ATCC 43300 (MRSA) was purchased in a freeze-dried form from the American Type Culture Collection (ATCC; Rockefeller, MD, USA). Species identification and susceptibility testing was performed with the Vitek 2 automated system (Bio Mérieux, Marcy I’Étoile, France). The phenotypic classification of the ATCC 43300 strain was further confirmed by methicillin-resistant determinant A (MecA) expression, which encodes PBP2a (penicillin-binding protein 2a) and mediates methicillin resistance (21). Recombinant MBD-14 was commercially available and purchased from EIAab Science Co., Ltd. (Wuhan, China). Pyrrolidine dithiocarbamate (PDTC), SB203580, antibodies against p-ERK, p-p38 MAPK, NF-κB p65, NF-κB p-P65, p-IκBα and GAPDH were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

The murine osteoblastic cell line, MC3T3-E1, is a non-transformed cell line established from newborn mouse calvaria. These cells exhibit the osteoblastic phenotype, as evidenced by the expression of ALPase (22), synthesis of the extracellular matrix (ECM) components, such as osteocalcin and type-1 collagen, and their ability to mineralize the ECM (22). MC3T3-E1 cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Gibco, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO₂ in air at 37°C. Cells were trypsinized and cultivated for an additional 2 days before use at an appropriate density. When the mono-layers reached approximately 85% confluence, stimulation experiments were performed in serum-free DMEM containing ascorbic acid in a humidified atmosphere with 5% CO₂ at 37°C as previously described (23).

The production of bacterial culture supernatants was carried out as previously described by Gläser et al (24). In this method, ATCC 43300 bacteria were grown in BBL Trypticase Soy Broth (TSB; BD Biosciences, Franklin Lakes, NJ, USA) under shaking conditions at 37°C until they reached an optical density of 1.0; 1 ml of this culture was then added to 9 ml TSB and the resulting culture was incubated for 24 h in 50-ml centrifuge tubes (Corning Inc., NY, USA) without shaking. Subsequently, the bacteria were centrifuged at 5,000 × g for 15 min and then cell-free conditioned medium was prepared by filtering the supernatant through a 0.22-μm pore-size filter (Tullagreen, Carrigtwohill, County Cork, Ireland) (25). The resulting supernatants were used for stimulation experiments. Stimulation experiments with TSB medium were performed to analyze the potentially unexpected effects of the medium. The concentration of SAS was determined by the number of S. aureus bacteria in the resulting culture incubated for 24 h as previously described (19). The number of colony-forming units (CFUs) was counted and expressed relative to the amount of TSB for planktonic bacteria in 50 ml centrifuge tubes (CFUs/ml). Mouse OBs in the monolayer culture were exposed to various concentrations of SAS (diluted 1:10, 1:20, 1:40 and 1:80, i.e., the number of OBs vs. the concentration of bacterial exoproducts from the number of Staphylococcus aureus).

A Cell Counting kit-8 (Beyotime, Shanghai, China) was used to examine cell viability according to the manufacturer’s instructions. MC3T3-E1 cells were seeded at 1×10⁴ cells/well in 96-well culture plates (Corning Inc.) and were then treated with SAS at various concentrations at 37°C in a humidified incubator containing 95% air and 5% CO₂. After 12, 24, 48 and 72 h, the 10 μl CCK-8 solution was added to each well and incubated for 1 h in an incubator. The absorbance was measured using a Synergy HT multidetection microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm as previously described (26,27).

At various time points, the cells treated with SAS at various concentrations were harvested and total RNA was isolated using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions with an additional DNase I (Fermentas)-treatment step to eliminate residual genomic DNA. Total RNA (1 μg) was reverse transcribed using a Fermentas RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Real-time PCR was performed on an ABI 7500 Fast machine (Applied Biosystems, Courtaboeuf, France) using Roche FastStart Universal SYBR-Green Master (Rox) (Roche Applied Science, Indianapolis, IN, USA). The reactions were performed using cDNA templates and specific forward and reverse primers (Table I). Refer to Table I for the detailed annealing temperature which was used according to the different primers. The specificity of the amplification reaction was determined by analyzing the melting curves. The relative amount of target gene expression was normalized to β-actin as an internal control. The quantification of gene expression was based on the cycle threshold value of each sample, which was calculated as the average of 3 replicate measurements for each sample analyzed as previously described (12,28).

The collected supernatants from the different stimulation experiments were examined by MBD-14, DEFb14 ELISA kit (EIAab Science Co., Ltd.) according to the manufacturer’s instructions. ELISA was performed on each sample in trip-licate. The optical density of each sample was measured with a Synergy HT multidetection microplate reader (BioTek Instruments) at a wavelength of 450 nm. The examination of TNF-α and IL-6 levels was performed according to the standard protocols of the ELISA kit (EIAab Science Co., Ltd.).

Cells in a 6-well plate were harvested under different conditions and washed with PBS 3 times. Protein was extracted using the Total protein Extraction kit according to the manufacturer’s instructions (ProMab Biotechnologies, Inc., Richmond, CA, USA). Proteins were quantified using the BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Samples containing equal amounts of protein (40 μg/lane) were run on 12% polyacrylamide gels, transferred onto nitrocellulose membranes (Whatman, Dassel, Germany) and blocked for 1 h with 5% non-fat milk in TBST (Tris-buffered solution containing 0.1% Tween-20) at room temperature. The membranes were then soaked with primary antibodies overnight at 4°C followed by secondary antibody incubation for 1 h at room temperature. Finally, the membranes were reacted with enhanced chemiluminescence (ECL) reagents and band densities were determined using Gel Pro 4.0 analyzer software (Media Cybernetics Inc., Bethesda, MD, USA) and the integrated optical density (IOD) was calculated as previously described (29).

The results are expressed as the means ± SEM derived from at least 3 independent experiments. Statistical analysis between groups was performed by one-way ANOVA. The level of p<0.05 was considered to indicate a statistically significant difference. SPSS software (version 19.0; IBM, Chicago, IL, USA) was used for statistical analysis.

ATCC 43300 exhibited a low level of susceptibility to penicillin (MIC, 64 μg/ml) and recombinant MBD-14 exhibited a strong antimicrobial activity against ATCC 43300 (MIC, 8 μg/ml). According to a low level susceptibility to penicillin and MecA gene expression, ATCC 43300 was the methicillin-resistant strain (21). In addition, the amount of bacteria in the resulting culture was calculated as approximately 3.5±0.6×10⁹ CFUs/ml using the spread plate method.

In order to determine the optimal proportion and action time of the mixed agent which could contribute to the inflammatory reaction with low cytotoxic response, a cytotoxicity assay was conducted to investigate the effects of culture with various concentrations of SAS on OBs at different time points (12, 24, 48 and 72 h). We first evaluated the effect of various concentrations of SAS on cell viability. OBs were treated with SAS for 12, 24, 48 and 72 h, at the following concentrations: diluted at 1:10, 1:20, 1:40 and 1:80. Throughout the incubation period, cell proliferation was analyzed using a CCK-8 kit. At 72 h, treatment with 10 μg/ml LPS was used as the positive control to evaluate whether the cell proliferation was already affected. All investigated treatment ratios from 1:20 to 1:80 induced less OB proliferation compared to the untreated group (OB group), as shown in Fig. 1 (p<0.05). In the OB group in which OBs without SAS stimulation were cultured for 72 h, the proliferation of cells measured by CCK-8 assay was highest and normalized to 100% (negative control). At 12 h, the proliferation of cells was the highest at the 1:10 treatment ratio, followed by the 1:20 treatment ratio. However, no significant difference was observed (p>0.05). A lower cell number was consistently observed in the groups treated with higher concentrations than those treated with the concentration of 1:10 and 1:20 (p<0.05). Moreover, the lowest cell numbers were observed in the group treated with the concentration of 1:80 (p<0.05), and the same findings were observed at 24, 48 and 72 h (Fig. 1A). When the cells were cultured with the same concentrations of bacterium-free supernatant for different periods of time, the levels of cell proliferation positively correlated with the duration (Fig. 1B). The proliferation of cells was the highest at 12 h, followed by 24 h. However, no significant difference was observed (p>0.05). Furthermore, the lowest number of OBs was observed in the group treated for 72 h, followed by the group treated for 48 h. The difference between these 2 groups was significant. The above findings regarding the different treatment times were observed at different concentrations (Fig. 1B).

Subsequently, the level of the pro-inflammatory cytokines, TNF-α and IL-6, was assessed by ELISA at different time points (12, 24, 48 and 72 h) when the cells were incubated with various concentrations of SAS. At 72 h, treatment with 10 μg/ml LPS was used as the positive control to evaluate whether the model of acute inflammation for OBs has been established. When the cells were cultured with different doses of bacterium-free supernatant, the levels of TNF-α and IL-6 positively correlated with the concentrations of bacterium-free supernatant and the duration. The levels of TNF-α in the SAS + OB and LPS + OB groups treated with different concentrations for different periods of time were significantly higher than those of the OB group at 72 h (negative control). As shown in Fig. 2A, the release of TNF-α with SAS stimulation was the lowest at the concentration of 1:10 at 12 h, followed by the 1:20, 1:40 and 1:80 concentrations. Moreover, a significant difference was observed between the 1:10 and 1:20 concentrations, and the highest level of inflammatory cytokines was observed at the concentration of 1:80. At 24, 48 and 72 h, the levels of TNF-α showed a similar tendency to those of TNF-α at 12 h (Fig. 2A). Obviously, at the concentration of 1:20, there was statistically significant difference in the level of inflammatory cytokines between 12 and 24 h. In addition, no statistically significant differences were observed between the group treated with the concentration of 1:20 at 24 h and the LPS + OB group (positive control) (Fig. 2B). The SAS + OB group treated at the concentration of 1:20, which increased the level of TNF-α at 24 h showed similar abilities compared with the LPS + OB group, suggesting an effective stimulation of the OBs. The variation trend of IL-6 showed a similarity with that of TNF-α (Fig. 2C and D). In conclusion, the results of CCK-8 assay and ELISA for TNF-α and IL-6 showed that the optimal ratio of the mixed agent was 1:20 and the optimal treatment time was 24 h.

To confirm the constitutive expression of MBD-14 and determine whether mouse OBs are a suitable model to examine the induction of MBD-14 following exposure to bacterial supernatant and LPS, the protein level of secreted MBD-14 in cell culture supernatants was examined by ELISA. Cells stimulated with SAS (1:20) were incubated for 12, 24 and 48 h and SAS significantly increased the release of MBD-14 compared to the untreated controls (OB group) (Fig. 3A) (p<0.05). The level of MBD-14 at 12 h was the lowest at all time points following stimulation. In addition, bacterial stimulation increased the level of MBD-14 by up to 3-fold, similar to the OB group after 24 h (p<0.05). The SAS-induced MBD-14 secretion level peaked within 48 h (approximately 60 ng/ml) (Fig. 3A) (p<0.05). Fig. 3B demonstrates the higher level of MBD-14 induction following stimulation with SAS compared to treatment with LPS at 24 and 48 h (p<0.05). This upregulation was more pronounced when SAS was used appropriately as a stimulus.

The p38 and other intracellular MAPK pathways have been implicated in the regulation of HBD-3 expression, in a number of bacterial stimulation models in primary keratinocytes (25). To examine the overall level of p38 and ERK activation (phosphorylation) in OBs challenged with S. aureus secreted bacterial exoproducts, the cells lysed at various time points following exposure to SAS were used for the detection of the phosphorylated form of p38 and ERK by immunoblot analysis (Figs. 4 and 5A and B). Real-time PCR was used to demonstrate the mRNA expression of p38 MAPK and ERK upon stimulation with SAS (Fig. 5C and D). Furthermore, the amount of MBD-14 released from OBs exposed to bacterial supernatants was then examined by ELISA (Fig. 9D). As shown in Figs. 4 and 5, cells without SAS stimulation contained a discernible level of activated p38 MAPK at the baseline. However, the mRNA expression and the phosphorylation of p38 MAPK gradually increased within 12 h and remained elevated for at least 48 h with SAS (1:20) and 10 μg/ml LPS stimulation, a striking contrast to that of ERK at 3 time points (Figs. 4 and 5). Moreover, the treatment of OBs with LPS increased p38 MAPK and ERK activation within 12 h, and this level of activation persisted for at least 48 h. Subsequently, the contribution of p38 MAPK activation to the SAS-induced release of MBD-14 was investigated using the selective inhibitor, SB203580, which specifically inhibits the phosphorylation of the downstream kinases of p38 isoforms α and β. Pre-treatment of the cells with SB203580 for 30 min prior to exposure to SAS significantly inhibited the SAS-induced phosphorylation and mRNA expression of p38 MAPK, and significantly attenuated the release of MBD-14 (Figs. 8 and 9A, D and E). After using SB203580, the amount of MBD-14 released decreased to almost 20 ng/ml at 24 h and 30 ng/ml at 48 h, which was significantly lower than the levels in the the OB + SAS group (p<0.05) (Fig. 9D). These results suggest that the p38 MAPK signaling pathway is involved in the SAS-induced release of MBD-14, whereas ERK is not.

NF-κB has been shown to be involved in the transcriptional activation of pro-inflammatory cytokines, including IL-6 and TNF-α, in murine OBs exposed to extracellular stimuli (20). To determine whether bacterial exoproducts affect the activity of the NF-κB pathway in OBs, the cells lysed at various time points following exposure to SAS were used for the detection of the phosphorylated form of the inhibitory subunit of NF-κBα (IκBα) and NF-κB by western blot analysis (Figs. 6 and 7A–C). We examined whether SAS increased the mRNA expression of NF-κB p65 by real-time PCR (Fig. 7D). Within 12 h following treatment with SAS (1:20) and 10 μg/ml LPS, the mRNA expression of NF-κB p65 and the phosphorylation of IκBα and NF-κB p65 slightly increased to the basal level. At later time points, the SAS-induced phosphorylation and mRNA expression of NF-κB p65 and IκBα increased within 24 h and reached a maximal level at 48 h. Moreover, treatment of the cells with LPS increased NF-κB activation within 12 h, and this level of activation persisted for at least 48 h (Figs. 6 and 7A–C). Subsequently, we examined the role of the NF-κB signaling pathway in the SAS-induced MBD-14 expression. The downregulation of the NF-κB pathway by the relative inhibitor, PDTC, was verified by western blot analysis and real-time PCR (Figs. 8 and 9F). The cells were pre-treated for 30 min with 20 μM PDTC and then treated with SAS for an additional 12, 24 and 48 h, followed by the measurement of the phosphorylation and mRNA expression of NF-κB p65. Pre-treatment of cells with PDTC markedly inhibited the SAS-induced phosphorylation of NF-κB p65 at the different time points (Figs. 8 and 9B and C). Correspondingly, the amount of MBD-14 released decreased to 24±2.1 ng/ml at 24 h and 34±2.8 ng/ml at 48 h following pre-treatment with PDTC, which was far lower than the level in the OB + SAS group (p<0.05) (Fig. 9D). These results suggest that NF-κB mediates the SAS-induced MBD-14 expression.

To determine whether the SAS-induced activation of the p38 MAPK pathway is mediated through NF-κB, the cells were pre-treated for 30 min with the inhibitor, PDTC, and then treated with SAS for an additional 12, 24 and 48 h, followed by the measurement of the phosphorylation and mRNA expression of p38 MAPK. PDTC did not significantly alter the phosphorylation and mRNA expression of p38 MAPK in response to SAS treatment (p<0.05) (Figs. 8 and 9E). These results suggest that the NF-κB pathway does not regulate p38 MAPK activation. In order to determine whether the SAS-induced activation of NF-κB is mediated through the p38 MAPK pathway, the cells were pre-treated for 30 min with SB203580 and then treated with SAS for different periods of time, followed by the measurement of the phosphorylation and mRNA expression of NF-κB p65. Subsequently, SB203580 did not inhibit the activation of NF-κB p65 in response to SAS treatment (Figs. 8 and 9F). These results suggest that the p38 MAPK pathway does not regulate NF-κB activation.