All animal experimental protocols were approved by the Animal Care Committee of Nanjing University of Chinese Medicine (Nanjing, China) and were performed in accordance with the Guide for Care and Use of Laboratory Animals. A total of 10 Sprague-Dawley male rats (weighing 8–10 g), were obtained from Experimental Animal Center of Nanjing University of Chinese Medicine (Nanjing, China) within 24 h post-birth and kept in an incubator under 12-h light/dark cycle with a humidity of 45–75% at 19–27°C, with free access to food and water.

The calvaria was dissected from surface-sterilized rats and subsequently soaked in 75% ethanol for 5–10 min at 4°C. Isolation of calvaria osteoblasts was performed as previously described (19,20). Briefly, the frontal and parietal bone was separated, cut into fragments (1 mm³), digested in 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C for 15 min and the precipitates following centrifugation at 750 × g and 4°C for 5 min were treated with collagenase II (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 5 min for further digestion (19). Precipitated cells were subsequently suspended in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 15% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂ for 24 h. Cells were then transferred to DMEM with 20% FBS. Medium was replaced every 2 days. For ICA (95.4%; cat. no I1286; Sigma-Aldrich; Merck KGaA) treatment, ICA was added into the cell cultures prior to incubation at concentration of 0, 10, and 20 µg/l at 37°C in 5% CO₂ for 24 h. Each experiment was performed in triplicate.

An MTT (Sigma-Aldrich; Merck KGaA) assay was performed to detect cell viability as previously described (16). Osteoblasts (50 cells/well) were seeded into 96-well plates and maintained in DMEM supplemented with 20% FBS (Gibco; Thermo Fisher Scientific) at 37°C in 5% CO₂ for 24 h prior to treatment with 10 µg/l and 20 µg/l ICA for 5 days. The supernatant of cell cultures was subsequently discarded and 20 µl MTT solution (5 mg/ml) was added into each well and cells were incubated for a further 4 h. 150 µl DMSO was added and the optical density at an absorbance of 490 nm was determined using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

Osteoblasts (5×10⁴ cells/ml) were seeded into 6-well culture plates until 90% confluence. Cells were then treated with ICA for 48 h and harvested, fixed with 4% paraformaldehyde at 4°C for 4 h, and stained with propidium iodide (PI) for 30 min in the dark at room temperature. For cell cycle analysis, the at G0/G1, S and G2/M phase distribution of 10,000 cells was determined using the BD FACS Calibur™ flow cytometer equipped with CellQuest Pro 5.1 software (BD Biosciences, San Jose, CA, USA). Each experiment was performed in triplicate.

Osteoblasts were seeded into 6-well plates (1×10⁵ cells/well) and cultured in DMEM at 37°C in 5% CO₂ for 48 h and treated as aforementioned. Cells were harvested with 0.25% trypsin (Sigma-Aldrich; Merck KGaA) at 37°C in 5% CO₂ for 20 min for apoptotic analysis using the Annexin V apoptosis detection kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were stained with Annexin V and PI for 30 min in the dark at room temperature followed by BD FACS Calibur™ flow cytometry analysis using CellQuest Pro 5.1 software (BD Biosciences). Annexin V positive and PI negative stained cells indicated early apoptosis.

Following incubation with ICA (10 µg/l) for 1, 2, 3 and 4 days, calvaria osteoblasts were prepared for ALP staining. A total of 1×10⁴ cells/well were placed into 24-well plates and incubated in the aforementioned conditions until 85% confluence. Cells were subsequently harvested, fixed using 4% paraformaldehyde at 4°C for 30 min and stained for the analysis of ALP activity with an ALP activity assay kit (BioVision, Inc., Milpitas, CA, USA) according to manufacturer's protocol. Images of stained cells were captured using an Olympus BX51 inverted fluorescent microscope (magnification, ×40; Olympus Corporation, Tokyo, Japan). Additionally, the ALP activity of cell cultures was analyzed using an ELISA kit (cat. no. K422; BioVision, Inc.) according to manufacturers' instruction.

For the in vitro visualization of nodular patterns and calcium deposition, osteoblasts were stained with Alizarin red S after 21 days of culture in DMEM as previously described (20,21). Briefly, 1,000 cells/well were placed into 24-well plates to reach to 80% confluence. Cells were subsequently harvested, fixed using 4% paraformaldehyde at 4°C and stained with 2% Alizarin red S (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Cells were washed with distilled water prior to observation of plate samples for calcium deposition, which indicates bone nodule formation or osteoblast mineralization, using an Olympus BX51 inverted fluorescent microscope (magnification, ×40; Olympus Corporation).

For proteomics analysis, osteoblasts (1×10⁶ cells/ml) were transferred to a flask and allowed to reach 80% confluence prior to the addition of ICA (10 µg/l) at 37°C in 5% CO₂ for 48 h. The secretory proteins from conditioned cell cultures of osteoblasts were prepared for proteomics as previously described (22,23). In brief, the supernatants of the ICA-treated osteoblasts were collected and gathered using TCA-acetone solution (1:3) at −20°C overnight. Precipitates from centrifugation (750 × g at 4°C for 5 min) were subsequently washed with acetone, air-dried, quantified using the Bradford assay method (Applygen Technologies, Inc., Beijing, China) (24) and stored at −80°C.

2-DE was conducted as described previously (23). Immobilized pH gradient (IPG) strips (17 cm; pH 3–10; GE Healthcare Life Sciences, Uppsala, Sweden) were rehydrated with 150 µg protein for 12–16 h at room temperature, followed by isoelectric focusing (IEF, 60,000 Vh) in a Protean IEF cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The IPG strips were subsequently equilibrated as previously described (25) and transferred onto a 12% polyacrylamide gel (Beyotime Institute of Biotechnology, Nanjing, China), which was subjected to the second dimension (200 V, room temperature) in a Protean II XI cell (Bio-Rad Laboratories, Inc.) for 7 h at 6°C. Polyacrylamide gels were washed and silver stained using a Fast Silver Stain kit according to the manufacturer's protocols (Beyotime Institute of Biotechnology). Finally, the gels were scanned and analyzed using an ImageScanner^™ (GE Healthcare Life Sciences) transmission scan with ImageMaster version 5.0 gel image analysis (GE Healthcare Life Sciences). The silver-stained spots with more than 2-fold change in spot intensity and novel spots among groups were considered to be upregulated DEPs. Spots with less than 1-fold change were considered downregulated DEPs. Each experiment was performed in triplicate.

A total of 60 DEP spots were manually cut from gels and digested with trypsin (Gibco; Thermo Fisher Scientific, Inc.) in 96-well plates, according to previously described methods (18). DEP spots were subsequently excised from the polyacrylamide gels. Excised bands were then destained using 50% acetonitrile and 50 mM ammonium bicarbonate at 37°C for 30 min and digested using trypsin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 16 h. DEPs were subsequently extracted with 50% acetonitrile and 0.1% trifluoroacetic acid 3 times. Gel spot extracts (1.5 µl) were placed into new 96-well plates, vacuum dried and stored at −80°C prior to MS analysis using a MALDI-TOF/MS system (Bruker Corporation, Ettlingen, Germany) according to instructions as previously described (22,26).

MS peptide mass fingerprinting (PMF) data was obtained using Mascot Distiller software (v.2.3.2; Matrix Science Ltd., London, UK) (22), and searches of PMF peptide sequences were performed based on the international protein index (IPI) rat FASTA database (v3.31; 41,251 sequences; 21,545,744 residues; ftp://ftp.ebi.ac.uk/pub/databases/IPI). Sequence alignments were generated using the CLC Free Workbench software package (version 4.0.3; Qiagen, Inc., Valencia, CA, USA), and protein properties were identified as previously described (27).

In order to investigate the DEP-associated pathways and Gene Ontology (GO) Consortiums, DEP sequences (peptides) were analyzed with the Protein ANalysis THrough Evolutionary Relationships (PANTHER) pathway database (http://www.pantherdb.org/) (28,29). Proteins were classified according to their function and were annotated with ontology terms (PANTHER pathways, GO terms and PANTHER protein class) and sequences are assigned to PANTHER pathways.

Western blot analysis was performed to validate expression of several identified potential DEPs. Cellular proteins of calvaria osteoblasts were extracted, quantified as aforementioned. Proteins were subsequently separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Membranes were blocked with 5% milk for 1 h at 4°C, probed with the primary antibodies against β-actin (cat. no. ab8227; 1:1,000; Abcam, Cambridge, MA, USA), serpin family F member 1 (cat. no. ab14993; PEDF; 1:1,000; Abcam), signal transducing adaptor molecule (cat. no. ab48015; STAM; 1:1,000; Abcam), c-Myc (cat. no. ab32072; 1:1,000: Abcam), protein disulfide isomerase family A, member 3 (cat. no. ab13506; PDIA3; 1:1,000; Abcam), heat shock protein family A member 4 (cat. no. ab2787; HSP70; 1:1,000; Abcam), and nuclear protein, co-activator of histone transcription (cat. no. ab70595; NPAT; 1:1,000; Abcam) overnight at 4°C. Membranes were subsequently incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. no. ab6721; 1:10,000, Abcam) for 1 h at room temperature. Bands were subsequently visualized using an enhanced chemiluminescence detection reagent (Beyotime Institute of Biotechnology) and were subjected to densitometric analysis with QuantiScan software (v.3.0; BIOSOFT, Cambridge, UK).

Experimental data was analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). and is expressed as the mean ± standard deviation. Differences among groups were determined using one-way analysis of variance (ANOVA) followed by Fisher's Least Significant Difference for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Prior to ICA treatment, isolated calvaria osteoblasts were characterized by ALP and Alizarin red S staining (Fig. 1). At day 3, cells were stained with an ALP assay kit and were observed to have purple stained nuclei accompanied by red-brown stained membrane and cytoplasm (Fig. 1A). Cells were stained with Alizarin red S at day 21 to detect calcium and an irregular red-orange stain was observed, indicating the formation of mineralized bone nodules and the deposition of calcium-rich hydroxyapatite (Fig. 1B).

Osteoblasts were treated with ICA to evaluate the effect of ICA on cell function. Osteoblasts treated with 10 µg/l ICA for 48 h exhibited the highest ALP activity (P<0.05; Fig. 2A). Cells treated with 10 µg/l ICA demonstrated higher ALP activity compared with cells treated with 20 µg/l ICA at 24, 48 and 72 h time intervals (P<0.05). For Alizarin red S staining analysis, it was confirmed that osteoblasts treated with 10 µg/l ICA had brighter irregular red-orange staining than 20 µg/l ICA-treated cells (Fig. 2B). These results suggested that 10 µg/l ICA concentration was the optimum treatment compared with 20 µg/l for ICA-mediated ALP activation and calcium deposition in osteoblasts.

The MTT assay confirmed that ICA promoted the cell viability of osteoblast in a dose- and time-dependent manner (Fig. 3A). Cells treated with 10 µg/l ICA had significantly increased cell viability at 72 h following ICA incubation (P<0.05). Notably, osteoblasts treated with 20 µg/l ICA had decreased cell viability compared with the 10 µg/l ICA treatment (P<0.05).

Cell cycle distribution analysis revealed that ICA treatment increased the percentage of cells in the S and G2/M phases, and reduced the percentage in the G0/G1 phase (Fig. 3B). As expected, there was also an increase in the percentage of cells in the S and G2/M phase and a decrease in the G0/G1 phase in osteoblasts treated with 10 µg/l ICA compared with those treated with 20 µg/l ICA (P<0.05). Cell apoptosis analysis revealed that ICA inhibited cell apoptosis (Fig. 3C). Osteoblasts treated with 10 µg/l ICA had a significantly lower apoptotic rate when compared with the control and 20 µg/l ICA treated cells (P<0.05). Taken together, it was concluded that ICA increased cell viability and inhibited cell apoptosis. Furthermore, it was determined that the optimum ICA concentration for osteoblast viability was 10 µg/l.

Proteomic analysis revealed 60 proteins points that were differentially expressed (with more than two-fold change in spot intensity) or specifically expressed (expression specifically induced by ICA) in osteoblasts treated with 10 µg/l ICA, compared with the control with >750 silver-stained spots (Fig. 4). Bioinformatics analysis identified 56 DEP sequences, which were matched to reference sequences in databases using the CLC Free Workbench software package. Identified DEPS included 22 upregulated DEPs, including STAM binding protein, insulin-like growth factor-binding protein 2 (IGFBP-2) precursor and PEDF. Eight downregulated DEPs were identified, including phosphoglycerate mutase 1 and myosin-1 and succinate dehydrogenase subunit A. A total of 24 specially expressed proteins were identified in ICA-treated osteoblasts, including heat shock proteins [heat shock cognate 71 kDa protein (HSPA8) and heat shock protein, mitochondrial precursor], myc-associated factor X (Max), nuclear protein ataxia-telangiectasia (NPAT) and PDIA3 precursor. Glutaredoxin 5 and translationally controlled tumor protein were DEPs that were lost in ICA treated cells (Table I). A total of four downregulated DEPs in ICA treated osteoblasts were not aligned in the CLC Free Workbench software package.

Sequences of these 56 DEPs were subjected to PANTHER pathway database analysis (29,30). DEP enrichment analysis for PANTHER pathways, GO terms (biological processes, molecular function and cellular component) and PANTHER protein classification were performed (Fig. 5). Results revealed that these DEPs were associated with PANTHER pathways, including receptor (PC00197), isomerase (PC00135), calcium-binding protein (PC00060) and signaling molecule (PC00207); enriched into GO terms including ‘transporter activity’ (GO: 0005215), ‘catalytic activity’ (GO: 0003824), ‘immune system process’ (GO: 0002376) and ‘cell part’ (GO: 0044464); annotated into pathways including Fas (P00020), fibroblast growth factor (FGF; P00021), p53 (P00059) and apoptosis (P04398).

Previous reports have demonstrated the association of gene dysregulation with osteoporosis or bone mineral content accumulation, including PEDF (30,31) and PDIA3 (32). Furthermore, NPAT, c-Myc and several other proteins are thought to be involved in the regulation of histone transcription (32,33), calcium metabolism (34) and cell proliferation (35,36). Max associates and interacts with c-Myc, thus forming the c-Myc-Max complex, which has core activating roles in gene expression. Inhibition of c-Myc-Max dimerization formation results in the inhibition of cancer cell proliferation (37,38). HSPA8 is a gene that encodes for an important member of HSP70 protein family (39). Therefore, the expression of HSP70 was investigated. In the present study, Max was identified as a protein that was specifically expressed in response to ICA administration. The expression of c-Myc protein and several identified DEPs in ICA (10 and 20 µg/l) treated osteoblasts was subsequently detected. ICA treatment was demonstrated to upregulate the expression of PEDF, STAM, c-Myc, PDIA3, HSP70 and NPAT. Expression of these DEPs were higher in cells treated with 10 µg/l ICA compared with control cells (P<0.05; Fig. 6). In addition, the expression levels of PEDF, PDIA3, HSP70 and NPAT proteins exhibited by the control cells were not significantly different compared with cells treated with 20 µg/l ICA. These findings were in accordance with the bioinformatics analysis results.