Male C57Bl/6 mice (age, 12 months) were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. The MLO-Y4 mouse-derived osteocytic cell line (derived from long bones of mice) was purchased from PythonBio (http://www.pythonbiotech.com). Fetal bovine serum (FBS), calf serum (CS), and α-minimum essential medium (α-MEM) were purchased from Thermo Fisher Scientific, Inc. A Cell Counting Kit-8 (CCK-8) assay was purchased from Dojindo Molecular Technologies, Inc. The ROS assay kit was purchased from Beyotime Institute of Biotechnology. Primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (product code ab9485), sclerostin (product code ab63097), and Sirt1 (product code ab12193) were purchased from Abcam. Goat anti-rabbit secondary antibody conjugated with Cy3 (cat. no. SA00009-2) was purchased from ProteinTech Group, Inc. Horseradish peroxidase (HRP)-conjugated secondary anti-bodies (product code FD0155) and protease inhibitor tablets were purchased from Fude Biological Technology Co. Ltd. Apocynin, which is an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, N-acetyl-L-cysteine (NAC), which is an ROS scavenger, fluorescein diacetate (FDA) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich; Merck KGaA. The Sirt1 activator, SRT3025, was purchased from Selleck Chemicals.

AOPPs-BSA was prepared as previously described (12,24). Briefly, 20 mg/ml BSA was incubated (at 37°C for 30 min) with 40 mM hypochlorous acid (HOCl) and diluted using phosphate-buffered saline (PBS, pH 7.4). To remove free HOCl, the mixed solution was dialyzed in PBS at 4°C for 24 h. The controls used were BSA and PBS alone. Furthermore, a Detoxi-Gel column (Pierce; Thermo Fisher Scientific, Inc.) was used to remove contaminated endotoxins from all the samples. The endotoxin levels in the obtained samples were measured using a Limulus Amebocyte Lysate kit (Sigma; Merck KGaA) and a level of <0.05 ng/mg was considered as acceptable. Content of AOPPs in the sample was determined as previously described (12).

In total, 56 male C57Bl/6 mice (age, 12 months; body weight, 33.5±3.1 g) were used. All mice were maintained at 23°C and 50-70% humidity with a 12-h light/dark cycle and were provided with free access to food and water. All animal experiments were approved (approval no. NFYY-2018-68) by the Laboratory Animal Care and Use Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China).

The animal study consisted of two independent experiments. For the AOPPs-BSA interventional experiment, 28 mice matched by body weight were randomized into four groups (7 mice in each group) and subjected to the following treatments: Group 1, intraperitoneal (i.p.) injection of vehicle (PBS, pH 7.4); Group 2, i.p. injection of native BSA at 50 mg/kg/day; Group 3, i.p. injection of AOPPs-BSA at 50 mg/kg/day; and Group 4, i.p. injection of AOPPs-BSA at 100 mg/kg/day. For the blocking experiment, the remaining 28 mice were randomized into four groups and subjected to treatments as follows: Group 1, i.p. injection of AOPPs-BSA alone at 100 mg/kg/day; Group 2, i.p. injection of AOPPs-BSA at 100 mg/kg/day and apocynin at 100 mg/kg/day in drinking water; Group 3, i.p. injection of AOPPs-BSA at 100 mg/kg/day and NAC at 100 mg/kg/day in drinking water; Group 4, i.p. injection of AOPPs-BSA at 100 mg/kg/day and SRT3025 at 50 mg/kg/day in drinking water. After a treatment duration of 16 weeks, mice in each group were anesthetized using isoflurane (4% for induction, and 2.5% for maintenance), and blood samples were collected from the retroorbital vein (~500 µl blood was obtained from each mouse). Mice were then immediately sacrificed by standard cervical dislocation to avoid suffering. Subsequently, plasma was separated and stored at -80°C. The tibia, femur and lumbar vertebra specimens were also obtained for further assessments.

The trabecular microstructure of L4 vertebral bodies and left proximal tibias were analyzed using micro-CT (µCT80; SCANCO Medical AG) at a resolution of 10 µm. The volume of interest (VOI) was defined as the entire trabecular bone region in the L4 vertebral body, and the trabecular bone region 100 slices downstream from the proximal growth plate of the tibia. The trabecular microstructure morphometric parameters, including bone volume over total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were obtained via VOI analysis. All µCT analyses were performed in accordance with the guidelines of the American Society for Bone and Mineral Research (25).

Tibial bone samples were fixed in 2% paraformaldehyde at 25°C for 24 h, decalcified in 0.5 M EDTA (pH 7.5) for 3 weeks, and then embedded in paraffin. Osteoblast number and osteoclast area were measured on hematoxylin and eosin (H&E)-stained sections (5 µm) and tartrate-resistant acid phosphatase (TRAP)-stained sections (5 µm), respectively. For H&E-staining, paraffin sections were stained with hematoxylin for 2 min at 37°C, followed by eosin for 5 min at 37°C. For TRAP staining, paraffin sections were first reacted for TRAP activity (37°C, 50 min) and then counterstained at 37°C for 5 min with light green SF yellowfish solution (Merck KGaA,).

For immunohistochemical staining, paraffin sections (5 µm) were de-waxed, rehydrated, incubated in antigen retrieval solution, and washed with Tris-buffered saline (TBS). Endogenous superoxidase was then eliminated using 0.3% H₂O₂. TBS containing 2% BSA was used to block the non-specific antibody binding (37°C, 60 min). Then, sections were incubated overnight at 4°C with primary antibodies against sclerostin (product code ab63097; 1:50) and Sirt1 (product code ab12193; 1:100; both from Abcam), followed by incubation (37°C, 60 min) with biotinylated secondary antibodies (1:2,500; cat. no. ZB-2306; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). Proteins were visualized under a light microscope using brown pigments via a standard diaminobenzidine protocol. Subsequently, slides were lightly counterstained with hematoxylin.

Serum levels of N-terminal propeptide of type 1 procollagen (P1NP) and C-terminal crosslinked telopeptides of type I collagen (CTX-I) were measured using ELISA kits (CSB-E12775m and CSB-E12782m, respectively; Cusabio Technology LLC), following the manufacturer's instructions. Serum levels of total superoxide dismutase (t-SOD) and malondialdehyde (MDA) were quantified using commercial reagent kits (cat. nos. A001-1-2 and A003-1-2, respectively; Nanjing Jiancheng Bioengineering Institute).

MLO-Y4 cells were cultured in growth medium containing α-MEM, 2.5% CS, 2.5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were incubated at 37°C in an atmosphere of 5% CO₂. The medium was changed every 2 days, and the cells were passaged after reaching 80% confluence.

During the experiment, MLO-Y4 cells were treated with various concentrations of AOPPs-BSA (from 0 to 200 µg/ml) for different durations (from 0 to 24 h), as well as co-incubated with apocynin, NAC, or SRT3025, dissolved in dimethyl sulfoxide (DMSO).

Subsequently, cell viability at different concentrations of AOPPs-BSA (50,100, 200 and 400 µg/ml) was qualitatively assessed using the fluorescein diacetate (FDA) staining assay. After washing MLO-Y4 cells three times in PBS, 5 µg/ml FDA (dissolved in DMSO) was added, and the cells were incubated at 37°C for 10 min. Images of the live cells were captured using a model IX71 fluorescence microscope (Olympus Corporation). To quantitatively determine the effect of AOPPs-BSA on cell viability, a CCK-8 assay was performed following the manufacturer's protocol. MLO-Y4 cells were washed three times using PBS, and 10 µl CCK-8 solution was added to each well. Cells were incubated at 37°C for 1 h and the absorbance was measured at 450 nm under a microplate reader (BioTek Instruments, Inc.).

According to the manufacturer's instructions, intracellular ROS levels were measured in MLO-Y4 cells using a ROS assay kit (cat. no. S0033M; Beyotime Biotechnology) containing 2,7,-dichlorofluorescein diacetate (DCFH-DA) as the probe. MLO-Y4 cells under different treatment conditions (AOPPs-BSA of 0 to 200 µg/ml) were incubated with 10 µM DCFH-DA for 30 min at 37°C. The fluorescence intensity was measured using a SpectraMax M5 microplate reader (Molecular Devices, LLC), and the fluorescence images were captured following ROS staining.

Total RNA of MLO-Y4 cells or femoral bone homogenate was extracted using an RNA extraction kit (Takara Bio, Inc.) and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was obtained from total RNA (50 ng), using a PrimeScript™ RT reagent kit (Takara Bio, Inc.). To quantify mRNA expression, the obtained cDNA was amplified using TB Green^® Premix Ex Taq™ (TliRNaseH Plus; Takara Bio, Inc.), and qPCR was performed using a LightCycler480 system (Roche Diagnostics), following the manufacturer's instructions. Thermocycling conditions were set as follows: Incubation at 95°C for 30 sec, amplification at 95°C for 5 sec, 60°C for 30 sec for a total of 40 cycles, 95°C for 5 sec, with a final extension at 60°C for 60 sec. The expressions levels of Sclerostin (SOST) and SIRT1 genes were measured, with GAPDH as the reference gene. The 2^−ΔΔCq method was used for the measurement of gene expression (26). The primer sequences are listed in Table I.

To obtain the total cell protein, cells were rinsed with PBS at 4°C and lysed using a cell lysis buffer (Hangzhou Fude Biological Technology Co., Ltd.). To collect the protein, the cell lysate was centrifuged at a speed of 1,2000 × g for 30 min at 4°C. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Subsequently, the proteins (20 µg/lane) were separated via 6-10% SDS-PAGE, and transferred to PVDF membranes (EMD Millipore). Furthermore, the membranes were blocked (60 min at 37°C) using 5% BSA in TBS containing 0.1% Tween-20, and incubated overnight at 4°C with primary antibodies. The primary antibodies were directed against GAPDH (1:5,000 dilution), sclerostin (1:1,000 dilution) and Sirt1 (1:2,000 dilution). The proteins bound to the membrane were detected ECL reagent (Bio-Rad Laboratories, Inc.) after incubation with goat anti-rabbit IgG-HRP conjugated secondary antibody (product code ab205718; 1:2,000 dilution; Abcam) at 37°C for 1 h. The integrated density of all protein bands was analyzed using ImageJ software (1.52v; National Institutes of Health).

MLO-Y4 cells were washed three times using PBS and fixed for 15 min at room temperature using 4% paraformaldehyde. Following another three washes with PBS, the cells were permeabilized for 20 min at room temperature, using 1% Triton X-100/PBS solution. Then, the cells were blocked with 5% BSA in PBS for 30 min at 37°C. The cells were incubated overnight at 4°C with primary rabbit anti-mouse sclerostin antibody or Sirt1 antibody (both 1:50 dilution). Primary antibodies were not added in the negative control. The following day, the primary immunoreaction was detected by incubation at 4°C for 12 h with a Cy3-conjugated goat anti-rabbit secondary antibody (1:200 dilution). Finally, the cells were counterstained using Fluoroshield mounting medium with DAPI (37°C, 5 min). Images were captured using IX71 fluorescence microscope (Olympus Corporation).

Data are presented as the mean ± SEM. All statistical analyses were performed using the SPSS version 22.0 (IBM Corp.). For each independent in vitro experiment, ≥3 technical replicates were analyzed. Differences between groups in vivo and in vitro were evaluated using one-way ANOVA, followed by post hoc least significant difference (LSD) test. For all statistical tests, P<0.05 was considered to indicate a statistically significant difference.

The present study first measured the effect of AOPPs on bone mass and microstructure in aging mice. Compared with PBS or BSA, 50 mg/ml AOPPs-BSA significantly decreased the bone mineral density (BMD) of L4 vertebral body, and this effect was more notable in 100 mg/ml AOPPs-treated mice. However, no marked difference was observed in femoral BMD of different groups. (Fig. 1A and B). Micro-CT analysis revealed that treatment with AOPPs-BSA significantly aggravated the trabecular microstructure in aging mice. Compared with PBS or BSA-treated mice, AOPPs-treated mice displayed a significant decrease in BV/TV, Tb.N, and Tb.Th but an increase in Tb.Sp in both L4 vertebral bodies and proximal tibias. Furthermore, the degeneration of bone microstructure in the high-dose AOPPs-BSA group was greater than that in the low-dose group. (Fig. 1C-L).

To understand the effect of AOPPs on bone turnover in aging mice, the serum level of a bone formation marker (P1NP), and a bone resorption marker (CTX-1) were measured in mice of different groups. Consistent with our previous study (24), the plasma P1NP concentration was significantly decreased in mice treated with AOPPs-BSA, while the CTX-1 concentration was increased (Fig. 1M and N). Along with this finding, there was a significant decrease in the number of osteoblasts and an increase in the osteoclast area observed in the cortical diaphysis of mice treated with AOPPs-BSA, as evident by the H&E and TRAP staining, respectively (Fig. 1O-R). These data indicated that AOPPs accelerated bone deterioration in aging mice by suppressing bone formation and promoting bone resorption.

Sclerostin, a WNT/β-catenin inhibitor, is predominantly secreted by osteocytes (5). This protein decreases the bone mass by promoting bone resorption and suppressing bone formation (5). In the present study, it was revealed that AOPP-treated mice displayed decreased bone forming activity but increased bone resorbing activity, which was consistent with our previous study (24). These results indicated a probable involvement of sclerostin in AOPP-induced bone loss. Given that plasma sclerostin increases with age (27), the effect of AOPPs on sclerostin expression was therefore examined in aging mice. As presented in Fig. 2A, the SOST mRNA expression in the AOPP-treated groups was higher compared with that in PBS- or BSA-treated groups. An increased expression of sclerostin protein in mice challenged with AOPPs was also observed with immunohistochemical staining (Fig. 2B and C).

OS is regarded as a key promoting factor of osteoporosis (28). Previous studies have reported that AOPPs increase the OS status in a number of animal models (16,21,22,29). The present study evaluated the impact of AOPPs on the OS status of aging mice by testing the plasma AOPPs, MDA and t-SOD levels in femoral bone. Compared with mice treated with PBS or BSA, mice treated with AOPPs-BSA exhibited higher AOPPs and MDA levels but lower t-SOD levels (Fig. 2D-F). These results indicated that AOPPs enhanced OS in aging mice.

Sirt1 regulates sclerostin expression in osteocytes (30), and under OS status, the abundance and deacetylating activity of Sirt1 is compromised (31). Therefore, it was investigated whether there was a fluctuation of Sirt1 expression under challenge of AOPPs. As was revealed by RT-qPCR and immunohistochemical staining, Sirt1 mRNA and protein expression levels were significantly decreased in AOPP-treated mice compared with those in PBS- or BSA-treated mice (Fig. 2G-I).

To investigate the effect of AOPPs on the expression of sclerostin in vitro, an osteocytic cell line, MLO-Y4, was used. First, the cytotoxic effect of AOPPs-BSA on MLO-Y4 cells was tested. As previously described (22,32), the viability of MLO-Y4 cells treated with different doses of AOPPs, ranging between 50-400 µg/ml, was examined. FDA staining revealed that there was only a slight decrease in the viability of cells treated with 50-200 µg/ml of AOPPs-BSA. However, cells treated with 400 µg/ml AOPPs displayed a sharp decrease in fluorescence intensity, indicating a high cytotoxic effect of AOPPs-BSA at this concentration (Fig. 3A). Furthermore, a CCK-8 assay was performed to quantify the influence of AOPPs-BSA on the viability of MLO-Y4 cells. Consistent with the FDA staining, AOPPs-BSA up to 200 µg/ml did not markedly affect cellular activity, while the absorbance value was noticeably lower in the 400 µg/ml AOPP-treated group, suggesting compromised cell viability in this group (Fig. 3B).

To evaluate the effect of AOPPs on the expression of sclerostin in MLO-Y4 cells, the cells were incubated for 24 h with AOPPs-BSA (ranging between 50-200 µg/ml). According to RT-qPCR analysis, the SOST mRNA expression was nearly 0.5-fold higher in the 50 µg/ml AOPPs-BSA group compared with that in the PBS group. As the concentration of AOPPs-BSA increased, the SOST mRNA expression levels were also increased (Fig. 3C). However, compared to the PBS group, SOST mRNA levels did not notably change in the BSA-treated group. The results from the western blot analysis and immunofluorescence staining further indicated that treatment with AOPPs upregulated the expression of sclerostin in MLO-Y4 cells in a dose-dependent manner (Fig. 3D and E).

Previous, including the research group of the present authors, have reported that AOPPs significantly increase the cellular ROS levels in numerous pathological processes by activating NADPH oxidases (21,22,33). Therefore, the present study examined the ROS levels in MLO-Y4 cells treated with AOPPs. Compared with PBS, BSA did not markedly affect DCFH fluorescence, which represented the intracellular ROS levels. However, 50 µg/ml AOPPs-BSA notably increased the cellular fluorescence intensity. In addition, the AOPPs-BSA-induced ROS effect was more evident in the 100- and 200-µg/ml groups (Fig. 4A and B). To investigate whether NADPH oxidases were involved in AOPP-induced ROS accumulation, MLO-Y4 cells were co-incubated with apocynin, an NADPH oxidase inhibitor. As presented in Fig. 4C, apocynin successfully decreased the ROS increase triggered by AOPPs.

Evidence from previous studies has suggested that Sirt1 directly decreases the expression of sclerostin in osteocytes via epigenetic regulation (30,34,35). In addition, OS decreases Sirt1 expression in multiple diseases (31). Thus, it was investigated whether AOPPs downregulated Sirt1 expression in MLO-Y4 cells. As revealed in Fig. 4D, AOPPs significantly decreased the mRNA expression levels of Sirt1 in a dose-dependent manner. This effect of AOPPs was further confirmed at the protein expression level, as detected via western blotting (Fig. 4E) and immunofluorescence staining (Fig. 4F). However, when the cells were co-incubated with apocynin, or a ROS scavenger, such as NAC, the aforementioned effect of AOPPs was largely blocked (Fig. 4G). These results indicated that AOPPs downregulated the expression level of Sirt1 in MLO-Y4 cells by triggering NADPH oxidase-dependent ROS accumulation.

To verify the roles of NADPH oxidases, ROS, and Sirt1 in the AOPP-induced expression of sclerostin, MLO-Y4 cells treated with AOPPs were co-incubated with apocynin, NAC, and a Sirt1 activator, SRT3025. The western blotting results demonstrated that the AOPP-induced expression of sclerostin was largely abrogated by apocynin (Fig. 4H) and NAC (Fig. 4I), whereas the expression of this protein was significantly, but not completely, diminished by SRT3025 (Fig. 4J). These results indicated that the NADPH oxidase-dependent and ROS-triggered downregulation of Sirt1 was involved in the AOPP-induced upregulation of sclerostin.

To further confirm the mechanism of AOPP-induced bone deterioration in vivo, AOPP-treated mice were co-treated with apocynin, NAC, or SRT3025. The results of the BMD measurement results identified that apocynin, NAC, and SRT3025 effectively ameliorated the loss of bone mass in L4 vertebral bodies in mice treated with AOPPs-BSA (Fig. 5A). However, the BMD of the femoral specimens was not markedly different among the various groups (Fig. 5B). Apocynin, NAC, and SRT3025 also largely alleviated the bone microstructural degeneration induced by AOPPs. As indicated by the micro-CT analyses results, the BV/TV, Tb.N and Tb.Th of L4 vertebral bodies were notably higher in the AOPPs-BSA + apocynin, AOPPs-BSA + NAC or AOPPs-BSA + SRT3025 groups compared with in the AOPPs-BSA group. However, Tb.Sp in these three groups was reduced (Fig. 5C-G). Moreover, the same microstructural changes were observed in the proximal tibias (Fig. 5H-L).

The effects of apocynin, NAC, and SRT3025 on the expression of sclerostin in the cortical bone of AOPP-treated mice were also evaluated. The RT-qPCR results demonstrated that the mRNA expression levels of SOST were significantly lower in the AOPPs-BSA + apocynin, AOPPs-BSA + NAC, and AOPPs + SRT3025 groups compared with in the AOPPs-BSA group (Fig. 5M). This result was also confirmed at the protein level via immunohistochemical staining (Fig. 5N and O).