Maackiain dampens osteoclastogenesis via attenuating RANKL‐stimulated NF‐κB signalling pathway and NFATc1 activity

Abstract Osteolytic diseases are typified by over‐enhanced formation and resorbing function of osteoclasts and have a major impact on human health. Inhibition of osteoclastic differentiation and function is a key strategy for clinical therapy of osteolytic conditions. Maackiain is a natural compound extracted from Sophora flavescens, which has been applied to anti‐allergic and anti‐tumour treatments. The present results showed that Maackiain could restrain receptor activator of nuclear factor‐κB ligand (RANKL)‐stimulated osteoclast formation and hydroxyapatite resorption dose‐dependently, and interrupt the structures of F‐actin belts in the mature osteoclasts. It also repressed the expressions of osteoclast‐specific genes and proteins. Furthermore, Maackiain could inhibit RANKL‐stimulated NF‐κB and calcium signalling pathways, and dampen Nuclear factor of activated T cell cytoplasmic 1 activity, protein expression and translocation into the nucleus. These results revealed that Maackiain may have a potential therapeutic effect on osteoclast‐related disorders.


| INTRODUC TI ON
Osteolytic diseases are caused by the hyperactive differentiation and resorbing function of osteoclasts 1 which are coupled with osteoblasts to maintain healthy bone homeostasis. 2 Enhanced bone resorption will result in osteolytic conditions such as osteoporosis, Paget's disease and osteonecrosis. [3][4][5] Especially in postmenopausal women with osteoporosis due to oestrogen deficiency, serious complications such as fragility fractures could occur under slight external force, which imposes a huge burden socio-economically. 3 However, the current clinical methods for preventing and treating osteoporosis are limited and often have severe side effects. 6,7 Hence, discovering novel natural compounds for osteoporosis treatments is necessary.
Bone homeostasis is maintained by coupling activities of osteoclasts and osteoblasts. 8 As bone-forming cells, osteoblasts are responsible for forming new mineralized bone. Osteoclasts are bone-resorbing cells originated from hematopoietic stem cells. The differentiation and maturation of osteoclasts are inseparable from two important regulators, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). 9 Via binding to RANK on the surface of osteoclast precursor cells, RANKL mediates the differentiation and resorptive function of osteoclasts. 10 The binding of RANKL and RANK activates several signalling pathways to initiate the differentiation of osteoclast precursor cells into mature osteoclasts, importantly, NF-κB signalling pathway and MAPK signalling pathway. 11,12 Nuclear factor of activated T cell cytoplasmic 1 (NFATc1) acts as a master transcriptional factor in the process of osteoclast differentiation, 13 which controls the expression of downstream osteoclast-related genes, including Ctsk (encoding cathepsin K), Ctr (encoding calcitonin receptor), Acp5 (encoding TRAcP), and c-fos (encoding c-Fos), ultimately leading to the formation of mature multinucleated osteoclasts. Moreover, the initiation of the calcium signalling pathway can facilitate the self-expansion and translocation of NFATc1 into the nucleus. 14,15 Maackiain is a compound extracted from the roots of Sophora flavescens, a traditional Chinese herb. Recent studies revealed that alkaloids from S. flavescens can suppress osteoclast activity and tartrate-resistant acid phosphatase (TRAcP) activity through RANKL-induced NF-κB signalling pathway. 16 Another S. flavescens extract named Sophocarpine has also shown its inhibitory effect on osteoclast formation and bone-resorbing function by inhibiting the NF-κB signalling pathway. 17 It seems that S. flavescens extracts are promising agents for treating osteoclastic diseases.
As reported, Maackiain can dampen human monoamine oxidase B (MAO-B) enzyme for the treatment of disorders like Alzheimer disease, Parkinson's disease and depression. 18 Maackiain has also been found to have functions of anti-allergy, 19 anti-mosquito, 20 anti-tumour 21 and pro-apoptosis. 22 However, it has not been reported whether Maackiain can affect RANKL-stimulated osteoclastogenesis.
The present study investigated the role of Maackiain in osteoclast formation and function using in vitro experiments. It was found that Maackiain can restrain the differentiation and bone-resorbing function of osteoclasts. This inhibitory effect was attributed to that Maackiain blocked RANKL-stimulated NF-κB and calcium signalling pathways, and reduced NFATc1 activity, protein expression and translocation into the nucleus.

| Materials and regents
Maackiain with a purity of ≥98% (Cat#CFN99746) was commercially obtained from Wuhan ChemFaces Technology Co.,

| Isolation and purification of osteoclast precursors
Bone marrow macrophages (BMMs), the osteoclast precursor cells, were isolated from long bones of C57BL/6J female mice and then purified as described. 24 The method was approved by

| Osteoclast differentiation and TRAcP staining
Osteoclastogenesis assay was used routinely as reported. 25 In brief, BMMs were seeded into 96-well plates at a density of 6 × 10 3 cells per well. The next day, the adherent BMMs were stimulated with RANKL at a concentration of 50 ng/mL to induce osteoclastogenesis. Subsequently, various concentrations of Maackiain (0, 5, 10, 20, 30, 40 μmol/L) were added into each group of culture. Media was changed every 2 days until mature osteoclasts were formed.
Then, cells were fixed with 2.5% glutaraldehyde, gently washed with PBS for twice and stained with TRAcP staining solution at 37°C for about 40 minutes. The cells of each group were photographed using an inverted light microscope, and osteoclast-like cells with multi-nuclei (with more than three nuclei) were scored.

| Cell viability assay
BMMs were seeded into a 96-well plate at a density of 6 × 10 3 per well.
The next day, different concentrations of Maackiain (0, 5, 10, 20, 30, 40 μmol/L) were added and incubated for 2 days, then 20 μL of MTS reagent was added and incubated for 2 hours in dark. MTS absorbance at 490 nm was measured using a microplate reader.

| Hydroxyapatite resorption assay
In order to detect the bone-resorbing function of osteoclasts, the resorption pits were observed on hydroxyapatite plates. BMMs were seeded into 6-well plates at a density of 8 × 10 4 per well and induced with 50 ng/mL RANKL. After mature osteoclasts were observed under a microscope, cells were detached using cell dissociation solution (Sigma-Aldrich) and then seeded into a 96-well plate covered with hydroxyapatite at the bottom (Corning). Cells were continued to be stimulated with 50 ng/mL RANKL in the presence or absence of 40 μmol/L of Maackiain. After 48-hour incubation, cells were gently washed with PBS, half of the wells were stained with TRAcP solution, and the other half were washed with 10% bleach to expose resorption areas. An inverted light microscope and ImageJ software were applied to quantify the resorbing pits of hydroxyapatite as previously reported. 26

| Immunofluorescent staining
BMMs were seeded onto coverslips of 24-well, and RANKL was added as described above until mature osteoclasts were formed, with the presence or absence of Maackiain (40 μmol/L). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% (v/v) Triton X-100 for 5 minutes, followed by being blocked with 3% BSA at room temperature for 10 minutes. The cells were incubated with anti-vinculin or anti-NFATc1 antibody at 4°C, followed by the incubation with a secondary anti-mouse IgG conjugated with FITC (Thermo Fisher). Then, the cells were co-stained with rhodamine phalloidin and Hoechst 33258 for 20 minutes. After the staining, the coverslips were mounted with the ProLong Gold Antifade Mountant and visualized using a confocal microscope (Nikon Corporation) as previously described. 27

| mRNA isolation and Real-time PCR analysis
BMMs were seeded into 6-well plates, and RANKL at a concentration of 50 ng/mL was added after the adherence, with or without different concentrations of Maackiain for 5 days. After osteoclasts were formed, TRIzol™ reagent and PureLink™ RNA Mini Kit (Thermo Fisher) were used to isolate total RNA, followed by reverse transcription of RNA into single stranded cDNA using M-MLV reverse transcriptase with oligo-dT primer. Real-time PCR process was performed using the resulting cDNA, specific primers listed in Table 1 and PowerUP TM SYBR Green Master Mix (Thermo Fisher). The conditions for PCR were as below: Holding stage of 95°C for 5 minutes, PCR stage (40 cycles) of 95°C for 15 seconds, and annealing at 60°C for 60 seconds, Melt curve stage of 95°C for 15 seconds, 60°C for 60 seconds and 95°C for 15 seconds. The obtained data were analysed by ΔΔC t method.

| Western blot assay
BMMs were seeded into 6-well plates, followed by the stimulation with RANKL (50 ng/mL) for 0, 1, 3 and 5 days, in the presence or absence of Maackiain (40 μmol/L). Cells were collected and lysed with 120 μL of radioimmunoprecipitation assay buffer to extract total proteins. The proteins were loaded and separated using SDS-PAGE, transferred onto a nitrocellulose membrane and blocked with 5% skim milk for non-specific binding. Specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies were used for binding to proteins which were detected using enhanced chemiluminescence reagents (PerkinElmer) and visualized on an Image-quant LAS 4000 (GE Healthcare). The band intensities were quantified with ImageJ software.

TA B L E 1 Gene primer sequences
As to the protein expressions of osteoclast-related signalling pathways, BMMs were seeded at a density of 2.5 × 10 5 cells/well and cultured overnight. Cells were starved in serum-free medium for 2 hours, followed by being pre-treated with or without Maackiain (40 μmol/L) for 1 hour, and stimulated with RANKL (50 ng/mL) for various durations. Western blots were performed as above.

| Measurement of intracellular Ca 2+ oscillation
BMMs were seed into 48-well plates and cultured with complete α-MEM overnight. Cells were starved in serum-free medium for

| Statistic analysis
All data in present study were presented as mean ± standard deviation and statistically analysed with SPSS 17.0 software.
Student's t tests were used for the comparison between two sets and one-way ANOVA tests for the comparison among three or more groups. A P-value ≤.05 indicates a statistical significance.

| Maackiain attenuated RANKL-stimulated osteoclastogenesis
In order to clarify the inhibitory effect of Maackiain on osteoclast formation during RANKL induction process, gradient concentrations of Maackiain were applied to cell culture. The results in Figure 1A,B showed that Maackiain exhibited a suppressive effect on osteoclast differentiation from the concentration of 5 μmol/L. Additionally, cytotoxicity test indicated that it had no cell toxicity on BMM proliferation, as shown in Figure 1C. The chemical structure of Maackiain is shown in Figure 1D.

| Maackiain suppressed RANKL-induced osteoclast function
The resorbing pits of hydroxyapatite plate were analysed to evaluate the osteoclast function. As shown in Figure 2A,

| Maackiain suppressed RANKL-induced osteoclastic protein and gene expressions
Following RANKL stimulation and Maackiain intervention for different time-points, Western blot was utilized to detect the downstream protein expressions in the osteoclastic signalling pathways.
As shown in Figure 3A,

| Maackiain restrained RANKL-induced NF-κB signalling pathway
To elucidate the molecular mechanism of Maackiain's suppressive effect, a detailed time course of the RANKL stimulation was conducted, and Western blot analysis showed that RANKL stimulation increased the degradation of IκB-α and the phosphorylation of p65 as early as less than 30 minutes, while Maackiain had an inhibitory effect on the activation of NF-κB signalling pathway ( Figure 4).

| Maackiain attenuated the NFATc1 activity
As the master transcription factor during RANKL-stimulated osteoclastogenesis, NFATc1 could be activated by Ca 2+ signalling and then translocated into nucleus. Calcium oscillation assay indicated that Maackiain restrained the intensity of Ca 2+ flux ( Figure 5A). And the gene and protein expressions of NFATc1 had been also downregulated from Day 3 ( Figure 5B-D). Moreover, the co-staining of F I G U R E 3 Maackiain suppressed osteoclast-associated protein and gene expressions. A, Maackiain of 40 μmol/L inhibited c-Fos, Integrin β3, MMP9 and CTSK protein expressions. B, Quantitative analysis of band intensities relative to the intensity of β-actin. C, RT-PCR results showed that Maackiain inhibited osteoclast differentiation and bone resorption-related genes including Acp5, c-fos, Ctsk and Mmp9. n = 3; *P < .05, **P < .01, ***P < .001 NFATc1, nucleus and actin belts was applied to further gain insight into the nuclear translocation of NFATc1, and the observed reduction of NFATc1 both in cytoplasm and nucleus was shown in Figure 5E.

| D ISCUSS I ON
Over-activation of osteoclasts can lead to osteolytic conditions such as Paget's disease, osteoporosis and osteonecrosis. [3][4][5] The Therefore, we concluded that Maackiain could inhibit the formation and resorbing function of osteoclasts via inhibiting NF-κB signalling pathway and NFATc1 activity which results from blocking Ca 2+ signalling pathway ( Figure 6). These results indicated that Maackiain could be a potential drug against osteoclast-related disorders.

ACK N OWLED G EM ENTS
This study was supported in part by the Australian National Health

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

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