Galangin suppresses RANKL‐induced osteoclastogenesis via inhibiting MAPK and NF‐κB signalling pathways

Abstract Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti‐inflammation and anti‐oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose‐dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast‐specific genes, such as tartrate‐resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V‐ATPase d2) and dendritic cell–specific transmembrane protein (DC‐STAMP), were down‐regulated by galangin treatment. Furthermore, the results of the pit formation assay and F‐actin ring staining revealed impaired osteoclastic bone resorption in the galangin‐treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C‐Jun and C‐Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)‐induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF‐κB signalling pathways.

(TRAP), making new space for osteogenesis, and stimulate osteoblast differentiation. 5 In the majority of osteoporosis cases, osteoclasts are extensively activated, resulting in increased bone resorption, leading to bone mass loss when bone resorption activities exceed bone formation activities. Currently, oestrogen-replacement therapy and bisphosphonates are widely used in the clinic because of their effective inhibition of osteoclastogenesis and prevention of bone mass loss. 6 However, it has been reported that oestrogen-replacement therapy increases the risk of post-menopausal breast cancer, which makes such therapy a difficult choice for both patients and doctors. 7 The adverse effects of bisphosphonates present another challenge for the management of osteoporosis. 8,9 Osteonecrosis of the jaw is a serious side-effect related to the use of bisphosphonates, exposing patients to pain, soft tissue swelling, bone exposure and infection. Once osteonecrosis is established, treatment is difficult and is without well-established protocols that assure absolute therapeutic success. 10 Thus, searching for a safer and more effective therapeutic target is urgently needed.
Galangin (3,5,7-trihydroxyflavone, Figure 1A) is a natural bioflavonoid that is primarily extracted from the rhizomes of Alpinia officinarum, a herbal medicine that has been used in Asia for decades. 11 Previous studies have shown that the bioflavonoid compounds quercitrin and taxifolin inhibit osteoclasts. 12 Belonging to the family of bioflavonoids, galangin possesses antibacterial, 13 anti-inflammatory 14 and antiviral 15 activities, and inhibits a variety of tumour cells. [16][17][18] Galangin inhibits collagen-induced arthritis and prevents osteoclastic bone resorption through enhancement of osteoblast-induced TNF receptor superfamily member 11b (OPG) expression. 19 Thus, we were interested in whether galangin inhibits the functions of osteoclasts. The aim of this study was to evaluate the effects of galangin on the differentiation and bone resorption activity of osteoclasts, and to explore its underlying mechanisms in vivo and in vitro.
Then, the absorbance was measured at a wavelength of 570 nm.

| TRAP staining
Cells were seeded into 96-well plates, treated with 0, 3, 6 or 12 μmol/L galangin, and supplemented with 30 ng/mL M-CSF and 100 ng/mL RANKL (PeproTech, Rocky Hill, NJ, USA). The medium was replaced every other day for six days. The cells were fixed with 4% paraformaldehyde for 10 minutes, washed with phosphate-buffered saline (PBS) twice and stained for TRAP. The images of TRAP-positive multinucleated cells were counted, and the surface areas of osteoclasts per field were calculated using Image-Pro Plus software (Media Cybernetics).

| Actin ring staining
An F-actin staining kit (Millipore, Darmstadt, Germany) was used according to the manufacturer's protocol. BMMs were treated with 0, 3, 6 or 12 μmol/L galangin, supplemented with 30 ng/mL M-CSF and 100 ng/mL RANKL for six days. Then, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes. After rinsing with PBS three times, cells were treated with 100 μL iFluor 488-Phalloidin working solution for 40 minutes. Cells were rinsed with PBS three times, and then 6-diamidino-2-phenylindole (DAPI) staining solution was added for 5 minutes. Images were acquired under a fluorescence microscope (Eclipse TS100; Nikon).

| Bone resorption assays
Cells were seeded into 96-well plates containing 100μm bone slices (Rongzhi Haida Biotech Co., Ltd), treated with 0, 3, 6 or 12 μmol/L galangin and supplemented with 30 ng/mL M-CSF and 100 ng/mLf RANKL. Cells were removed by mechanical agitation and sonication until matured osteoclasts were formed. Bone resorption pits were observed under a scanning electron microscope (SEM; Field Environmental Instruments (FEI) Inc, Hillsboro, OR, USA) and the areas of pits were measured by Image-Pro Plus software.

| Immunofluorescence assay
BMMs were seeded (1 × 10 4 cells/well) in a 24-well plate and incubated in a basal medium for 24 hours. After pre-treatment with 12 μmol/L galangin for 30 mins, 100 ng/mL RANKL was added and the wells were cultured for 30 mins. Then, the cells were fixed with 4% PFA for 30 mins, rinsed with PBS, and 5% BSA was used for 2h at room temperature to block non-specific binding sites. Then, cells were incubated with anti-p65 antibody at 4℃ overnight. After using PBS washing three times, the cells were incubated for 2 hours at room temperature with secondary antibodies and stained with DAPI for 5 mins in the dark. Pictures of p65 nuclear translocation were obtained using an immunofluorescence microscope.

| RNA extraction and qRT-PCR experiments
Total RNA was extracted from osteoclasts using RNAiso Plus Reagent were analysed using ΔΔCT method.

| Western blot analysis
The cells were pre-treated with 12 µmol/L galangin for 1 hours.
Untreated cells were used as a control. Then, BMMs were stimulated with 30 ng/mL M-CSF and 100 ng/ml RANKL for 0, 5, 10, 20, 30 or 60 minutes, respectively. To investigate the dose-dependent effects of galangin, BMMs were pre-treated with different concentrations (0, 3, 6, 12 μmol/L) of galangin for 1 hours and then stimulated with RANKL for 30 minutes. To determine the effect of galangin on NFTAc1, C-Jun and C-Fos, BMMs were treated with 100 ng/mL RANKL with or without 6 µmol/L galangin for 3 days. Total cellular proteins were extracted from cultured cells using RIPA lysis buffer. Lysates were centrifuged at 12 000 g for 10 minutes at 4°C, and the supernatants were collected and mixed with SDS-sampling buffer, followed by incubation at 100°C for 5 minutes. Samples were then resolved by SDS-PAGE gels and transferred into nitrocellulose membranes via electroblotting.
Membranes were blocked with 5% skim milk for 2 hours and probed with primary antibodies overnight at 4°C. Membranes were then washed and incubated with HRP-conjugated secondary antibodies for 2 hours. Immunoreactivity detection was performed using a LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan), and the obtained images were analysed with ImageJ.

| Micro-CT scanning
The calvaria was separated and fixed with 4% paraformaldehyde for three days. The fixed calvaria samples were analysed using a highresolution μCT scanner (Scanco Microct u100, Switzerland). Image

| Histomorphometry analysis
The samples were decalcified in 10% EDTA for two weeks and embedded in paraffin. Histological sections were prepared for TRAP and HE staining. The sections were photographed, histomorphometric analyses of BV/TV (%) were performed, and the number of TRAP-position OCs and OC per bone surface (OC/BS) were determined utilizing ImageJ software.

| Statistical analysis
All data are presented as the means ± SD The differences between two groups were evaluated by unpaired, two-tailed Student's t tests, and one-way analysis of variance (ANOVA) with LSD tests were used for multiple comparisons, with P < .05 considered to be statistically significant. All experiments were repeated at least three times. Statistical analysis was performed using SPSS software version 19.0.

| Cytotoxic effects of galangin on bone marrowderived macrophages
The chemical formula of galangin is shown in Figure 1A. The MTT assay was performed to analyse the potential cytotoxicity of galangin against BMMs. As shown in Figure 1B

| Galangin inhibits RANKL-induced osteoclast formation in vitro
The differentiation of osteoclasts was inhibited by galangin treatment in a concentration-dependent manner. In the control group,   Figure 2C). Therefore, these results indicate that galangin suppressed the differentiation of osteoclasts.

| Galangin suppresses the bone resorptive activity of osteoclasts
Since galangin inhibited the formation of osteoclasts and the expression of osteoclast-specific genes, its effects on in vitro osteoclastic bone resorption were examined. Galangin treatment dose-dependently impaired the formation of mature F-actin rings ( Figure 3A). The treated osteoclasts showed smaller and fewer Factin rings than the control group ( Figure 3C). Consistent with these results, the areas of osteoclast-induced bone resorption pits dropped to 36.3 ± 2.31% (P < .001), 25.2 ± 0.85% (P < .001) and 5.14 ± 1.73% (P < .001) of that in the control group in the 3, 6 and 12 μmol/L galangin-treated groups, respectively ( Figure 3B and 3D).
Collectively, our data show that galangin attenuated bone resorption and the formation of mature F-actin rings.

| Galangin inhibits RANKL-induced activation of the MAPK and NF-κB pathways
To further elucidate the mechanism underlying galangin-induced inhibition of suppression of osteoclast formation, we investigated the signalling pathways involved in osteoclastogenesis. Here, the galangintreated group showed the phosphorylation of IκBα and p65 was inhibited by galangin treatment, where the total IκBα level dropped rapidly after RANKL induction alone for 10 minutes and recovered at the However, the nuclear translocation of p65 was blocked by galangin treatment (Figure 4G, H). Therefore, the inhibitory effects of galangin on osteoclasts may involve the MAPK and NF-κB signalling pathways.

| Galangin prevents LPS-induced bone loss in vivo
To explore the potential protective effect of galangin in vivo, an  Figure 7A, B, C). In addition, the pathological examination of liver and kidney has been done to explore the side-effects of galangin in our mice models.
Interestingly, H&E staining revealed that galangin had no toxic effect on liver and kidney ( Figure 7D). Collectively, our data suggest that galangin protects against LPS-induced bone loss in vivo.

| D ISCUSS I ON
In our study, we identify and characterize the effects of galangin, a well-known component of traditional Chinese medicine, on the differentiation and the function of osteoclasts. We found that galangin inhibited multinucleation, the formation of F-actin rings and the bone resorptive activity of osteoclasts, which was confirmed by the reduced expression of osteoclast-specific genes, including TRAP, Ctsk, DC-STAMP and V-ATPase d2. Furthermore, F I G U R E 5 Galangin attenuates MAPK and NF-κB signalling pathways and NFATc1 activity in a dose-and time-dependent manner. (A-E) BMMs were pre-treated with different concentrations of galangin for 1 h and then stimulated with RANKL for 30 min. The protein expressions of p-p65/p65, p-p38/p38, p-ERK/ERK, p-JNK/JNK and actin were checked by using Western blot analysis. (F-I) BMMs were treated with RANKL combined with or without galangin for 3 days. The images of the Western blots representing the effect of galangin on C-Fos, C-Jun and NFATc1 from 0, 1 and 3 days and the ratios of intensity of C-Fos, C-Jun and NFATc1 relative to actin were calculated using ImageJ software. Data are represented as the mean ± SD *P < .05, **P < .01, ***P < .001, compared with control group induced bone loss may be attributed to the increased activation of osteoclasts. In our previous study, we found that galangin could suppress osteosarcoma cells by inhibiting their proliferation and invasion and accelerating their apoptosis, and the mechanism may be associated with the inhibition of PI3K and its downstream signalling pathway. 28 Daniel Branstetter reported that the targeting of the bone microenvironment by inhibition of osteoclastogenesis prevents tumour-induced osteolysis and subsequent skeletal complications. 29 We speculate that galangin has dual functions in the inhibition of osteosarcoma cells, as well as osteoclasts in the process of inhibiting invasion and metastasis of osteosarcoma.
Those results indicate galangin may inhibit lung metastasis of osteosarcoma by inhibiting the function of osteoclasts, which needs to be further explored. In addition, galangin has no obvious effect on the proliferation of MC3T3-E1 osteoblast cells in the range of F I G U R E 7 Histological analysis of the effect of galangin on LPS-induced bone loss in vivo. (A) TRAP (magnification, 100× or 200×) staining, respectively, of calvaria sections. (B-C) The number of TRAP-positive cells and OcS/BS% were measured using ImageJ software. (D) H&E staining (magnification, 100× or 400×) revealed that galangin had no toxic effect on liver and kidney. Data are represented as the mean ± SD ***P < .001, compared with control group 0-25μM, but it can promote the differentiation and bone mineralization of MC3T3-E1 osteoblast cells in the range of 0-12μM through experiments of MTT assay, ALP staining and Alizarin Red Staining ( Figures S1, S2).
There are several limitations in this study. First, a positive control group, such as a bisphosphonates-treated group, was not included.
Secondly, the explanation for the influence on p38 and ERK rather than JNK signalling by galangin remains unclear, and the target molecule that galangin directly affects needs to be further explored.
Additionally, the LPS-inducted mouse calvarial model is not identical to the physio-pathologic processes of osteolysis in human patients.
Further experiments need to be conducted on large animals or humans to confirm the efficacy of galangin.
In conclusion, we found a novel pharmacological role of galangin, that is galangin could effectively suppress the development and progress of osteoporosis through suppression of osteoclastogenesis via inhibition of p38, ERK and NF-κB signalling pathways. We believe that galangin merits further studies and galangin has presented as a promising agent against osteoporosis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.