Asperpyrone A attenuates RANKL‐induced osteoclast formation through inhibiting NFATc1, Ca2+ signalling and oxidative stress

Abstract Imbalance of osteoblast and osteoclast in adult leads to a variety of bone‐related diseases, including osteoporosis. Thus, suppressing the activity of osteoclastic bone resorption becomes the main therapeutic strategy for osteoporosis. Asperpyrone A is a natural compound isolated from Aspergillus niger with various biological activities of antitumour, antimicrobial and antioxidant. The present study was designed to investigate the effects of Asperpyrone A on osteoclastogenesis and to explore its underlining mechanism. We found that Asperpyrone A inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner when the concentration reached 1 µm, and with no cytotoxicity until the concentration reached to 10 µm. In addition, Asperpyrone A down‐regulated the mRNA and protein expression of NFATc1, c‐fos and V‐ATPase‐d2, as well as the mRNA expression of TRAcP and Ctsk. Furthermore, Asperpyrone A strongly attenuated the RNAKL‐induced intracellular Ca2+ oscillations and ROS (reactive oxygen species) production in the process of osteoclastogenesis and suppressed the activation of MAPK and NF‐κB signalling pathways. Collectively, Asperpyrone A attenuates RANKL‐induced osteoclast formation via suppressing NFATc1, Ca2+ signalling and oxidative stress, as well as MAPK and NF‐κB signalling pathways, indicating that this compound may become a potential candidate drug for the prevention or treatment of osteoporosis.

the monocyte/macrophage haematopoietic lineage, osteoclast plays an essential role in bone resorption. Thus, suppressing or inhibiting the osteoclast formation or activity was concerned to be the therapeutic potential for preventing the development of osteoporosis. Although bisphosphonates (eg alendronate) are widely prescribed for senile osteoporosis through inhibiting osteoclast formation and activities, the adverse effects limited the use of this drug. 6 Therefore, screening and finding new natural compounds which had the effects on inhibiting osteoclastogenesis may serve as potential therapies to treat senile osteoporosis and have become an attractive research topics.
Two key cytokines involved in the process of osteoclastogenesis: (a) RANKL (receptor activator of NF-κB ligand) is an essential cytokine in the process of the osteoclast differentiation, which interacts with RANK to regulate signalling pathways such as NF-κB, MAPKs and calcium signalling in osteoclasts and then consequently stimulates the downstream factor-related osteoclastogenesis such as NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos (a protein of AP-1 transcription factor required). 7 (b) M-CSF (macrophage colonystimulating factor) is another crucial cytokine involved in the proliferation and differentiation of monocytes/macrophages, especially in the regulation of the survival and proliferation of pre-osteoclasts and mature osteoclasts. 8 Additionally, M-CSF also up-regulates RANK levels in BMMs (bone marrow macrophage cells) and then enhances the effects of RANKL stimulation on osteoclast differentiation. 9 The natural compound of Asperpyrone A was isolated from Aspergillus niger. As one of BNPs (Bis-naphtho-γ-pyrones), it has biological activities including antitumour, antimicrobial and antioxidant. 10,11 Based on our initial compound screening, we found that Asperpyrone A was a candidate drug for attenuating osteoclast formation. Therefore, we investigated whether Asperpyrone A could inhibit osteoclast formation or activation and explored its effects on RANKL-induced signalling pathways such as MAPK, NF-κB, Ca 2+ signalling and ROS products in the present study. This study also provides evidence that Asperpyrone A may become a potential candidate drug for the prevention or treatment of osteoporosis.
Recombinant M-CSF was produced from R&D Systems, and RANKL protein (GST-rRNAKL) was expressed and purified as previously described. 12 The MTS cytotoxicity assay and luciferase report assay were purchased from Promega. The antibodies of phosphorylated ERK (p-ERK), NFATc1, V-ATPase-d2, c-fos, IκB-α and JNK were obtained from Santa Cruz and the antibodies of P38, phosphorylated P-38 (P-P38), phosphorylated JNK(p-JNK), c-fos and ERK from Cell Signaling Technology.

| Cell culture
Six-week-old C57BL/6J mice were used for bone marrow macrophage cells (BMMs) isolation. After flushing the femur and tibia by α-MEM medium, the cells were cultured in the complete culture medium (10% FBS and 100 U/mL penicillin, 100 g/mL streptomycin and 2 mmol/L Lglutamine) and supplied with M-CSF (50 ng/mL). The BMMs were passaged when they reached 70%-80% confluence, and the 3rd passage was used for further research. RAW264.7 cells were purchased from the American Type Culture Collection and cultured in complete culture medium without M-CSF. Then, the RAW264.7 cells were prepared for the luciferase reporter assay by stably constructed of the NF-kB-or the NFATc1-responsive luciferase reporter. 13

| Osteoclastogenesis assay and compounds screening
Bone marrow macrophage cells were seeded into 96-well plates at a density of 6000 cells/well and cultured with complete medium supplemented with M-CSF (50 ng/mL) and RANKL (50 ng/mL) for 5 days. TRAcP (tartrate-resistant acid phosphatase) staining was used for osteoclastogenesis assay. Natural compound screening was performed to detect whether they can inhibit RANKL-induced osteoclastogenesis using BMM culture (the compounds varying concentrations from 0.5 μmol/L to 10 μmol/L).

| Cytotoxicity assays
MTS was used to detect the cell cytotoxicity of candidate compound Asperpyrone A. BMMs were seeded into a 96-well plate at

| Osteoblast cell culture and differentiation assays
The osteoblast cells were obtained from the calvaria of neonatal mice as previously described. 14 In briefly, collagenase (0.1%) and dispase (0.2%) were used to digest the calvaria tissue, and then, the isolated cells were cultured in DMEM with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin. The 3rd passage was used for osteoblast differentiation assay. Osteoblast cells were seeded in 48-well plate and cultured with 50 µg/mL ascorbic acid, 10 mmol/L β-glycerophosphate and 10 −7 mmol/L dexamethasone (DXM). The cells were treated with BMP, 2.5 and 5 μmol/L Asperpyrone A, respectively. ALP staining was performed for bone differentiation assay on day 7.

| Luciferase reporter assays
The RAW264.7 cells were used for luciferase reporter assays as the previous study. 13 In brief, the RAW264.7 cells with either the NF-kB-or the NFATc1-responsive luciferase reporter were prepared and seeded into 48-well plates, respectively. RANKL stimulation (50 ng/ mL) followed 1 hour after pre-treated with various concentrations of Asperpyrone A. Luciferase activity was detected according to the protocol of luciferase reporter assays (Promega).

| Reverse transcription (RT)-PCR analysis
The BMMs were stimulated with M-CSF (50 ng/mL) and RANKL (50 ng/mL) in the presence or not of Asperpyrone A for 1, 3 and 5 days, respectively; then, Trizol was used to isolate the total RNA of cells. The cDNA was synthesized from the RNA template, and the quantitative real-time PCR reactions were performed. The Ct value (cycle threshold) was used to assess the gene expression according to the 2 −ΔΔt method. 15 The expression of β-actin and B2M mRNA was used for normalization. The detailed information of the primers used in RT-PCR analysis was shown in Table 1.

| Western blotting
The BMMs were cultured in 6-well plates with M-CSF (50 ng/mL)

| Intracellular Ca 2+ oscillation assay
Ca 2+ oscillations were investigated by Fluo4-AM Kit (Thermo Fisher Scientific) as previously reported. 16 In brief, the BMMs were seeded into a 48-well plate, and 2.5 or 5 μmol/L of Asperpyrone A was

| Intracellular ROS production detection
As described previously, 17 the BMMs were pre-treated with Asperpyrone A (2.5 or 5 µmol/L) or not, followed by M-CSF and RANKL (50 ng/mL), and then incubated in H2DCFDA for 1 hour.
As DCF (2',7'-dichlorofluorescein) converted from H2DCFDA when oxidation showed highly fluorescent, confocal microscope (NIKON A1Si) was used to detect the fluorescence of DCF in the cells and the mean fluorescence intensity (ROI) of ROS-positive cells was analysed using Image J software.

| Statistical analysis
All the results are presented as the mean ± standard deviation (SD), One-way ANOVA test was used for the comparing the means of all the groups, followed by Bonferroni's post hoc test for every two groups analysis. Statistical software IBM SPSS for Windows (version 21) was used to perform all the statistical analyses, and GraphPad Prism 5 (version5.01) was used to prepare the bar figures. P < .05 was considered as significant differences.

| Asperpyrone A inhibited RANKL-induced osteoclast formation
The results of compounds screening showed that Asperpyrone A ( Figure 1A) Figure 1D).
Interestingly, the Asperpyrone A exhibited no significant inhibitory effect on osteoclast activity as the bone resorption assay showed that no more resorption area was found when the concentration of Asperpyrone A reached 5 μmol/L ( Figure 1E). Thus, Asperpyrone A mainly affects osteoclast formation.

| Asperpyrone A attenuated the expression of the osteoclast-related genes
We then explored the expression of osteoclast-specific marker genes in BMMs during osteoclast formation with or without the presence of Asperpyrone A. We found that treatment with 5 μmol/L of Asperpyrone A down-regulated the mRNAs expression of NFATc1, c-fos, V-ATPase-d2 (Vacuolar-type H+-ATPase d2), TRAcP and Ctsk in BMMs during osteoclast formation induced by RANKL, and even 2.5 μmol/L of Asperpyrone A inhibited c-fos, V-ATPase-d2 and TRAcP mRNA expression (Figure 2).

| Asperpyrone A down-regulated osteoclastrelated proteins and NFATc1 activity
The BMMs, the results showed that NFATc1 activity in RAW264.7 cells was also suppressed by Asperpyrone A either with 2.5 or 5 μmol/L concentration ( Figure 3B).

| Asperpyrone A down-regulated RANKLinduced MAPK and NF-κB signalling pathways
To further understand the mechanism of Asperpyrone A on suppressing RANKL-induced osteoclast differentiation in BMMs, MAPK F I G U R E 3 Asperpyrone A downregulates osteoclast-related proteins and NFATc1 activity. A, Western blot was used to detect the protein expression of NFATc1, c-FOS and V-ATPase-d2 in BMMS when pre-treated with or without 2.5 and 5 μmol/L Asperpyrone A. B, NFAT luciferase activity was investigated when pre-treated with varying concentration of Asperpyrone A in RAW264.7 cells (n = 3). *P < .05, **P < .01 compared with the positive group (with RNAKL but without Asperpyrone A treated)

| Asperpyrone A inhibited RANKL-induced Ca 2+ oscillations
RANKL stimulates Ca 2+ oscillations in BMM cells through activation of the Ca 2+ signal pathways. In the present study, we discovered that the intensity of Ca 2+ oscillations in BMMs increased and Asperpyrone A had the inhibit effectory during RANKL-induced osteoclast formation ( Figure 6). It appears that both 2.5 and 5 μmol/L concentration of Asperpyrone A exhibited a similar efficacy.

| Asperpyrone A suppressed RANKL-induced intracellular ROS products
As RANKL stimulation increases ROS production in BMMs during osteoclast differentiation, 17

| Asperpyrone A had no effect on the osteoblast differentiation
The result of ALP staining showed that there was no significant difference between Asperpyrone A (2.5 and 5 μmol/L) and control group ( Figure S1), indicating that Asperpyrone A did not affect the osteoblast differentiation.

| D ISCUSS I ON
As RANKL has been identified as one of the critical cytokines that regulates osteoclast formation and activity, 18,19 inhibiting RANKL-induced osteoclastogenesis was considered to be a potential therapeutic strategy for osteoporosis. For example, Zhou et al demonstrated that dihydroartemisinin suppressed both osteoclast formation and resorption in vitro, as well as reversed the bone loss in ovariectomized mice. 13 Song et al showed that eriodictyol was potentially useful for the prevention of osteoporosis through inhibiting osteoclast formation and function. 16 Achyranthes bidentata polysaccharide, berberine sulphate, nitidine chloride, artesunate and so on were also found to be potential therapeutic candidates for the prevention or treatment of osteoporosis. [20][21][22][23] Other studies also investigated the osteoporotic effects of compounds such as magnolol and polysaccharides. [24][25][26][27][28] As one of BNPs, the compound of Asperpyrone A was isolated from Aspergillus niger with various biological activities including antitumour, antimicrobial and antioxidant. 10,11 Its effects on attenuating RANKL-induced osteoclast formation were identified by our F I G U R E 5 Asperpyrone A suppresses NF-κB signalling pathway. A, The results of Western blot showed that IκBα protein degradation was attenuated in BMMs when pre-treated with Asperpyrone A. B, NF-κB luciferase activity was investigated when pre-treated with varying concentration of Asperpyrone A in RAW264.7 cells (n = 3). *P < .05, compared with the positive group (with RNAKL but without Asperpyrone A treated) compound screening assay using TRAcP staining. Therefore, we further investigated the effects of Asperpyrone A on suppressing RANKL-induced osteoclast formation and its cellular mechanisms in the present study. The findings demonstrated that Asperpyrone A significantly decreased both the number and the size of osteoclast, but had no inhibitory effect on the osteoclast function, which indicated Asperpyrone A mainly affects osteoclast formation but not osteoclast activity. In addition, the results of MTS assay exhibited no cytotoxicity on BMM cells until the concentration reached 10 μmol/L. Furthermore, the result of ALP staining showed that the Asperpyrone A had no effect on osteoblast differentiation.
These findings suggest that Asperpyrone A has a major effect on osteoclasts and could be a potential candidate anti-resorptive drug for osteoporosis.
Then, we explored the mechanisms by which Asperpyrone A inhibited osteoclast formation. NFATc1 and c-fos were identified as the critical regulator in the process of osteoclast formation. 29 In addition, NFATc1 can amplify its effectiveness via improving other osteoclastogenesis-related transcription factors in an autoamplification loop, including c-fos, NF-κB and NFATc2. 30   Previous studies showed that natural compounds not only suppressed the osteoclast formation in vitro, but also reversed the bone loss in ovariectomized (OVX) model mice. [47][48][49] One of the limitations in this study was that the animal experiments had not been performed. However, based on the evidence of Asperpyrone A inhibited osteoclast formation and did not affect osteoblast differentiation, it is highly possible that Asperpyrone A should be effective for the treatment of osteoclast -related conditions such as OVX-induced osteoporosis.
Taken together, our study had shown that natural compound

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.