Osteogenic Activities of Trifolirhizin as a Bioactive Compound for the Differentiation of Osteogenic Cells

Plant extracts are widely used as traditional medicines. Sophora flavescens Aiton-derived natural compounds exert various beneficial effects, such as anti-inflammatory, anticancer, antioxidant, and antiregenerative activities, through their bioactive compounds, including flavonoids and alkaloids. In the present study, we investigated the biological effects of an S. flavescens-derived flavonoid, trifolirhizin (trifol), on the stimulation of osteogenic processes during osteoblast differentiation. Trifol (>98% purity) was successfully isolated from the root of S. flavescens and characterized. Trifol did not exhibit cellular toxicity in osteogenic cells, but promoted alkaline phosphatase (ALP) staining and activity, with enhanced expression of the osteoblast differentiation markers, including Alp, ColI, and Bsp. Trifol induced nuclear runt-related transcription factor 2 (RUNX2) expression during the differentiation of osteogenic cells, and concomitantly stimulated the major osteogenic signaling proteins, including GSK3β, β-catenin, and Smad1/5/8. Among the mitogen-activated protein kinases (MAPKs), Trifol activated JNK, but not ERK1/2 and p38. Trifol also increased the osteoblast-mediated bone-forming phenotypes, including transmigration, F-actin polymerization, and mineral apposition, during osteoblast differentiation. Overall, trifol exhibits bioactive activities related to osteogenic processes via differentiation, migration, and mineralization. Collectively, these results suggest that trifol may serve as an effective phytomedicine for bone diseases such as osteoporosis.


Introduction
Dysregulation in bone metabolism and remodeling are features of serious bone diseases, such as osteoporosis and periodontitis [1].Dysfunction in the proliferation, migration, and differentiation of osteogenic cells leads to deficient and excessive bone formation [2].Mesenchymal progenitor cells differentiate into osteoblasts [3].Mesenchymal progenitor cells differentiate into osteoblasts [3].Osteoblasts are bone-forming cells required for bone synthesis and mineralization during bone development and remodeling [3,4].Mesenchymal progenitor cells undergo differentiation into osteocytes, and the bone matrix eventually surrounds the osteocytes and becomes calcified [5][6][7].Osteoblast differentiation is induced and regulated by multicellular signaling.The Wnt family member 3a (Wnt3a), bone morphogenetic protein 2 (BMP2), and mitogen-activated protein kinase (MAPKs)mediated signaling pathways are mainly responsible for controlling the nuclear expression and activity of runt-related transcription factor 2 (RUNX2), which is a master protein for osteoblast differentiation [8][9][10].Consequently, osteoblasts physiologically produce and secrete different molecules, such as collagen, growth factors, alkaline phosphatase (ALP), and numerous non-collagenous proteins.Osteoblast dysfunction is associated with pathological bone loss [5,6].
In the present study, trifol (>98% purity) was isolated from the roots of S. flavescens.In this study, the pharmacological effects of trifol on osteogenic processes, intracellular signaling, and matrix mineralization during the differentiation of osteogenic MC3T3-E1 cells in vitro were evaluated.

Trifol Increases Osteoblast Differentiation and the Levels of Osteogenic Marker Genes
We investigated whether trifol induced cytotoxicity and osteoblast differentiation in osteogenic MC3T3-E1 cells.In the MTT assay, trifol exerted no cytotoxicity at concentrations up to 50 µM (Figure 2A).As a result, low concentrations (1-10 µM) of trifol, which were found to be non-cytotoxic, were used in subsequent experiments.ALP activity is the main phenotypic marker of osteoblastic differentiation.The effect of trifol on the differentiation of osteogenic cells was determined based on ALP activity.In the ALP staining and activity assays, ALP activity was found to increase in trifol-treated cells compared to cells treated with OS alone (Figure 2B,C).The osteogenic effects of trifol were validated by detecting osteogenic marker genes in trifol-treated cells.Trifol increased the mRNA levels of Alp,  ing and activity assays, ALP activity was found to increase in trifol-treated cells com pared to cells treated with OS alone (Figure 2B,C).The osteogenic effects of trifol were validated by detecting osteogenic marker genes in trifol-treated cells.Trifol increased the mRNA levels of Alp, collagen type I (ColI), and bone sialoprotein (Bsp) compared to OS alone (Figure 2D-F).These data suggest that trifol exhibits biological activity in osteo genic cells.

Trifol Enhances Osteogenic Phenotype and Mineralization in Osteoblast Differentiation
Osteogenic processes induce migration and cytoskeletal changes, leading to osteoblast maturation and bone formation.Trifol was found to increase transmigration across the extracellular matrix (ECM) (Figure 3A,B).Thereafter, cytoskeletal changes were observed using an intravital multiphoton microscope (IMPM).Trifol significantly promoted morphological changes during osteoblast differentiation on Matrigel-coated culture plates (Figure 3C,D).Finally, we investigated whether trifol accelerated mineralization based on the accumulation of calcium deposits.Using alizarin red S (ARS) staining, trifol was found to increase mineralization during osteoblast maturation (Figure 3E,F).These data suggest that trifol induces bone-forming phenotypes, such as cell migration, morphological changes, and calcium deposits, during osteoblast differentiation.
ed morphological changes during osteoblast differentiation on Matrigel-coated culture plates (Figure 3C,D).Finally, we investigated whether trifol accelerated mineralization based on the accumulation of calcium deposits.Using alizarin red S (ARS) staining, trifol was found to increase mineralization during osteoblast maturation (Figure 3E,F).These data suggest that trifol induces bone-forming phenotypes, such as cell migration, morphological changes, and calcium deposits, during osteoblast differentiation.

Trifol Enhances Nuclear RUNX2 Expression and Osteogenic Signaling Proteins in Osteoblast Differentiation
RUNX2 is a master transcription factor involved in osteogenic gene expression and differentiation.RUNX2 expression levels were determined using Western blot analysis.Trifol increased the total RUNX2 expression compared to the control, but did not alter the RUNX2 expression compared to OS (Figure 4A), suggesting that trifol increases the nuclear RUNX2 expression, but not total RUNX2 expression.The expression of RUNX2 was further examined by means of an immunofluorescence assay using PI, a nuclear marker.Nuclear RUNX2 levels were significantly enhanced in trifol-treated cells (Figure 4B).
Trifol increased the total RUNX2 expression compared to the control, but did not alter the RUNX2 expression compared to OS (Figure 4A), suggesting that trifol increases the nuclear RUNX2 expression, but not total RUNX2 expression.The expression of RUNX2 was further examined by means of an immunofluorescence assay using PI, a nuclear marker.Nuclear RUNX2 levels were significantly enhanced in trifol-treated cells (Figure 4B).Expression and localization of RUNX2 due to trifol in osteogenic cells.(A) After osteogenic cells were cultured with OS for 3 days, protein levels of RUNX2 were detected using Western blot analysis, and the levels were standardized using the level of total β-actin, which served as a We proceeded to investigate the cellular signaling proteins involved in nuclear RUNX2 expression.Compared with OS alone, trifol stimulated GSK3β phosphorylation, β-catenin dephosphorylation, β-catenin expression, and Smad1/5/8 phosphorylation, but did not affect Wnt3a or BMP2 expression (Figure 5A,B).In addition, the phosphorylation levels of JNK were significantly elevated in trifol-treated cells (Figure 5C); however, those of ERK1/2 and p38 remained unchanged in the trifol-treated cells (Figure 5C).These data suggest that trifol regulates the major osteogenic signaling proteins, p-GSK3β, β-catenin, p-Smad1/5/8, and p-JNK, to exert its biological activities in osteogenic cells.
loading control.(B,C) At 3 days, RUNX2 levels in the nucleus were monitored via an immunoflu orescence analysis with a RUNX2 antibody (green) and a nuclear marker, PI (red).The arrows in dicate the nuclei.Scale bar: 50 μm (B).Relative levels are displayed in a bar graph (C).OS: osteo genic supplement medium containing 50 μg/mL L-ascorbic acid and 10 mM β-glycerophosphate Data are expressed as the mean ± SEM. * p < 0.05: statistically significant compared to the control.# p < 0.05: statistically significant compared to OS.Data represent the results of three experiments.
We proceeded to investigate the cellular signaling proteins involved in nuclear RUNX2 expression.Compared with OS alone, trifol stimulated GSK3β phosphorylation β-catenin dephosphorylation, β-catenin expression, and Smad1/5/8 phosphorylation, bu did not affect Wnt3a or BMP2 expression (Figure 5A,B).In addition, the phosphorylation levels of JNK were significantly elevated in trifol-treated cells (Figure 5C); however those of ERK1/2 and p38 remained unchanged in the trifol-treated cells (Figure 5C) These data suggest that trifol regulates the major osteogenic signaling proteins, p-GSK3β β-catenin, p-Smad1/5/8, and p-JNK, to exert its biological activities in osteogenic cells.were detected using Western blot analysis.The levels were standardized using the level of total β-actin, which served as a loading control.(C) Phosphorylation levels of MAPKs, including ERK1/2, JNK, and p38, were detected using Western blot analysis.The levels were standardized using those of total ERK1/2, JNK, and p38, respectively, which served as loading controls.OS: osteogenic supplement medium containing 50 µg/mL L-ascorbic acid and 10 mM β-glycerophosphate.Data represent the results of three experiments.*, p < 0.05 compared with the control; #, p < 0.05 compared with OS.Data represent the results of three experiments.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 15 (B), were detected using Western blot analysis.The levels were standardized using the level of total β-actin, which served as a loading control.(C) Phosphorylation levels of MAPKs, including ERK1/2, JNK, and p38, were detected using Western blot analysis.The levels were standardized using those of total ERK1/2, JNK, and p38, respectively, which served as loading controls.OS: osteogenic supplement medium containing 50 μg/mL L-ascorbic acid and 10 mM β-glycerophosphate.Data represent the results of three experiments.*, p < 0.05 compared with the control; #, p < 0.05 compared with OS.Data represent the results of three experiments.

Discussion
In the present study, we investigated the effects of trifol on osteoblast differentiation and maturation.We originally reported novel evidence for bioactive activities related to osteogenic processes via differentiation, migration, and mineralization by a flavonoid S. flavescens-derived trifol.The development, formation, remodeling, and repair of bones are mediated by the osteogenic processes involved in osteoblast differentiation [5,[30][31][32][33].Osteoblast dysfunction causes pathological conditions in bone diseases, such as osteoporosis, Paget's disease, and osteonecrosis [34,35].Currently, the treatment of bone diseases is limited by side effects and high costs [13,[36][37][38].Several plant-derived compounds have been demonstrated to play a stimulating role in the differentiation and mineralization of osteoblasts, leading to improvement of bone diseases and clinical applications [5,6,[37][38][39][40][41][42][43].Based on the findings of the present study, the flavonoid derived from S. flavescens, trifol, promotes osteoblast differentiation and mineralization by increasing osteogenic activity via the stimulation of key proteins (Smad1/5/8 and β-catenin) involved in Wnt3a and BMP2 osteogenic signaling.To date, the intracellular signaling and mechanisms underlying the biological function of trifol in osteogenicity have not been reported.These findings suggest that trifol may be a novel drug source for the treatment of bone diseases such as osteoporosis.
Osteoblast differentiation stimulates the activity and expression of the ALP enzyme, a well-known early osteoblast differentiation marker that induces the hydrolysis of inorganic pyrophosphate and organic phosphomonoesters [5,6,44,45].With increased ALP activity, collagen and non-collagenous proteins promote calcium deposition and hydroxyapatite crystallization on the extracellular matrix for mineralization and bone formation [46,47].Our results show that trifol stimulated ALP activity and expression without inducing cytotoxicity.Trifol also upregulated the expression of the osteogenic genes Alp, ColI, and Bsp.RUNX2 is a key regulator of osteogenic processes and increases the expression of various osteogenic genes, including Alp, ColI, and Bsp, leading to the differentiation and maturation of osteogenic cells [48][49][50][51][52][53].The present study demonstrates that trifol promotes the accumulation of RUNX2 in the nucleus, which aligns with the increased expression of Alp, ColI, and Bsp induced by trifol.These findings suggest that trifol promotes the differentiation of osteogenic cells by increasing ALP activity and mineralized tissue proteins via nuclear RUNX2 accumulation.
Migration phenotypes induce movement into specific niches during osteogenic processes, leading to attachment, osteoid formation, and mineralization [1,[54][55][56][57].The present study demonstrates that trifol accelerates transmigration to the extracellular matrix.The cytoskeletal changes induced by F-actin polymerization promote osteogenic processes [58,59].In addition, trifol-induced morphological changes are mediated by F-actin polymerization.Osteoblast maturation results in bone matrix mineralization via calcium deposits in the extracellular matrix.Based on our findings, trifol promotes mineralization during osteoblast maturation, which is consistent with the trifol-induced increase in ALP enzyme activity.ALP knockout animals exhibit abnormal mineral apposition and bone fractures [45].These findings suggest that trifol exerts anabolic activities that accelerate bone formation and mineral apposition.
The differentiation and mineralization of osteoblasts are regulated by RUNX2, which is activated and increased by the osteogenic BMP2, Wnt3, and MAPKs (ERK1/2, JNK, and p38) signaling pathways [60,61].The binding of BMP2 to BMP2 receptors initiates the canonical Smad1/5/8 and non-canonical MAPKs pathways, and induces RUNX2 activity and expression in the nucleus [62].The binding of Wnt3a to Frizzled and LRP5/6 receptors induces the degradation of GSK3β and the stabilization of β-catenin, which is translocated into the nucleus and leads to RUNX2 expression in the nucleus [63,64].Our findings demonstrate that trifol stimulates BMP2 and Wnt3 signaling pathway proteins by promoting Smad1/5/8 phosphorylation, JNK phosphorylation, and β-catenin stabilization.A previous study revealed that trifol induces cytotoxicity and apoptosis of gastric cancer cells by inhibiting the Ras/Raf/MEK/ERK pathway [65].However, trifol did not affect ERK phosphorylation.The different effects of trifol on ERK could be attributed to the different concentrations and cell types used in the in vitro experimental system.Our data also showed that trifol did not cause cytotoxicity in osteogenic cells, unlike in gastric cancer cells.Therefore, trifol exhibits osteogenic activities by regulating RUNX2 via the stimulation of osteogenic signaling proteins in osteogenic cells.Recently, we reported the osteogenic effects of a secoiridoid glycoside, trifloroside (TriFs) from Gentianae Scabrae Radix roots on osteogenic cells [66].Compared with trfiol, TriFs has noncytotoxic effects at low concentrations, but increases cell growth at high concen-trations on osteogenic cells.TriFs increased JNK and p38 phosphorylation but not AKT and ERK phosphorylation.TriFs also increased the total expression of RUNX2 in osteo-blast differentiation.Therefore, trifol exhibits osteogenic activity by regulating RUNX2 through stimulation of osteogenic signaling proteins in osteogenic cells in a manner dif-ferent from previously reported trifol.

Plant Material and Isolation
NMR spectroscopy was conducted using a JEOL ECX-500 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 1 H (500 MHz) and 13 C (125 MHz).HPLC was performed using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA).Silica gel 60 (70-230 mesh/230-400 mesh ASTM, Merck, Darmstadt, Germany), ODS-A (YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography.S. flavescens Aiton was purchased from a commercial herbal medicine company.A voucher specimen was deposited in the Natural Products Bank of the National Institute for Korean Medicine Development (NIKOM, Gyeongsan, Republic of Korea).

Cytotoxicity
Cytotoxicity in osteogenic cells was analyzed using an MTT assay.Live cells were treated with MTT solution, and the absorbance of formazan was recorded on a Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific) at 540 nm.

ALP Staining and Activity Assays
Osteogenic cells were differentiated for 7 days, and ALP staining and activity were performed as previously described [66].

Total RNA Isolation and RT-PCR Analysis
After osteogenic differentiation for 5 days, the TRIzol reagent (Life Technologies, Gaithersburg, MD, USA) was used to extract the total RNA.Thereafter, reverse transcription of RNA into single-stranded cDNA was performed using AccuPower RT PreMix with oligo (dT) 15 primers (Bioneer Corporation, Daejeon, Republic of Korea).The band intensities of the PCR products were quantified under ultraviolet exposure (ProteinSimple Inc., Santa Clara, CA, USA) as previously described [67].

Western Blot Analysis
The cells were lysed, and the concentration of the isolated protein was determined using Bradford reagent (Bio-Rad, Hercules, CA, USA).Equal amounts of protein from each sample were separated via SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA).After blocking with 5% skim milk in 1×TBS containing 0.05% Tween-20 (TBST), washing with TBST, and incubation with specific primary antibodies overnight at 4 • C, the membranes were washed with TBST and incubated with horseradish-peroxidase-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch, West Grove, PA, USA).Protein images were monitored using a ProteinSimple Detection System (ProteinSimple Inc., Santa Clara, CA, USA).

Migration Assay
Cell migration assays were performed using Matrigel-coated membranes in a Boyden chamber as previously described [66].Matrigel solution (Corning Life Sciences, Tewksbury, MA, USA) and 0.5% crystal violet (Sigma-Aldrich) were used.Images were captured using a light microscope.

Phalloidin and DRAQ5 Staining
F-actin polymerization in osteogenic cells was detected using phalloidin staining (Thermo Fisher Scientific), and the nuclei were detected using DRAQ5 (Thermo Fisher Scientific).The images were captured using an IMPM (Leica Microsystems) at the KBSI.

ARS Staining Assay
Mineralization by calcium deposits in the extracellular matrix was performed using ARS dye, as previously described [66].After osteogenic cell differentiation was induced, ARS staining was performed, and images of the stained mineralization were observed using a scanner.

Statistical Analysis
The data were analyzed using Prism Version 5 (GraphPad Software Inc., San Diego, CA, USA) and are presented as the mean ± standard error of the mean (SEM).p < 0.05 was considered statistically significant, as determined using Student's unpaired t-test.

Conclusions
In conclusion, S. flavescens-derived trifol was found to accelerate osteoblast differentiation and maturation by stimulating osteogenic processes via the osteogenic signaling pathways.Notably, the dysregulation of osteogenic processes leads to abnormal bone formation in bone diseases [34,35].Although in vivo experiments should be performed to further investigate trifol-induced bone formation, our in vitro data provide new evidence for the use of trifol as a bone-protective compound and bone healing accelerant through its promotion of bone-forming activities.

Figure 2 .
Figure 2. Cytotoxicity and osteoblast differentiation due to trifol in osteogenic cells.(A) Osteogenic cells were treated with the indicated concentrations of trifol for 24 h.Cell toxicity (%) was analyzed using an MTT assay.(B,C) Cells were treated in OS with the indicated concentrations of trifol for 7 days.ALP levels were measured using ALP staining (B) and activity (C) assays.(D-F) Cells were treated in OS with the indicated concentrations of trifol for 7 days.The mRNA levels of Alp (D), Col (E), and Bsp (F) were detected using PCR; the relative fold is shown as a bar graph.OS: osteogenic supplement medium containing 50 μg/mL L-ascorbic acid and 10 mM β-glycerophosphate.Data are expressed as the mean ± SEM. * p < 0.05: statistically significant compared to the control.#, p < 0.05: statistically significant compared to OS.Data represent the results of three experiments.

Figure 2 .
Figure 2. Cytotoxicity and osteoblast differentiation due to trifol in osteogenic cells.(A) Osteogenic cells were treated with the indicated concentrations of trifol for 24 h.Cell toxicity (%) was analyzed using an MTT assay.(B,C) Cells were treated in OS with the indicated concentrations of trifol for 7 days.ALP levels were measured using ALP staining (B) and activity (C) assays.(D-F) Cells were treated in OS with the indicated concentrations of trifol for 7 days.The mRNA levels of Alp (D), ColI (E), and Bsp (F) were detected using PCR; the relative fold is shown as a bar graph.OS: osteogenic supplement medium containing 50 µg/mL L-ascorbic acid and 10 mM β-glycerophosphate.Data are expressed as the mean ± SEM. * p < 0.05: statistically significant compared to the control.#, p < 0.05: statistically significant compared to OS.Data represent the results of three experiments.

Figure 3 .
Figure 3. Promotion of osteogenic phenotypes and maturation by trifol in osteogenic cells.(A,B) Cells were treated in OS with the indicated concentrations of trifol for 3 days, and cell migration was measured using a Matrigel-coated membrane in a Boyden chamber.Scale bar: 50 μm (A); the relative levels are displayed as a bar graph (B).(C,D) At 3 days, F-actin polymerization was measured using phalloidin (green) and DRAQ5 (red) staining assay.Scale bar: 100 μm.(C); the relative levels are displayed as a bar graph (D).(E,F) At 14 days, mineralization was visualized using ARS staining (E); the relative levels are displayed as a bar graph (F).OS: osteogenic supplement medium containing 50 μg/mL L-ascorbic acid and 10 mM β-glycerophosphate.*, p < 0.05 compared with the control; #, p < 0.05 compared with OS.Data represent the results of three experiments.

Figure 3 .
Figure 3. Promotion of osteogenic phenotypes and maturation by trifol in osteogenic cells.(A,B) Cells were treated in OS with the indicated concentrations of trifol for 3 days, and cell migration was measured using a Matrigel-coated membrane in a Boyden chamber.Scale bar: 50 µm (A); the relative levels are displayed as a bar graph (B).(C,D) At 3 days, F-actin polymerization was measured using phalloidin (green) and DRAQ5 (red) staining assay.Scale bar: 100 µm.(C); the relative levels are displayed as a bar graph (D).(E,F) At 14 days, mineralization was visualized using ARS staining (E); the relative levels are displayed as a bar graph (F).OS: osteogenic supplement medium containing 50 µg/mL L-ascorbic acid and 10 mM β-glycerophosphate.*, p < 0.05 compared with the control; #, p < 0.05 compared with OS.Data represent the results of three experiments.

Figure 4 .
Figure 4.Expression and localization of RUNX2 due to trifol in osteogenic cells.(A) After osteogenic cells were cultured with OS for 3 days, protein levels of RUNX2 were detected using Western blot analysis, and the levels were standardized using the level of total β-actin, which served as a

Figure 4 .
Figure 4.Expression and localization of RUNX2 due to trifol in osteogenic cells.(A) After osteogenic cells were cultured with OS for 3 days, protein levels of RUNX2 were detected using Western blot analysis, and the levels were standardized using the level of total β-actin, which served as a loading control.(B,C) At 3 days, RUNX2 levels in the nucleus were monitored via an immunofluorescence analysis with a RUNX2 antibody (green) and a nuclear marker, PI (red).The arrows indicate the nuclei.Scale bar: 50 µm (B).Relative levels are displayed in a bar graph (C).OS: osteogenic supplement medium containing 50 µg/mL L-ascorbic acid and 10 mM β-glycerophosphate.Data are expressed as the mean ± SEM. * p < 0.05: statistically significant compared to the control.#, p < 0.05: statistically significant compared to OS.Data represent the results of three experiments.

Figure 6 .
Figure 6.Trifol promotes osteoblast differentiation by stimulating osteogenic signaling proteins (A,B) After cells were treated in OS with trifol in the absence or presence of Noggin, PKF, and SP600.At 7 days, ALP levels were measured using ALP activity assay (A).At 14 days, mineralization was measured using ARS staining assay (B).(C) Schematic showing trifol-mediated osteoblast

Figure 6 .
Figure 6.Trifol promotes osteoblast differentiation by stimulating osteogenic signaling proteins (A,B) After cells were treated in OS with trifol in the absence or presence of Noggin, PKF, and SP600.At