merck Millipore) for 5 min. The slides were then incubated with LC3 primary antibody (L8918; 1:1,000; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature and incubated with secondary antibody (fluorescence FITC; KC-RB-095; 1:100; Kangcheng Biotech, Shanghai, China) for 1 h at room temperature. DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) was added to the cells in the dark for 5 min. Following washing with PBS, the glass slides were sealed with seal sheet containing fluorescence quenching agent. A laser-scanning confocal fluorescence microscope (Olympus BX60; Olympus, Tokyo, Japan) was used to capture images.

All experiments were repeated at least three times with similar results. Representative data are given. Statistical analysis was performed using paired Student's t-tests. P<0.05 was considered to indicate a statistically significant difference. All data were analyzed with SPSS version 17.0 (SPSS Inc., Chicago, IL, USA).

To analyze the effects of dioscin on MC3T3-E1 cell proliferation, the MC3T3-E1 cells were incubated with dioscin at various concentrations (0.1, 0.5, 1, 5, 10 and 25 µg/ml) and cell proliferation was detected using MTT assays. As demonstrated in Fig. 1A, the results showed that dioscin concentrations of 0.5, 1, 5, 10 and 25 µg/ml significantly promoted MC3T3-E1 cell proliferation in 72 h in a concentration-dependent manner, compared with the control cells.

ALP, a representative marker of the early stage of osteoblast differentiation, is known to be important in the initiation of mineralization during bone formation. Osteoblastic differentiation was characterized by the activity of ALP. LC3B is a marker of autophagy. The present study analyzed the expression of ALP and LC3B in the MC3T3-E1 cells in response to dioscin. As demonstrated in Fig. 1B-D, dioscin concentrations of 0.5, 1, 5, 10 and 25 µg/ml promoted the expression of ALP and inhibited the expression of LC3B in MC3T3-E1 cells, in a concentration-dependent manner.

ASPP2 has been reported to be associated with autophagy and to regulate the expression of NF-κβ. In the present study, the effects of dioscin on the expression of ASPP2 and NF-κβ in MC3T3-E1 cells were detected. As demonstrated in Fig. 2, dioscin concentrations of 1, 5, 10 and 25 µg/ml decreased the expression of ASPP2 and promoted the expression of NF-κβ in the MC3T3-E1 cells, in a concentration-dependent manner.

A previous study showed that ASPP2 can inhibit autophagy through the p53-independent pathway (12) and NF-κβ was reported to be target protein of ASPP2, with two sites at residues 236–253 and 293–313 (17). To determine the effects of the ASPP2/NF-κβ pathway in dioscin-induced osteoblastic cell differentiation and proliferation, CAPE was used to inhibit the activation of NF-κB, and osteoblastic cell differentiation and proliferation were examined using western blot analysis and an MTT assay. As demonstrated in Fig. 3A, CAPE treatment inhibited the expression of p-NF-κβ, but did not affect the expression of ASPP2. CAPE reversed dioscin-induced osteoblastic cell differentiation by repressing the expression of ALP and BGP. CAPE also attenuated dioscin-induced osteoblastic cell proliferation (Fig. 3B). These results indicated that dioscin induced osteoblastic cell proliferation and differentiation by increasing the expression of p-NF-κβ.

The present study subsequently examined whether ASPP2/NF-κβ is involved in the dioscin-regulated autophagy of MC3T3-E1 cells. The MC3T3-E1 cells were treated with 1 µM CAPE, an inhibitor of NF-κβ activation, and with 10 µM dioscin for 72 h to determine the effect of the ASPP2/NF-κβ pathway on autophagy. As demonstrated in Fig. 3A, CAPE attenuated the dioscin-repressed expression of LC3B, and the immunofluorescence assay revealed that dioscin decreased autophagosome formation, which was reversed by CAPE (Fig. 4). These results indicated that dioscin repressed autophagy via the NF-κβ pathway in the MC3T3-E1 cells.