Escherichia coli LPS (serotype 055:B5), β-glycerophosphate (β-GP), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The protease inhibitor cocktail, and the selective mitogen-activated protein kinase (MAPK) inhibitors, SP600125 and PD98059 were purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Rockville, MD, USA). Antibodies to c-Jun N-terminal kinase 1 (JNK1), extracellular signal-regulated kinase 1 (ERK1), alkaline phosphatase (ALP) and caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to Runx2, osterix (OSX), osteocalcin (OCN), Bax, Bcl-2 and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MC3T3-E1 cells, an osteoblast-like cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Other chemicals and reagents used in this study were of analytical grade.

MC3T3-E1 cells were grown in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin solution, 10 mM HEPES solution and incubated at 37°C in 5% CO₂ humidified air. To examine the effect of SP600125 on osteoblast differentiation stimulated with LPS, MC3T3-E1 cells at a density of 5×10⁴ cells/cm² were cultured in osteogenic differentiation medium (DMEM with 10% FBS, 10 mM HEPES, 50 μg/ml L-ascorbic acid and 5 mM β-GP) for 2 days. On differentiation day 3, following pretreatment with or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS at the indicated time points.

Cell metabolism was measured using MTT assay. MC3T3-E1 cells at a density of 5×10³ cells/cm² were plated in 96-well culture plates and cultured in 0.2 ml osteogenic differentiation medium. Following pretreatment with 5, 10, 15, 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS for 1 and 3 days. At the end of treatment, 20 μl MTT (5 mg/ml) was added to each well. The cells were cultured for an additional 4 h, the supernatant was removed, and 100 μl dimethyl sulfoxide (DMSO) was added to each well. After agitation for 3 min, absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). The experiment was performed in triplicate.

MC3T3-E1 cells at 5×10⁵ cells/cm² were seeded in 100-mm diameter culture dishes and cultured in osteogenic differentiation medium for 2 days. On differentiation day 3, following pretreatment with 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS in osteogenic differentiation medium for 1 and 2 days. The cells were detached aided by a cell scraper in phosphate-buffered saline (PBS), and then centrifuged at 16,000 × g for 5 min. Cell pellets were placed in 300 μl of lysis buffer (Cytobuster protein extraction reagent; Novagen, Darmstadt, Germany) with 1X protease inhibitor cocktail. The ALP activity in the lysates was determined by the measurement of p-nitrophenyl phosphate (pNPP) using a commercial assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, cell lysate of each sample was added to the assay buffer (2 mM MgCl₂, 50 mM Tris-HCl/pH 9.8) containing 2.5 mM pNPP. After 30-min incubation at 37°C, the reaction was terminated by the addition of 0.2 M NaOH. Absorbance of the reaction mixture was measured by spectrophotometry (Eppendorf BioSpectrometer; Eppendorf AG, Hamburg, Germany) at 405 nm. A standard curve was also generated using a series of diluted colored p-nitrophenol (pNP) with known concentrations. The concentration was calculated by projecting the optical densities on the standard curve. Total protein concentrations were quantified by spectrophotometry (Eppendorf BioSpectrometer). The relative ALP activity was defined as micromoles of pNPP hydrolyzed per minute per 1 g of total protein (18).

MC3T3-E1 cells at a density of 5×10⁵ cells/cm² were seeded in 100-mm diameter culture dishes and cultured in osteogenic differentiation medium for 2 days. On differentiation day 3, following pretreatment with 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS in osteogenic differentiation medium for 1 and 2 days. The activity of caspase-3 was determined using the caspase-3 activity kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Briefly, the cells were detached aided by a cell scraper in PBS buffer, then centrifuged at 16,000 × g for 5 min and the cell pellets were placed in 300 μl of lysis buffer (cytobuster protein extraction reagent) with 1X protease inhibitor cocktail. Total protein concentrations were quantified by spectrophotometry (Eppendorf BioSpectrometer). The cell lysates of each sample were then incubated with a mixture provided in the kit, containing caspase-3 assay buffer, Ac-DEVD-pNA, the substrate of caspase-3. After 2-h incubation at 37°C, the optical density (coloration) resulting from the cleavage of the substrate and the release of pNA, was detected and quantified by spectrophotometry (Eppendorf BioSpectrometer) at 405 nm. A standard curve was also generated using a series of diluted pNA with known concentrations. The concentration was calculated by projecting the optical densities on the standard curve. The relative caspase-3 activity was defined as nanomoles of pNA per minute per 1 mg of total protein. All the experiments were carried out in triplicate.

Total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY, USA) and quantified by spectrophotometry. After isolation, 3 μg total RNA from each sample was reverse transcribed (RT) utilizing the HiFi-MMLV cDNA kit (Beijing CoWin Biotech, Beijing, China) according to the manufacturer’s instructions. The primer sequences of osteoblast-associated genes, caspase-3, Bax, Bcl-2, JNK1 and ERK1 (Sangon Biotech Co., Ltd., Shanghai, China) and annealing temperatures used in this study are listed in Table I. Quantitative PCR (qPCR) was performed with a RealSYBR Mixture (Beijing CoWin Biotech) according to the manufacturer’s instructions. The qPCR reactions were performed in the ABI PRISM 7300 sequence detection system (Applied Biosystems, Grand Island, NY, USA). In each reaction, 1 μl cDNA, 10 μl 2X RealSYBR Mixture, and 0.25 μM forward and reverse primer in a total volume of 20 μl were used. The reaction conditions used were: 1 cycle of 95°C for 5 min followed by 40 cycles of 95°C for 15 sec, annealing temperature for 30 sec and extension at 72°C for 30 sec. qPCR for each sample was run in triplicate. β-actin was used as an internal control, and the results were analyzed using the standard 2^−ΔΔCt method as previously described (20).

At the end of treatment, cell culture medium was aspirated and the cells were detached in PBS by scraping. Detached cells were centrifuged at 15,000 × g at 4°C for 15 min. Cell pellets were then lysed in 300 μl lysis buffer (Cytobuster protein extraction reagent; Novagen) with 25 mM NaF, 1 mM Na₃VO₄, and 1X protease inhibitor cocktail. Protein concentrations were quantified by spectrophotometry. For western blotting, an equal amount of protein from each sample was loaded on SDS-PAGE and electrotransferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% (w/v) bovine serum albumin in TBST [10 mM Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20, pH 7.5] for 1 h at room temperature, and incubated with primary antibodies incubated with primary antibodies: rabbit polyclonal anti-JNK, phosphorylated JNK, β-actin, goat polyclonal anti-OSX at dilutions of 1:500 (Santa Cruz Biotechnology, Inc.); rabbit polyclonal anti-ERK1/2 and phosphorylated ERK1/2 at dilutions of 1:1,000 (Cell Signaling Technology, Inc.). The secondary antibodies used for detection were horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) and HRP-conjugated rabbit anti-goat IgG (both from Santa Cruz Biotechnology, Inc.). Chemiluminescence ECL (Beyotime Institute of Biotechnology) was used to detect immunoreactive protein signals. Protein signals were then visualized on film and scanned and quantified using the ImageJ software (National Institutes of Health Image, Bethesda, MA, USA). For re-probing, PVDF membranes were stripped with 0.2 M NaOH for 10 min before blocking with another primary antibody. The expression of molecules of interest was determined relative to β-actin.

Data are presented as the means ± standard deviation (SD) for three or more independent experiments. Significant differences were determined using factorial analysis of variance (ANOVA). Statistical analysis was performed using SPSS13.0 software. P<0.05 was regarded significant and shown with an asterisk in the bar diagrams.

Following pretreatment with 5, 10, 15, 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS in the presence of SP600125 for 1 and 3 days. MTT assays showed that SP600125 at 50 μM significantly restored cell metabolism downregulated by LPS in MC3T3-E1 cells compared to the remainig treated cultures (P<0.01) (Fig. 1).

Following pretreatment with 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS in the presence of SP600125 for 1 and 2 days. The downregulated ALP activity in MC3T3-E1 cells induced by LPS was significantly restored by SP600125 compared to the non-treated culture at 1 and 2 days (P<0.01) (Fig. 2).

Cells were pretreated with 25 and 50 μM SP600125 or without SP600125 for 2 h, and then with 100 and 1,000 ng/ml LPS or without LPS in the presence of SP600125 for 1 day. The upregulated caspase-3 activity in MC3T3-E1 cells induced by LPS was significantly downregulated by SP600125 compared to the non-treated culture at 1 day (P<0.01) (Fig. 3).

Effect of SP600125 on mRNA expression levels of osteoblast-specific genes such as Runx2, ALP, BSP, OSX, OCN and Col1α1 in MC3T3-E1 cells stimulated with LPS was determined by the qPCR experiment. The results showed that SP600125 has restored the downregulated expression of early-stage osteoblast marker genes such as Runx2, ALP and BSP in MC3T3-E1 cells induced by LPS compared to the non-treated culture at 1 day. However, the expression of late-stage genes such as OSX, OCN and Col1α1 was not affected (Fig. 4A and C). This result suggested that SP600125 may play a protective role in early-stage osteoblast differentiation in MC3T3-E1 cells induced by LPS.

Following pretreatment with 10, 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS in the presence of SP600125 for 1 day. The protein expression of early-stage osteoblast markers such as Runx2 and ALP in MC3T3-E1 cells inhibited by LPS was significantly enhanced by SP600125 compared to the non-treated culture at 1 day, whereas the protein expression of late-stage marker genes such as OSX and OCN was not affected by SP600125 (Fig. 5).

Following pretreatment with 10, 25 and 50 μM SP600125 or without SP600125 for 2 h, the cells were treated with 100 and 1,000 ng/ml LPS or without LPS in the presence of SP600125 for 1 and 2 day. The upregulated mRNA and protein expression levels of caspase-3 and Bax in MC3T3-E1 cells induced by LPS were significantly decreased by SP600125 compared to the non-treated culture, whereas the downregulated expression of Bcl-2 was significantly enhanced by SP600125 (Figs. 6–8).

After pretreatment with PD98059 alone, or treatment with PD98059 for 2 h followed by treatment SP600125 for 2 h, the cells were treated with or without LPS for 30 min or 1 day in the presence of MAPK inhibitors. The results showed that PD98059 attenuated phosphorylation of ERK1/2 induced by LPS. Additional treatment with SP600125 showed that phosphorylation of ERK1/2 was significantly enhanced. SP600125 significantly enhanced the protein expression of ERK1, but decreased JNK1 expression in MC3T3-E1 cells induced by LPS after SP600125 treatment for 24 h (Figs. 9 and 10). SP600125 >10 μM was shown to completely decrease the protein expression of JNK1 compared to the non-treated cultures in MC3T3-E1 cells stimulated with LPS.