Biomolecules & Therapeutics 2024; 32(2): 205-213  https://doi.org/10.4062/biomolther.2023.067
Hydroxychavicol Inhibits In Vitro Osteoclastogenesis via the Suppression of NF-κB Signaling Pathway
Sirada Srihirun1, Satarat Mathithiphark2, Chareerut Phruksaniyom1, Pitchanun Kongphanich3, Wisutthaporn Inthanop3, Thanaporn Sriwantana4, Salunya Tancharoen1, Nathawut Sibmooh4 and Pornpun Vivithanaporn4,*
1Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok 10400,
2Faculty of Allied Health Sciences, Burapha University, Chonburi 20131,
3Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400,
4Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Samut Prakan 10540, Thailand
*E-mail: pornpun.viv@mahidol.edu
Tel: +6628395206, Fax: +6622011611
Received: March 27, 2023; Revised: July 15, 2023; Accepted: July 31, 2023; Published online: February 1, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.
Keywords: Hydroxychavicol, Osteoclast, RAW264.7, Bone resorption, Osteoporosis, NF-κB
INTRODUCTION

Bone, a mineralized connective tissue, is dynamically remodeled by the balance between bone-forming osteoblasts and bone-resorbing osteoclasts (Delaisse et al., 2020). The excessive bone-resorbing activity of osteoclasts plays a vital role in the pathogenesis of osteoporosis, leading to an increased risk of bone fracture (Bi et al., 2017). Therefore, the inhibition of the bone-resorbing activity of osteoclasts is a crucial target of drugs for the treatment of osteoporosis. Osteoclasts are specialized bone-resorbing cells differentiated from myeloid/monocyte lineage. The differentiation of osteoclasts is mainly induced by RANKL (Udagawa et al., 2021). The binding of RANKL to the RANK receptor promotes the activation of the IκB kinase complex (IKK), the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway, and mitogen-activated kinases (MAPKs), including p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) (Park et al., 2017). The activation of this signaling activates the transcription factors, including NF-κB, c-Fos, and c-Jun, that are required for the expression of the nuclear factor of activated T-cells 1 (NFATc1) at an early stage of osteoclastogenesis. NFATc1 is a master transcription factor for osteoclastogenesis (Kim and Kim, 2014). NFATc1 promotes the expression of genes required for osteoclast differentiation and function, such as tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), matrix metalloproteinase 9 (MMP-9), and cathepsin K.

Hydroxychavicol is a primary phenolic compound in the leaves of Piper betle (Betel) with various pharmacological effects, including antimicrobial, anti-cancer, and anti-platelet effects (Chang et al., 2007; Sharma et al., 2009; Ali et al., 2010; Gundala et al., 2014). In addition, hydroxychavicol promoted bone formation in vitro and inhibited bone loss induced by glucocorticoids in animals (Mishra et al., 2021). Hydroxychavicol has an anti-inflammatory effect by inhibiting cytokines production induced NF-κB signaling pathway (Seo et al., 2022). Since NF-κB plays a vital role in RANKL-induced osteoclast formation, we investigated the effects of hydroxychavicol on RANKL-induced osteoclast formation, function, and its potential mechanism in RAW264.7 cells.

MATERIALS AND METHODS

Ethic statement

This study was approved by the ethical committee of the Faculty of Dentistry/Faculty of Pharmacy, Mahidol University (COA.No.MU-DT/PY-IRB 2022/054.1710) according to the Declaration of Helsinki. Human PBMCs were isolated from the whole blood of healthy subjects (n=3). All participants provided written informed consent before enrolling in this study.

Cell culture

RAW264.7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 37°C in a 5% CO2 incubator. Cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). Osteoclast formation was induced by 50 ng/mL RANKL (R&D systems, Minneapolis, MN, USA).

Human PBMCs were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). The fresh whole blood was layered over Histopaque-1077 (1:1) and centrifuged at 400×g for 30 min at room temperature without a break. The PBMCs were collected and washed with PBS. Subsequently, cells were counted and plated in DMEM, 10% FBS, 1% penicillin-streptomycin, and 25 ng/mL human M-CSF (R&D systems). After expansion for 3 days, cells were detached with cell scrappers and seeded for osteoclast differentiation.

Cell viability assay

RAW264.7 cells were seeded at 5×103 cells in 96 well plates. Hydroxychavicol (HY-N1887) was purchased from Medchem Express (Monmouth Junction, NJ, USA). Hydroxychavicol was dissolved in DMSO at 100 mM and kept at –80°C until use. Hydroxychavicol at concentrations of 0.1-50 μM was added in RAW264.7 cells for 4 days. The final concentrations of DMSO (vehicle control) were 0.1-0.5%, depending on the maximum concentration of hydroxychavicol. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT, Sigma-Aldrich) was added to cells and incubated for 2 h to determine cell viability. After adding the dimethyl sulfoxide (DMSO) to dissolve the formazan crystal, the optical density was measured at 562 nm.

TRAP staining assay

RAW264.7 cells were seeded at 5×103 cells in 96-well plates. After overnight incubation, hydroxychavicol (2.5-10 μM) and 50 ng/mL RANKL were added in RAW264.7 cells and retreated on day 3. On day 4, cells were fixed with 10% formalin-neutral buffer (30 mM NaH2PO4 and 45 mM Na2HPO4) at room temperature for 5 min. Osteoclast formation was stained by TRAP staining kits according to the manufacturer’s instructions (Cosmo Bio, Carlsbad, CA, USA). TRAP-positive multinucleated cells (more than three nuclei) were counted and photographed by inverted microscopy (Olympus CKX53), equipped with an Olympus DP22 color camera (Olympus Corporation, Tokyo, Japan).

In separate experiments, human PBMCs were seeded at 3×104 cells in 96-well plates in the presence of 25 ng/mL M-CSF. After overnight incubation, hydroxychavicol (2.5-10 μM) 25 ng/mL human M-CSF and 50 ng/mL human RANKL was added in RAW264.7 cells and retreated on day 3 and day 5. Osteoclast formation was stained by TRAP staining kits using the same methods as described previously.

F-actin ring formation assay

RAW264.7 cells were seeded at 1×104 cells in 8-well chamber slides (Ibidi, Fitchburg, WI, USA). Hydroxychavicol and RANKL were treated using the same methods as described previously. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min. F-actin rings were stained with Phalloidin-iFluor 488 (Abcam, Cambridge, MA, USA) for 1 h and counterstained with Prolong gold anti-fade with DAPI (Cell Signaling, Danvers, MA, USA). The spiral scan of the confocal microscope captured the F-actin rings, and the green fluorescence intensity was quantified by Leica Las X software (Leica, Berlin, Germany).

Bone resorption assay

RAW264.7 cells were seeded at 5×103 cells on bovine bone slides (Boneslide, Jelling, Denmark) in 96-well plates. Hydroxychavicol was treated as described previously. Osteoclast formation was induced by 50 ng/mL RANKL for 7 days. The cells were removed from the bone slides by sonication in 1 M NH4OH for 20 min. A scan electron microscope photographed pit formation on bone slides. The resorptive bone area (pit area) was quantified by Image-Pro software version 7 (Media cybernetics, Rockville, MD, USA).

Quantitative real-time PCR

RAW264.7 cells were seeded at 1×105 cells in 6-well plates, and total RNA was extracted and purified on day 4 by an RNA purification kit according to the manufacturer’s protocol (Favorgen Biotech Corp., Ping Tung, Taiwan). The complementary DNA was synthesized from 0.5 mg of total RNA by the iScript cDNA synthesis kit (Bio-rad, Hercules, CA, USA). The primer sequences of cathepsin K, DC-STAMP, MMP-9, and GAPDH are shown in Table 1. Quantitative real-time PCR was measured using KAPA SYBR fast (KAPA biosystem, Wilmington, MA, USA) on Bio-rad real-time PCR CFX96 (Bio-rad). The gene expression values were calculated from the differences between Ct of the target’s gene with GAPDH and expressed as relative fold change (the 2-ΔΔCT method). The value of undifferentiated cells was defined as 1.

Table 1 Sequence of primers

Target genePrimerSequences
Cathepsin KForward5’-GAAGAAGACTCACCAGAAGCAG-3’
Reverse5’-TCCAGGTTATGGGCAGAGATT-3’
DC-STAMPForward5’-GGGGACTTATGTGTTTCCACG-3’
Reverse5’-ACAAAGCAACAGACTCCCAAAT-3’
MMP-9Forward5’-CTGGACAGCCAGACACTAAAG-3’
Reverse5’-CTCGCGGCAAGTCTTCAGAG-3’
GAPDHForward5’-AGGTCGGTGTGAACGGATTTG-3’
Reverse5’-TGTAGACCATGTACTTGAGGTCA-3’


Western blot

For the expression of transcription factors (NFATc1, c-Fos, and c-Jun), RAW264.7 cells in 6-well plates were treated with hydroxychavicol and 50 ng/mL RANKL for 18 h. For NF-κB signaling pathway, cells were treated for 5, 10, and 15 min. The cells were washed with PBS and lysed in protein lysis buffer (50 mM Tris, 150 mM NaCl, and 0.5% NP-40) with proteinase inhibitor cocktail III (1:500, Calbiochem, La Jolla, CA, USA). Protein was loaded on 10% polyacrylamide gel and transferred to the nitrocellulose membrane. The membranes were blocked with 5% skimmed milk for 1 h and subsequently incubated with primary antibodies, including NFATc1 (Cell Signaling, #8032), c-Fos (Cell Signaling, #2250), c-Jun (Cell Signaling, #9165), β-tubulin (Cell Signaling, #86298), p-p65 (Abcam, #ab86299), p65 (Abcam, #ab16502), P-Ikbα (Cell Signaling, #2859) and Ikbα (Cell Signaling, #9242) at 4°C overnight. The corresponding horseradish peroxidase-conjugated secondary antibodies were incubated for 2 h and followed by enhanced chemiluminescence (ECL) detection (Bio-Rad). Band density was quantified by Amersham Imagequant 800 (GE Healthcare, Piscataway, NJ, USA).

NF-κB nuclear translocation

RAW264.7 cells were seeded on coverslips in a 48-well plate. Hydroxychavicol (10 μM) and RANKL (50 ng/mL) were added into cells and incubated for 15 min. After treatment, coverslips were washed with PBS. Cells were fixed with 4% paraformaldehyde (10 min, room temperature), permeabilized with 0.1% Triton X-100 (10 min, room temperature), and blocked with 5% bovine serum albumin (1 h, room temperature). The p65 antibody was incubated at 4°C overnight and further incubated with a FITC-conjugated secondary antibody for 2 h. The samples were mounted with Prolong gold anti-fade with DAPI (Cell Signaling). The 5 representative fluorescence images were captured on Leica confocal microscope (Leica). The regions of interest (ROI) of FITC and DAPI were created by Leica Las X software (Leica). The DAPI staining was used to define the nuclear ROI. The percentages of p65 nuclear translocation were calculated by dividing FITC fluorescence intensity in the nucleus with whole cells (nucleus and cytoplasm).

Statistical analysis

Data are presented as means ± standard error mean (SEM). Data processing and statistical analysis were done by GraphPad Prism software Version 4 (GraphPad Software Inc., San Diego, CA, USA). The statistical differences (p<0.05) were evaluated by using one-way ANOVA with Tukey’s multiple comparisons.

RESULTS

The cytotoxicity of hydroxychavicol

The cytotoxicity of hydroxychavicol was tested by MTT assays in RAW264.7 cells for 4 days. Hydroxychavicol at 50 μM significantly decreased cell viability (Fig. 1). Therefore, non-toxic concentrations of hydroxychavicol at 2.5-10 μM was used in further experiments.

Figure 1. The cytotoxicity of hydroxychavicol on RAW264.7 cells. Hydroxychavicol (0.1-50 μM) was incubated in RAW264.7 cells for 4 days. Cell viability assay was measured by MTT assays. Data are mean ± SEM. *p<0.05 tested by one-way ANOVA with Tukey’s multiple comparisons compared with a control group (n=3).

Hydroxychavicol attenuates the formation and function of osteoclasts induced by RANKL

To investigate the effect of hydroxychavicol on osteoclastogenesis, TRAP-positive multinucleate cells were used as a marker of osteoclast formation (Boyle et al., 2003). TRAP-positive multinucleated cells were generated in response to RANKL stimulation (Fig. 2A). Hydroxychavicol suppressed RANKL-induced osteoclast formation in a concentration-dependent manner (Fig. 2A, 2B). Hydroxychavicol at concentrations of 7.5 and 10 μM significantly decreased the number of TRAP-positive cells. The formation of the F-actin ring, a unique bone resorption structure of mature osteoclasts (Han et al., 2019), was stained with Phalloidin-iFluor 488. Mature osteoclasts were identified by assessing the appearance of the F-actin ring with multi nuclei (stained with DAPI). RANKL stimulated the formation of a clearly defined F-actin ring with multi-nuclei (Fig. 2C, 2D). Cells treated with hydroxychavicol (7.5 and 10 μM) showed a smaller size of RANKL-induced F-actin ring formation with few nuclei. To confirm hydroxychavicol’s effect on bone resorptive activity of osteoclast, we measured pit formation on bone slides by scanning electron microscope. On day 7, hydroxychavicol at 7.5 and 10 μM significantly decreased the resorptive bone area induced by osteoclasts (Fig. 2E, 2F).

Figure 2. Hydroxychavicol decreases osteoclast formation and bone resorption. RAW264.7 cells were treated with RANKL (50 ng/mL) and 0.1% DMSO (vehicle control), hydroxychavicol for 4 days. (A) Representative pictures of TRAP-positive osteoclasts treated with indicated concentrations of hydroxychavicol (scale bar=100 μm). (B) The numbers of TRAP-positive osteoclasts (≥3 nuclei) were quantitively analyzed. (C) Representative immunofluorescence of mature osteoclasts for F-actin ring and nuclei (scale bar=100 μm). (D) Quantification of F-actin was measured by of fluorescence intensity by Leica Las X software. (E) Representative pictures of resorptive pits (scale bar=50 μm). (F) The area of resorption pits was quantified by Image-Pro software. Data are mean ± SEM. *p<0.05 tested by one-way ANOVA with Tukey’s multiple comparisons compared with a vehicle control group (n=3).

Hydroxychavicol decreases the expression of osteoclast-specific genes

The induction of osteoclast-specific genes, including DC-STAMP, cathepsin K, and MMP-9, are essential for osteoclast maturation (Rashid et al., 2023). DC-STAMP plays a key role in the fusion of mononuclear precursor cells into multinuclear osteoclasts (Langdahl, 2021). RANKL promoted DC-STAMP expression, which was markedly attenuated by 10 μM hydroxychavicol (Fig. 3A). Cathepsin K and MMP-9 are bone-resorptive enzymes secreted by osteoclasts in resorptive lacuna for bone mineralization (Vaananen et al., 2000). RANKL upregulated the expression of cathepsin K and MMP-9, while hydroxychavicol suppressed the expression of cathepsin K and MMP-9 (Fig. 3B, 3C).

Figure 3. Hydroxychavicol inhibits NF-κB signaling induced by RANKL. (A) Representative western blots of phospho-p65 (p-p65), p65, phospho-Iκβα (p-Iκβα), Iκβα, and β-tubulin in RAW264.7 cells after being treated with RANKL (50 ng/mL) and 0.1% DMSO (vehicle control), or 10 μM hydroxychavicol for 5, 15, and 30 min. Quantitative assessment of band intensity ratio of (B) p-p65 with p65, (C) p-Iκβα with Iκβα. (C) Representative pictures of nuclear translocation of P-65 subunit of NF-κB in cells after being treated without RANKL, with RANKL (50 ng/mL) + 0.1% DMSO (vehicle control), or 10 μM hydroxychavicol for 15 min. (D) Quantitative assessment of nuclear NF-κB p65 levels. The nuclear NF-κB translocation percentages were calculated by dividing FITC fluorescence intensity in the nucleus with whole cells (nucleus + cytoplasm). Data are mean ± SEM. *p<0.05 tested by t-test compared with a control group (vehicle control) (n=3).

Hydroxychavicol inhibits NF-κB signaling induced by RANKL

The NF-κB pathway is a major downstream signaling induced by RANKL in the early phase of osteoclast formation (Boyce et al., 2015). The phosphorylation of Iκβα induced by RANKL is rapidly degraded by the proteasomal pathway, which allows translocation of p65/p50 NF-κB subunit into the nucleus. To elucidate the inhibitory mechanism of hydroxychavicol on osteoclasts, the effect of hydroxychavicol on the RANKL-induced NF-κB pathway was investigated. RANKL rapidly induced the phosphorylation of Iκβα at 5 min and subsequently degradation (Fig. 4). Simultaneously, p65 was phosphorylated by RANKL. Hydroxychavicol (10 μM) inhibited phosphorylation of p65 and Iκβα at 5 min (Fig. 4A, 4B). The immunofluorescent assay revealed that hydroxychavicol inhibited nuclear translocation of p65 at 15 min after stimulation with RANKL (Fig. 4C, 4D).

Figure 4. Hydroxychavicol decreases RANKL-induced expression of osteoclast differentiation genes. RAW264.7 cells were differentiated to osteoclasts by 50 ng/mL RANKL for 4 days in the absence or presence of hydroxychavicol at 7.5 and 10 µM. The mRNA levels of DC-STAMP (A), cathepsin K (B) and MMP-9 (C) were determined using real-time PCR. Data are mean ± SEM. *p<0.05 tested by one-way ANOVA with Tukey’s multiple comparisons compared with the control group (n=3).

Hydroxychavicol abrogates RANKL-induced downstream expression of c-Fos, c-Jun, and NFATc1

c-Fos, c-Jun, and NFATc1 are important transcription factors for osteoclastogenesis (Rolph and Das, 2020). The activation of c-Fos and c-Jun by RANKL induces the expression of NFATc1, which subsequently turns on the expression of osteoclast-specific genes (Takayanagi, 2007). Therefore, the effect of hydroxychavicol on the expression of c-Fos, c-Jun, and NFATc1 induced by RANKL was examined by western blotting. RANKL induced the expression of c-Fos, c-Jun, and NFATc1 was suppressed by hydroxychavicol (Fig. 5).

Figure 5. Hydroxychavicol decreases RANKL-induced expression of c-Fos, c-Jun, and NFATc1. (A) Representative western blots of c-Fos, c-Jun, NFATc1, and β-tubulin in osteoclasts after being treated with RANKL (50 ng/mL) and 0.1% DMSO (vehicle control), hydroxychavicol at 7.5 and 10 μM for 18 h. Quantitative assessment of band intensity ratio of c-Fos (B), c-Jun (C), and NFATc1 (D). Data are mean ± SEM. *p<0.05 tested by one-way ANOVA with Tukey’s multiple comparisons compared with a control group (n=3).

Hydroxychavicol decreased osteoclast differentiation from human PBMCs

The anti-osteoclastogenic effect of hydroxychavicol was confirmed in osteoclasts generated from human PBMCs. RANKL induced the formation of multinucleated TRAP-positive osteoclasts after stimulation for 7 days. Hydroxychavicol at 15 and 20 μM inhibited RANKL-induced osteoclasts from human PBMCs (Fig. 6).

Figure 6. Hydroxychavicol decreases RANKL-induce osteoclast differentiation from human peripheral mononuclear cells (PBMCs). Representative pictures of osteoclasts after being treated 25 ng/mL M-CSF without RANKL (A), 0.2% DMSO (vehicle control) with 25 ng/mL M-CSF and 50 ng/mL RANKL (B), hydroxychavicol at 10 μM (C), 15 μM (D) and 20 μM (E) with 25 ng/mL M-CSF and 50 ng/mL RANKL for 7 days. TRAP-positive cells were stained with a TRAP staining kit and photographed (scale bar=100 μm). (F) Quantitative assessment of TRAP-positive osteoclasts (nuclei ≥3). Data are mean ± SEM. *p<0.05 tested by one-way ANOVA with Tukey’s multiple comparisons compared with a vehicle control group (n=3).
DISCUSSION

Our results demonstrated that hydroxychavicol inhibits osteoclastogenesis in RAW264.7 cells and human PBMCs. Hydroxychavicol exhibits a marked inhibition of osteoclastogenesis, as seen by the decrease of TRAP-positive osteoclasts and F-actin formation. The anti-osteoclastogenic effect of hydroxychavicol is parallel with the reduction of bone-resorptive pits and the expression of osteoclast-specific genes. Finally, hydroxychavicol inhibits osteoclastogenesis by suppressing the nuclear translocation of NF-κB induced by RANKL (Fig. 7).

Figure 7. Schematic representation of the potential anti-osteoclastogenic mechanism of hydroxychavicol. Hydroxychavicol inhibits RANKL-induced nuclear translocation of NF-κB resulting in a transcriptional suppression of the key transcription factor NFATc1. These actions subsequently inhibit the expression of NFATC1’s target genes including TRAP, cathepsin K, DC-STAMP and MMP-9.

RAW264.7 is a standard model for in vitro generation of osteoclasts (Collin-Osdoby and Osdoby, 2012). Osteoclasts generated from RAW264.7 cells mimicked the morphology and function of mature osteoclasts, including multinucleated TRAP-positive cells, F-actin ring structure, and bone-resorbing activity (Kong et al., 2019). Due to the expression of the RANK receptor, RAW264.7 cells were differentiated independently from the macrophage-colony stimulating factor (M-CSF) (Cuetara et al., 2006;Song et al., 2019). In our study, RANKL induced the differentiation of RAW264.7 to mature osteoclasts with bone resorptive function.

Our result showed that hydroxychavicol inhibited RANKL-induced TRAP-positive multinucleated cells differentiated from RAW264.7 and human PBMCs. Because hydroxychavicol significantly decreased the viability of RAW264.7 cells and human PBMCs at 50 μM, hydroxychavicol at 10 to 20 μM effectively suppressed osteoclast differentiation from RAW264.7 and human PBMCs without a cytotoxic effect. Consistent with our TRAP-stained results, hydroxychavicol also decreased F-actin ring formation and the expression of osteoclast-specific genes. In addition to osteoclast formation, hydroxychavicol exhibited a significant reduction in resorption pits induced by osteoclasts, suggesting the effect of hydroxychavicol on osteoclast function.

NFATc1 regulates the early phase of RANKL-induced osteoclast differentiation by inducing the expression of genes associated with both osteoclast formation and inorganic bone matrix degradation (Kim and Kim, 2014). Exogenous overexpression of NFATc1 induced osteoclast formation in bone marrow-derived macrophages in the absence of RANKL, while NFATc1 knockout caused osteopetrosis in mice (Aliprantis et al., 2008; Kim et al., 2008). In addition to NFATc1, the expression of osteoclast-specific genes requires cooperation with other transcription factors, including c-Fos and c-Jun, which are downstream transcription factors activated by the MAPK pathway (Zhao et al., 2007; Lee et al., 2018). In our study, hydroxychavicol suppressed NFATc1, c-Fos, and c-Jun expression induced by RANKL at 18 h. This result indicates the effect of hydroxychavicol on the early pathway of RANKL-induced osteoclast formation.

Hydroxychavicol has been reported to inhibit NF-κB nuclear translocation by blocking the phosphorylation of Iκβα induced by lipopolysaccharide, leading to the decreased expression of iNOS, COX-2, and TNF-α in RAW264.7 cells (Sarkar et al., 2008; Seo et al., 2022). Nuclear translocation of NF-κB is a key downstream signaling pathway induced by RANKL for osteoclastogenesis (Abu-Amer, 2013). The nuclear translocation of p65/p50 NF-κB subunits is regulated by IκB proteins (Scott et al., 1993). Upon activation of RANKL to its receptor, IκB proteins are rapidly phosphorylated by IκB kinase (IKK), leading to proteasome degradation, which allows the translocation of the phospho-p65 subunit into the nucleus (Palombella et al., 1994; Mercurio et al., 1997). In our study, at 5 min after stimulation by RANKL, hydroxychavicol suppressed the phosphorylation of p65 and Iκβα, which subsequently inhibited nuclear translocation of p65 at 15 min. This result suggested that the anti-osteoclastic effect of hydroxychavicol is mediated at least in part by the inhibition of NF-κB pathway. A molecular docking study suggested that hydroxychavicol has an affinity to bind to the inside of the IKK binding pocket (Maslikah et al., 2023). This suggests that IKK may be a target of hydroxychavicol; however, the possibility of interrupting the binding of RANKL to receptor by hydroxychavicol cannot be excluded. In addition, the impact of hydroxychavicol on the other early downstream activation of the RANK receptors, such as mitogen-activated protein kinase (MAPK) pathway, should be further investigated.

Natural product-based anti-resorptive molecules have emerged as an alternative treatment for osteoporosis (Martiniakova et al., 2020). Due to the incidences of side effects, the long-term use of current anti-resorptive drugs such as estrogen replacement, bisphosphonates, RANKL inhibitors, and calcitonin are still debated (Adler, 2018; Langdahl, 2021). Hydroxychavicol and eugenol, major polyphenolic compounds in leaves of Piper betle, have been well-documented for pharmacological effects (Biswas et al., 2022). Eugenol (3-methoxy-4-hydroxyallybenzene) inhibited RANKL-induced osteoclast formation by inhibiting NF-κB and MAPK pathways (Deepak et al., 2015). Our study demonstrated that hydroxychavicol at a concentration of 10 μM effectively inhibited osteoclast formation and function compared with 200 μM of eugenol, suggesting the superior potency of hydroxychavicol on osteoclasts. Hydroxychavicol has previously been reported to attenuate glucocorticoid-induced osteoporosis in animals due to enhanced bone formation (Mishra et al., 2021). In addition to bone formation, hydroxychavicol’s effect on bone may be associated with the inhibition of osteoclast function. Although our data provide the in vitro effect of hydroxychavicol on osteoclasts, further investigation should be conducted in vivo and clinical studies to confirm the clinical benefit of hydroxychavicol on bone diseases.

ACKNOWLEDGMENTS

This research study is supported by Specific League Funds from Mahidol University and Dean’s Research Novice Award of the Faculty of Medicine Ramathibodi Hospital, Mahidol University.

We acknowledge the facilities and technical assistance of the Research office, Faculty of Dentistry, Mahidol University. We would like to thank Mr. Sarut Thairat for his technical assistance for confocal microscopy.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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