Isoschaftoside Inhibits Osteoclastogenesis by Suppressing the NF-κB and MAPK Pathways

Abstract

Bone remodeling is regulated by the equilibrium between bone formation and resorption. However, excessive bone resorption by osteoclasts disrupts bone homeostasis and contributes to several bone-related disorders. Hematopoietic lineage cells differentiate into multinucleated osteoclasts through osteoclastogenesis, a process driven by M-CSF and RANKL. Multiple signaling pathways are involved in osteoclast maturation. RANKL-induced activation of NF-κB has been recognized as a crucial mechanism in osteoclast differentiation. Additionally, MAPK signaling activated by RANKL plays a significant role in this process. The stimulation of NF-κB and MAPK by RANKL enhances the transcription of numerous genes associated with osteoclastogenesis. Isoschaftoside (ISH), a C-glycosyl flavonoid, can be isolated from Viola yedoensis. The herb Viola yedoensis has traditionally been used to manage inflammation-related diseases, and its crude extracts have demonstrated pharmacological effects. Nonetheless, the influence of ISH on osteoclastogenesis has not been documented previously. In this study, we explored the effect of ISH on osteoclastogenesis. Our findings indicate that ISH attenuates RANKL-induced activation of NF-κB and MAPK; moreover, ISH inhibits osteoclastogenesis and bone resorption stimulated by RANKL and M-CSF.

INTRODUCTION

Bone remodeling is precisely regulated by maintaining a balance between bone formation and resorption. However, increased bone resorption mediated by osteoclasts—multinucleated cells originating from hematopoietic cells—disrupts bone homeostasis and contributes to bone-related diseases such as osteoporosis (Wang et al., 2022). Osteoclasts are generated from hematopoietic stem cells and macrophages through stimulation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) (Mun et al., 2020). These signaling molecules interact with their respective receptors, RANK and tumor necrosis factor receptor-associated factor (TRAF6), on the cell surface. Following activation, the receptor-ligand binding initiates the differentiation of premature osteoclasts (pre-OCs) into mature osteoclasts (OCs) (Park et al., 2017). Additional factors contributing to osteoclastogenesis include lipopolysaccharide (LPS) and pro-inflammatory cytokines such as IL-1 and TNF-α (Silva et al., 2013). These stimuli activate the NF-κB pathway and mitogen-activated protein kinase (MAPK) pathways. Both pathways are recognized as central regulators of osteoclast formation. The nuclear factor kappa B (NF-κB) signaling pathway orchestrates osteoclast and osteoblast formation and becomes activated in osteoclast precursors in response to the essential cytokine RANKL; RANKL is also involved in controlling osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors (Boyce et al., 2023). Moreover, RANKL and certain pro-inflammatory cytokines initiate NF-κB and downstream pathways, including c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), thereby promoting osteoclast formation and activity (Rhee et al., 2018). The MAPKs, specifically c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an essential role in osteoclastogenesis, cellular metabolism, and functional activation (Lee et al., 2018). In addition, MAPK signaling in conjunction with M-CSF influences the proliferation of osteoclast precursors, and, along with RANKL stimulation, further supports osteoclast differentiation (Lee et al., 2018).

Activation of the MAPK pathway by RANKL induces the expression of multiple genes critical for osteoclastogenesis. RANKL-mediated JNK phosphorylation stimulates expression of c-Fos and NFATc1 (Lee et al., 2018). In particular, NFATc1 is a central transcription factor that upregulates genes encoding TRAP, cathepsin K, E-cadherin, DC-STAMP, and osteoclast stimulatory transmembrane protein (OC-STAMP) (Lee et al., 2018). RANKL-induced ERK activation additionally modulates the expression of matrix metalloproteinase 9 (MMP-9) (Lee et al., 2018; Raggatt and Partridge, 2010). Therefore, agents capable of inhibiting the MAPK-RANKL signaling pathway are considered promising candidates for the treatment of bone-related diseases.

Isoschaftoside (ISH) is a C-glycosyl flavonoid extracted from Viola yedoensis (Lee et al., 2016).The herb Viola yedoensis has been employed in the treatment of inflammation-related conditions, including swelling, boils, and chronic hepatitis. Additionally, crude extracts of Viola yedoensis have demonstrated pharmacological activities such as anti-HIV and anticoagulant effects (Zhou et al., 2009; Wang et al., 2008; Zhang et al., 2024). Nevertheless, the role of ISH in osteoclastogenesis remains unexplored. In this study, we evaluated the potential of ISH to regulate osteoclastogenesis. Our findings indicate that ISH inhibits RANKL-induced NF-κB and MAPK activation and further suppresses osteoclastogenesis and bone resorption induced by RANKL and M-CSF.

MATERIALS AND METHODS

Chemical and reagents

Recombinant RANKL and M-CSF were purchased from BioLegend (BioLegend, CA, USA). MTT formazan (M2003) were purchased from Sigma-Aldrich (Sigma-Aldrich, MO, USA). Antibodies against phospho-JNK (9255), JNK (9252), phospho-ERK (9101), ERK (9102), phospho-p38 (9211), p38 (9212), phospho-NF-κB (3033), NF-κB (8242), and lamin B (13435) were purchased from Cell Signaling Technology Inc. (Cell Signaling). β-actin (sc-47778) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Methanol-d4 and DMSO-d6 were used as NMR solvents (Cambridge Isotope Laboratories, USA). HPLC grade water and acetonitrile were purchased from Fisher Scientific Korea Ltd. (Seoul Korea). The 1H– and 13C–NMR spectra were acquired using Bruker AVANCE III at 600 MHz (Bruker Co., Ltd., Germany). Waters 2695 HPLC system with photodiode array detector (Agilent technologies, USA) was used to confirm the purity of compound.

Preparation of compound

Isoschaftoside (ISH) was isolated from Viola yedoensis and provided by Dr. Ki Yong Lee (College of Pharmacy, Korea University). The structure of ISH (Fig. 1A) was determined based on 1H-, 13C NMR and ESI-MS spectra with reference to previously published paper (Jeong et al., 2016) and can be seen in supplementary materials of previous literature (Lee et al., 2024).

Fig. 1.

ISH inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells. (A) Structure of ISH. (B) RAW 264.7 cell viability of ISH. (C) RAW 264.7 cells were incubated with medium or RANKL (50 ng/mL) or RANKL and ISH (5, 10 μM) for 5 days, then stained for TRAP expression. TRAP-positive cells were photographed and representative images are shown (magnification, ×100). (D) TRAP positive cells with over than 3 nuclei were counted as mature osteoclasts. (E) RAW 264.7 cells were incubated with medium or RANKL (50 ng/mL) or RANKL and ISH (5, 10 μM) for 3, 4, 5 days, then TRAP activity was measured. (F) RAW 264.7 cells were incubated with medium or RANKL (50 ng/mL) or RANKL and ISH (5, 10 μM) for 5 days. After incubation, cells were removed and the resorption pits formed on the surface were observed by light microscopy (magnification, ×200) and measured by ImageJ. (G) Bone resorption areas were quantified. ^#p<0.05, ^##p<0.01 compare to control (medium only). *p<0.05, **p<0.01 compared to control (RANKL only).

Cell culture and isolation of bone marrow-derived macrophages

Murine macrophage RAW 264.7 cells were cultured in DMEM high glucose supplemented with 10% FBS and 1% antibiotics. Bone marrow-derived macrophages (BMMs) were isolated from 10 weeks old male C57BL/6 mice. BMMs were cultured with minimal essential media (α-MEM) containing 10% FBS, 1% antibiotics and 30 ng/mL M-CSF.

Osteoclast differentiation

RAW264.7 cells (1×10³ cells/mL) and BMMs (1×10³ cells/mL) were seeded onto 24 well plate. Conditioned medium for osteoclast formation was maintained with α-MEM containing 10% FBS and 1% antibiotics with 50 ng/mL recombinant RANKL for 5 days. The BMMs were co-treated with 50 ng/mL of RANKL and 30 ng/mL of M-CSF for 5 days.

Tartrate-resistant acid phosphatase (TRAP) staining and activity assay

The RAW264.7 cells and BMMs were stimulated by RANKL or LPS for 5 days, then were washed with PBS and fixed with 4% formaldehyde and 0.1% Triton X-100 for 10 min at room temperature. The cells were stained for TRAP expression using an acid phosphatase kit (Merck, Darmstadt, Germany), and the supernatant was used to measure TRAP activity with an ELISA plate reader at 405 nm.

Pit formation assay (bone resorption)

RAW264.7 cells (1×10³ cells/mL) were seeded to 24 well pit formation assay plate (Corning, NY, USA). The ISH was pre-incubated for 24 h and then RANKL was added to cells. After 5 days, pit formation assay plates were incubated with 10% of bleach solution for 5 min at 37°C. After aspirating out bleach solution, the wells were washed with PBS. The bone resorption area was observed by light microscopy and measured by ImageJ (NIH, USA).

Preparation of cytosolic and nuclear fraction

RAW264.7 cells were washed twice and scraped with PBS. The cell suspension was incubated with buffer A (10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 300 mM saccharose, 0.1% NP-40 and 0.5 mM PMSF) and centrifuged for 1 min at 9300g (4°C). The supernatants (cytosolic fraction) were picked up and the cell pellets were resuspended and incubated for 5 min (4°C) in buffer B (20 mM HEPES-KOH pH 7.9, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT). These were immediately centrifuged at 17,000 g for 5 min (4°C) and the supernatants (nuclear fraction) were recovered.

Western blot analysis

RAW 264.7 cells were harvested from 60 mm dishes for protein extraction. Protein concentrations were measured by BCA protein assay kit (Pierce, Rockford, IL, USA). 10 μg of proteins were separated at 8% to 10% of SDS-PAGE gel and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in 0.05% TBST for 1 h and incubated with primary antibodies at 4°C for overnight. The membranes were incubated with horseradish peroxidase conjugated secondary antibodies (1:1000), then washed with TBST and reacted with ECL reagent (Pierce) for protein band visualization. The intensities of bands were analyzed by ImageQuant LAS 4000.

Electrophoretic mobility shift assay (EMSA)

RAW 264.7 cells were harvested and nuclear fraction was extracted as mentioned above. NF-κB/DNA binding activity was detected by DIG gel shift kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol. For the formation of DNA-protein complexes, 10 μg of nuclear extracts were incubated with 2 μg of DIG-labeled oligonucleotide (NF-κB forward: 5’-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGGGAGGCGTGG-3’, NF-κB reverse: 5’-CCACGCCTCCCCCTGGAAAGTCCCCAGCGGAAAGTCCCTTGTAACAA-3’) for 30 min at room temperature. The DNA-protein complexes were separated by 6% nondenatured polyacrylamide gel using 1×TBE as a running buffer. After electrophoresis, the gels were transferred to nylon membranes and detected with CSPD and a chemiluminescent substrate. Signal intensity was quantified by and image analyzer (LAS 4000).

Immunofluorescence assay

RAW 264.7 cells (1×10² cells/mL) were seeded onto 4 well chambers in the presence or absence of ISH. After 24 h, cells were stimulated by RANKL for 1 h. Then the cells were washed with PBS and fixed with 4% formaldehyde containing 0.1% triton X-100 for 15 min at room temperature. After blocking by 5% BSA for 1 h, cells were incubated with phospho-NF-κB antibody for 1 h and washed with PBS containing 0.1% Tween 20. Then secondary antibody (Alexa Fluor 488; Thermo Scientific) was incubated for 1 h. The nucleus was stained by Hoechst for 5 min at 37°C. The result was analyzed using software NIS-Elements BR.

Reverse-transcription PCR analysis

RAW264.7 cells (2×10⁵ cells/mL) were seeded to 60 mm dishes for analysis of mRNA expression. After experimental treatment, total RNA was isolated using Trizol reagent (Life Technologies, CA, USA). For the synthesis of complementary DNA, 1 μg of total RNA was prepared by AccuPower Rocket Script cycle RT premix (Bioneer, Daejeon, Korea). Then, cDNA was conducted to PCR using the primer of NFATc1, c-Fos, Cathepsin K and GAPDH. The PCR conditions were optimized to primer or efficiency of target gene amplification. Amplified mRNA products were run on the 2% agarose gel and detected by Vilber Lourmat.

F-actin ring formation assay

After pre-treatment of ISH (10 μM) for 24 h, RAW 264.7 cells (1×10³ cells/mL) were incubated with 50 ng/mL of RANKL for 5 days (24 well plates). After 5 days, cells were washed with PBS and fixed with 4% formaldehyde containing 0.1% triton X-100 for 10 min. And the cells were incubated with Alexa flour 594 phalloidin (Thermo Scientific) diluted in 5% BSA for 30 min at room temperature. For nuclear staining, cells were incubated with Hoechst for 5 min. Fluorescence signals were taken and analyzed by NIS-Elements BR software (Nikon).

Statistical analysis

Each experiment was repeated at least three times. Experimental data was expressed as means ± standard deviation (SD). Statistical significance of the p value was obtained ANOVA and Student-Newman-Keul tests (^#p<0.05, ^##p<0.01, *p<0.05, **p<0.01).

RESULTS

This study aimed to assess the effects of Isoschaftoside (ISH) on osteoclastogenesis activated by the RANKL signaling pathway. We utilized RAW 264.7 cells (murine macrophages) and bone marrow-derived macrophages (BMMs), which represent an established in vitro model system for osteoclast differentiation.

ISH inhibits RANKL-induced osteoclastogenesis

To identify a non-toxic concentration of ISH, we conducted an MTT assay (Fig. 1B), establishing 10 μM as the maximum dose. Osteoclasts are characterized by the production of enzymes such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and matrix metalloproteinase-9 (MMP-9) (Sharma et al., 2021). As TRAP is a widely accepted marker for osteoclastogenesis, we utilized TRAP staining, enumerated TRAP-positive cells, and evaluated TRAP activity to determine the impact of ISH on osteoclast differentiation. Initially, RAW 264.7 cells were treated with increasing concentrations of ISH alongside RANKL and allowed to differentiate into osteoclasts for up to 5 days. As depicted in Fig. 1C, the formation of osteoclasts was markedly reduced following pretreatment with ISH. The quantification of TRAP-positive cells (Fig. 1D) and assessment of TRAP activity (Fig. 1E) were consistent with the staining observations. Next, we examined whether ISH influences bone resorption. Bone resorption area was assessed using a pit assay with a calcium phosphate-coated Corning Osteo Assay kit. RAW 264.7 cells were cultured with varying concentrations of ISH in the presence of RANKL for 5 days. RANKL promoted bone resorption; however, pretreatment with ISH led to a decrease in osteoclast pit area (Fig. 1F, 1G). Collectively, these findings demonstrate that ISH exerts an inhibitory effect on RANKL-induced osteoclastogenesis and bone resorption.

ISH suppresses the osteoclastogenesis of BMMs and LPS-induced osteoclastogenesis in RAW 264.7 cells

Bone marrow-derived macrophages (BMMs) can also differentiate into osteoclasts following stimulation with M-CSF and RANKL (Quinn and Gillespie, 2005). To determine whether ISH inhibits the osteoclastogenesis of BMMs, BMMs were treated with varying concentrations of ISH in the presence of M-CSF/RANKL and cultured for up to 5 days to permit osteoclast differentiation. As depicted in Fig. 2A, pretreatment with ISH led to a marked reduction in BMM differentiation into osteoclasts in a dose-dependent manner. Quantification of TRAP-positive cells (Fig. 2B) and assessment of TRAP activity (Fig. 2C) were consistent with the TRAP staining outcomes. Because LPS is also known to induce osteoclast formation (Lv et al., 2025), we further evaluated the impact of ISH on LPS-induced osteoclastogenesis. RAW 264.7 cells were exposed to different concentrations of ISH alongside LPS and cultured for up to 5 days for osteoclast differentiation. As shown in Fig. 2D, ISH pretreatment significantly inhibited osteoclast formation in these cells. Counting TRAP-positive cells (Fig. 2E) and measuring TRAP activity (Fig. 2F) confirmed the findings from TRAP staining. Collectively, these results demonstrate that ISH inhibits both M-CSF/RANKL-induced osteoclast formation in BMMs and LPS-induced osteoclastogenesis in RAW 264.7 cells.

Fig. 2.

ISH inhibits M-CSF/RANKL-induced osteoclastogenesis in BMMs and LPS-induced osteoclastogenesis in RAW 264.7 cells. (A) BMMs were incubated with M-CSF (30 ng/mL) or M-CSF/RANKL (50 ng/mL) or M-CSF/RANKL and ISH (5, 10 μM) for 5 days, then stained for TRAP expression. TRAP-positive cells were photographed and representative images are shown (magnification, ×100). (B) TRAP positive cells with over than 3 nuclei were counted as mature osteoclasts. (C) BMMs were incubated with M-CSF (30 ng/mL) or M-CSF/RANKL (50 ng/mL) or M-CSF/RANKL and ISH (5, 10 μM) for 3, 4, 5 days, then TRAP activity was measured. (D) RAW 264.7 cells were incubated with medium or LPS (100 ng/mL) or LPS and ISH (5, 10 μM) for 5 days, then stained for TRAP expression. TRAP-positive cells were photographed and representative images are shown (magnification, ×100). (E) TRAP positive cells with over than 3 nuclei were counted as mature osteoclasts. (F) RAW 264.7 cells were incubated with medium or LPS (100 ng/mL) or LPS and ISH (5, 10 μM) for 3, 4, 5 days, then TRAP activity was measured. ^#p<0.05, ^##p<0.01 compared to control (medium only). *p<0.05, **p<0.01 compared to M-CSF/RANKL-induced BMMs or LPS-induced RAW 264.7 cells.

ISH inhibits the activation of the NF-kB pathway

Multiple signaling pathways are implicated in osteoclast maturation. RANKL-induced NF-κB activation, in particular, is crucial for osteoclast differentiation (Boyce et al., 2005). Activation of NF-κB enhances the expression of genes involved in osteoclastogenesis and bone resorption (Abu-Amer, 2013; Asagiri and Takayanagi, 2007). Viola yedoensis has previously been shown to inhibit both NF-κB and MAPK pathways (Jeong et al., 2016). To elucidate whether ISH influences RANKL-induced NF-κB activation, RAW 264.7 cells were treated with graded concentrations of ISH and RANKL for 1 h. We then examined NF-κB nuclear translocation by immunofluorescence assay. As displayed in Fig. 3A and 3B, phosphorylated NF-κB nuclear translocation was reduced by ISH in a dose-dependent fashion. To further validate these findings, we performed western blot analysis. RANKL increased phosphorylated NF-κB protein levels, whereas ISH reduced levels in both total cell lysates and nuclear extracts (Fig. 3C). In addition, EMSA demonstrated that ISH suppressed NF-κB-DNA binding activity by blocking the formation of the NF-κB-DNA complex (Fig. 3D). These findings suggest that the suppressive effects of ISH on osteoclast maturation and bone resorption are closely linked to the inhibition of the NF-κB pathway.

Fig. 3.

ISH inhibits the activation of the NF-κB pathway. (A) After RAW 264.7 cells were pre-treated with ISH (5, 10 μM) for 24 h, RANKL (50 ng/mL) was added and incubated for 60 min. Localization of p-NF-κB (green) was visualized with fluorescence microscope (magnification, ×100). Cells were stained with Hoechst for the nuclei (blue). (B) Co-localized fluorescent cells were counted. (C) After RAW 264.7 cells were pre-treated with ISH (5, 10 μM) for 24 h, RANKL (50 ng/mL) was added and incubated for 60 min. The protein expression of p-NF-κB and NF-κB in nuclei fraction or total cell extraction was analyzed by western blot. (D) NF-κB/DNA binding activity was observed by EMSA. ^##p<0.01 compared to control (medium only). **p<0.01 compared to RANKL-induced RAW 264.7 cells.

ISH suppresses the MAPK pathway

Because RANKL-induced MAPK activation regulates the expression of numerous genes involved in osteoclast differentiation, we examined whether ISH inhibits osteoclastogenesis by suppressing the MAPK pathway. Our results demonstrated that ISH decreased the phosphorylation levels of JNK and ERK in RAW 264.7 cells, while p-p38 levels remained unaffected (Fig. 4A). This finding suggests that the inhibitory effect of ISH on osteoclastogenesis is associated with suppression of the MAPK pathway. The expression of their downstream genes, including NFATc1, c-Fos, and Cathepsin K, was also reduced at the transcriptional level (Fig. 4B).

Fig. 4.

ISH suppresses the MAPK pathway and impairs F-actin ring formation in RAW 264.7 cells. (A) After RAW 264.7 cells were pre-treated with ISH (1, 2.5, 5, 10 μM) for 24 h, RANKL (50 ng/mL) was added and incubated for 60 min. The protein expression of MAPKs was analyzed by western blot. (B) After RAW 264.7 cells were pre-treated with ISH (10 μM) for 24 h, RANKL (50 ng/mL) was added and incubated for 60 min. The mRNA expression of downstream molecules of MAPKs was observed by RT-PCR. (C) After RAW 264.7 cells were pre-treated with ISH (5, 10 μM) for 24 h, RANKL (50 ng/mL) was added and incubated for 5 days. Representative fluorescence images of osteoclasts stained with Alexa flour 594 phalloidin (red) to visualize F-actin rings and Hoechst (blue) for nuclear staining were observed with fluorescence microscope (magnification, 200×).

ISH impairs F-actin ring formation in RAW 264.7 cells

A podosome belt is a dynamic, actin-rich structure found in osteoclasts. The core of each podosome features a filamentous actin (F-actin) ring structure. This belt anchors osteoclasts to the bone surface, establishing a sealing zone (Georgess et al., 2014). Consequently, F-actin ring formation is a critical event for bone resorption in mature osteoclasts. We assessed whether ISH influences F-actin ring generation. RAW 264.7 cells were incubated with RANKL in the presence or absence of ISH for 5 days. F-actin rings were observed in RAW 264.7 cells treated with RANKL; however, their formation was diminished upon ISH treatment (Fig. 4C). These results demonstrate that ISH suppresses F-actin ring formation in RAW 264.7 cells.

DISCUSSION

Bone remodeling is a fundamental process necessary for maintaining skeletal health. To preserve bone homeostasis, coordination between osteoclast and osteoblast activity is required (Raggatt and Partridge, 2010; Roodman, 2004). However, excessive bone resorption mediated by osteoclasts leads to bone disorders such as osteoporosis (Wang et al., 2022). Thus, identifying safe and effective inhibitors of bone loss remains crucial. The herb Viola yedoensis has been traditionally utilized for inflammatory diseases (Zhou et al., 2009; Wang et al., 2008; Zhang et al., 2024). Crude extract of Viola yedoensis has been shown to possess anti-inflammatory effects by suppressing HO-1, NF-κB, and MAPK signaling pathways in RAW 264.7 cells (Lee et al., 2016). Since both NF-κB and MAPK signaling pathways participate in osteoclastogenesis, we evaluated whether ISH, a C-glycosyl flavonoid derived from Viola yedoensis, could regulate RANKL- or M-CSF/RANKL-induced osteoclast differentiation.

First, we examined whether ISH modulates osteoclast formation. Our results show that ISH inhibits RANKL-induced osteoclast formation in RAW 264.7 cells. Quantification of TRAP-positive cells and assessment of TRAP activity demonstrated the inhibitory effect of ISH on osteoclastogenesis. Differentiation of RAW264.7 cells into mature osteoclasts in response to RANK/RANKL signaling typically requires up to 5 days. To assess the effect of early ISH intervention on this process, we added ISH at 1, 2, and 3 days following initial RANKL stimulation of RAW264.7 cells. Notably, ISH inhibited osteoclastogenesis even when administered 3 days after RANKL treatment. These findings suggest that ISH targets an early stage in the pathway leading to RANKL-mediated osteoclastogenesis. As osteoclasts contribute to bone resorption, the pit assay serves as a standard approach to evaluate osteoclast function by measuring hydroxyapatite resorption (Asagiri and Takayanagi, 2007). We therefore performed a pit assay to investigate the inhibitory effect of ISH on osteoclast activity. The pit assay results confirmed that ISH reduced both bone resorption and osteoclast formation. Collectively, these observations indicate that ISH suppresses RANKL-induced osteoclast formation and bone resorption. As illustrated in Fig. 2, ISH also inhibited osteoclastogenesis induced by M-CSF/RANKL in primary bone marrow-derived macrophages (BMMs) and suppressed LPS-induced osteoclast formation in RAW 264.7 cells.

Next, we sought to clarify the underlying mechanisms. Osteoclast maturation is regulated by several signaling pathways. RANKL-induced NF-κB activation is recognized as a critical mediator of osteoclast differentiation. Activated NF-κB enhances the transcription of genes associated with osteoclastogenesis and bone resorption (Abu-Amer, 2013; Asagiri and Takayanagi, 2007). Given previous reports that Viola yedoensis suppresses both NF-κB and MAPK pathways (Lee et al., 2016), we hypothesized that the inhibitory effect of ISH on osteoclastogenesis may involve these pathways. As activated NF-κB translocates to the nucleus to promote downstream gene expression, we evaluated whether ISH can block NF-κB nuclear translocation. Immunofluorescence analysis revealed that ISH pre-treatment reduced the number of cells exhibiting nuclear localization of NF-κB (green). In addition, electrophoretic mobility shift assay (EMSA) demonstrated that ISH decreased the formation of NF-κB-DNA complexes. Furthermore, ISH reduced the levels of phosphorylated NF-κB in total cell lysates and nuclear extracts (Fig. 3C, 3D). These findings collectively indicate that ISH inhibits osteoclast maturation and bone resorption by blocking NF-κB activation.

Previous studies have demonstrated that MAPK signaling, activated by RANKL induction, also triggers osteoclast differentiation (Lee et al., 2018). RANKL-induced MAPK activation facilitates the expression of several genes implicated in osteoclastogenesis (Chang et al., 2008; Cuetara et al., 2006). Therefore, we assessed whether ISH impedes osteoclast formation through the inhibition of the MAPK pathway. As presented in Fig. 4, ISH inhibited the activation of JNK and ERK, and it further decreased the mRNA expression of NFATc1, c-Fos, and Cathepsin K, which are regulated by JNK and ERK. Although JNK, ERK, and p38 all belong to the MAPK family, the lack of response in p-p38 suggests that ISH may have distinct molecular targets or regulatory mechanisms for each pathway; further investigation is required to elucidate these differences. Additionally, we evaluated the inhibitory effect of ISH on F-actin ring formation, a critical process for bone resorption by mature osteoclasts, and found that ISH suppressed F-actin ring development.

Overall, our data suggest that ISH inhibits osteoclastogenesis in RAW264.7 cells and BMMs by suppressing both the NF-κB and MAPK signaling pathways (Fig. 5). Based on these findings, ISH emerges as a promising therapeutic candidate for bone-related diseases.

Fig. 5.

Schematic diagram shows how ISH inhibits osteoclastogenesis by suppressing the NF-κB and MAPK pathways. RANK, Receptor activator of the NF-κB; RANKL, Receptor activator of the NF-κB ligand; TRAF6, Tumor necrosis factor receptor-associated factor 6; MAPK, Mitogen-activated protein kinase; TAK1, Transforming growth factor-beta-activated kinase 1; ERK, Extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IκB, Inhibitor of nuclear factor kappa B; NFATc1, Nuclear factor activated T-cells 1; TRAP, Tartrate-resistant acid phosphatase.