Introduction

The destruction of bone and cartilage is the major cause of progressive disabilities culminating in the crippling of patients suffering from rheumatoid arthritis (RA). Extensive bone erosion is often seen as marginal joint erosions radiographically, and it is predictive of a poor prognosis. Prevention of the development of bone destruction is an important objective in the treatment of RA.

Osteoclasts are the cell type primarily responsible for resorption during both physiologic bone turnover and inflammatory bone disease1. They are formed by the fusion of mononuclear cells derived from osteoclast precursors. Bone-resorbing osteoclasts exhibit highly polarized morphological features such as actin rings, clear zones, and ruffled borders, which are markers of functional osteoclasts. The resorption lacuna is formed by targeted secretion of protons and proteases from the ruffled border after tight attachment of osteoclasts to mineralized bone surfaces, which is mediated by integrin receptors expressed on the cell surface following polarization2. Multiple kinds of inflammatory cytokines are involved in the regulation of osteoclasts, including IL-1β.

Aromatic aminoketone (SY0916) had been demonstrated to be a new type of PAF receptor antagonist and is considered a novel anti-inflammation drug. The anti-inflammatory effects of SY0916 have been shown by our previous studies both in vivo and in vitro3. Notably, we found that SY0916 prevented bone erosion from occurring in CIA rats4. However, we did not study the mechanism underlying this effect of SY0916. The aim of the present study was to investigate the effects and mechanism of SY0916 on bone destruction in vitro.

Materials and methods

Animals

Male C57BL/6 mice (H-2d, 18±2 g, 6–8 weeks old) were provided by the Experimental Animal Center, Chinese Academy of Medical Sciences & Peking Union Medical College (SPF, certificate No SCXK2004-0001). All animals were housed in groups under a 12-h light/dark regime (lights on from 7:00 to 19:00) at 23±2 °C prior to the experiments, and were given standard laboratory chow and tap water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee before any actual experimentation began.

Reagents

Rh IL-1β, M-CSF, and sRANKL were purchased from Peprotech. FITC-phalloidin, MTT, and 1,25-dihydroxyvitamin D3 were purchased from Sigma. ELISA kits for murine MMP-9 were from R&D systems. αMEM and FCS were from Hyclon. TRIzol reagent was purchased from Promega. SYBR® PrimeScriptTM RT-PCR kit was from Takara. Annexin V-FITC kit for apoptosis detection was from BAOSAI Reagent.

Test compound

SY0916 was synthesized as described previously3, its structure is shown in Figure 1. The purity was more than 99% upon HPLC analysis. SY0916 was prepared in a 0.01 mol/L stock solution with DMSO and stored at -20 °C. Before using, the stock solution was diluted to the appropriate concentration in αMEM.

Figure 1
figure 1

The structure of SY0916, MW=386.31.

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Cell culture

The MC3T3-E1 cell line (an osteoblastic cell line derived from mice) was purchased from the cell center at the Chinese Academy of Medical Sciences & Peking Union Medical College. MC3T3-E1 cells were cultured in αMEM containing 10% FCS at 37 °C in 5% CO2. When the cells were confluent (after approximately 3 d in culture), a mixture of 0.05% trypsin and 0.02% EDTA was used to passage the cells. The collected cells were then seeded on culture dishes.

The preparation of bone marrow cells

The femur and tibia were collected from 6–8 week-old C57BL/6 mice sacrificed under sterile conditions. A single cell suspension was prepared by gently pressing bone marrow segments through a glass injector. Bone marrow cells were separated from red blood cells by centrifugation at 790×g for 20 min in a Histpopaque 1077 centrifuge. The collected cells were washed twice and seeded on culture plates in αMEM containing 10% FCS at 37 °C in 5% CO2 for about 6 h and then collected.

Induction and purification of osteoclasts

MC3T3-E1 cells (1×106 cells/mL) and bone marrow cells (2×107 cells/mL) were co-cultured in αMEM containing 10% FCS in 100 mm tissue culture dishes (15 mL/dish) precoated with 2.5 mL of 0.2% collagen gel matrix. After 2 d, half of the medium was changed with fresh medium. Subsequently, 1,25-dihydroxyvitamin D3 (10−8 mol/L) and M-CSF (50 ng/mL) were added to the media as stimulants. After culturing for 5 d, the non-adherent cells were removed and the mononuclear cells attached to the osteoblastic cell layer were recovered as pOCs by gentle pipetting with fresh αMEM. pOCs were collected by centrifugation (110×g for 5 min) and 2×105 cells were placed in 48 well culture plates, which were then cultured with M-CSF (50 ng/mL) and sRANKL (50 ng/mL)5. OCLs were formed within 48 h of culturing, which then differentiated to osteoclasts after stimulation with sRANKL (100 ng/mL) for 24 h.

MTT assay for the proliferation of OCLs

OCLs were placed in 96-well culture plates and pre-cultured for 4 h. Then, the incubation continued for another 48 h with SY0916 at concentrations ranging from 0.01 to 10 μmol/L. Proliferation of OCLs was determined by MTT assay. MTT was added to each well for 4 h before the addition of DMSO, and the absorbance value of each well was measured at 570 nm.

Determination of the activity and function of osteoclasts

Purified osteoclasts were obtained according to the method presented above. Sterile bone slices (2 cm2) and coverslips were pre-positioned in the plates. Except for the blank control group (without the addition of IL-1β), IL-1β (280 ng/mL) was added into each group. SY0916 was added to experimental groups at concentrations ranging from 0.01 to 10 μmol/L. After culturing for 3 d, the coverslips were dislodged and the number of osteoclasts with more than three nuclei were measured by TRAP staining following the protocol provided by the TRAP staining kit (Sigma)6. Each group was divided in three parallel wells and five random fields were counted for each well. The bone slices were stained by toluidine blue as reported previously and the functioning of osteoclasts was determined by measuring the area of bone resorption pits. Images of osteoclasts and bone slices were captured by a digital camera. The osteoclast count and the bone lacuna area were analyzed by Image 5.0 software.

ELISA method for MMP-9 expression assay

Osteoclasts were placed in 48-well culture plates and pre-cultured for 4 h. Except for the blank control group (without the addition of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 (from 0.01 to 10 μmol/L) was added to the experimental groups. Subsequently, all groups were incubated for 48 h. Then, the cell supernatants were collected and the level of MMP-9 was measured by an ELISA kit according to the manufacturer's protocol.

Determination of osteoclasts apoptosis by AnnexinV-FITC kit

Osteoclasts were placed in 48 well culture plates and pre-cultured for 4 h. Except for the blank control group (without the addition of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 (from 0.01 to 10 μmol/L) was added to the experimental groups. All groups were incubated for 72 h. The cells were washed twice with cold PBS and resuspended in 1× binding buffer at a concentration of 1×106 cells/mL. Subsequently, 100 μL of cell suspension was transferred to a 5-mL culture tube. Ten μL of Annexin V-FITC and 5 μL of PI were added to the cell suspension7. The tube was vortexed gently and incubated for 30 min at room temperature (20–25 °C) in the dark. Then, 400 μL of 1×binding buffer was added into each tube. The cells were immediately analyzed by flow cytometry.

Real-time PCR for RANK, RANKL, OPG, and MMP-9 gene expression

Except for the blank control group (without the addition of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 (from 0.01 to 10 μmol/L) was added to the experimental groups. All groups were incubated for 15 h. To detect the mRNA expression of RANK and MMP-9, total cellular RNA was extracted from 1×105 osteoclasts. To detect the mRNA expression of RANKL and OPG, total cellular RNA was extracted from 5×106 MC3T3-E1 cells stimulated with IL-1β. The RNA was extracted with TRIzol according to a previously reported method8. DNA synthesis was performed following the protocol of the SYBR® PrimeScriptTM RT-PCR kit. Real-time PCR for β-actin, RANKL, OPG, RANK, and MMP-9 was performed in triplicate following the manufacturer's protocol. The samples were placed into 8 stripe tubes (Axygen). PCR amplifications were performed using the ABI PRISM 7000HT Real-Time PCR system. Thermal cycling conditions were 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 31 s at 60 °C. The primers used for the reaction are shown in Table 1. Gene expression was measured by relative quantitation9, which compared the threshold cycles (Ct) of the sample of interest to the Ct generated by a reference sample that was referred to as the calibrator (non-stimulated cells that were incubated for the same time period as cells in the experimental groups). Gene expression was normalized to β-actin expression by subtraction of Ct to provide ΔCt values. The ΔΔCt was calculated as the difference between ΔCt values for IL-1β-stimulated and non-stimulated cells. The relative difference in the expression of the gene of interest between stimulated and unstimulated cells was determined using the equation of 2−ΔΔCt. This equation was considered valid only if the amplification efficiencies of the gene of interest and β-actin were approximately equal. To confirm the validity of the method, we performed the reaction over a range of DNA template concentrations and the resulting slope of the log of the template DNA concentration plotted against the ΔCt calculated for β-actin and each gene of interest was less than 0.1 (data not shown).

Table 1 Primer sequences used in real-time RT-PCR.
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Statistical method for analysis

All values were expressed as the mean±SD of at least three independent experiments. Statistical analysis was carried out using SPSS-13.0 software. One-way ANOVA with post hoc analysis by an LSD test or by the Dunnett's C test was applied, which compared treatment groups to a specific control group. A value of P<0.05 was considered statistically significant.

Results

The effect of SY0916 on the proliferation of OCLs

The proliferation of OCLs was significantly decreased by SY0916 between 0.01 and 10 μmol/L. The value of OD: 0.40±0.07 (0.01 μmol/L), 0.30±0.08b (0.1 μmol/L), 0.39±0.06b (1 μmol/L), 0.31±0.09b (10 μmol/L), 0.48±0.05 (control), n=5, bP<0.05 vs control group.

The effect of SY0916 on the activity and function of osteoclasts

IL-1β significantly increased the number of osteoclasts and bone resorption pits. Typical photos of osteoclasts stained by TRAP and bone slices stained by toluidine are shown in Figure 2. IL-1β also increased the total area and average area of bone resorption pits, which was in accordance with previous reports10, 11. SY0916 significantly decreased both the total area and average area of bone resorption pits at concentrations ranging from 0.01 to 10 μmol/L. In addition, SY0916 also inhibited the number of osteoclasts at 0.01–10 μmol/L (Table 2).

Figure 2
figure 2

The photographs of osteoclasts stained by TRAP and bone resorption pits stained by toluidine blue. Sterile bone slices (2-cm2) and coverslips were pre-positioned in the plates. Except for the blank control group (without the stimulator of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 at 0.01–10 μmol/L were added into the experimental groups. After being cultured for 3 d, osteoclasts were measured by TRAP staining. The bone slices were stained by toluidine blue. Images of osteoclasts and bone slices were captured by the digital camera of the microscope. (A) The typical photographs of the osteoclasts (TRAP stain, ×400). (B) The typical photographs of bone slices (toluidine blue stain, ×400). a: blank control group; b: IL-1β stimulated group; c: SY0916 10 μmol/L group.

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Table 2 Effect of SY0916 on the activity and function of osteoclasts. Except for the blank control group (without the stimulator of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 at 0.01–10 μmol/L were added into the experimental groups. After culturing for 3 d, the coverslips were dislodged and the number of osteoclasts with more than three nuclei was measured. The function of osteoclasts was determined by measuring the number and area of bone resorption pits. Images of osteoclasts and bone slices were captured on the digital camera and analyzed by Image 5.0 software. Values were expressed as mean±SD. n=5. bP<0.05 vs blank control group. eP<0.05 vs IL-1β group.
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The effect of SY0916 on the MMP-9 secretion of osteoclasts

The level of MMP-9 in osteoclasts increased approximately 2-fold after stimulation with IL-1β for 48 h. The increase in MMP-9 was also observed in osteoclasts after stimulation with IL-1β for 24 h, but it was not a significant difference. SY0916 notably inhibited the expression of MMP-9 in osteoclasts stimulated by IL-1β at 0.1–10 μmol/L (Figure 3).

Figure 3
figure 3

Effect of SY0916 on the secretion of MMP-9 in osteoclasts. Osteoclasts were placed on the 48-well culture plates and pre-cultured for 4 h. Except for the blank control group (without the stimulator of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 at 0.01–10 μmol/L were added int o the experimental groups. Then the incubation continued for another 48 h. The cell supernatants were collected and the level of MMP-9 was measured by ELISA kit according to the manufacturer's protocol. Values were expressed as mean±SD. n=3. bP<0.05 vs blank control group. eP<0.05 vs IL-1β group.

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The effect of SY0916 on the apoptosis of osteoclasts

Under normal conditions, osteoclasts extensively undergo apoptosis after five days in culture. However, IL-1β inhibited the apoptosis of osteoclasts and extended their lifespan. SY0916 significantly blocked the effect of IL-1β at 0.01–10 μmol/L (Figure 4).

Figure 4
figure 4

Effect of SY0916 on the apoptosis of osteoclasts. Except for the blank control group (without the stimulator of IL-1β), IL-1β (280 ng/mL) was added into each group and SY0916 at 0.01–10 μmol/L were added into the experimental groups. After being cultured, the cells were resuspend in 1×Binding Buffer at a concentration of 1×106 cells/mL. 100 μL (1×105 cells) was transfered into a 5 mL culture tube, then added 10 μL of Annexin V-FITC and 5 μL of PI. The tubes were gently vortexed and incubated for 30 min at room temperature in dark. Four hundred μL of 1×Binding Buffer was added into each tube. All samples were analyzed on a FACS flow cytometry (EPICS XLHP). Apoptotic cells, viable cells and necrotic cells were detected as An+/PI− cells, An−/PI− cells and PI+ cells, respectively. The An+/PI− cells represent the annexin-V positive and propidium iodide negative cells, that is, the apoptosis cells. The experiment was repeated three times and the typical result was presented.

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The effect of SY0916 on the gene expression of RANK, RANKL, OPG, and MMP-9

IL-1β significantly increased the mRNA expression of RANK, RANKL, OPG, and MMP-9. IL-1β stimulation increased the mRNA expression of RANKL and OPG by approximately 6-fold and 2-fold, respectively, which caused the ratio of RANKL to OPG to significantly increase in MC3T3-E1 cells. SY0916 between 0.01 and 10 μmol/L significantly inhibited the mRNA expression of MMP-9 and RANK in osteoclasts. In addition, SY0916 inhibited the expression of RANKL and OPG in MC3T3-E1 cells, whereas the inhibition of the OPG gene was weaker. As a result, the ratio of RANKL to OPG in MC3T3-E1 cells was significantly decreased (Figure 5A–5B).

Figure 5
figure 5

Effect of SY0916 on gene expression of RANKL and OPG in MC3T3-E1 cells and gene expression of RANK and MMP-9 in osteoclasts. SYBR® PrimeScriptTMRT-PCR kit was applied for cDNA synthesis and real-time PCR for β-actin, RANKL, OPG, RANK, MMP-9. The detection was performed in triplicate following the protocol of the kit. Gene expression was measured by relative quantitation. The relative difference in the genes of interest expression between stimulated and unstimulated cells was determined using the equation of 2−ΔΔCt. Results were expressed as fold increase in gene expression by IL-1β stimulated cells compared to unstimulated (media alone) cells. The y-axis showed the means of 2−ΔΔCt of each group, which reflected the fold increase in the genes of interest expression. The x-axis showed the treatment given to each group. (A) Effect of SY0916 on the gene expression of RANK and MMP-9 in osteoclasts; (B) Effect of SY0916 on the gene expression of RANKL and OPG in MC3T3-E1 cells. bP<0.05 vs blank control, eP<0.05 vs IL-1β.

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Discussion

SY0916 was developed as a novel type of anti-inflammatory drug. In a previous pharmacodynamic study, Wang et al found that SY0916 treatment had a beneficial effect in CIA rats, especially in preventing bone erosion in this model4. However, little was known about the mechanism underlying the compound's effect on bone protection.

Osteoclasts, the only somatic cells with bone-resorbing capacity, play a critical role in the pathogenesis of bone destructive disorders such as rheumatoid arthritis and osteoporosis12. Therefore, osteoclasts were chosen as the study targets for this research. The present study showed that SY0916 concentrations from 0.1 to 10 μmol/L could significantly inhibit the proliferative and functional responses of osteoclasts stimulated by IL-1β. Furthermore, Wang et al also observed the effect of SY0916 on the apoptosis of osteoclasts. The result showed that SY0916 between 0.01 and 10 μmol/L significantly decreased the lifespan of osteoclasts after treatment with IL-1β. Therefore, the inhibitory effect of SY0916 on osteoclasts may be a critical pathway associated with its prevention of bone destruction.

The system constituted by RANKL, OPG, and RANK plays an important role in bone metabolism. RANKL and OPG are expressed by osteoblasts, whereas RANK is expressed by osteoclasts. RANKL and RANK affect bone metabolism by stimulating osteoclast differentiation, function and survival. In contrast, OPG is the decoy receptor for RANKL. OPG inhibits RANKL function by competing with RANK for RANKL13, 14. In the case of increased levels of RANKL, patients present with symptoms characteristic of abnormal bone resorption, which appear similar to osteoporosis15. In the case of increased OPG levels, patients present with symptoms of hyperosteogeny or osteosclerosis. SY0916 directly inhibit the function of osteoclasts. Meanwhile, we further investigated the influence of SY0916 on gene expression of OPG, RANK, and RANKL. These results showed that SY0916 significantly inhibited IL-1β-mediated increases in mRNA expression of RANKL and RANK in osteoblasts and osteoclasts, respectively. Noticeably, we found that the ratio of RANKL to OPG was significantly decreased, although OPG mRNA expression in osteoblasts was also decreased by SY0916. Therefore, we concluded that the mechanism underlying the effects of SY0916 on osteoclastic bone destruction involved regulation of the balance between RANKL and OPG, and the inhibition of RANK.

It is well known that matrix metalloproteinase 9 (MMP-9) is responsible for bone matrix degradation and is highly expressed both at early stages of osteoclast development and in mature osteoclasts. Moreover, many researchers have reported that RANKL stimulated MMP-9 gene expression in osteoclasts through TRAF616. In the present study, we found that SY0916 could inhibit the expression of MMP-9 induced by IL-1β at the level of both RNA and protein. The inhibitory effect of SY0916 on MMP-9 expression was in line with that of SY0916's effect on RANKL expression.

In conclusion, our study indicated that SY0916 could inhibit the inflammatory diseases associated with bone destruction by restoring the normal state of osteoclasts, which included the generation, activation, functioning and apoptosis of osteoclasts. In addition, the regulation of the RANKL-OPG-RANK system was also involved in this process. The regulation of a RANKL associated signal transduction network influenced protein expression that was associated with bone destruction, which included increases in MMP-9. In summary, this study is an important contribution to elucidating the mechanism underlying the effect of SY0916 on bone protection.

Author contribution

Wen-jie WANG and Lin WANG designed the research; Lin WANG, Shan-ying PENG, Ping LI, and Yang LIU performed the research; Lin WANG, Shan-ying PENG, and Ping LI analyzed the data; Lin WANG and Wen-jie WANG wrote the paper.