Novel Osteoblastogenic Activity of Magnolia kobus: The Pharmacological Potential for Osteoporosis

Abstract

Magnolia kobus (M. kobus) has long been used to treat nasal congestion, allergic rhinitis, and sinusitis. In the current study, we demonstrate the effects and underlying mechanisms of M. kobus flower water extract (ME) and ME-derived constituent magnolin on in vitro osteoblastogenic and anti-osteoclastogenic responses. Treatment with ME or magnolin markedly enhanced the osteoblast differentiation and mineralization in MC3T3-E1 pre-osteoblasts. This osteoblastogenic activity of ME or magnolin was closely associated with upregulation of osteoblast-specific molecules, including RUNX2, DLX5, OSX, alkaline phosphatase, collagen type I, and osteopontin, as well as the activation of mitogen-activated protein kinase (MAPK) signaling pathways. Concurrently, magnolin inhibited osteoclast differentiation through inactivating MAPK pathways and downregulating NFATc1, c-Fos, tartrate-resistant acid phosphatase, and cathepsin K in RANKL-treated RAW264.7 cells. These observations suggest that ME and magnolin have pharmacological potential for the treatment and prevention of metabolic bone disorders, including osteoporosis.

1. Introduction

Bone tissue is a complex and dynamic organ system consisting of the extracellular matrix (ECM), including proteins, proteoglycans, and carbohydrates, and diverse cell types such as osteoblasts, osteoclasts, and osteocytes. This cyclic process involves continuous and coordinated interplay events between cells and the ECM, where osteoblasts induce bone formation, while osteoclasts control bone resorption, perpetuating the iterative course of bone regeneration and homeostasis [1,2]. As a pre-osteoblastic lineage derived from mouse calvaria, MC3T3-E1 cells exhibit osteoblastic characteristics including proliferation, differentiation, and mineralization in response to ascorbic acid and β-glycerophosphate stimulation [3,4]. Ascorbic acid is known to be an essential substance for synthesizing collagen, an intercellular binding molecule that forms bones, teeth, muscles, vascular tissue, and ligaments [5,6]. β-glycerophosphate promotes osteoblast differentiation and contributes to providing the calcium and phosphorus needed for mineral replacement in cells [6]. Osteoblast differentiation is coordinately regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPKs), and by the expression of key osteoblastic molecules, including runt-related transcription factor 2 (RUNX2), distal-less homeobox 5 (DLX5), osterix (OSX), alkaline phosphatase (ALP), collagen type I, osteopontin (OPN), and osteocalcin (OCN). These molecules are interconnected and work cooperatively at different stages of osteoblast development and bone matrix deposition [7,8,9,10]. In addition, osteoclasts are multinucleated cells that are differentiated from the mononuclear cells of the monocyte and macrophage lineages. Receptor activator of nuclear factor κB ligand (RANKL) stimulation induces osteoclast differentiation through activating phosphoinositide 3 kinase (PI3K)/Akt, MAPKs, nuclear factor-κB (NF-κB), nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and c-Fos-dependent mechanisms [11,12], with differentiation markers including RANK, tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK), as well as NFATc1 and c-Fos. Disrupting the coordinated balance between osteoblast and osteoclast activities fails to maintain bone homeostasis, leading to a variety of bone diseases including osteoporosis; therefore, understanding the cellular and molecular mechanisms of bone remodeling and homeostasis can provide novel therapeutic strategies and targets in bone diseases.

Natural remedies are gaining global prominence as conventional or adjunctive strategies for promoting health and preventing disease. It is widely appreciated that natural remedies show minimal side effects and favorable safety profiles. Many plant-derived natural products such as terpenoids (ursolic acid, costunolide, osthole, etc.), flavonoids (icariin, puerarin, naringin, etc.), polyphenols (vanilic acid, resveratrol, cinnamic acid, etc.), alkaloids (berberine, harmine, sanguinarine, etc.), and lignans (schisandrin, pinoresinol, sesamin, etc.) have been reported to regulate osteoblastic and/or osteoclastic activities by multiple signaling pathways, including MAPKs, PI3K/Akt, and NF-κB [13,14,15,16].

M. kobus is a large deciduous tree found in the Republic of Korea and Japan that grows 5–10 m tall and belongs to the Magnoliaceae family [17]. Dried flower buds of M. kobus have long been used for treating sinusitis and allergic rhinitis [18]. Magnolin (1-(3,4-dimethoxyphenyl)tetrahydro-4-(3,4,5-trimethoxyphenyl)-furo [3,4-c]furan) is an active ingredient isolated from M. kobus that is reported to be abundant in its flower buds and barks [19,20]. The magnolin content in M. kobus flower water extract has been found to be approximately 1.5% [21]. Previous studies indicate that magnolin suppresses the production of tumor necrosis factor-α (TNF-α), nitric oxide, and prostaglandin E₂, and inhibits the activation of MAPK [19,22,23]. Although the diverse pharmacological properties of M. kobus flower buds and magnolin have been studied, the roles and molecular mechanisms of M. kobus on bone formation are poorly understood. Therefore, in this study, we intended to examine the effects and molecular mechanisms of M. kobus flower water extract (ME) and the ME-derived component magnolin on the differentiation of MC3T3-E1 pre-osteoblasts and RAW264.7 pre-osteoclasts.

2. Results

2.1. ME and Magnolin Enhance Osteoblastogenesis in MC3T3-E1 Cells

We first examined the effect of ME or the ME-derived component magnolin on cell viability in MC3T3-E1 cells. As shown in Figure 1A (left panel), treatment with ME (25–200 μg/mL) did not affect cell viability, except for the case of the highest concentration used in this experiment (400 μg/mL). Similarly, magnolin showed no cytotoxicity at concentrations lower than 20 μM (Figure 1A, right panel). In addition, the proliferative response of MC3T3-E1 cells to serum stimulation was not changed by treatment with ME or magnolin, except for when the highest concentration was used, similarly to the cell viability experiments (Figure 1B). We next examined the effect of ME or magnolin on osteogenic differentiation medium (DM)-cultured MC3T3-E1 cell responses. Neither ME nor magnolin altered the osteogenic DM-induced cell proliferation, except for when higher concentrations (200–400 μg/mL and 20–40 μM, respectively) were used (Figure 1C). To rule out the possibility that ME- and magnolin-mediated changes in cell viability and proliferation affected the process and results of cell differentiation, in many subsequent experiments, the effects of ME and magnolin were examined at concentrations up to 100 μg/mL and 10 μM, respectively.

Alkaline phosphatase (ALP) is one of the critical enzymes involved in the initial phases of osteoblastic differentiation, which functions to regulate calcium and phosphate metabolism and is prominently found in the extracellular matrix (ECM) and cells of calcified tissues [24]. Treatment with ME or magnolin dose-dependently promoted ALP expression in osteogenic DM-treated MC3T3-E1 cells (Figure 1D). ME- and magnolin-mediated upregulation of ALP expression is well correlated with elevated ALP activity (Figure 1E). These findings demonstrate that ME and magnolin may exert osteoblastogenic activity through enhanced ALP expression and activity.

Furthermore, magnolin dose-dependently inhibited RANKL-induced TRAP-positive multinucleated cell formation and expression of TRAP and CTSK, the key markers for osteoclasts (Figure A1). This anti-osteoclastogenic activity of magnolin might have been mediated through the suppression of osteoclastogenesis-related transcription factors such as NFATc1 and c-Fos (Figure A2). Taken together, these results demonstrate that ME-derived magnolin possesses both osteoblastogenic and anti-osteoclastogenic activities.

2.2. ME and Magnolin Elevate Mineralization in MC3T3-E1 Cells

Mineralization of bone is the key process by which calcium and phosphate ions are deposited in the ECM, initiating the formation of hydroxyapatite crystals [25]. The degree of mineralization was photometrically quantified using alizarin red S, a plant-based dye with a high affinity for calcium [26,27]. As shown in Figure 2, treatment with ME or magnolin dose-dependently enhanced the matrix mineralization in DM-treated MC3T3-E1 cells. Collectively, these findings strongly suggest that ME and magnolin possess osteoblastogenic activity (Figure 1 and Figure 2).

2.3. ME and Magnolin Promote the Expression of Osteoblastogenic Transcription Factors in MC3T3-E1 Cells

To understand the molecular mechanisms involved in ME- and magnolin-induced osteoblast differentiation, we examined the changes in the levels of RUNX2, DLX5, and OSX, which are pivotal transcription factors that regulate osteoblast differentiation and bone formation. As shown in Figure 3, treatment with ME and magnolin markedly induced the protein expression of RUNX2, DLX5, and OSX in DM-treated MC3T3-E1 cells. These observations suggest that enhanced expression of osteoblastogenic transcription factors might mediate the ability of ME and magnolin to induce osteoblast differentiation.

2.4. ME and Magnolin Induce Osteoblastogenic Markers in MC3T3-E1 Cells

Based on ME- and magnolin-mediated induction of osteoblastogenic transcription factors, we then examined the changes in the levels of osteoblast-differentiation-related proteins such as ALP, collagen type I, OCN, and OPN, which are crucial markers of osteoblast differentiation [10,28]. Although the enhancement of ME and magnolin in Col1a1 and OPN mRNA expression manifested slightly differently, treatment with ME and magnolin significantly elevated the mRNA expression of Alp, Col1a1, and OPN but not that of Ocn (Figure 4), suggesting the putative regulation of early- and intermediate-stage osteoblast differentiation markers.

2.5. ME and Magnolin Enhance Osteoblast Differentiation by Activating p38 MAPK-Dependent Signaling Pathways in MC3T3-E1 Cells

To further elucidate the molecular mechanisms of ME and magnolin in promoting osteoblast differentiation, we examined their effects on the phosphorylation of MAPK signaling pathways [29,30]. As shown in Figure 5, treatment with ME and magnolin significantly enhanced the DM-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, but there was also a minor ME-mediated increase in the phosphorylation of JNK.

Although we cannot exclude the possibility that ME- and magnolin-enhanced osteoblast differentiation is associated with downstream molecules of various signaling pathways such as transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP)/Smad signaling, Wnt/β-catenin signaling, and fibroblast growth factor (FGF)/FGFR signaling, and we while determined that ME and magnolin enhanced the phosphorylation of MAPKs with slightly different potencies, these data suggest that the osteoblastogenic properties of ME and magnolin might be mediated, at least partly, by the activation of MAPK-dependent signaling pathways [1,7].

To determine the contributions of ERK and p38 MAPK activity to ME- and magnolin-mediated osteoblast differentiation, we examined the changes in mineralization in the presence of PD98059 (an inhibitor of the ERK pathway) or SB203580 (an inhibitor of the p38 MAPK pathway). Pretreatment with PD98059 did not alter the ability of ME and magnolin to enhance DM-induced mineralization. However, SB203580 completely abrogated the enhancement of ME and magnolin in mineralization to the levels observed in response to DM induction, indicating the possible involvement of p38 MAPK activity in ME- and magnolin-mediated mineralization (Figure 6). These findings demonstrate that the p38 MAPK-dependent signaling pathway may mediate the enhancement of ME and magnolin in mineralization associated with osteoblast differentiation.

On the other hand, in RAW264.7 cells, magnolin inhibited RANKL-induced activation of MAPKs and NF-κB pathways, which are crucial signaling cascades for osteoclast differentiation (Figure A3 and Figure A4). As shown in Figure A3, magnolin dose-dependently suppressed the phosphorylation of ERK, JNK, and p38 MAPK in response to RANKL stimulation, and abrogated RANKL-induced phosphorylation of IκB kinase (IKK)α/β and IκBα, leading to inhibition of IκBα degradation and subsequent NF-κB activation (Figure A4). Taken together, these findings suggest that magnolin might exert both osteoblastogenic and anti-osteoclastogenic activities by modulating MAPK signaling pathways.

3. Discussion

In the current study, we report that M. kobus flower water extract (ME) and magnolin enhance DM-induced osteoblast differentiation and mineralization in MC3T3-E1 cells and reduce RANKL-induced osteoclast differentiation in RAW264.7 cells. The mechanisms of these osteoblastogenic activities involve the activation of MAPK signaling pathways, including p38 MAPK, and the upregulation of osteogenesis-related molecules, including RUNX2, DLX5, OSX, ALP, collagen type I, and OPN. Magnolin’s enhancing effects and molecular mechanisms on MCT3T3-E1 cell responses are quite similar to the patterns of ME, demonstrating that magnolin might be responsible for the osteoblastogenic activity of ME. Concurrently, in RANKL-treated RAW264.7 cells, magnolin inhibits the activation of MAPK and NF-κB signaling pathways and the expression of osteoclastogenesis-associated molecules such as NFATc1, c-Fos, TRAP, and CTSK. This study represents the first description of the osteoblastogenic and anti-osteoclastogenic activities of M. kobus and magnolin, and their pharmacological potential for managing and treating bone diseases such as osteoporosis. However, to overcome the limitations of findings obtained from mouse cell lines, further research is needed to elucidate molecular mechanisms underlying human bone metabolism and to identify therapeutic targets and strategies for human bone diseases using experimental system based on human cells.

Bone remodeling and homeostasis are coordinately controlled by three main types of cells, osteoblasts, osteoclasts, and osteocytes, which are indispensable for the maintenance of bone strength and integrity. Orchestrated regulation of signal transduction networks and osteogenesis-related molecules such as transcription factors, hormones, and growth factors is essential for osteoblast differentiation, which represents the early stage of bone formation and homeostasis [31,32]. Activation of several key signaling pathways, including Wnt/β-catenin, TGF-β/BMP/Smad, FGF/phosphoinositide 3 kinase/Akt or Raf/ERK, parathyroid hormone/protein kinase A, and hedgehog signaling, has been reported to regulate the expression of osteoblast-differentiation-related transcription factors and proteins [1,9,33,34]. Therefore, an integrative understanding of osteoblast-differentiation-related signaling networks and changes in gene expression may help to develop potential targets and therapeutic strategies for treating bone diseases.

Among the main downstream signaling pathways associated with osteoblast-differentiation-related signaling networks, MAPK signaling pathways including ERK, p38 MAPK, and JNK have been reported to regulate the expression of key transcription factors such as RUNX2, DLX5, and OSX [35]. RUNX2 and DLX5 act as master regulators for the early stages of osteoblast differentiation, while OSX, downstream of RUNX2, regulates the later stages of osteoblast maturation [36]. These transcription factors act in coordination to drive the expression of osteoblast-differentiation-related marker proteins such as ALP, collagen type I, OPN, and OCN, which are essential for ECM formation and mineralization associated with bone formation and strength. Our findings indicate that ME- and magnolin-mediated activation of the p38 MAPK pathway and upregulation of early/intermediate osteoblast differentiation markers such as RUNX2, DLX5, OSX, ALP, collagen type I, and OPN may be, at least in part, involved in osteoblast differentiation and mineralization.

It has recently been reported that in lipopolysaccharide-treated HGF-1 cells and RANKL-treated RAW264.7 cells, M. kobus water extract possesses anti-inflammatory and anti-osteoclastogenic activities by regulating Toll-like receptor 4/NF-κB-dependent inflammatory cytokines and TNF receptor-associated factor 6/MAPK-dependent osteoclast-differentiation-related protein expression, respectively, suggesting the pharmacological potential for periodontal disease management based on inflammation and bone resorption [21]. In this study, magnolin significantly inhibited the RANKL-induced activation of MAPK and NF-κB-signaling, resulting in the suppression of TRAP and CTSK expression.

To our knowledge, there are no previous studies of the effects of M. kobus on osteoblast differentiation. Although its integrative roles and molecular mechanisms in bone formation and remodeling need to be further explored, our findings demonstrate the osteoblastogenic and anti-osteoclastogenic activities of M. kobus and magnolin via putative MAPK-dependent mechanisms, warranting further investigations of M. kobus for the treatment and prevention of bone diseases associated with osteoporosis.

4. Materials and Methods

4.1. Reagents

M. kobus extract (ME) was provided by NUON Co. (Seongnam, Republic of Korea) and only the standardized ME containing approximately 1.5% magnolin was used for consistent quality and effect. The preparation methods of ME were as follows: The flower buds of M. kobus were washed with water and dried in air. The dried samples were subjected to extraction with distilled water at room temperature for 48 h, followed by concentration using a rotary evaporator and a subsequent freeze-drying process to obtain ME [17,21]. Magnolin (SML2438) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and stock solutions of ME and magnolin were dissolved in 100% dimethyl sulfoxide (DMSO, D1370) (Duchefa Biochemie, Haarlem, The Netherlands). The final concentration of DMSO in all experimental controls and treatments was 0.1% to avoid impacting cellular responses, including cytotoxicity and differentiation. The following agents were obtained from commercial sources: anti-DLX5 (sc-398150), anti-cathepsin K (sc-48353), and anti-osterix (sc-393325) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-RUNX2 (12556), anti-NFAT2 (NFATc1, 8032), anti-c-Fos (2250), anti-phospho-ERK (T202/Y204) (9101), anti-phospho-p38 MAPK (T180/Y182) (9215), anti-phospho-JNK (T183/Y185) (9251), anti-ERK (9102), anti-p38 MAPK (9212), anti-JNK (9252), anti-phospho-IKKα/β (2697), anti-IKKβ (8943), anti-phospho-IκBα (2859), anti-IκBα (4814), anti-actin (4970) antibodies, and rabbit and mouse IgG-horseradish peroxidase conjugates (Cell Signaling Technology, Beverly, MA, USA); and PD98059 (S1177) and SB203580 (S1076) (Selleck Chemicals, Houston, TX, USA).

4.2. Cell Culture Conditions

MC3T3-E1 subclone 4 (CRL-2593) and RAW264.7 (TIB-71) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MC3T3-El cells were grown in 10% fetal bovine serum (FBS)–minimum essential medium (MEM) alpha (A10490-01) with 1% antibiotics (100 U/mL penicillin–100 μg/mL streptomycin, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and used within passage 16 for all experiments [37]. In order to induce osteoblast differentiation, MC3T3-E1 cells were cultured in osteogenic differentiation medium (DM) consisting of MEM alpha supplemented with 10% FBS, 1% antibiotics, 10 mM β-glycerophosphate (G9422) (Sigma-Aldrich), and 50 µg/mL ascorbic acid (A4544) (Sigma-Aldrich). RAW264.7 cells were cultured in 10% FBS–Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) with 1% antibiotics. For osteoclast differentiation, RAW264.7 cells were treated with MEM alpha supplemented with 10% FBS, 1% antibiotics, and 25 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA).

4.3. Cell Viability and Proliferation

MC3T3-E1 cells were seeded at a density of 5 × 10³ cells/well in 96-well plates and incubated in 10% FBS-MEM alpha for 3 days. After 3 days, cells were treated with different concentrations of ME or magnolin for 24 h in serum-free media. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [38]. Following the culture for 24 h, the supernatant was removed and 100 µL of MTT solution (0.5 mg/mL, Duchefa Biochemie) was added to each well. After incubation for 1 h, the supernatant was aspirated and dissolved in 100 µL of DMSO per well. The absorbance at 540 nm was measured by using the SpectraMAX 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Changes in cell proliferation were determined using the CellTiter 96^® AQueous One Solution reagent (Promega, Madison, WI, USA) [39]. MC3T3-E1 cells (5 × 10³ cells/well in 96-well plates) were incubated in 10% FBS-MEM alpha for 3 days. After 3 days, cells were treated with ME or magnolin for 24 h in 10% FBS-MEM alpha. For the osteoblast differentiation experiments, MC3T3-E1 cells (1 × 10⁵ cells/well in 6-well plates) were incubated in 10% FBS-MEM alpha for 3 days. After 3 days, cells were treated with ME or magnolin for 21 days in osteogenic DM (10% FBS-MEM alpha containing 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid). The culture medium was exchanged every third day. On the twenty-first day, the absorbance at 490 nm was measured using the SpectraMAX 190 microplate reader. For osteoclast differentiation experiments, RAW264.7 cells were treated with magnolin for 3 days in 10% FBS MEM alpha containing 25 ng/mL RANKL.

4.4. RNA Purification and Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)

RNA was isolated and determined using the TRIzol^® reagent and the NanoDrop^TM 2000 spectrophotometer (Thermo Fisher Scientific). cDNA synthesis was carried out using the SuperScript^® III first-strand synthesis system (Thermo Fisher Scientific), followed by the TOPreal™ SYBR Green Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR) kit (Enzynomics, Daejeon, Republic of Korea) and CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA).

Primer sets for Ocn were forward 5’-CTGACCTCACAGATCCCAAGC-3’ and reverse 5’-TGGTCTGATAGCTCGTCACAAG-3’; for OPN(Spp1) were forward 5’-ATCTCACCATTCGGATGAGTCT-3’ and reverse 5’-TGTAGGGACGATTGGAGTGAAA; for Alp were forward 5’-CCAACTCTTTTGTGCCAGAGA-3’ and reverse 5’-GGCTACATTGGTGTTGAGCTTTT-3’; for Col1a1 were forward 5’-GCTCCTCTTAGGGGCCACT-3’ and reverse 5’-ATTGGGGACCCTTAGGCCAT-3’; and for glyceraldehydes-3-phosphate dehydrogenase (Gapdh) were forward 5’-ACCACAGTCCATGCCATCAC-3’ and reverse 5’-CCACCACCCTGTTGCTGTAG-3’ [40,41,42].

4.5. Western Blot Analysis

MC3T3-E1 cells were seeded at a density of 2 × 10⁴ cells/well in 6-well plates and incubated for 5 days in 10% FBS-MEM alpha. They were then cultured in osteogenic DM (10% FBS-MEM alpha containing 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid) treated with various concentrations of ME or magnolin for the indicated times. RAW264.7 cells were cultured in 10% FBS-MEM alpha containing 25 ng/mL RANKL in the presence or absence of magnolin. Cells were rinsed twice with phosphate-buffered saline (PBS, pH 7.4) and lysed with RIPA buffer (Sigma-Aldrich) including 1% protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Cell lysates were subjected to Western blot analysis, as previously described [43,44]. Band intensities were integrated and quantified using NIH ImageJ version 1.51j8 software.

4.6. Alkaline Phosphatase (ALP) Staining and Assay

MC3T3-E1 cells were cultured in osteogenic DM with various concentrations of ME or magnolin for fourteen days, with the culture medium exchanged every third day. On the fourteenth day, cells were fixed in 4% formaldehyde solution for ALP staining or lysed to measure ALP activity directly. Cells were stained with the TRACP and ALP double-stain kit (Takara bio, Kusatsu, Shiga, Japan). Purple-stained ALP was observed and photographed using a CKX53 inverted microscope (Olympus, Tokyo, Japan). In addition, ALP activity was determined using the ALP assay kit (Abcam, Cambridge, UK) and the SpectraMAX 190 microplate reader (Molecular Devices).

4.7. Alizarin Red S Staining and Mineralization Assay

After culturing MC3T3-E1 cells to full confluence, they were cultured in osteogenic DM treated with various concentrations of ME or magnolin for 21 days in the presence or absence of PD98059 (25 μM) and SB203580 (5 μM). The culture medium was exchanged every third day. On the twenty-first day, cells were washed with PBS, fixed in 4% formaldehyde solution (Sigma-Aldrich) for 15 min, and rinsed with distilled water. Cells were stained with alizarin red S (ARS, Science cell, Carlsbad, CA, USA) solution for 1 h, washed with distilled water, and observed using a CKX53 inverted microscope. For quantitative analysis, the stained dye was eluted with 10% cetylpyridinium chloride (Sigma-Aldrich) for 30 min and measured at 562 nm using the SpectraMAX 190 microplate reader [45].

4.8. Tartrate-Resistant Acid Phosphatase (TRAP) Staining Assay

RAW264.7 cells were seeded at a density of 5 × 10⁴ cells/well in 6-well plates and incubated for 24 h. Cells were pretreated with magnolin in 10% FBS-MEM alpha for 1 h, followed by undergoing RANKL (25 ng/mL) stimulation for 3 days. On the third day, cells were washed with PBS, and fixed in 4% formaldehyde solution for 15 min at room temperature, and washed with distilled water. Cells were stained with TRACP and ALP double-stain kit (Takara bio), according to the manufacturer’s instructions. TRAP-positive cells were observed and photographed using a CKX53 light microscope.

4.9. Statistical Analysis

Results were expressed as the mean ± standard deviation from at least three different experiments. Statistical analysis was conducted using one-way ANOVA followed by Dunnett’s test (GraphPad Prism 10.4.1) (GraphPad Software Inc., San Diego, CA, USA), with statistical significance considered when p < 0.05.