Tectorigenin inhibits RANKL‐induced osteoclastogenesis via suppression of NF‐κB signalling and decreases bone loss in ovariectomized C57BL/6

Abstract Metabolism of bone is regulated by the balance between osteoblast‐mediated bone formation and osteoclast‐mediated bone resorption. Activation of osteoclasts could lead to osteoporosis. Thus, inhibiting the activity of osteoclasts becomes an available strategy for the treatment of osteoporosis. Tectorigenin is an extract of Belamcanda chinensis In the present study, the anti‐osteoclastogenesis effects of tectorigenin were investigated in vitro and in vivo. The results showed preventive and therapeutic effects of tectorigenin at concentrations of 0, 10, 40, and 80 μmol/L in the maturation and activation of osteoclasts. A signalling study also indicated that tectorigenin treatment reduces activation of NF‐κB signalling in osteoclastogenesis. Animal experiment demonstrated that tectorigenin treatment (1‐10 mg/kg, abdominal injection every 3 days) significantly inhibits bone loss in ovariectomized C57BL/6. Our data suggest that tectorigenin is a potential pharmacological choice for osteoporosis.

Tectorigenin (Chemical Abstracts Service number 548-77-6, C16H12O6, molecular weight, 300.26) is an extract of Belamcanda chinensis of the iris species. Its potential usage in anti-inflammatory and antioxidant activity was reported before, 13,14 and more specifically, its anti-inflammatory effect in osteoarthritis and NF-κB signalling blockage. 15 In this study, the effect of tectorigenin on RANKL-induced NF-κB activation and osteoclastogenesis was investigated in vitro, plus its anti-osteoporosis action in an ovariectomized (OVX) mice model. β-actin (SC-47778) antibodies was purchased from Santa Cruz Biotechnology.

| Cell Counting Kit (CCK-8) assay
CCK-8 was used to evaluate the cytotoxicity of tectorigenin according to the manufacturer's instruction. Briefly, bone marrow mononuclear (BMM) cells or RAW264.7 cells were seeded in 96-well plates at a density of 3000/well. After incubation with concentrations of 0, 10, 40, 80, or 160 μmol/L of tectorigenin for 24 hour or 48 hour, cells were incubated with 10 μL CCK-8 for 4 hour. The optical density (OD) was read at a wavelength of 450 nm.

| BMM cells isolation and cell treatment
Bone marrow mononuclear cells were prepared as other previous studies. 16,17 In brief, 6-week-old C57/BL6 mice were sacrificed. Cells extracts from femurs and tibias of one mouse were incubated in cell culture (α-MEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin) with 30 ng/mL M-CSF in one T-75 cm 2 flask. The medium was changed every 2 or 3 days. When the cells reached 90% confluence, cells were washed with PBS and trypsinized at 37°C with 5% CO 2 for 30 minutes for harvest. The harvested cells were classified as BMM cells.
Cells were seeded in plates for analysis. Subconfluent cells were pretreated with tectorigenin at concentrations of 0, 10, 40, or 80 μmol/L for 1 hour then incubated with or without RANKL (25, 50, or 100 ng/mL) according to the study design.
All medium used for BMM cells in this study contained 30 ng/mL M-CSF, while medium for RAW264.7 contained no M-CSF.

| Osteoclastogenesis analysis
Bone marrow mononuclear cells were seeded in 24-well plates (5 × 10 5 cells/well) and treated with 0, 10, 40, or 80 μmol/L of tectorigenin in the presence of RANKL (50 ng/mL) for 4 days. RAW264.7 cells were also treated with tectorigenin (0, 10, 40, 80 μmol/L) in the presence of RANKL (50 ng/mL) for 4 days. Cells were then fixed and stained for TRAP assay according to the manufacturer's protocol. Subconfluent cells were incubated with tectorigenin at concentrations of 0, 10, 40, or 80 μmol/L in the presence of RANKL (50 ng/mL) for 7 days. The medium was changed every 3 days. After the treatment, the plates were washed with 10% bleach solution and air-dried at RT. Osteoclast resorption area was observed using a light microscope.

| RNA extraction and real-time PCR
Bone marrow mononuclear cells or RAW264.7 cells were seeded in 12-well plates at a density of 1 × 10 6 cells/well and treated with tectorigenin at concentrations of 0, 10

| Protein extraction and Western blot analysis
Bone marrow mononuclear cells were used for Western blot analysis. Cells were pretreated with 0, 10, 40, or 80 μmol/L of tectorigenin for 1 hour, and then stimulated with 50 ng/mL RANKL for 1 hour for detection of NF-κB p65, phosphor-NF-κB p65, IκBα, and phosphor-IκBα. We used radioimmunoprecipitation assay (RIPA) containing protease and phosphatase inhibitors to prepare cell extract. Equal amounts of cell extract were separated by 10% SDS-PAGE, and electro-transferred to polyvinylidene difluoride membranes. After blocking with 5% bull serum albumin (BSA, Sigma-Aldrich, St, Louis, MO, USA) for 2 hours, the membranes were blotted with primary antibodies at 4°C overnight, then incubated for 1 hour with secondary antibody. After that, signals were detected using West Dura Extended Duration Substrate with exposure to X-ray film.

| Luciferase reporter gene analysis
RAW264.7 cells were used in this experiment. Cells were first stably transfected with an NF-κB luciferase reporter construct (Promega, Madison, WI) firstly. Then, transfected cells were pretreated with tectorigenin (0, 10, 40, or 80 μmol/L) for 1 hour and then stimulated with RANKL (50 ng/mL) for 6 hour. After that, the luciferase assay system (Promega) was used to measure the luciferase activity.

| Immunofluorescence microscopy
Coexpression of NF-κB p65 was carried out using fluorochromeconjugated antibodies. RAW264.7 cells cultured 24-well plate (5000/well) were fixed in 4% paraformaldehyde for 10 minutes, and permeabilized for 5 minutes with 0.1% v/v Triton X-100. Cells were incubated with primary antibody (sc-8008) at 4°C overnight, washed, and then incubated with fluorochrome-conjugated secondary antibody for 2 hour in the dark. Coverslips were mounted onto glass slides using DAPI-containing mounting medium.

| Induction of osteoporosis in mice
A mice model of osteoporosis was developed by bilateral ovariectomies in 6-week-old female C57BL/6 as previously described in other studies. 18,19 One week after surgery, all the mice were divided into four groups with 10 mice per group: the mice that underwent sham surgery were grouped as Sham; mice with bilateral ovariectomies were divided randomly into three groups: OVX, OVX + Tectorigenin 1 mg/kg, and OVX + Tectorigenin 10 mg/kg. Mice in OVX and Sham groups were treated with vehicle. Mice in OVX + Tectorigenin 1 mg/kg received low dose of tectorigenin (1 mg/kg), while OVX + Tectorigenin 10 mg/kg was treated by high dose of tectorigenin (10 mg/kg). All the tectorigenin or vehicle was delivered by abdominal injection every 3 days. All mice were sacrificed by excess amount of chloral hydrate after 6-week treatment.
The distal femurs were preserved and fixed in 4% paraformaldehyde solution. The timeline of animal experiments is shown in  were measured by CT Analyzer software.

| Histological analysis
Samples fixed with 4% paraformaldehyde solution were decalcified and embedded in paraffin, sectioned at 5 μm thickness. The sections were stained with haematoxylin and eosin (H&E) and TRAP.

| Statistical analysis
The results are presented as mean ± standard deviation of three or more experiments. Statistical differences were performed with SPSS 12.0 version. One-way ANOVA with a subsequent post hoc Tukey's test was used for multiple comparisons. P < 0.05 is considered to be significant with statistical meaning.

| Cytotoxicity of tectorigenin and preventive effect on RANKL-induced osteoclast differentiation and bone resorption in vitro
To  Figure 1C, tectorigenin significantly reduced resorption area stimulated by RANKL.

| Effect of tectorigenin on RANKL-stimulated osteoclast-specific gene expression
To investigate the effect of tectorigenin on RANKL-stimulated osteoclast-specific gene expression, BMM cells and RAW264.7 were pretreated with tectorigenin at concentrations of 0, 10, 40, or 80 μmol/L for 1 hour, and then stimulated by RANKL (50 ng/mL) for 48 hour. Up-regulation of osteoclast-specific genes, including TRAP, NFATc1, cathepsin K (Cts K), and MMP9, were investigated by Realtime PCR. As shown in Figure 2, expression of TRAP, NFATc1, Cts K, and MMP9 was down-regulated by tectorigenin treatment. Combined data in Figure 1, the data demonstrated that tectorigenin exhibited a preventive effect on osteoclastogenesis.

| Therapeutic effect of tectorigenin on RANKLinduced osteoclast differentiation in vitro
To determine whether tectorigenin could also help in treating RANKL-induced osteoclast differentiation, BMM cells were stimulated by RANKL (50 ng/mL) for the first 4 days until osteoclasts were observed. Cells were then incubated with tectorigenin at concentrations of 0, 10, 40, or 80 μmol/L with RANKL (50 ng/mL) for another 4 days. Tartrate-resistant acid phosphatase stain showed that tectorigenin reduced osteoclast differentiation induced by RANKL stimulation ( Figure 2B). Our results demonstrated that tectorigenin showed a therapeutic role when the differentiation of osteoclasts was already initiated by RANKL.

| Effect of tectorigenin on RANKL-induced activation of NF-κB signalling in vitro
Considering the importance of NF-κB signalling in osteoclast differentiation, 20  The luciferase reporter gene assay had a similar result ( Figure 3B).
The increased transcriptional activity of NF-κB induced by RANKL was greatly decreased by tectorigenin treatment with a dose-dependent manner. Furthermore, the location and concentration of NF-κB p65 was visualized using immunofluorescence microscopic analysis, and this kind of inhibition was also observed ( Figure 3C). These results indicate that tectorigenin inhibits RANKL-induced activation of NF-κB signalling in vitro.

| Effect of tectorigenin on OVX-induced bone loss in vivo
To investigate effects of tectorigenin on OVX-induced bone loss in vivo, we developed mice model of osteoporosis by bilateral Furthermore, TRAP staining also indicated decreased number of osteoclasts between trabeculars ( Figure 5). Collectively, these data indicated that tectorigenin reduced OVX-induced bone loss in vivo.

| DISCUSSION
Balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption induces bone turnover and remodelling. 4 Thus, inhibiting the activity of osteoclasts becomes an available strategy for the treatment of osteoporosis. We report that Phosphorylation and protein level of NF-κB p65 and IκBα were measured by Western blot. B, RAW264.7 cells were pretreated with similar concentration of tectorigenin for 1 h, and then stimulated with 50 ng/mL RANKL for 6 h for luciferase reporter gene assay. C, RAW264.7 cells were pretreated with tectorigenin (80 μmol/L) for 2 h, and then stimulated with 50 ng/mL RANKL for 30 min for immunofluorescence microscopic analysis. Significance was calculated by a one-way ANOVA with a post hoc Tukey's multiple comparisons test. **P < 0.01, ***P < 0.001 vs RANKL+ 0 μmol/L concentration tectorigenin treated group F I G U R E 5 Experimental timeline and histological analysis of in vivo effect of tectorigenin. The distal femurs were decalcified and embedded in paraffin, sectioned at 5 μm thickness. The sections were stained with haematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP). The red arrows in TRAP stained sections illustrates the red-stained osteoclasts F I G U R E 4 Effect of tectorigenin on ovariectomized (OVX)-induced bone loss in vivo. Mice in OVX and Sham groups were treated with vehicle. Mice in OVX + Tectorigenin 1 mg/kg received low dose of tectorigenin (1 mg/kg), while OVX + Tectorigenin 10 mg/kg was treated by high dose of tectorigenin (10 mg/kg). All tectorigenin or vehicle was delivered by abdominal injection every 3 d. The distal femurs were subjected to Micro-CT to evaluate the bone loss level. Bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N.), and trabecular separation (Tb.Sp.) were measured by CT Analyzer software. Significance was calculated by a one-way ANOVA with a post hoc Tukey's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 vs OVX group obvious protective effect on OVX-induced bone loss. Histological analysis also showed less osteoclasts in the tectorigenin treated mice.
NF-κB signalling is one of the major pathways involved in osteoclastogenesis. 6 When the signalling is activated by RANKL, phosphorylation of NF-κB p65 and IκBα and degradation of IκBα contribute to the activation and nuclear translocation of NF-κB p65. NF-κB p65 could enhance osteoclast-specific gene expression, thus resulting in osteoclast differentiation and maturation. 17,22 A study by Rong Zeng et al 23 indicated that alternative NF-κB signalling also plays a role in controlling the independent processes of osteoclast mitochondrial biogenesis. NF-κB signalling is involved in immune responses and function regulation of osteoclasts as well. 24 NF-κB signalling has already been one important target for osteoporotic pharmacological intervention. 22,25 It has been reported in other studies that tectorigenin inhibit activation of NF-κB on other cells. 15,26,27 In this study, we evaluated the activation of NF-κB signalling in the tectorigenin treatment on BMM cells. Our data demonstrated that tectorigenin could inhibit degradation of IκBα and phosphorylation of NF-κB p65 and IκBα induced by RANKL stimulation at non-cytotoxicity concentrations. Furthermore, transcription analysis revealed a similar result.
The traditional pharmacologic therapy consists of drugs like teriparatide or bisphosphonate, of which long-term safety still remains unknown. 9,28,29 Here, we reported for the first that tectorigenin exerts anti-osteoporosis effects in vivo and inhibits RANKL-induced osteoclastogenesis in vitro. Thus, our study suggests that tectorigenin has a therapeutic potential for osteoporosis.

| CONCLUSION
We demonstrate that tectorigenin inhibits RANKL-induced osteoclastogenesis via suppression of NF-κB signalling in vitro and could also reduce bone resorption in ovariectomized C57BL/6. These findings suggest that tectorigenin is a potential pharmacological choice for osteoporosis.

CONF LICT OF I NTEREST
No conflicts of interest have been reported by the authors or by any individuals in control of the content of this article.

AUTHOR CONTRIBU TI ON
Chiyuan Ma, Kai Xu, and Jiahong Meng contributed equally to this study. All authors listed were involved in the study and preparation of the manuscript. All conference has been list at the end of the paper with mark in the manuscript. All data supporting the conclu-