Antiosteoporotic activity of nodakenetin, a coumarin compound from Angelica decursiva, by activation of the Wnt/β-catenin signaling pathway

Abstract

Angelica decursiva (Umbelliferae) is a medicinal plant widely used to treat colds, coughs and fevers in Korea, Japan, and mainland China. The anti-inflammatory activity of nodakenetin, a furano-coumarin compound from A. decursiva, has been reported, although, the antiosteoporotic activity remains unknown. This study sought to investigate the antiosteoporotic activity and precise mechanism of action of nodakenetin in vitro cell culture and in vivo bone remodeling models. The transcriptional activity of nodakenetin on the Wnt signaling pathway was assessed using the TOPflash/FOPflash assay. The effect of nodakenetin on the osteoblast differentiation was measured using Alizarin red staining and alkaline phosphatase (ALP) activity. Western blotting and real-time RT-PCR were used to assess the effect of nodakenetin on the expression of markers related to Wnt/β-catenin pathway and osteoblast differentiation. The in vivo antiosteoporotic activity of nodakenetin was assessed using an ovariectomized (OVX)-induced bone loss mouse model. Nodakenetin activated the Wnt/β-catenin pathway through regulation of DKK1, β-catenin and other target proteins of the Wnt/β-catenin pathway in HEK293 and MC3T3-E1 cells. Nodakenetin induced the differentiation of MC3T3-E1 cells as shown by enhanced Alizarin red staining and ALP activity. Induction of osteoblast differentiation was related to upregulated expression of bone formation biomarkers such as bone morphogenic proteins and Runx2. Oral administration of nodakenetin in the OVX mouse model effectively protected the deterioration of bone microstructure in OVX mice. Nodakenetin exhibits antiosteoporotic activity in vitro and in vivo through the activation of the Wnt/β-catenin pathway and subsequent induction of osteoblast differentiation.

Graphical abstract

Abbreviations

ALP

alkaline phosphatase

BMP-2/4

bone morphogenic protein-2/4

BMD

bone mineral density

BV/TV

bone volume

Cbfa1

core binding factor a1

DMEM

Dulbecco's modified Eagle medium

DKK1

Dickkopf-1

FBS

fetal bovine serum

GSK3β

glycogen synthase kinase-3β

LEF

lymphoid enhancer factor

LDL

low-density lipoprotein

MEM-α

minium essential medium-α

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NF-kB

nuclear factor kB

p-NPP

p-nitrophenylphosphate

OVX

ovariectomized

PBS

phosphate-buffered saline

RANKL

receptor for activation of nuclear factor kB ligand

Runx2

runt-related transcription factor 2

RT-PCR

reverse transcription-polymerase chain reaction

SRB

sulforhodamine B

TCF

T-cell factor

Tb. No

trabecular number

Tb. Sp

trabecular separation

1. Introduction

Osteoporosis is characterized by decreased bone density and increased bone fragility, which subsequently causes substantial morbidity in older people.^1,2 Osteoporosis is related to a homeostatic imbalance between bone formation and resorption, and the etiology is attributed to factors such as aging, changing endocrine conditions (menopause), systemic inflammation, and poor nutritional status.^2,3

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption.⁴ Osteoblasts are derived from mesenchymal stem cells and induce bone formation and the increase in bone mass by secretion of osteoids and inhibition of osteoclast activity, whereas osteoclasts are multinucleated cells differentiated from monocyte/macrophage precursors and are involved in bone resorption. The differentiation and activation of osteoclasts are modulated by receptor for activation of nuclear factor kB (NF-kB) ligand (RANKL). RANKL is expressed on the surface of osteoblast-lineage cells and interacts with its receptor RANK on osteoclast precursors. RANKL stimulates the resorptive activity of osteoclast by targeting the mature osteoclasts.⁵ Disruption of this bone homeostasis can lead to osteoporosis.

Osteoporosis treatment often involves antiresorptive drugs, such as estrogen, selective estrogen receptor modulators, and bisphosphonates, which work by inhibiting osteoclast function.⁶ Although these drugs have shown positive results, their prolonged use is restricted because of many detrimental side effects, such as gastrointestinal intolerance, breast cancer and uterine bleeding.6, 7, 8 Therefore, the development of new therapies is essential. A promising strategy is to stimulate osteoblastic bone formation rather than solely inhibit osteoclast activity. In line with this approach, we focused on natural products to identify compounds with better therapeutic efficacy and fewer side effects.

To discover novel anabolic drugs for treating osteoporosis from natural products, we screened natural compounds for their ability to activate the Wnt/β-catenin signaling pathway. This pathway plays a key role in regulating bone mass through various mechanisms, including the renewal of stem cells, stimulation of preosteoblast replication, induction of osteoblastogenesis, and inhibition of osteoblast apoptosis.^9,10 The activation efficacy of the Wnt/β-catenin signaling pathway was tested using a TOPflash reporter gene assay, and nodakenetin (Fig. 1) from Angelica decursiva Franch. & Sav. (Umbelliferae) exhibited significant activity among the tested compounds. A. decursiva is a medicinal plant widely distributed in Korea, Japan and mainland China, and has been traditionally used to treat colds, coughs and fevers. Various classes of compounds such as pyrano-coumarins, furano-coumarins and lignans have been isolated from A. decursiva.¹¹ Nodakenetin is a furano-coumarin compound with reported anti-inflammatory activity.¹²

Fig. 1.

Chemical structure of nodakenetin.

The Wnt/β-catenin signaling pathway plays a pivotal role in bone formation through its interaction with bone morphogenic proteins (BMPs) and activation of key osteogenic genes, including runt-related transcription factor 2 (Runx2). The synergy between BMP and Wnt signaling leads to enhanced expression of osteogenic genes such as Dlx 5, Msx2, and Runx2, which are essential for osteoblast differentiation. BMPs activate the SMAD pathway, which directly regulates these genes, while Wnt/β-catenin signaling further enhances their transcriptional activation. TCF/LEF1, acting as a downstream transcription factor of the Wnt/β-catenin signaling pathway recruits coactivators and enhances the expression of these osteogenic genes.^10,13 Herein, we demonstrated that nodakenetin exhibited antiosteoporotic activity in both in vitro cell culture and in vivo bone remodeling models, showing that its mechanism is mediated by the activation of the Wnt/β-catenin signaling pathway.

2. Material and methods

2.1. Chemicals

Dulbecco's modified Eagle medium (DMEM), minium essential medium alpha (MEM-α, GIBCO, Custom Product, Catalog No. A1049001), fetal bovine serum (FBS), antibiotics-antimycotics solution and trypsin-EDTA were purchased from Invitrogen Co. (Grand Island, NY, USA). Goat antirabbit IgG-HRP, goat anti-mouse IgG-HRP, goat anti-goat IgG-HRP, cyclin D1, survivin, BMP2, β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against p-β-catenin, ERK1/2, p-ERK1/2, DKK1, p-GSK3β(Y216), p-GSK3α/β(S21/9) and GSK3β were purchased from Cell Signaling Technology (Beverly, MA, USA). Gene-specific primers were synthesized by Bioneer (Daejeon, Korea). AMV reverse transcriptase, dNTP mixture, random primer, RNasin, and Taq polymerase were purchased from Promega (Madison, WI, USA). L-ascorbic acid, β-glycerophosphate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. Nodakenetin was isolated from Angelica decursiva according to a previous report.¹⁴

2.2. Cell culture

Mouse calvaria MC3T3-E1 cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), were cultured in α-MEM supplemented with 10 % heat-inactivated fetal bovine sera, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. Cells were incubated at 37 °C and 5 % CO₂ in a humidified atmosphere.

2.3. Transfection and luciferase reporter gene assay

Transient transfections were conducted using Lipofectamine 2000 transfection reagent (Invitrogen, Waltham, MA, USA). Human embryonic kidney (HEK293) cells were seeded in 48-well plates, and then transfected with 0.1 μg of a luciferase reporter plasmid (TOPflash or FOPflash) and 0.005 μg of the Renilla luciferase vector for normalization. Cells were also cotransfected with 0.02 μg of the pcDNA β-catenin expression vector and 0.004 μg of the TCF4 expression vector to activate the Wnt signaling pathway. After 24 h transfection, cells were treated with a test compound. After incubation for an additional 24 h, cells were lysed and the lysate assessed in a dual luciferase activity assay using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Luciferase activity was measured and calculated relative to that of the vehicle control.

2.4. Sulforhodamine B (SRB) assay

Cells were seeded in 96-well plates at 1 × 10⁴ cells/well, and compounds diluted to various concentrations in medium were added to the cells and then incubated for 72 h. Cells were fixed with 10 % trichloroacetic acid solution for 30 min at 4 °C, washed five times with tap water, and air dried. Cells were stained with 0.4 % SRB in 1 % acetic acid solution for 30 min at room temperature. After washing to remove unbound dye and drying, stained cells were dissolved in 10 mM Tris (pH 10.0), and the absorbance measured at 515 nm. Cell viability was calculated by comparison with absorbance of the vehicle-treated control group. The concentration of 50 % cell survival (IC₅₀) was determined by nonlinear regression analysis using Tablecurve software.

2.5. MTT cell viability assay

Cells were seeded in 96-well plates at 1 × 10⁴ cells/well, and compounds diluted to various concentrations in medium were added to the cells with continued incubation for 72 h. MTT (5 mg/mL in PBS) was added to the media (at a final concentration of 500 μg/mL) and further incubated for 4 h. The media was discarded, and 200 μL of dimethyl sulfoxide was added to each well to dissolve the formazan. The absorbance was measured at 570 nm. The IC₅₀ was determined by nonlinear regression analysis using Tablecurve software.

2.6. Mineralization assay

Cells were seeded in 48-well plates at 0.6 × 10⁴ cells/well and stimulated with 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate and incubated with or without various concentrations of nodakenetin for 10 days. Cells were washed twice with PBS and fixed with 70 % ethanol for 30 min. Fixed cells were stained with 2 % Alizarin red S solution (pH 4.0) for 5 min. The plate was washed several times using distilled water, and cells were then observed under a microscope. Cells were solubilized with 10 % cetylpyridinium chloride for 15 min and the absorbance measured at 570 nm for quantification of the dye intensity.

2.7. Alkaline phosphatase (ALP) activity

MC3T3-E1 cells were incubated in osteogenic medium containing 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate. Cells were seeded in 48-well plates at 0.6 × 10⁴ cells/well and incubated with or without various concentrations of nodakenetin for 6 days. Cells were washed with phosphate-buffered saline (PBS), fixed using 70 % ethanol for 5 min, and then extracted in lysis solution (10 mM Tris and 0.1 % Triton X-100 buffer, pH 7.5). Enzymatic activity was determined using p-nitrophenylphosphate (p-NPP) as a substrate. The color change of p-NPP to p-nitrophenol was measured at 405 nm. Protein concentrations in the cell lysates were measured using the Bradford assay at 595 nm.

2.8. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR)

MC3T3-E1 cells were seeded in 100 mm² dishes (8 × 10⁴ cells/mL) and exposed to various concentrations of nodakenetin for 12 h. Total cellular RNA was extracted with TRIzol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's recommendations. Total RNA (1 μg) was reverse-transcribed using oligo-(dT)15 primers and avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA). Real-time PCR was conducted with the MiniOpticon system (Bio- Rad, Hercules, CA, USA), using reverse transcription product, iQ™ SYBRs Green Supermix (Bio-Rad) 5 μL, and primers to a total volume of 20 μL. The standard thermal cycler conditions were employed: 20 s at 95 °C, 40 cycles of 20 s at 95 °C, 20 s at 56 °C, and 30 s at 72 °C, followed by 1 min at 95 °C, and 1 min at 55 °C. The threshold cycle (Ct), the fractional cycle number where the amount of amplified target gene reaches a fixed threshold, was determined using MJ Opticon Monitor software. Mean Ct values for each transcript were normalized by dividing them by the mean Ct value of the β-actin transcript for that sample. Normalized transcript levels were expressed relatively to samples obtained from controls. The following primers were used: β-catenin F5ʹ-TGCAGATCTTGGACTGGACA-3ʹ; β-catenin R5ʹ-AAGAACGGTAGCTGGGATCA-3ʹ; BMP-2 F5ʹ-TGAGGATTAGCAGGTCTTTG-3ʹ; BMP-2 R5ʹ-CACAACCATGTCCTGATAAT-3ʹ; Runx2 F5ʹ-AAGTGCGGTGCAAACTTTCT-3ʹ; Runx2 R5ʹ-TCTCGGTGGCTGGTAGTGA-3ʹ; β-actin F5ʹ-ACCAGAGGCATACAGGGACA-3ʹ; and β-actin R5ʹ-CTAAGGCCAACCGTGAAAG-3ʹ. Gene-specific primers were synthesized by Bioneer (Daejeon, Korea).

2.9. Western blot analysis

Cultured cells were collected in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, and 0.5 % NP-40 with protease and phosphatase inhibitor cocktails. After 10 min of centrifugation (15,000 rpm), supernatant containing protein was collected. The protein concentration was measured using the bicinchoninic acid method. Immunoblotting was performed via a standard protocol. Western blot results were visualized using enhanced chemiluminescence solution (Intron, Daejeon, Korea) on LAS-4000 (Fuji Film Corp., Tokyo, Japan).

2.10. Animal study

Female ICR mice (18–20 g, 8 weeks old) were purchased from Central Laboratory Animal Inc. (Seoul, Korea) and housed under standard laboratory conditions with free access to food and water. The temperature was regulated to 22 °C ± 2 °C, and a 12 h light/dark schedule was maintained. Mice were allowed 1 week for acclimatization to the laboratory environment. All animal experiments were performed according to the Institutional Animal Care and Use Committee Guidelines of Seoul National University (permission number: SNU-140425-5).

At 9 weeks of age, the mice were bilaterally ovariectomized (OVX); eight mice underwent sham operation. After 1 week of recovery from surgery, the OVX mice were randomly divided into four groups of eight mice each: the OVX control, 17β-estradiol (E2) (10 μg/kg), and nodakenetin (50 or 100 mg/kg) groups. Nodakenetin was orally administered in distilled water (0.2 mL) for 12 weeks, and the same volume of distilled water was used for the sham and OVX control groups. Sham, OVX, and E2 were administered through an oral gavage. After 12 weeks of treatment, the animals were euthanized. Femur bones were dissected and divested of soft tissue for trabecular microarchitecture analysis.

2.11. Analysis of bone microarchitecture

The bone microarchitecture of the femur was assessed using microcomputed tomography (μCT system, SkyScan 1076, Aart-selaar, Belgium) 0.6–2.1 mm from the growth plate. The X-ray source was set at 50 kV and 200 μA and filtered using a 0.5 mm aluminum filter. The scanning angular rotation was 180° with angular steps of 0.5°. The voxel size was 8.9 μm. The morphometric index of the bone region was measured from microtomographic data using three-dimensional (3D) imagery. Bone morphometric parameters, including bone mineral density (BMD), bone volume (BV/TV), trabecular number (Tb. No.) and trabecular separation (Tb. Sp), were calculated with the CTan software (SkyScan, Kontich, Belgium).

2.12. Statistical analysis

All experiments were repeated at least three times. Data are presented as the mean ± SD for the indicated number of independently performed experiments. The results were evaluated using one-way analysis of variation (ANOVA) coupled with Dunnett's t-test in Tablecurve software for comparisons with the vehicle. Significant differences were defined as p-values less than 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

3. Results

3.1. Effect of nodakenetin on the β-catenin/TCF transcription activity

Wnt/β-catenin signaling is known to be involved in the preosteoblast to osteoblast differentiation. We examined the transcriptional activity of nodakenetin on the Wnt signaling pathway using the TOPflash (TCF reporter plasmid)/FOPflash (mutant TCF-binding sites) assay. HEK293 cells with TOPflash gene plasmid, enabled production of luciferase in response to β-catenin-mediated TCF/LEF transcriptional activation; the FOPflash gene plasmid was used as a negative control.

Cells transfected with plasmids bearing TCF4 and β-catenin genes exhibited significant activation of TCF/LEF transcriptional activity following treatment with nodakenetin in a concentration-dependent manner up to 50 μM, however, the FOPflash activity was hardly affected by nodakenetin (Fig. 2A). When H₂O and EtOH extracts were also tested for comparison, H₂O extract showed significant activity whereas EtOH extract showed minimum activity (Supplementary Fig. 1). The cytotoxicity of nodakenetin was tested with the SRB assay (Fig. 2B), which showed no cytotoxic activity even at higher concentrations (100 μM). These results suggest that nodakenetin selectively activates endogenous Wnt signaling by activation of Wnt/β-catenin/TCF responsive transcriptional activity without inducing cytotoxicity.

Fig. 2.

Effect of nodakenetin on the β-catenin/TCF transcription activity in HEK293 cells (A) β-catenin/TCF responsive transcription by nodakenetin. HEK293 cells were transiently transfected with β-catenin, TCF4, TOPflash and Renilla for 24 h and treated with nodakenetin (6.25, 12.5, 25 and 50 μM) for 24 h. Luciferase activity was estimated using the Dual Luciferase Reporter Assay System (Promega) and expressed as relative units. Transfection efficiency was normalized by Renilla (*p < 0.05, **p < 0.01). (B) Cytotoxicity effect of nodakenetin. HEK293 cells were treated indicated concentrations of nodakenetin for 72 h. Cell viability was measured using the SRB assay.

3.2. Effect of nodakenetin on the Wnt signaling pathway in HEK293 cells

Western blotting was performed to evaluate the effect of nodakenetin on the expression of Wnt signaling-related proteins (Fig. 3A). Treatment with nodakenetin decreased the level of DKK1, a negative regulator of Wnt signaling, but increased that of β-catenin, a crucial transcriptional coregulator, in a concentration-dependent manner. The level of p-β-catenin was also decreased, and this indicates that the degradation of β-catenin was decreased.

Fig. 3.

Effect of nodakenetin on the Wnt signaling pathway in HEK293 cells (A) Effect of nodakenetin on the protein expressions of Wnt signaling components. HEK293 cells were treated with indicated concentrations of nodakenetin for 24 h, and the expressions of Wnt signaling components were determined by Western blot analysis. β-actin was used as an internal standard. (B) Effect of nodakenetin on the protein expressions of Wnt signaling target proteins. HEK293 cells were treated with indicated concentrations of nodakenetin for 24 h, and the expressions of c-Myc, cyclin D1 and survivin were determined by Western blot analysis. β-actin was used as an internal standard.

Nodakenetin treatment had minimal effect on the level of GSK3β. However, the level of p-GSK3β (Tyr216) was increased by treatment with nodakenetin in a concentration-dependent manner, whereas that of p-GSK3β (Ser9) was decreased. Therefore, nodakenetin can activate Wnt signaling, which requires phosphorylation of GSK3β (Tyr216)¹⁵ and p-GSK3β (Ser9) is known to inactivate GSK3β activity.¹⁶ Moreover, the expression of c-Myc, cyclin D1, and survivin, which are all Wnt signaling target genes, was also increased by treatment with nodakenetin in a concentration-dependent manner (Fig. 3B).

3.3. Effect of nodakenetin on the Wnt signaling pathway in MC3T3-E1 cells

Western blotting was performed to evaluate the effect of nodakenetin on the Wnt signaling pathway in MC3T3-E1 (preosteoblast) cells. The cytotoxicity of nodakenetin against MC3T3-E1 cells was first tested using the MTT assay, and no cytotoxic activity was observed with up to 100 μM of nodakenetin treatment (Fig. 4A).

Fig. 4.

Effect of nodakenetin on the Wnt signaling pathway in MC3T3-E1 cells (A) Cytotoxicity effect of nodakenetin. MC3T3-E1 cells were treated indicated concentrations of nodakenetin for 72 h. Cell viability was measured using the MTT assay. (B, C) Effect of nodakenetin on Wnt signaling components and targets. (B) MC3T3-E1 cells were treated with indicated concentrations of nodakenetin for 24 h, and the protein expressions of Wnt signaling components and targets were determined by Western blot analysis. (C) MC3T3-E1 cells were treated with indicated concentrations of nodakenetin for 12 h, and the mRNA expressions of β-catenin were determined by real-time RT-PCR (*p < 0.05, **p < 0.01).

The expression of DKK1 was markedly decreased by treatment with nodakenetin in a concentration-dependent manner, whereas that of β-catenin was increased (Fig. 4B). The level of p-β-catenin (Ser 45), the predegradation form of β-catenin, also decreased with nodakenetin treatment. The expression of survivin, cyclin D1, and c-myc, the downstream target genes of Wnt signaling increased following nodakenetin treatment. The expression of β-catenin mRNA, determined via real-time RT-PCR, was increased with nodakenetin treatment (Fig. 4C). Collectively, these results show that nodakenetin activates Wnt signaling pathway even in preosteoblasts.

3.4. Effect of nodakenetin on osteoblast differentiation

Extracellular matrix mineralization is the main biomarker for osteoblastic maturation. Alizarin red S staining was used to evaluate the effects of nodakenetin osteoblast maturation.¹⁷ MC3T3-E1 cells were cultured with nodakenetin under differentiation conditions for 10 days. Calcification nodules were increased in a concentration-dependent manner in differentiated MC3T3-E1 cells (Fig. 5A). Interestingly, treatment with nodakenetin produced an increase in the number of calcification nodules even in undifferentiated cells. This result indicates that nodakenetin can self-induce mineralization of MC3T3-E1 cells.

Fig. 5.

Effect of nodakenetin on the differentiation of osteoblast in MC3T3-E1 cells (A) Enhancement of matrix mineralization by nodakenetin. Extracellular matrix (ECM) calcium deposits (bone nodule formation) for matrix mineralization were measured by Alizarin red S staining. Cells were cultured in 48-well plate for 10 days. For the osteoblast differentiation, the culture medium was changed to a fresh osteogenic medium containing 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid to initiate matrix mineralization (*p < 0.05, **p < 0.01 was considered statistically significant compared to differentiated MC3T3-E1 cells control. #p < 0.05, ##p < 0.01 was considered statistically significant compared to undifferentiated MC3T3-E1 cells control.). (B) Enhancement of ALP activity by nodakenetin. A colorimetric enzyme assay was used. Increasing effects of indicated concentration nodakenetin on ALP activity were examined in MC3T3-E1 cells on 6 days. Absorbance was measured at λ = 405 nm and compared with p-nitrophenol (PNP) standard. The ALP enzyme activity was expressed as nmol PNP/min/mg protein. The protein contents of lysates were determined by the Bradford method (*p < 0.05, **p < 0.01 was considered statistically significant compared to differentiated MC3T3-E1 cells control. #p < 0.05, ##p < 0.01 was considered statistically significant compared to undifferentiated MC3T3-E1 cells control.).

ALP activity is used as a histochemical biomarker for osteoblasts as ALP is expressed on the osteoblast cell surface.¹⁸ When MC3T3-E1 cells were cultured under differentiation conditions for 6 days, the ALP activity was increased by nodakenetin treatment in a concentration-dependent manner (Fig. 5). Furthermore, the ALP activity was significantly increased by nodakenetin treatment in undifferentiated cells. These results indicate that nodakenetin effectively stimulates osteoblast differentiation of MC3T3-E1 cells.

3.5. Effect of nodakenetin on the level of osteoblast differentiation related proteins

BMPs are the target proteins of Wnt signaling pathway, and regulate the differentiation of osteoblasts by stimulating bone formation.¹³ Runx2 is a crucial transcription factor for osteoblast differentiation, and BMP signaling is known to be required for Runx2-dependent osteoblast phenotype induction.¹⁹ To elucidate the underlying mechanism of osteoblast differentiation in nodakenetin treated MC3T3-E1 cells, the effect of nodakenetin on BMPs and Runx2 was determined using real-time RT-PCR and Western blot analysis.

Nodakenetin treatment significantly increased the levels of BMP2 and BMP4 proteins (Fig. 6A). In addition, nodakenetin treatment induced mRNA expression for BMP2 and Runx2 proteins in MC3T3-E1 cells (Fig. 6B). These results suggest that nodakenetin induced the differentiation of osteoblasts via BMP- and Runx2-related Wnt activation.

Fig. 6.

Effect of nodakenetin on the level of osteoblast differentiation related proteins (A) MC3T3-E1 cells were treated with indicated concentrations of nodakenetin for 24 h, and the protein expressions of Wnt signaling components and targets were determined by Western blot analysis. (B) MC3T3-E1 cells were treated with indicated concentrations of nodakenetin for 12 h, and the mRNA expressions of β-catenin and Runx2 were determined by real-time RT-PCR.

3.6. Effect of nodakenetin on bone loss in OVX-induced mouse model

To evaluate the in vivo antiosteoporotic activity of nodakenetin, a OVX-induced bone loss mouse model was employed. Increased body weight in mice in the OVX group was reversed by oral administration of nodakenetin for 12 weeks after ovariectomy (Fig. 7A). The architecture of the trabecular bone of the femur was observed using 3D-μCT. Trabecular bone architecture was destroyed in the OVX-induced group compared with that in the sham group (Fig. 7B). However, this damage was markedly recovered upon administration of nodakenetin in a dose-dependent manner. A protective effect on trabecular bone architecture under the same condition was confirmed in the estradiol (E2) group, as a positive control. The parameters of microstructural index were analyzed with 3D-μCT (Fig. 7C). The BMD of the OVX group was markedly reduced compared with that of the sham group. However, the BMD of the trabecular bone of the femur in nodakenetin-treated group was significantly and dose-dependently increased compared with that in the OVX group. The reduced BV/TV in the OVX group was significantly recovered in the nodakenetin-treated group. In addition, the reductions in the Tb. No. and Tb. Sp. In the OVX group were markedly recovered with nodakenetin treatment. These results indicate that administration of nodakenetin improved bone properties in the ovariectomized bone loss mouse model.

Fig. 7.

Effect of nodakenetin on bone loss in OVX-induced mouse model (A) Change in body weight over 12 weeks post-ovariectomy. (B) Effect of nodakenetin on bone 3D microCT image of distal femur in OVX mice. (C) Effect of nodakenetin on bone morphometric parameters BMD (g/cm2), BV/TV (%), Tb.No (1/mm), Tb. Sp (mm) as analyzed with micro-CT SkyScan CTAn software. The data are presented as the mean ± SD (n = 8) (*p < 0.05, **p < 0.01 was considered statistically significant compared to OVX group. #p < 0.05, ##p < 0.01 was considered statistically significant compared to sham group.).

4. Discussion

As life expectancy has increased, age-related diseases, such as osteoporosis, have emerged as a significant issue. This condition, characterized by decreased bone density and the deterioration of bone microarchitecture, leads to an increased risk of fractures, especially in the elderly population. Current treatments, primarily antiresorptive drugs like bisphosphonates, inhibit bone resorption but are only partly effective in improving bone mineral density and have limitations due to adverse effects, such as atypical femoral fractures and osteonecrosis of the jaw. Therefore, novel therapies are required, and anabolic (proformative) drugs have attracted wide attention in recent years. Osteoanabolic drugs could potentially effectively stimulate bone formation and provide a more comprehensive treatment approach for osteoporosis.^6,20,21

The frizzled family receptor and LDL coreceptor 5/6 are ligands for Wnt and are presented on the surface of the osteoblasts. Activation of these receptors induces signaling of the canonical Wnt/β-catenin pathway, which subsequently stimulates osteoblast activity.²² Therefore, small molecules that can induce Wnt/β-catenin pathway could be developed as anabolic drugs for osteoporosis. Accordingly, small molecules derived from natural products, such as active compounds found in medicinal plants, could be developed for this role. In this study, we identified nodakenetin, a furano-coumarin isolated from A. decursiva, as being able to activate Wnt/β-catenin signaling via screening with TOPflash reporter gene assay.

Nodakenetin activated the Wnt/β-catenin pathway through regulation of DKK1, β-catenin and other target proteins of Wnt/β-catenin pathway both in HEK293 and MC3T3-E1 cells (Fig. 3, Fig. 4). Increased ALP activity and calcium deposition showed that nodakenetin could induce differentiation of MC3T3-E1 cells (Fig. 5). Furthermore, the induction of osteoblast differentiation was related to increased expression of bone formation biomarkers such as BMPs and Runx2 (Fig. 6). The in vivo antiosteoporotic activity of nodakenetin was assessed using the OVX mouse model which has been widely used as a postmenopausal osteoporosis mimicking model.²³ The oral administration of nodakenetin effectively protected the deterioration of bone microstructure in OVX mice (Fig. 7).

Wnt/β-catenin pathway is diversely related with bone-related diseases,²⁴ and recently, DKK1 has been emerged as a crucial biomarker of these diseases.²⁵ We found that nodakenetin significantly down-regulated the level of DKK1 as well as activated Wnt/β-catenin pathway both in HEK293 and MC3T3-E1 cells.

The findings of the present study indicate that nodakenetin could be a novel anabolic therapeutic agent for osteoporosis, demonstrating efficacy in the activation of the Wnt/β-catenin signaling pathway, thereby promoting osteoblast differentiation and bone formation. Considering the limitations of existing antiresorptive therapies, nodakenetin represents a promising candidate that addresses the need for osteoanabolic agents capable of enhancing bone formation. Nevertheless, further investigations, including comprehensive toxicological evaluations, particularly focusing on potential acute and chronic toxicities, will be crucial to advance nodakenetin towards clinical application as a viable therapeutic option for osteoporosis.

5. Conclusions

The present study shows that nodakenetin exhibits antiosteoporotic activity in vitro and in vivo. The underlying mechanisms of action of nodakenetin correlated with induction of osteoblast differentiation through the activation of the Wnt/β-catenin pathway. Therefore, nodakenetin has a therapeutic potential as a lead compound in development of therapeutic agents for osteoporosis.

Author contributions

EJJ and JS designed this study, performed all the experiments and drafted the manuscript. HJP managed the project. JSC provided nodakenetin. SKL supervised the study. All authors have approved the final version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (NRF-2022R1A2C3005459 and RS-2023-00245570).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: