Mouse serum albumin (MSA), p38 inhibitor SB203580, JNK inhibitor SP600125, ERK inhibitor PD98059, N-acetylcysteine (NAC) and apocynin were obtained from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Trypsin-EDTA, fetal bovine serum (FBS), newborn calf serum, α-minimum essential medium (α-MEM) and penicillin-streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). TRIzol^® reagent was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The Prime Script^® One Step real time-polymerase chain reaction (RT-PCR) kit and SYBR Premix Ex Taq were obtained from Takara Biotechnology Co., Ltd. (Dalian, China). Radioimmunoprecipitation assay (RIPA) lysis buffer and phenylmethylsulfonyl fluoride (PMSF) were from Beyotime Institute of Biotechnology (Shanghai, China). The Detoxi-Gel column was from Pierce (Thermo Fisher Scientific, Inc.). Cell Death Detection enzyme-linked immunosorbent assay (ELISA)PLUS kit and Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit were obtained from Roche Life Science (Indianapolis, IN, USA). Rabbit anti-sclerostin (cat. no. sc-130258) and anti-RANKL (cat. no. sc-9073) antibodies were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-phosphorylated (P)-p38 (cat. no. #4631S), anti-p38 (cat. no. #9212S), anti-P-ERK 1/2 (cat. no. #9101S), anti-ERK 1/2 (cat. no. #9102S), anti-P-JNK (cat. no. #9251S) and anti-JNK (cat. no. #9252S) antibodies were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat. no. ab8245) antibody and the Cellular Reactive Species Detection Assay kit were purchased from Abcam (Cambridge, UK).

AOPPs-MSA was prepared in vitro by incubating 20 mg/ml MSA with 200 mM hypochlorous acid (HOCl) for 30 min at 37°C. The mixture was then dialyzed overnight against phosphate-buffered saline to remove any free HOCl. All prepared samples were passed through a Detoxi-Gel column to remove contaminated endotoxin. Endotoxin levels in the preparation were determined using the Limulus Amebocyte Lysate kit (Sigma-Aldrich; Merck Millipore) and were shown to be <0.025 EU/ml. AOPP content in the preparation was determined according to a previously published method (1). Briefly, 200 µl of the sample or chloramine-T (standard curve; Sigma-Aldrich; Merck Millipore) were plated in a 96-well plate and mixed with 20 µl acetic acid. The absorbance was read immediately at 340 nm using a microplate reader (Multiskan MK3; Thermo Fisher Scientific, Inc.). The content of AOPP in the AOPP-MSA preparation was 68.80±5.35 nmol/mg protein in AOPPs-MSA, and 0.27±0.03 nmol/mg protein in native MSA.

Mouse osteocyte-like MLO-Y4 cells were kindly provided by Dr. Ni Guoxin (Southern Medical University, Guangzhou, China). The cells were cultured on type I collagen-coated plates in α-MEM at 37°C in a humidified atmosphere containing 5% CO₂. The medium was supplemented with 5% newborn calf serum (CS), 5% FBS and 1% penicillin-streptomycin.

Cells were lysed with RIPA buffer containing 0.5 mM PMSF, and the lysates were centrifuged prior to determination of the protein concentration using a bicinchoninic acid kit (Sigma-Aldrich; Merck Millipore). A total of 30 µg of protein was loaded per sample channel, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% gels). The separated proteins were then transferred to polyvinylidene fluoride membranes. The membranes were blocked with blocking buffer (5% defatted milk) and incubated at room temperature for 1 h. The membranes were initially probed with anti-sclerostin (1:500 dilution), anti-RANKL (1:500 dilution), anti-P-p38, anti-P-ERK 1/2 and anti-P-JNK (all 1:1,000 dilution) primary antibodies, and incubated at 4°C overnight. Subsequently, membranes were stripped and reprobed with anti-ERK 1/2, anti-JNK, anti-p38 (all 1:1,000 dilution) or anti-GAPDH (1:500 dilution) for normalization and densitometric analysis. Blots were scanned and visualized using the Odyssey detector, as mentioned below. After extensive washing with Tris-buffered saline/Tween 20 (TBS-T), the membranes were incubated for 1 h at room temperature with the secondary antibody [IRDye^® 680LT-conjugated donkey anti-rabbit immunoglobulin G (IgG) (H + L) antibody; cat. no. P/N 925–68023, 1:20,000 dilution). Images were detected using the Odyssey detector (LI-COR Biosciences, Lincoln, NE, USA).

Total RNA was extracted from the lysed cells using TRIzol^® reagent. Aliquots of each RNA extraction were then reverse transcribed simultaneously into cDNA using the PrimeScript^® One Step RT-PCR kit, according to the manufacturer's protocol. RT-qPCR was performed in a total volume of 20 µl in duplicate using the SYBR^® Premix Ex Taq kit and Fast Real-time PCR system 7300 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR reaction mixture contained 2X SYBR^® Premix Ex Taq™ (10 µl), PCR forward primer (10 µM; 0.4 µl), PCR reverse primer (10 µM; 0.4 µl), ROX Reference Dye II (50x; 0.4 µl), RT reaction solution (cDNA) (2.0 µl) and double-distilled H₂O (6.8 µl). The PCR thermocycling conditions were as follows: Stage 1: 95°C for 30 sec, repeat one time; Stage 2: 95°C for 5 sec, 60°C for 34 sec, repeat 40 times; Stage 3: 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec, repeat one time. The mRNA value for each gene was normalized relative to the mouse GAPDH mRNA levels in the same RNA samples, and the results were quantified using the 2^−ΔΔCT method (16). Primer sequences were as follows: GAPDH, forward 5′-ATGGCCTTCCGTGTTCCTAC-3′, reverse 5′-CACCTTCTTGATGTCATCATACTTG-3′; sclerostin (gene name, SOST), forward 5′-GCCTCCTCAGGAACTAGAGAAC-3′, reverse 5′-TACTCGGACACGTCTTTGGTG-3′; and RANKL, forward 5′-TGTACTTTCGAGCGCATGATG-3′ and reverse 5′-AGGCTTGTTTCATCCTCCTG-3′.

Intracellular ROS were detected in MLO-Y4 cells using the Cellular Reactive Species Detection Assay kit (Deep Red Fluorescence). Briefly, ROS assay solution (100 µl/well) was added to the treated or untreated cells at a density of 3,000 cells per well, and the cells were incubated in a 5% CO₂ atmosphere at 37°C in an incubator for 1 h. The fluorescence signal was monitored at an excitation wavelength of 650 nm and an emission wavelength of 675 nm. Untreated cells were used to determine background fluorescence. Data were expressed as percentage of controls.

To detect apoptosis, the Cell Death Detection ELISAPLUS kit was used. Briefly, MLO-Y4 cells were seeded in 96-well plates at a density of 3,000 cells/well and were incubated overnight in α-MEM supplemented with 5% FBS and 5% CS at 37°C in a humidified atmosphere containing 5% CO₂. On the following day, the cells were treated with either MSA (control) or various concentrations of AOPPs for 60 min, or with 200 µg/ml AOPPs for the indicated time. The cells were then lysed, and the supernatant was analyzed using the Cell Death Detection ELISAPLUS kit, according to the manufacturer's protocol.

Another apoptosis analysis was conducted using the Annexin V/PI assay. The Annexin V-FITC Apoptosis Detection kit was used in accordance with the manufacturer's protocol. Briefly, MLO-Y4 cells were serum-starved for 24 h, and were pretreated for 30 min at 37°C with JNK inhibitor SP600125 (30 µM), p38 inhibitor SB203580 (30 µM), ROS scavenger NAC (10 µM) and NADPH oxidase inhibitor apocynin (100 µM), prior to incubation with the indicated concentration of AOPPs-MSA for 24 h. Cells were trypsinized and double-stained with FITC-conjugated Annexin V and PI. Cells were analyzed using a flow cytometer (Beckman Coulter, Brea, CA, USA).

All experiments were performed in triplicate. Data are presented as the mean ± standard deviation. One-way analysis of variance was performed, followed by a least significant difference (LSD) method when P<0.05 for multiple comparisons. Statistical analyses were conducted using SPSS 19.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study initially investigated whether AOPPs induced apoptosis of osteocytic MLO-Y4 cells (Fig. 1). As determined by Cell Death Detection ELISA kit and Annexin V/PI staining, treatment of the cells with AOPPs significantly induced apoptosis in a dose- and time-dependent manner (Fig. 1A and B). Subsequently, the present study examined the effects of JNK inhibitor SP600125 (30 µM), p38 inhibitor SB203580 (30 µM), ROS scavenger NAC (10 µM) and NADPH oxidase inhibitor apocynin (100 µM) on AOPPs-induced apoptosis. Pretreatment of the cells with these agents significantly reversed AOPPs-induced apoptosis (Fig. 1B).

ROS generation was detected using the Cellular Reactive Species Detection Assay kit (Fig. 2). Treatment of MLO-Y4 cells with AOPPs induced a significant increase in intracellular ROS production, in a dose- and time-dependent manner (Fig. 2B). Conversely, AOPPs-induced ROS generation was suppressed by NAC pretreatment, but not by SP600125 or SB203580 treatment. In addition, the NADPH oxidase inhibitor apocynin exhibited inhibitory action on ROS generation (Fig. 2A and B).

Sclerostin and RANKL are associated with bone remodeling by regulating osteoblastogenesis and osteoclastogenesis. Therefore, the present study examined the effects of AOPPs on the expression levels of sclerostin and RANKL in MLO-Y4 cells. As shown in Fig. 3, exposure of MLO-Y4 cells to AOPPs significantly increased the expression of sclerostin, at the protein and mRNA levels (Fig. 3A-C), in a dose- and time-dependent manner. In addition, the effects of AOPPs on the expression levels of RANKL were detected by western blotting and RT-qPCR. Treatment with AOPPs also increased the expression levels of RANKL (Fig. 3A, B and D). However, treatment of the cells with medium alone or MSA did not exert any effects on the expression levels of sclerostin and RANKL.

The extent and duration of MAPK activation serves a key role in regulating cell functions (17,18). As shown in Fig. 4, activation of the JNK, p38 and ERK MAPKs was detected by western blotting, using phosphorylation level as an index of enzyme activity. Treatment of MLO-Y4 cells with AOPPs markedly induced the activation of JNK and p38, but not ERK, in a dose- and time-dependent manner. To further clarify the involvement of the activation of MAPKs in AOPPs-induced upregulation of sclerostin and RANKL, MLO-Y4 cells were pretreated with PD98059 (30 µM), SP600125 (30 µM) and SB203580 (30 µM) for 30 min, and were then co-treated with AOPPs (200 µg/ml) for the indicated durations. As shown in Fig. 5, SP600125 and SB203580, but not PD98059, significantly inhibited AOPPs-induced upregulation of sclerostin and RANKL, as confirmed by western blotting. These results indicate that AOPPs-induced upregulation of sclerostin and RANKL is associated with activation of JNK and p38 MAPK. As expected, NAC and apocynin effectively suppressed AOPPs-induced upregulation of sclerostin and RANKL (Fig. 5).