DE (Figure 1a, molecular weight = 263.38 Da, Product No.: HB-01665, a purity of > 98% (w/w)) was purchased from Huashite Biology (Huashite Industrial Biotechnology Co., Ltd., Wuhan, Hubei, China). It was prepared in dimethyl sulfoxide (DMSO, Product No.: D2650-50ML, Sigma-Aldrich, St Louis, MO, USA) and stored at −20 °C. Phosphate-buffered saline (PBS, Product No.: P4417-100TAB), paraformaldehyde (Product No.: 158,127-3 KG), 17b-Estradiol (E2; product number: E8875), ethylenediaminetetraacetic acid (EDTA, Product No.: EDS-1KG), NaOH (Product No.: S8045-1 KG), glycerol (Product No.: G5516-4L), hematoxylin (Product No.: HHS16), and eosin (Product No.: HT110116), were purchased from Sigma-Aldrich. The cell counting kit-8 (CCK-8; Product No.: C0038) and alkaline phosphatase (ALP) staining kit (Product No.: P0321S) were obtained from Beyotime Biotechnology (Beyotime Institute of Biotechnology Co, Ltd, Shanghai, China). Minimal essential medium (MEM, Product No.: 11,095-072) and fetal bovine serum (FBS, Product No.: 10,099-141) were purchased from Gibco (Thermo Fisher Scientific Inc., Grand Island, NY, USA). L-lysine (Product No.: B877236) and sodium periodate (Product No.: S817518) were obtained from Macklin reagent (Macklin Biochemical Co., Ltd, Shanghai, China). The PrimeScript™ RT reagent kit (Product No.: RR047A) and RNAiso Plus (Product No.: 9109) and were obtained from TaKaRa (Takara Biomedical Technology Co., Ltd., Otsu, Shiga, Japan). Other chemical reagents were purchased from Sigma–Aldrich.

The mice were anesthetized with sodium pentobarbital (40 mg/kg) and sacrificed by cervical dislocation. Bone samples were fixed in 4% (w/v) paraformaldehyde overnight. The distal femora were measured by micro-CT scan (Scanco Medical, Brüttisellen, Zurich, Switzerland) with a voltage of 55 kVp, an intensity of 200 mA, a resolution of 5 mm per pixel and an exposure of 230 m. The three-dimensional structure and morphometry were reconstructed by three-dimensional model visualization software (mCT Ray V4.2) and data analyzed by data analysis software (mCT Evaluation Program V6.6) for trabecular bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), trabecular separation (Tb. Sp).

Bone specimens were fixed with 4% paraformaldehyde overnight. The samples were then decalcified at 4 °C (pH 7.3) for 10 days in a decalcification solution (1.45% (w/v) EDTA, 1.25% (w/v) NaOH, and 1.5% (v/v) glycerol). After being embedded in paraffin, the bone tissue was sectioned into 5 μm sections with a paraffin microtome (RM2125RTS, Leica Biosystems, Wetzlar, Germany). Deparaffinized tissue sections were washed with PBS and blocked with 3% (w/v) bovine serum albumin (BSA) and 0.4% (v/v) Triton X-100. Then, sections were incubated for 16 h at 4 °C with the following primary antibodies: runt-related transcription factor 2 (Runx2, Product No.: A2851, dilution rate: 1:100, ABclonal Technology Co., Ltd., Wuhan, Hubei, China), osteocalcin (Ocn, Product No.: A6205, dilution rate: 1:150, ABclonal), and β-catenin (Product No.: 8480, dilution rate: 1:150, Cell Signaling Technology Inc., Danvers, MA, USA). The primary antibodies were removed by PBS, and the sections were incubated with secondary antibodies (Product No.: 8114, dilution rate: 1:150, CST) for 1 h at room temperature (RM). The secondary antibody was removed by PBS, and sections were incubated with DAB (3,3′-diaminobenzidine) and hematoxylin for double staining. The sections were dehydrated, covered with coverslips, and photographed using a ZEISS microscope (Axio Scope A1, Oberkochen, Germany). The integrated optical density (IOD)/area was used as a parameter to quantify staining intensity using the image pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA). The images were taken at ×10 magnification.

DE(1 and 10 μM)were treated with the C3H/10T1/2 cells for 12 h. The C3H/10T1/2 cells were fixed using 4% paraformaldehyde. β-catenin (51067-2-AP, 1:200, proteintech) and labeled with secondary antibodies for 1 h in the dark. After labeling, cells were incubated with DAPI for 5 min. Images were acquired with an Leica confocal microscope (STELLARIS 5, Leica, German). The β-catenin nuclear translocation were mesured.

The modified hematoxylin and eosin (H&E) staining was performed on deparaffinized bone tissue sections. For H&E staining evaluation, sum area of the trabecular positive region (red area) per maximum image was used in each distal femur as a parameter to quantify the trabecular area using the IPP 6.0 software. The images were taken at ×10 magnification.

The bone samples were removed from euthanized mice and fixed for 24 h at 4 °C with the solution containing 8% (w/v) paraformaldehyde, L-Lysine, and sodium periodate. Then, tissue specimens were decalcified for 10 days at 4 °C and dehydrated in 30% (w/v) sucrose for 24 h at RM. Finally, samples were prepared as 5 μm sagittal-oriented sections using a Leica frozen microtome (CM1850, Leica, Wetzlar, Germany). The sections were incubated with ALP solution from the ALP staining kit (C3206, Beyotime Institute of Biotechnology, China). For ALP staining, the sum of positive cells was quantified per maximum image in each distal femur using the IPP 6.0 software. The images were taken at ×10 magnification.

C3H/10T1/2 cells (a murine embryo fibroblast cell line, classic cell model of mesenchymal stem cells (MSCs)) were purchased from Stem Cell Bank (Code number: SCSP-506, Chinese Academy of Sciences, Shanghai, China). Cells were cultured in MEM and 10% FBS at 37 °C with 5% CO₂ for subculture. When cells reached 80% confluence, they were subcultured into the osteogenic medium with 10% FBS (MEM supplemented with 0.1 μmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 μmol/L ascorbic acid). At the same time, DE (1 and 10 μM) was added to the osteogenic medium. The cells were maintained in the osteogenic medium for 1 week, and the medium changed every 3 days. Human Bone Marrow-Mesenchymal Stem Cells (hBMSC) were purchased from Guangzhou Jennio Biotech CO., Ltd. Cells were cultured in DMEM and 10% FBS at 37 °C with 5% CO₂ for subculture. When cells reached 80% confluence, they were subcultured into the osteogenic medium with 10% FBS (DMEM supplemented with 0.1 μmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 μmol/L ascorbic acid). At the same time, DE (0.1 and 1 μM) was added to the osteogenic medium. The cells were maintained in the osteogenic medium for 1 week, and the medium changed every 3 days.

Cell proliferation was determined using a CCK-8 assay kit (C0037, Beyotime Institute of Biotechnology, China), following the manufacturer’s protocol. A density of 5 × 10⁴ cells/well was used to seed C3H/10T1/2 cells and hBMSC into 96-well plates. After treating the cells with DE (0.01–100 μM) for 7 days, a CCK-8 reagent was added to the wells and incubated at 37 °C for 4 h. Cell proliferation was measured by a microplate reader measuring the optical density absorbance at 450 nm.

Cells were fixed in 4% paraformaldehyde for 10 min at RM. After washing with PBS, C3H/10T1/2 cells were incubated with ALP staining buffer following the manufacturer’s protocol. To remove excess dye, cells were washed in PBS. IPP 6.0 software was used to measure ALP-positive cells pictured by microscope.

Cells treated with DE in the osteogenic differentiation medium for 7 days were then lysed immediately for 10 min in an SDS-loading buffer (62.5 mM Tris-HCl, 50 mM dithiothreitol, 2% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and pH 6.8) at 96 °C. The primary antibodies, i.e., osterix (Osx, AV31622, 1:500, Sigma), Runx2 (A2851, 1:2000, Abclonal), Osetocalcin(Ocn, ab93876, 1:500, Abcam, Cambridge, UK), GSK-3β (22104-1-AP, 1:500, Proteintech Group Inc., Wuhan, Hubei, China) and P-GSK-3β (Tyr216) (05-413, 1:1000, Merk, Darmstadt, German) were incubated at 4 °C overnight. The secondary anti-rabbit antibody was incubated for 1 h at RM, then visualized using western lighting plus enhanced chemiluminescence (0RT2755, 0RT2655, Perkin Elmer, Waltham, MA, USA). The quantitative gray value of WB bands was measured by ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA).

In vivo experiment, C3H/10T1/2 cells were rapidly extracted using RNAiso Plus and reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit. Quantitative polymerase chain reaction (PCR, RR420A, TaKaRa) analysis was performed with specific gene primers (Table 1) using the Thermal Cycler PCR System (ABI 9700, Applied Biosystems, Foster City, CA, USA). Quantifying RNA expression levels was performed using the 2^−ΔΔCT method, and Gapdh was used as a positive control.

All data were analyzed using GraphPad Prism version 9.0 software (GraphPad Software Inc., La Jolla, CA, USA). One-way analysis of variance and Tukey multiple comparison test were used to analyze the multiple comparisons in our data. The value was presented as the mean ± SD. The significance level was set at P < 0.05.

CCK-8 assays were performed (Figure 1b) to determine if DE (Figure 1a1-a2) affected the proliferation of C3H/10T1/2 cells and hBMSC. In an ordinary complete medium for 7 days, DE did not significantly affect the growth of C3H/10T1/2 cells at concentrations ranging from 10 nM to 50 μM, or that of hBMSCs from 100 nM to 10 μM.

C3H/10T1/2 cells and hBMSCs were cultured for 7 days in osteogenic differentiation medium supplemented with DE at concentrations of 1μM/10 μM and 0.1μM/1 μM, respectively. The ALP staining assay (Figure 1c-f) showed that DE (1 μM and 10 μM)/DE (0.1 μM and 1 μM) increased ALP positivity of C3H/10T1/2 cells/hBMSC in a dose-dependent manner (Figure 1c1-c3, d1-d3/1e1-e3,f1-f3). Based on the quantitative analysis of ALP positive cells (Figure 1g), DE (1 μM and 10 μM) stimulated ALP activity dose-dependently both in C3H/10T1/2 cells and hBMSC. DE (1 μM and 10 μM) was found to promote Runx2, Ocn, and Osx expressions associated with osteogenesis in C3H/10T1/2 cells (Figure 1h). DE (0.1 μM and 1 μM) was found to promote Runx2 and Osx expressions associated with osteogenesis in hBMSC (Figure 1h). The gray value of WB bands was measured as a measure of protein expression level, and results of Runx2, Ocn and Osx (Figure 1i) increased gradually with increasing DE concentrations, peaking at 10 μM with C3H/10T1/2 cells. The gray value of WB bands was measured as a measure of protein expression level, and results of Runx2 and Osx (Figure 1j) increased gradually with increasing DE concentrations, peaking at 10 μM with hBMSC. In vitro, DE promotes osteoblast differentiation.

We investigated the effects of DE on mRNA levels of osteogenic-related genes (Runx2, Ocn, Osx, and Col1α1) in osteogenesis-induced cultured C3H/10T1/2 cells for 7 days. Quantitative PCR analysis of Runx2 (Figure 2a), Ocn (Figure 2b), Osx (Figure 2c), and Col1α1 (Figure 2d) was performed, and total mRNA levels of osteogenic-related genes were increased in dose-dependent relationship with the exposure of DE (1 μM and 10 μM). As a result, the above results indicate that DE can effectively enhance osteogenic-related gene transcription in vitro.

Female C57/BL6 mice were ovariectomized and administered DE intragastrically or E2 via intraperitoneal injection for 1 month. Body weight (Supplementary Figures S1a,c) and uterus mass (Supplementary Figures S1b,d) were recorded in order to confirm the effects of ovariectomy validated by increased. In the mice, OVX was successfully body weight and decreased in uterus weight. An effect depending on the dose of DE or E2 on bone formation was evaluated (Supplementary Figure S2a) using the H&E staining assay. In a manner depending on DE dose, the increasing trabecular area was found in the OVX + DE group (10 mg/kg/day and 20 mg/kg/day) compared to the OVX group, with a peak of 20 mg/kg/day. As a positive group, the trabecular area in OVX + E2 was significantly increased compared with OVX group (Supplementary Figure S2a). Our intervention concentration of DE in vivo was set at 20 mg/kg/day.

As part of our investigation of DE’s effect on trabecular bone formation, micro-CT analysis was performed on the mice distal femur in sham, OVX, and OVX + DE (Figure 3a) group. As compared to OVX, OVX + DE mice had significantly higher Tb. Th (Figure 3b), Tb. N (Figure 3d), BV/TV (Figure 3e), and lower Tb. Sp (Figure 3c). H&E staining of the distal femur (Figure 3f) showed an increase in the trabecular area compared to the OVX group (Figure 3g). The distal femur was stained with ALP (Figure 3h). ALP-positive cells were more significant in OVX + DE mice than in OVX mice (Figure 3i). In the distal femur, IHC staining of Runx2 (Figure 3j) was performed, and the results indicated that the sum IOD for Runx2 (Figure 3k) increased in OVX + DE group compared to OVX mice. We conducted IHC staining of Ocn (Figure 3l) on the mice’s distal femur, and the results showed that Ocn’s sum IOD (Figure 3m) increased in OVX + DE group when compared with OVX mice. As a result, DE can prevent osteoporosis in mice induced by ovariectomy.

C3H/10T1/2 Cells were treated with DE for 7 days in an osteogenic medium. By WB (Figure 4a), we measured the levels of phosphorylation of GSK-3β (Tyr216) (a hallmark of GSK-3β activity) and β‐catenin. It was found that P-GSK-3β (Tyr216) was decreased and β-catenin levels were increased in osteogenesis-induced cultured C3H/10T1/2 cells with DE (1 μM and 10 μM). Immunofluorescence analysis demonstrated that DE (1 and 10 μM) promoted β-catenin nuclear translocation in C3H/10T1/2 cells (Figure 4d). This effect was confirmed by quantitative analysis (Figure 4e). Furthermore, IHC staining of β-catenin was performed on the mice’s distal femur (Figure 4f). The results indicated that β-catenin sum IODs increased in OVX + DE mice compared to OVX mice (Figure 4g). The critical role of β-catenin was confirmed by its knockdown (Figures 5c,d), which reversed the DE-mediated increases in ALP activity (Figures 5a,b) and Osx protein levels (Figures 5c,e), suggesting that the effect of DE on osteogenic differentiation depends on β-catenin. Based on these data, DE is shown to promote osteogenic differentiation and trabecular bone formation by preventing GSK-3β activity and β-catenin degradation.