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. 2022 Jul 7;26(16):4520–4529. doi: 10.1111/jcmm.17476

Effects of Scoparone on differentiation, adhesion, migration, autophagy and mineralization through the osteogenic signalling pathways

Kyung‐Ran Park 1, Bomi Kim 2, Joon Yeop Lee 2, Ho‐Jin Moon 3, Il Keun Kwon 3,4,✉, Hyung‐Mun Yun 5,✉
PMCID: PMC9357629  PMID: 35796406

Abstract

Scoparone (SCOP), an active and efficient coumarin compound derived from Artemisia capillaris Thunb, has been used as a traditional Chinese herbal medicine. Herein, we investigated the effects of SCOP on the osteogenic processes using MC3T3‐E1 pre‐osteoblasts in in vitro cell systems. SCOP (C11H10O4, > 99.17%) was purified and identified from A. capillaries. SCOP (0.1 to 100 μM concentrations) did not have cytotoxic effects in pre‐osteoblasts; however, it promoted alkaline phosphatase (ALP) staining and activity, and mineralized nodule formation under early and late osteogenic induction. SCOP elevated osteogenic signals through the bone morphogenetic protein 2 (BMP2)‐Smad1/5/8 pathway, leading to the increased expression of runt‐related transcription factor 2 (RUNX2) with its target protein, matrix metallopeptidase 13 (MMP13). SCOP also induced the non‐canonical BMP2‐MAPKs pathway, but not the Wnt3a‐β‐catenin pathway. Moreover, SCOP promoted autophagy, migration and adhesion under the osteogenic induction. Overall, the findings of this study demonstrated that SCOP has osteogenic effects associated with cell differentiation, adhesion, migration, autophagy and mineralization.

Keywords: Artemisia capillaris, autophagy, differentiation, mineralization, osteoblast, SCOP

1. INTRODUCTION

The Artemisia capillaris Thunb is a traditional herbal medicine which possesses remarkable therapeutic and protective effects in hepatic diseases. 1 , 2 , 3 A. capillaris contains coumarin and flavonoid compounds, which possess anti‐oxidant, anti‐inflammatory, anti‐cancer and anti‐osteoporosis activities. 4 , 5 , 6 , 7 , 8 Scoparone (6,7‐dimethoxycoumarin) (SCOP), which is primarily found in Artemisia plant roots, is a coumarin compound and has pharmacological properties relating to vasorelaxation or immunosuppression. 9 , 10 Recently, it has been discovered that SCOP suppresses inflammatory responses by inhibiting PI3K‐Akt signalling in chondrocytes, suggesting that SCOP may be a therapeutic compound in degenerative and bone diseases such as osteoarthritis. 11

Mesenchymal stem cells (MSCs) and pre‐osteoblasts differentiate, adhere and migrate to specific regions, leading to the bone‐specific protein secretion and matrix mineralization. 12 , 13 , 14 Osteoblast differentiation and maturation help multicellular organisms maintain bone tissue homeostasis and control skeletal development, formation and remodelling. 13 , 15 Osteoblast malfunction induces bone formation abnormalities, resulting in low bone mass and fragile fractures in bone diseases. 16 By regulating bone metabolism, anabolic drugs for bone diseases can improve osteoblast differentiation and bone formation; however, there are issues relating to drug safety and cost. 16 , 17 , 18 , 19 , 20 Currently, osteogenic compounds are of increasing interest, as these compounds have relatively fewer side effects and may be used for a longer period of time than previously used drugs for bone diseases. 21 , 22 Thus, it is worth investigating the identification of osteogenic compounds and their pharmacological effects on osteoblast differentiation, migration, adhesion and mineralization.

The purpose of this study was to examine the osteogenic roles of SCOP of dry, aboveground A. capillaris tissue in differentiation, migration, adhesion, autophagy and mineralization in in vitro cell systems using MC3T3‐E1 pre‐osteoblasts that have been studied in osteoblast differentiation. Our findings demonstrated that SCOP has bone‐forming activities, suggesting potential roles as an osteogenic compound.

2. METHODS

2.1. Plant material

Artemisia capillaris Thunb was purchased from Korean medicine Omniherb (www.omnishop.co.kr). The P149 voucher specimen was deposited in the Natural Products Bank (NIKOM). Column chromatography (CC) was conducted using silica gel (Merck, Darmstadt, Germany). 13 C Nuclear magnetic resonance (NMR) and 1H NMR spectra were analysed using JEOL ECX‐500 spectrometer (JEOL Ltd.). Agilent 1260 series (Agilent Technologies) with a C18 column (Phenomenex, United States of America Synergi 10 μ Hydro‐RP 80A, 10 μm, 4.6 mm × 250 mm) was used for high‐performance liquid chromatography (HPLC).

2.2. Culture and differentiation

MC3T3‐E1 pre‐osteoblasts (#CRL‐2593) were obtained from the ATCC (Manassas, VA). Pre‐osteoblasts were grown at 37°C, 5% CO2 and 95% air under a humidified atmosphere in α‐MEM without L‐AA (WELGEME, Inc.) containing 10% foetal bovine serum (FBS) and 1× Gibco antibiotic‐antimycotic (Thermo Fisher Scientific). OS containing 10% FBS, 1× Gibco antibiotic‐antimycotic, 50 μg/ml L‐AA (Sigma‐Aldrich) and 10 mM β‐GP (Sigma‐Aldrich) with SCOP was used to stimulate osteoblast differentiation. 0.1, 1, 5, 10, 20, 30, 40, 50 and 100 mM SCOP stocks were prepared with 100% dimethyl sulfoxide (DMSO) and diluted to a final concentration (1:1000 dilution, 0.1% DMSO); 0.1% DMSO was treated as a control. During osteoblast differentiation, OS was changed every 2 days as described previously. 15

2.3. Cell viability

Cell viability was analysed using MTT solution (Sigma‐Aldrich). Absorbance was detected at 540 nm using the Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific) as described previously. 23

2.4. ALP staining and activity assays

For ALP staining assay, the ALP reaction solution was treated according to the manufacturer's protocol (Takara Bio Inc., Japan), as described previously. 24 For ALP activity assay, cell lysates were obtained using alkaline phosphatase activity colorimetric assay kit (Biovision). The activity was detected at 405 nm using the spectrophotometer (Thermo Fisher Scientific) as described previously. 24

2.5. ARS staining assay

Cells were stained with 2% ARS solution (pH 4.2) (Sigma‐Aldrich) for 10 min. Stains were captured using a scanner, and absorbance was detected at 590 nm using the spectrophotometer (Thermo Fisher Scientific) as described previously. 24

2.6. Western blot analysis

Osteogenic‐ and autophagic‐protein levels were detected using Western blot analysis as described previously. 24 , 25 The antibodies used were: RUNX2 (1:2000, #12556), p‐Smad1/5/8 (1:1000, #13820), p‐p38 (1:2000, #9211S), p38 (1:2000, #9212), p‐ERK1/2 (1:3000, #9101), ERK1/2 (1:3000, #9102), p‐JNK (1:2000, #9251), JNK (1:2000, #9252), GSK3β (1:1000, #12456), p‐GSK3β (1:1000, #9336), β‐catenin (1:1000), Wnt3a (1:1000, #2721), LC3A/B (1:1000, #12741), Beclin1 (1:1000, #3495) from Cell Signaling Technology (Beverly, MA, USA); BMP2 (1: 500, #CSB‐PAO9419AORb) from CUSABIO (Houston); β‐actin (1:2000, #sc‐47,778) from Santa Cruz Biotechnology (Santa Cruz); HRP‐secondary antibodies (1:20,000) from Jackson ImmunoResearch (West Grove).

2.7. Immunofluorescence assay

Nuclear RUNX2 expression was analysed using immunofluorescence as described previously. 24 The following antibodies were used: RUNX2 (1:400, #12556, Cell Signaling Technology), and secondary antibody (Alexa‐Fluor 488, 1:500, Invitrogen). DAPI (4′,6‐diamidino‐2‐phenylindole) solution was used (Sigma‐Aldrich) for nucleus stains.

2.8. DAPGreen autophagy detection assay

Autophagy Detection Kit (Dojindo) was used for detecting autophagosome formation as described previously. 25

2.9. Cell adhesion assay

Adhesion was analysed using Matrigel‐coated plates (Corning Life Sciences). The crystal violet‐stained cells were monitored under a light microscope, and absorbance was detected at 540 nm using the spectrophotometer (Thermo Fisher Scientific) as described previously. 15

2.10. Cell migration assay

Migration across ECM was carried out using Boyden chamber in Matrigel (Corning Life Sciences)‐coated nuclear pore filters. The images obtained under a light microscope were quantified as previously described. 26

2.11. Statistical analysis

Prism Version 5 program from GraphPad Software, Inc. was used for statistical analysis. Significance in p < 0.05 was analysed by using a one‐way anova with post hoc analysis, and the differences were assessed by the Bonferroni test. All values were reported as mean ± S.E.M.

3. RESULTS

3.1. Purification of SCOP from the extracts of A. capillaris dry aboveground part

The dry aboveground part of A. capillaris Thunb (10 kg) was extracted in MeOH at room temperature for 3 days. The crude extract (629.08 g) was suspended in DW and organic solvents. EtOAc soluble fraction (81.81 g) was subjected to silica gel CC, and fraction 1 was subjected to silica gel CC eluted with mixtures of CHCl3‐MeOH to afford compound 1 (SCOP, 100.0 mg) (Figure 1A). 125 MHz, CDCl3, 13C NMR: δ 160.5 (C‐2), 152.5 (C‐7), 149.4 (C‐6), 145.8 (C‐9), 144.3 (C‐4), 112.6 (C‐10), 111.2 (C‐3), 108.9 (C‐5), 100.0 (C‐8), 56.2 (OMe), 55.9 (OMe) (Figure 1B). 500 MHz, CDCl3, 1H‐NMR: δ 7.94 (1H, d, J = 9.5 Hz, H‐4), 7.24 (1H, s, H‐5), 7.06 (1H, s, H‐8), 6.29 (1H, d, J = 9.5 Hz, H‐3), 3.86 (3H, s, ‐OCH3), 3.80 (3H, s, ‐OCH3) (Figure 1C). Figure 1D displays SCOP's HPLC and structure (>99.17% purity; molecular formula: C11H10O4) (Figure 1D).

FIGURE 1.

FIGURE 1

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Isolation of scoparone (SCOP) from Artemisia capillaris. (A) Schematic of the method used to isolate and purify SCOP from A. capillaris. (B and C) 13C‐NMR (125 MHz, CDCl3) (B) and 1H‐NMR (500 MHz, CDCl3) (C) spectra of SCOP. (D) HPLC of purified SCOP (D), and its chemical structure, purity, and molecular formula ((D) inset)

3.2. SCOP accelerates early and late osteoblast differentiation

Cell viability (%) was measured using the MTT assay to investigate whether SCOP induces cytotoxicity after treating SCOP for 24 h. As shown in Figure 2A, SCOP showed no cytotoxic effects against pre‐osteoblasts at 0.1–100 μM (Figure 2A). Next, we induced osteoblast differentiation using OS with SCOP (1–10 μM) for 24 h to examine whether SCOP possesses osteogenic effects. Alkaline phosphatase (ALP) staining was performed to detect pre‐osteoblast early differentiation. ALP staining images were captured by a scanner, which revealed that SCOP elevated osteoblast differentiation at 7 days (Figure 2B). ALP enzymatic activity was detected by a spectrophotometer, which validated SCOP‐mediated osteoblast differentiation (Figure 2C). An Alizarin Red S (ARS) staining assay was used to explore whether SCOP modulates matrix mineralization by the late osteoblast differentiation. We used a scanner to detect the matrix mineralization at 21 days after inducing osteoblast differentiation using OS with SCOP (110 μM). The matrix mineralization was generated, which showed that SCOP accelerated late osteoblast differentiation (Figure 2D). The osteogenic effects of SCOP on the differentiation were statistically validated by quantifying ARS staining (Figure 2E). In addition, we found that SCOP increased OPG/RANKL during osteoblast differentiation (Figure S1A).

FIGURE 2.

FIGURE 2

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Cytotoxic and osteogenic effects of SCOP on pre‐osteoblasts. (A) Viability was detected using the MTT assay after treatment with SCOP (0.1 ~ 100 μM) for 24 h in pre‐osteoblasts. (B and C) The early differentiation was detected at 7 days using the ALP staining (B) and activity (C) assays after incubation in OS with SCOP (1–10 μM). (D and E) The late differentiation was detected to analyse the mineralization using the ARS staining assay after incubation in OS with SCOP (1–10 μM) for 21 days (D), and the stains were quantitatively analysed using a spectrophotometer (E). Data are represented as mean ± S.E.M. *p < 0.05 and # p < 0.05 indicate statistical significance compared with the control and OS, respectively

3.3. SCOP promotes canonical BMP2‐Samd1/5/8 signalling and RUNX2 expression

We further examined the mechanism by which SCOP acts on osteoblast differentiation by detecting canonical BMP2 signalling using Western blot analysis. We observed that SCOP increased intracellular and secreted BMP2 protein levels, and induced Smad1/5/8 phosphorylation, which is a key canonical BMP2 signalling protein (Figure 3A and (Figure S1B). SCOP also promotes RUNX2 protein levels, which is a core transcription factor induced by BMP2 signalling in osteoblast differentiation, followed by MMP13 protein expression, which is a RUNX2‐target protein (Figure 3B). The increased RUNX2 expression in the nucleus in response to SCOP was also validated using an immunofluorescence assay (Figure 3C). In addition, we validated that SCOP‐stimulated early and late osteoblast differentiation was attenuated by a BMP2 inhibitor, Noggin (Figure S1C,D).

FIGURE 3.

FIGURE 3

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Effects of SCOP on canonical BMP2 signalling and RUNX2. (A) Western Blot analysis of BMP2, phospho‐Smad1/5/8 (p‐Smad1/5/8) and β‐Actin levels. (B) Western Blot analysis of RUNX2, MMP13 and β‐Actin levels. (C and D) Immunofluorescence assay to assess RUNX2 (green) levels in nucleus (blue). Images were detected using a fluorescence microscope. RUNX2 (%) was shown in a bar graph. (D). Data are mean ± S.E.M. *p < 0.05 and # p < 0.05 indicate statistical significance compared with the control and OS, respectively

3.4. SCOP promotes non‐canonical BMP2‐MAPKs signalling and autophagosome formation

We subsequently examined whether SCOP affects the BMP2‐mediated non‐canonical pathway. As shown in Figure 4A, SCOP activated the non‐canonical pathway proteins, ERK, p38 and JNK (Figure 4A). We also investigated Wnt3a signalling, which revealed that SCOP did not noticeably affect the signalling proteins, including Wnt3a expression, GSK3β phosphorylation and β‐catenin stabilization (Figure 4B). Furthermore, autophagy signalling was investigated, and Western blot analysis revealed that Beclin1 and LC3A/B were increased by 10 μM SCOP, but not by 1 μM SCOP (Figure 4C). In addition, autophagy was observed by using a DAPGreen autophagosome formation assay, which revealed that 10 μM SCOP increased autophagy through the increased formation of autophagic vacuoles (Figure 4D,E).

FIGURE 4.

FIGURE 4

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Effect of SCOP on non‐canonical BMP2 signalling, Wnt3a signalling and autophagy. (A) Western Blot analysis of phospho‐p38 (P‐P38), p38, phospho‐JNK (p‐JNK), JNK, phospho‐ERK1/2 (p‐ERK), ERK and β‐Actin levels. (B) Western Blot analysis of Wnt3a, phospho‐GSK3β (p‐GSKβ), GSK3β, β‐catenin and β‐Actin levels. (C) Western Blot analysis of Beclin‐1, LC3A/B and β‐Actin levels. (D and E) Autophagosome formation was analysed and the DAPGreen‐stained autophagosome intensity was shown in a bar graph (D), and images were detected using a fluorescence microscope (E). Data are mean ± S.E.M. *p < 0.05 and # p < 0.05 indicate statistical significance compared with the control and OS, respectively

3.5. SCOP promotes osteoblast adhesion and migration

We finally explored whether SCOP influences osteoblast migration and adhesion in osteogenic processes. Adhesion experiments performed using Matrigel‐coated culture plates showed that SCOP marginally accelerated cell adhesion at 1 μM, and by a significant amount at 10 μM (Figure 5A,B). A Boyden chamber assay also showed that SCOP significantly increased osteoblast migration across the membrane coated by Matrigel (Figure 5C,D).

FIGURE 5.

FIGURE 5

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Effect of SCOP on osteoblast adhesion and migration (A and B) Adhesion was analysed on ECM‐coated plates, and images were detected using a light microscope. (A) Adhesion was shown in a bar graph (B). (C and D) Migration was analysed using Boyden chamber in ECM‐coated membrane, and images were detected using a light microscope (C). Migration was shown in a bar graph (D). Data are mean ± S.E.M. *p < 0.05 and # p < 0.05 indicate statistical significance compared with the control and OS, respectively

4. DISCUSSION

Osteoblasts control bone tissue homeostasis throughout life, and the damage and loss of osteoblasts can lead to skeletal diseases. 21 , 27 Herein, we demonstrated the activities of SCOP on MC3T3E‐1 pre‐osteoblasts, which are commonly utilized for researching in vitro osteogenic processes. 28 Osteoblast differentiation increases ALP, while osteoblast maturation results in bone matrix mineralization, which are well‐known early and late osteoblast differentiation markers, respectively. 27 , 29 , 30 Herein, we found that SCOP enhances ALP and bone matrix mineralization during osteogenic processes. We also demonstrated that SCOP accelerates osteoblast adhesion and migration. Osteoblast differentiation, migration and adhesion to the blood, bone marrow, periosteum induce bone‐specific protein secretion and bone matrix mineralization to produce bone tissue. 12 , 13 , 14 Our findings, based on the previous papers and present results, indicate that SCOP has osteogenic effects by promoting differentiation, adhesion, migration and mineralization.

Osteogenic processes are tightly regulated through complex networks activated by distinct osteogenic signalling molecules. BMP2, a bone morphogenetic factor, stimulates the canonical Smad1/5/8 and the non‐canonical MAPKs pathways, which subsequently control the critical transcription factor RUNX2. These events result in the production of bone‐forming proteins such as osterix, collagen and ALP for osteoblast differentiation. 31 , 32 , 33 Herein, SCOP enhances BMP2 and activates Smad1/5/8, ERK, JNK and p38. SCOP also increases RUNX2 expression and increases production of the target protein, MMP13, which is associated with calcification and degradation of extracellular matrix (ECM), as well as cell migration and adhesion during osteoblast differentiation bone repair. 34 , 35 , 36 In osteoblast differentiation, there is a close relationship between the BMP2 and Wnt3a signalling pathways, including the effects of Wnt3a regulating BMP2 expression and its target proteins. 37 , 38 , 39 , 40 , 41 However, we found that SCOP did not affect Wnt3a expression, GSK3β phosphorylation or β‐catenin stabilization. Thus, our results suggest that SCOP accelerates differentiation and mineralization by promoting BMP2‐mediated osteogenic signalling.

Autophagy is a self‐degradative mechanism in which cells engulf defective organelles and proteins, allowing metabolic balance to be maintained. 42 Autophagy‐related components play a role in bone metabolism and bone diseases by regulating the survival and function of bone tissue cells. 43 , 44 , 45 , 46 , 47 There is increasing evidence that shows that osteoblast differentiation and maturation are stimulated by autophagic process. 44 , 48 It was also reported that endoplasmic reticulum stress is induced by defective autophagy in osteoblasts, resulting in significant bone loss. 49 Recently, natural compound‐induced autophagy has been shown to enhance osteoblast differentiation and maturation, as well as ameliorate bone diseases. 50 , 51 , 52 Here, we demonstrate that high‐dose SCOP elevates autophagosome formation with increased Beclin‐1 and LC3A/B levels. It was reported that BMP2 induces autophagy to facilitate stem cell differentiation. 53 MAPK‐induced autophagy prevents MC3T3‐E1 pre‐osteoblasts from entering apoptosis. 54 Thus, our results suggest that SCOP‐induced BMP2 signalling is also involved in the autophagy pathway in osteogenic processes.

In conclusion, the findings of this study show for the first time that SCOP extracted from A. capillaris enhances the osteogenic processes—osteoblast differentiation, adhesion, migration, autophagy and mineralization through the BMP2 signalling pathways. However, in future studies, it will be necessary to compare human and murine osteoblasts, as they may behave differently in different species. Our data suggest that SCOP is a potential coumarin for developing an anabolic medication to improve osteoblast differentiation.

AUTHOR CONTRIBUTIONS

Kyung‐Ran Park: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); validation (equal); visualization (equal); writing – original draft (equal). Bomi Kim: Investigation (equal); methodology (equal); resources (equal); software (equal); validation (equal). JoonYeop Lee: Investigation (equal); methodology (equal); resources (equal); software (equal); validation (equal). Ho‐Jin Moon: Resources (equal). Il Keun Kwon: Funding acquisition (equal); resources (equal); writing – review and editing (equal). Hyung‐Mun Yun: Conceptualization (equal); funding acquisition (equal); project administration (equal); writing – review and editing (equal).

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

Supporting information

Figure S1

Click here for additional data file. (86.7KB, pdf)

ACKNOWLEDGEMENT

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF‐2020R1A2C2011937). This work was supported by the National Research Foundation of Korea [NRF] grant funded by the Korea government (MSIP) (NRF‐2022R1C1C1003491).

Park K‐R, Kim B, Lee JY, Moon H‐J, Kwon IK, Yun H‐M. Effects of Scoparone on differentiation, adhesion, migration, autophagy and mineralization through the osteogenic signalling pathways. J Cell Mol Med. 2022;26:4520‐4529. doi: 10.1111/jcmm.17476

Contributor Information

Il Keun Kwon, Email: kwoni@khu.ac.kr.

Hyung‐Mun Yun, Email: yunhm@khu.ac.kr.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

REFERENCES

  • 1. Han JM, Kim HG, Choi MK, et al. Artemisia capillaris extract protects against bile duct ligation‐induced liver fibrosis in rats. Exp Toxicol Pathol. 2013;65:837‐844. doi: 10.1016/j.etp.2012.12.002 [DOI] [PubMed] [Google Scholar]
  • 2. He CS, Yue HY, Xu J, et al. Protective effects of capillary artemisia polysaccharide on oxidative injury to the liver in rats with obstructive jaundice. Exp Ther Med. 2012;4:645‐648. doi: 10.3892/etm.2012.666 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Lee HS, Kim HH, Ku SK. Hepatoprotective effects of Artemisiae Capillaris Herba and Picrorrhiza rhizoma combinations on carbon tetrachloride‐induced subacute liver damage in rats. Nutr Res. 2008;28:270‐277. doi: 10.1016/j.nutres.2008.02.001 [DOI] [PubMed] [Google Scholar]
  • 4. Jung HA, Lee HJ, Kim YA, et al. Antioxidant activity of Artemisia capillaris Thunberg. Food Sci Biotechnol. 2004;13:328‐331. [Google Scholar]
  • 5. Cha JD, Moon SE, Kim HY, Cha IH, Lee KY. Essential oil of Artemisia capillaris induces apoptosis in KB cells via mitochondrial stress and caspase activation mediated by MAPK‐stimulated signaling pathway. J Food Sci. 2009;74:T75‐T81. doi: 10.1111/j.1750-3841.2009.01355.x [DOI] [PubMed] [Google Scholar]
  • 6. Chu CY, Tseng TH, Hwang JM, Chou FP, Wang CJ. Protective effects of capillarisin on tert‐butylhydroperoxide‐induced oxidative damage in rat primary hepatocytes. Arch Toxicol. 1999;73:263‐268. doi: 10.1007/s002040050615 [DOI] [PubMed] [Google Scholar]
  • 7. Lee CJ, Shim KS, Ma JY. Artemisia capillaris alleviates bone loss by stimulating osteoblast mineralization and suppressing osteoclast differentiation and bone resorption. Am J Chin Med. 2016;44:1675‐1691. doi: 10.1142/S0192415X16500944 [DOI] [PubMed] [Google Scholar]
  • 8. Lee SH, Lee JY, Kwon YI, Jang HD. Anti‐osteoclastic activity of Artemisia capillaris Thunb. Extract depends upon attenuation of osteoclast differentiation and bone resorption‐associated acidification due to chlorogenic acid, hyperoside, and Scoparone. Int J Mol Sci. 2017;18:322. doi: 10.3390/ijms18020322 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Huang HC, Chu SH, Chao PDL. Vasorelaxants from Chinese herbs, emodin and Scoparone, possess immunosuppressive properties. Eur J Pharmacol. 1991;198:211‐213. [DOI] [PubMed] [Google Scholar]
  • 10. Huang HC, Lee CR, Weng YI, Lee MC, Lee YT. Vasodilator effect of Scoparone (6,7‐dimethoxycoumarin) from a Chinese herb. Eur J Pharmacol. 1992;218:123‐128. doi: 10.1016/0014-2999(92)90155-w [DOI] [PubMed] [Google Scholar]
  • 11. Lu C, Li YQ, Hu SY, Cai YZ, Yang Z, Peng K. Scoparone prevents IL‐1 beta‐induced inflammatory response in human osteoarthritis chondrocytes through the PI3K/Akt/NF‐kappa B pathway. Biomed Pharmacother. 2018;106:1169‐1174. doi: 10.1016/j.biopha.2018.07.062 [DOI] [PubMed] [Google Scholar]
  • 12. Karsenty G, Kronenberg HM, Settembre C. Genetic control of bone formation. Annu Rev Cell Dev Biol. 2009;25:629‐648. doi: 10.1146/annurev.cellbio.042308.113308 [DOI] [PubMed] [Google Scholar]
  • 13. Zheng X, Dai J, Zhang H, Ge Z. MicroRNA‐221 promotes cell proliferation, migration, and differentiation by regulation of ZFPM2 in osteoblasts. Braz J Med Biol Res. 2018;51:e7574. doi: 10.1590/1414-431X20187574 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 14. Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell. 2002;2:389‐406. doi: 10.1016/s1534-5807(02)00157-0 [DOI] [PubMed] [Google Scholar]
  • 15. Park KR, Leem HH, Cho M, Kang SW, Yun HM. Effects of the amide alkaloid piperyline on apoptosis, autophagy, and differentiation of pre‐osteoblasts. Phytomedicine. 2020;79:153347. doi: 10.1016/j.phymed.2020.153347 [DOI] [PubMed] [Google Scholar]
  • 16. Marie PJ. Osteoblast dysfunctions in bone diseases: from cellular and molecular mechanisms to therapeutic strategies. Cell Mol Life Sci. 2015;72:1347‐1361. doi: 10.1007/s00018-014-1801-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Lee WC, Guntur AR, Long F, Rosen CJ. Energy metabolism of the osteoblast: implications for osteoporosis. Endocr Rev. 2017;38:255‐266. doi: 10.1210/er.2017-00064 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Russow G, Jahn D, Appelt J, Märdian S, Tsitsilonis S, Keller J. Anabolic therapies in osteoporosis and bone regeneration. Int J Mol Sci. 2018;20:83. doi: 10.3390/ijms20010083 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Kawai M, Mödder UI, Khosla S, Rosen CJ. Emerging therapeutic opportunities for skeletal restoration. Nat Rev Drug Discov. 2011;10:141‐156. doi: 10.1038/nrd3299 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Marie PJ, Kassem M. Osteoblasts in osteoporosis: past, emerging, and future anabolic targets. Eur J Endocrinol. 2011;165:1‐10. doi: 10.1530/EJE-11-0132 [DOI] [PubMed] [Google Scholar]
  • 21. An J, Yang H, Zhang Q, et al. Natural products for treatment of osteoporosis: the effects and mechanisms on promoting osteoblast‐mediated bone formation. Life Sci. 2016;147:46‐58. doi: 10.1016/j.lfs.2016.01.024 [DOI] [PubMed] [Google Scholar]
  • 22. Liu Y, Liu JP, Xia Y. Chinese herbal medicines for treating osteoporosis. Cochrane Database Syst Rev. 2014;3:CD005467. doi: 10.1002/14651858.cd005467.pub2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Park KR, Yun HM. RANKL‐induced osteoclastogenesis in bone marrow‐derived macrophages is suppressed by cisapride. Toxicology. 2019;422:95‐101. doi: 10.1016/j.tox.2019.05.010 [DOI] [PubMed] [Google Scholar]
  • 24. Park KR, Kim S, Cho M, Kang SW, Yun HM. Effects of PIN on osteoblast differentiation and matrix mineralization through runt‐related transcription factor. Int J Mol Sci. 2020;21:9579. doi: 10.3390/ijms21249579 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Park KR, Leem HH, Kwon YJ, Kwon IK, Hong JT, Yun HM. Falcarindiol stimulates apoptotic and autophagic cell death to attenuate cell proliferation, cell division, and metastasis through the PI3K/AKT/mTOR/p70S6K pathway in human oral squamous cell carcinomas. Am J Chin Med. 2021;50:295‐311. doi: 10.1142/S0192415X22500112 [DOI] [PubMed] [Google Scholar]
  • 26. Park KR, Park JE, Kim B, Kwon IK, Hong JT, Yun HM. Calycosin‐7‐O‐beta‐glucoside isolated from Astragalus membranaceus promotes osteogenesis and mineralization in human mesenchymal stem cells. Int J Mol Sci. 2021;22:11362. doi: 10.3390/ijms222111362 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Guntur AR, Rosen CJ. The skeleton: a multi‐functional complex organ: new insights into osteoblasts and their role in bone formation: the central role of PI3kinase. J Endocrinol. 2011;211:123‐130. doi: 10.1530/JOE-11-0175 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Wang D, Christensen K, Chawla K, Xiao G, Krebsbach PH, Franceschi RT. Isolation and characterization of MC3T3‐E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J Bone Miner Res. 1999;14:893‐903. doi: 10.1359/jbmr.1999.14.6.893 [DOI] [PubMed] [Google Scholar]
  • 29. Lee HS, Jung EY, Bae SH, Kwon KH, Kim JM, Suh HJ. Stimulation of osteoblastic differentiation and mineralization in MC3T3‐E1 cells by yeast hydrolysate. Phytother Res. 2011;25:716‐723. doi: 10.1002/ptr.3328 [DOI] [PubMed] [Google Scholar]
  • 30. Kim MB, Song Y, Hwang JK. Kirenol stimulates osteoblast differentiation through activation of the BMP and Wnt/beta‐catenin signaling pathways in MC3T3‐E1 cells. Fitoterapia. 2014;98:59‐65. doi: 10.1016/j.fitote.2014.07.013 [DOI] [PubMed] [Google Scholar]
  • 31. Kern B, Shen J, Starbuck M, Karsenty G. Cbfa1 contributes to the osteoblast‐specific expression of type I collagen genes. J Biol Chem. 2001;276:7101‐7107. doi: 10.1074/jbc.M006215200 [DOI] [PubMed] [Google Scholar]
  • 32. Chen G, Deng C, Li YP. TGF‐beta and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci. 2012;8:272‐288. doi: 10.7150/ijbs.2929 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. Artigas N, Ureña C, Rodríguez‐Carballo E, Rosa JL, Ventura F. Mitogen‐activated protein kinase (MAPK)‐regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program. J Biol Chem. 2014;289:27105‐27117. doi: 10.1074/jbc.M114.576793 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Nishimura R, Wakabayashi M, Hata K, et al. Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association with transcription factor Runx2 during endochondral ossification. J Biol Chem. 2012;287:33179‐33190. doi: 10.1074/jbc.M111.337063 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Toriseva M, Laato M, Carpén O, et al. MMP‐13 regulates growth of wound granulation tissue and modulates gene expression signatures involved in inflammation, proteolysis, and cell viability. PLOS One. 2012;7:e42596. doi: 10.1371/journal.pone.0042596 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Yamagiwa H, Tokunaga K, Hayami T, et al. Expression of metalloproteinase‐13 (Collagenase‐3) is induced during fracture healing in mice. Bone. 1999;25:197‐203. doi: 10.1016/s8756-3282(99)00157-x [DOI] [PubMed] [Google Scholar]
  • 37. Zhang R, Oyajobi BO, Harris SE, et al. Wnt/beta‐catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts. Bone. 2013;52:145‐156. doi: 10.1016/j.bone.2012.09.029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Rawadi G, Vayssière B, Dunn F, Baron R, Roman‐Roman S. BMP‐2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res. 2003;18:1842‐1853. doi: 10.1359/jbmr.2003.18.10.1842 [DOI] [PubMed] [Google Scholar]
  • 39. Fukuda T, Kokabu S, Ohte S, et al. Canonical Wnts and BMPs cooperatively induce osteoblastic differentiation through a GSK3beta‐dependent and beta‐catenin‐independent mechanism. Differentiation. 2010;80:46‐52. doi: 10.1016/j.diff.2010.05.002 [DOI] [PubMed] [Google Scholar]
  • 40. Sato MM, Nakashima A, Nashimoto M, Yawaka Y, Tamura M. Bone morphogenetic protein‐2 enhances Wnt/beta‐catenin signaling‐induced osteoprotegerin expression. Genes CellsGTC1258. 2009;14:141‐153. doi: 10.1111/j.1365-2443.2008.01258.x [DOI] [PubMed] [Google Scholar]
  • 41. Chen Y, Whetstone HC, Youn A, et al. Beta‐catenin signaling pathway is crucial for bone morphogenetic protein 2 to induce new bone formation. J Biol Chem. 2007;282:526‐533. doi: 10.1074/jbc.M602700200 [DOI] [PubMed] [Google Scholar]
  • 42. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726‐734. doi: 10.1038/nrc1692 [DOI] [PubMed] [Google Scholar]
  • 43. Smith M, Wilkinson S. ER homeostasis and autophagy. Essays Biochem. 2017;61:625‐635. doi: 10.1042/EBC20170092 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. di Giacomo V, Cataldi A, Sancilio S. Biological factors, metals, and biomaterials regulating osteogenesis through autophagy. Int J Mol Sci. 2020;21(8):2789. doi: 10.3390/ijms21082789 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Darcy A, Meltzer M, Miller J, et al. A novel library screen identifies immunosuppressors that promote osteoblast differentiation. Bone. 2012;50:1294‐1303. doi: 10.1016/j.bone.2012.03.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. Zhang LS, Guo YF, Liu YZ, et al. Pathway‐based genome‐wide association analysis identified the importance of regulation‐of‐autophagy pathway for ultradistal radius BMD. J Bone Miner Res. 2010;25:1572‐1580. doi: 10.1002/jbmr.36 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Zhao X, Huang B, Wang H, et al. A functional autophagy pathway is essential for BMP9‐induced osteogenic differentiation of mesenchymal stem cells (MSCs). Am J Transl Res. 2021;13:4233‐4250. [PMC free article] [PubMed] [Google Scholar]
  • 48. Shapiro IM, Layfield R, Lotz M, Settembre C, Whitehouse C. Boning up on autophagy: the role of autophagy in skeletal biology. Autophagy. 2014;10:7‐19. doi: 10.4161/auto.26679 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49. Li H, Li D, Ma Z, et al. Defective autophagy in osteoblasts induces endoplasmic reticulum stress and causes remarkable bone loss. Autophagy. 2018;14:1726‐1741. doi: 10.1080/15548627.2018.1483807 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50. Kim IR, Kim SE, Baek HS, et al. The role of kaempferol‐induced autophagy on differentiation and mineralization of osteoblastic MC3T3‐E1 cells. BMC Complement Altern Med. 2016;16:333. doi: 10.1186/s12906-016-1320-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51. Li W, Zhang S, Liu J, Liu Y, Liang Q. Vitamin K2 stimulates MC3T3E1 osteoblast differentiation and mineralization through autophagy induction. Mol Med Rep. 2019;19:3676‐3684. doi: 10.3892/mmr.2019.10040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Wang NN, Xu PC, Wu RJ, et al. Timosaponin BII improved osteoporosis caused by hyperglycemia through promoting autophagy of osteoblasts via suppressing the mTOR/NF kappa B signaling pathway. Free Radic Biol Med. 2021;171:112‐123. doi: 10.1016/j.freeradbiomed.2021.05.014 [DOI] [PubMed] [Google Scholar]
  • 53. Cai B, Zheng Y, Yan J, Wang J, Liu X, Yin G. BMP2‐mediated PTEN enhancement promotes differentiation of hair follicle stem cells by inducing autophagy. Exp Cell Res. 2019;385:111647. doi: 10.1016/j.yexcr.2019.111647 [DOI] [PubMed] [Google Scholar]
  • 54. Wei M, Duan D, Liu Y, Wang Z, Li Z. Autophagy may protect MC3T3‐E1 cells from fluoride‐induced apoptosis. Mol Med Rep. 2014;9:2309‐2315. doi: 10.3892/mmr.2014.2079 [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Figure S1

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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