Cinobufotalin prevents bone loss induced by ovariectomy in mice through the BMPs/SMAD and Wnt/β-catenin signaling pathways

Abstract

Background

Osteoporosis is a chronic bone disease characterized by bone loss and decreased bone strength. However, current anti-resorptive drugs carry a risk of various complications. The deep learning-based efficacy prediction system (DLEPS) is a forecasting tool that can effectively compete in drug screening and prediction based on gene expression changes. This study aimed to explore the protective effect and potential mechanisms of cinobufotalin (CB), a traditional Chinese medicine (TCM), on bone loss.

Methods

DLEPS was employed for screening anti-osteoporotic agents according to gene profile changes in primary osteoporosis. Micro-CT, histological and morphological analysis were applied for the bone protective detection of CB, and the osteogenic differentiation/function in human bone marrow mesenchymal stem cells (hBMMSCs) were also investigated. The underlying mechanism was verified using qRT-PCR, Western blot (WB), immunofluorescence (IF), etc.

Results

A safe concentration (0.25 mg/kg in vivo, 0.05 μM in vitro) of CB could effectively preserve bone mass in estrogen deficiency-induced bone loss and promote osteogenic differentiation/function of hBMMSCs. Both BMPs/SMAD and Wnt/β-catenin signaling pathways participated in CB-induced osteogenic differentiation, further regulating the expression of osteogenesis-associated factors, and ultimately promoting osteogenesis.

Conclusion

Our study demonstrated that CB could significantly reverse estrogen deficiency-induced bone loss, further promoting osteogenic differentiation/function of hBMMSCs, with BMPs/SMAD and Wnt/β-catenin signaling pathways involved.

INTRODUCTION

Osteoporosis is a systemic chronic bone disease characterized by low bone mass and degeneration of the bone microstructure.¹ The disruption of bone homeostasis between bone resorption and bone formation can ultimately lead to decreased bone strength and an increased fracture risk.² The primary manifestations of osteoporosis in the oral cavity include jaw fracture and aggravated alveolar bone resorption. Thus, a sparse array of trabeculae covered by a thin and porous bone cortex is a challenge to prosthodontic and implant treatment.³

To preserve normal bone homeostasis and avoid bone metabolic diseases, the treatment options for osteoporosis have mainly focused on: (1) the inhibition of bone resorption; (2) promotion of bone formation; and (3) dual-action therapy.⁴ Bone resorption inhibition agents (selective estrogen receptor modulators [SERMs]) have been central to mainstream therapeutic strategies over the past two decades.⁵ However, complications that increase the risk of various diseases (endometrial cancer, osteonecrosis of the jaw, and systemic infection) should not be ignored.⁶ The other therapies, bone formation promoting (anabolic) agents and dual-action agents (strontium ranelate and romosozumab), are not recommended due to their high price and complex complications (cardiovascular and cerebrovascular diseases).^7–10 Therefore, the development of new osteogenic drugs is urgently needed to strengthen the bone microstructure, restore patient bone mass, and minimize drug complications.

A critical aspect of new drug development is the identification of a drug's interaction with its target (drug-target interactions, DTI).^11,12 The traditional DTI exploration process has several disadvantages, including ambiguity of purpose, low feasibility, heavy workload, high capital cost, and long experimental period. By contrast, virtual screening (VS) has several advantages.¹³ VS can be used to assist drug discovery studies by calculating unknown biological DTIs, typically using the physicochemical and structural properties of compounds and targets, as well as experimentally validated biological information to generate predictive models.¹⁴ In VS, DLEPS is an efficient neural network tool that can predict the characteristic changes of gene expression profiles induced by small molecules.¹⁵ These gene profile changes, identified from CMap or predicted by DLEPS containing a high-resolution and high-specificity schema matching algorithm, can be used to screen for compounds against various diseases through disparate gene signatures and subsequently identify drug candidates.¹⁶ Thus, the novel method of VS offered by DLEPS provides a unique exploration pathway for drug discovery, making drug development and functional repurposing processes more accurate, economical, and efficient to better meet clinical requirements.

Mesenchymal stem cells (MSCs) are capable of multi-lineage differentiation and cellular migration; these characteristics and their paracrine functions and low immunogenicity risk¹⁷ thus represent an essential cellular source of bone tissue engineering (BTE) known to be involved in bone homeostasis maintenance.¹⁸ Numerous complex signaling mechanisms, such as bone morphogenetic proteins (BMPs),¹⁹ TGF-β/Smad,²⁰ Wnt/β-catenin²¹ and Notch^22,23 pathways, have been identified in the osteogenic differentiation of MSCs. In our previous research, we investigated the human MSC gene expression profiles in the ArrayExpress database. Senescence (GSE35956), primary osteoporosis (GSE35957) and cellular senescence (GSE35958) were selected for DLEPS identification. Finally, we identified a list of small bioactive molecules of Western medicine compounds that can reverse the genetic changes in osteoporosis. The representative compound ataluren (ATA) was screened to determine its effect on bone formation, and revealed the regulatory mechanism of osteogenesis promotion in hBMMSCs.

In this study, primary osteoporosis gene signatures obtained from a public database (ArrayExpress) were utilized for microarrays and high-throughput sequencing data.¹⁸ A natural compound library (L6000, 2719 molecules) was used as input for DLEPS to calculate anti-osteoporosis scores and select natural compounds with the highest ranking. DLEPS indicated that cinobufotalin (CB; Hua-chan-su) was a natural compound that could be used as an anti-osteoporosis drug candidate. CB is a TCM, and there are records of a proposed use of Chan-Su (extractions from skin secretions of Bufo gargarizans) to treat malignant swelling as early as 2000 years ago, during the Tang Dynasty.²⁴ CB is the effective component of Chan-Su, and CB injections have been approved by the China Food and Drug Administration (CFDA) as a form of modern medicine.²⁵ Moreover, CB has been used to treat cancer in various organs,^26–28 resulting in substantial improvements and reducing the side effects of chemotherapy. However, there is a lack of research on the effects of CB on bone metabolism, in particular on MSC differentiation and function.

The present study evaluated the restorative effect of CB on bone loss in a mouse model of OVX-induced osteoporosis. We also explored the effects of CB on osteogenic differentiation and function in hBMMSCs in vitro, revealing possible mechanisms of signal regulation. Our findings provide a novel mechanism for the pharmacological action of CB that supports its use in modern medicine, and also provide additional theoretical and empirical data to support the transformation of TCM into translational medicine.

MATERIALS AND METHODS Data collection

The microarray database reference datasets obtained from the ArrayExpress database (available at: https://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD-35959; Login number: E-GEOD-35959)²⁹ were acquired and downloaded by searching for the keyword ‘osteoporosis’. In this investigation, the natural compound library (L6000) was adopted to screen for the novel osteoporosis treatment agents.

Drug repositioning processed by DLEPS

DLEPS is a forecasting tool that can effectively complete matches and predictions of various drugs. DLEPS was applied to predict anti-osteoporosis agents.¹⁶ The anti-osteoporosis score was calculated by DLEPS based on the differentially expressed genes (DEGs) originating from an earlier study.³⁰ DLEPS training was executed by applying natural compound-induced changes in transcriptional profiles from the L6000 library, which collects compounds from animal and plant extracts with specific constituents, most of which are traditional Chinese medical ingredients.³¹

Cell culture and osteogenic induction

Primary hBMMSCs were purchased from Scien-Cell (Carlsbad, CA, USA). Cells were cultured in proliferation medium (PM) containing α-minimum essential medium (α-MEM) (Gibco, NY, USA), containing 10% (v/v) fetal bovine serum (FBS) (ExCell Bio, HK, PRC), and 1% (v/v) penicillin/streptomycin (Gibco) at 37°C and 5% CO₂. The induction of osteogenesis was performed using osteogenic induction medium (OM), containing α-MEM with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 10 nM dexamethasone (Sigma, MO, USA), 200 μM vitamin C (Sigma), and 10 mM β-glycerophosphate (β-GP, Sigma). The medium was refreshed every 3 days.

Animal experimental and ethical statements

Female C57BL/6 mice reared under specific pathogen-free (SPF) conditions were obtained from Charles River Laboratory Animal Technology Co., Ltd. Authorization of all animal experiments was awarded by the Peking University School of Medicine Institutional Committee for Animal Care and Use (LA2021040).

Based on the validation of previous research,^32,33 a mouse model of classical ovariectomy-induced bone loss mimicking estrogen deficiency was constructed (Figure S2A). The experimental groups and designs were as follows: (1) SHAM+PBS; (2) OVX + PBS; (3) OVX + CB_Low (low dose); and (4) OVX + CB_High (high dose). Following general anesthesia, surgical bilateral OVX or a sham operation was performed (Figure S2A). Three months later, drug administration (see below for drugs and dosages) was initiated and continued for 2 months, via intraperitoneal injection performed once every other day. A phosphate buffer solution (PBS) containing 1% DMSO (Sigma) was injected at a dose of 200 μL/mouse. Low and high doses of cinobufalin were calculated as 0.25 mg/kg (0.25 μg/g) and 0.5 mg/kg (0.5 μg/g), respectively. The average body weight of the mice was 38–40 g, and approximately 10 μg/mouse and 20 μg/mouse were delivered for the low and high dose administrations.

Micro-computed tomography examination

The mouse femoral specimens were scanned using a high-resolution micro-computed tomographer of the Inveon MM System (Siemens, Berlin, GER). The region of interest (ROI), defined as 1 mm proximal to the epiphysis, underwent subsequent imaging analysis. Imaging analysis parameters, including bone mineral density (mg/cm³, BMD), bone volume/total volume (%, BV/TV), bone surface area/bone volume (1/mm, BS/BV), trabecular number (1/mm, Tb.N), trabecular thickness (mm, Tb.Th), and trabecular separation (mm, Tb.Sp), were processed by an Inveon Research Workplace (Siemens).

Bone histological examination and histomorphometric measurements

Histomorphometric measurements were applied using automated image analysis software (Image-Pro Plus 6.0, Media Cybernetics, MD, USA). The epiphyseal region (above the epiphyseal plate) and diaphysial region (2 mm below the epiphyseal plate) were selected as the ROIs. The ratio of trabecular bone area and marrow cavity area within both epiphyses (%, eTb/Te, eMC/Te) or diaphyses (%, dTb/Te, dMC/Te) were calculated. As the active area of bone development and growth, the thickness of the epiphyseal plate at the apex (mm, Th.ePA) was also measured and was regarded as an indicator of bone development.

The mice were injected with calcein (10 mg/kg) 7 days before euthanasia (only one injection before euthanasia) and alizarin-3-methyliminodiacetic acid (10 mg/kg body weight) 2 days before euthanasia (only one injection before euthanasia). A quantitative analysis was performed using automated image analysis software (Image-Pro Plus 6.0), and the value of the mineral apposition rate (MAR) represented the rate of new bone formation.

Cell proliferation and CB cytotoxicity assay

The effect of cytotoxicity of CB on hBMMSC survival rates was evaluated using a Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, PRC). hBMMSCs (2.0 × 10³ per well) were cultured in clear 96-well plates (Corning, NY, USA) containing 100 μL medium.

Osteogenic differentiation assay

Alkaline phosphatase kit (ALP, CoWin Biotech, Guangzhou, PRC) and Alizarin Red Staining kit (ARS, Sigma-Aldrich, MO, USA) staining were used as osteoblast differentiation markers at 7th and 14th day of osteogenic induction culture.

Quantitative real-time PCR (qRT-PCR)

The reverse transcription of complementary DNA (cDNA) was performed using a PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan) and gene-specific primers (Sangon, Shanghai, PRC). qRT-PCR was performed using SYBR Green Master Mix (YEASEN, Shanghai, PRC) on an ABI Prism 7700 RT-PCR System (Thermo Fisher Scientific, MA, USA). The primer sequences used in the amplification are presented in Table S1.

Western blot analysis

Primary antibodies against BMP-2 (1:2000; Abcam, Cambridge, UK), phosphor-SMAD 1/5/9 (1:2000; Abcam, Cambridge, UK), Smad 1/5/9 (1:1000; Abcam, Cambridge, UK), GSK3-β (1:5000; Abcam, Cambridge, UK), β-Catenin (1:5000; Proteintech Group, Chicago, USA), BGLAP (1:2000; ABclonal, Wuhan, PRC), ALP (1:2000; Proteintech Group, Chicago, USA), and RUNX2 (1:1000; Abcam, Cambridge, UK) were incubated with the membrane overnight for molecular probing. The membranes were then subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000; Beyotime) at 37°C for 2 h. Finally, Super Signal West Pico Substrate (Thermo) was used to visualize the protein bands via enhanced chemiluminescence. A quantification analysis was performed using Image J 16.0v software, with the final result measured based on GAPDH normalization. Each test was repeated three times.

Immunofluorescent cytochemistry

Permeation was performed using 0.04% Triton X-100 for 20 min, followed by blocking in 5% bovine serum albumin (15 min at 37°C). Primary antibodies against ALP (1: 100) and RUNX2 (1: 100) were applied before an incubation with a combination of secondary antibodies using luciferin conjugated IgG(s). An automatic quantitative pathological imaging system (Vectra Polaris™, PerkinElmer, MA, USA) was applied to scan for target protein expression (DAPI, blue, 350 nm; FITC, green, 488 nm; CY5, red, 595 nm).

Statistical analysis

SPSS 26.0 (IBM, NY, USA) was used for data analysis. Comparisons between two groups were calculated using Student's t test if the Shapiro–Wilk normality test was passed, otherwise, a Mann–Whitney test was used. One-way ANOVA was conducted to identify differences among more than two groups. A p value <0.05 was considered statistically significant.

RESULTS Cinobufotalin: DLEPS drug screening and repositioning

The up-regulated and down-regulated genes associated with primary osteoporosis in human MSCs (E-GEOD-35959)¹⁵ (Table S2 for the list of genes) were identified using the public ArrayExpress database. To predict the efficacy score, these genes were used as the input value in DLEPS (REF https://www.nature.com/articles/s41587-021-00946-z); the analysis was executed using the natural product library (Table S3 for the list of natural compounds). As a result, the top-ranked small molecules were selected from the DLEPS prediction and provided for testing as a treatment for osteoporosis. The scatter plot of the osteoporosis effectiveness scores using 100 or 200 up/down genes is shown in Figure 1A. The top ranked small molecules are clustered in the upper right position. The heat map presents the top-ranking effectiveness score for the natural compounds associated with osteoporosis treatment (Figure 1B).

Based on a literature search, we excluded ingredients that had been reported as follows: bone loss treatment-related drugs (Oroxylin A,³⁴ Nomilin,³⁵ Phenylalanine,³⁶ Prunetin,³⁷ Medicagol³⁸); flavonoid and isoflavone-related substances (Cosmosiin,³⁹ 7-methoxy-8-hydroxy-4-phenylcoumarin,⁴⁰ Epimedoside A⁴¹). Subsequently, traditional Chinese medicines that had been reported to be therapeutically effective or with larger toxic side effects were excluded. Finally, two candidates, cinobufotalin and dictamnine, used as anti-cancer drugs in clinical practice,⁴² were selected to be candidate drugs in our study. Since both drug candidates have a certain cytotoxicity and higher effectiveness score in osteoporosis, CB was preferentially verified in further validation experiments.

Determination of cinobufotalin toxicity

We first treated OVX mice with low doses (0.25 mg/kg) and high doses (0.5 mg/kg) of CB for 2 months. The organ tissue sections of the mice were then observed, and no abnormalities were found (Figure 2A). In addition, cellular morphology (Figure 2B) showed no obvious abnormality after treatment with 0.01 μM CB. However, 12 h after treatment with 10 μM CB, cell numbers decreased and cell volume increased, and at 24 h the cells were shrunken, and exhibited an apoptotic state.

Compared to the PM group (Figure 2C), no significant differences were observed in the proliferative ability of hBMMSCs treated with 1% DMSO, 0.01 μM, and 0.05 μM CB; however, the proliferation of hBMMSCs presented varying degrees of inhibition after 0.25 μM and 1.25 μM CB treatment, with 1.25 μM CB showing a larger inhibitory effect on cellular proliferation, especially after the 4th day. Together, the cell culture and CCK-8 assay indicated that CB below 0.25 μM was not significantly cytotoxic to hBMMSC proliferation.

Cinobufotalin prevents bone loss in OVX mice

Femurs were harvested for imageological and morphological examinations, and analysis of related parameters (Figure 3). Imageological data (Figure 3A,D) showed that compared with the SHAM+PBS group, the BMD, BV/TV, Tb.N and Tb.Th all declined in the OVX + PBS group compared with the SHAM+PBS group, whereas BS/BV and Tb.Sp were trending upwards, indicating that 3 months after OVX surgery, the bone mass of the femur was significantly decreased and the osteoporosis model was successfully constructed. Compared with the OVX + PBS group, CB reversed the decline in BMD, BV/TV, Tb.N, and Tb.Th, but the values remained lower than the SHAM+PBS group. Compared with the OVX + PBS group, elevated BS/BV and Tb.Sp also declined following CB intervention, but remained higher than in the SHAM+PBS group. Moreover, a low dose of CB appears to have a more pronounced effect than a high dose, because the bone histomorphometric parameters (BMD, BV/TV, Tb.N, Tb.Th, BS/BV and Tb.Sp) showed more remarkable variation with the low dose. It can be inferred that CB restores bone mass in OVX surgery-induced osteoporosis, and a low dose of CB is more effective.

From histomorphometric measurements (Figure 3B,E,F), we observed that the proportion of trabecular bone in the epiphysis decreased after OVX, accompanied by an increase in the bone marrow cavity. Similar trends were also present in the diaphysis. It was further confirmed that OVX-induced bone loss was more pervasive in the epiphysis. More importantly, CB increased the proportion of trabecular bone in both the epiphysis and diaphysis, with a better restorative effect seen with a low dose of CB. Additionally, the thickness of the epiphyseal plate apex (another crucial indicator of bone growth⁴³) (Figure 3B,G) and MAR (Figure 3C,H) displayed similar results. Therefore, CB significantly reversed OVX-induced bone loss, especially in the epiphyseal region, and a low dose of CB was more effective.

Cinobufotalin promotes the osteogenic differentiation of hBMMSCs in vitro

ALP and ARS staining (Figure 4A,B) showed that, compared with the OM group, the OM + CB (all doses) group significantly enhanced ALP activity. Similar trends were also shown with ARS staining. However, ALP activity and the number of mineralized nodules both exhibited a downward trend when CB was greater than 0.05 μM (Figure 4C,D), showing that a dose of 0.05 μM CB can significantly promote the osteogenic differentiation of hBMMSCs in vitro and enhance the function of mineralized nodule formation. Hence, 0.05 μM CB was selected to be the intervention dose.

qRT-PCR showed that the genes relevant to osteogenic differentiation were all distinctly up-regulated following intervention with 0.05 μM CB (Figure 4E). Moreover, the fluorescence intensity of ALP and RUNX2 also showed an obviously elevation after 0.05 μM CB treatment (Figure 4F). Therefore, the promotional effect of CB (0.05 μM) on hBMMSC osteogenic differentiation and function likely occurred through the regulation of BGLAP, ALP, and RUNX2.

The promotional effect of cinobufotalin on hBMMSC osteogenic differentiation and function is likely mediated via the BMP/SMAD and Wnt/β-Catenin signaling pathways

Western blotting revealed that the levels of BMP-2, GSK3-β, and β-Catenin expression and the level of p-SMAD 1/5/9 phosphorylation were all up-regulated after induction of hBMMSC osteogenic differentiation (Figure 5A,B). OM induction can also increase the expression of the osteogenic differentiation-related factors BGLAP and RUNX2. Intriguingly, BMP/SMAD and GSK3-β/β-Catenin signaling all showed obvious enhancement to various degrees when 0.05 μM CB was involved (Figure 5A,B). Together, the above results led us to infer that the promotion of the osteogenic differentiation and function hBMMSCs by CB likely occurred via the BMP/SMAD and Wnt/β-Catenin signaling pathways.

Cinobufotalin reversed the suppression of osteogenic differentiation induced by BMP inhibitors (Noggin) in hBMMSCs

The specific BMP inhibitor Noggin (1.5 μg/mL) was tested in the osteogenic intervention. BMP-2 and phosphorylated SMAD 1/5/9 were severely inhibited by Noggin (1.5 μg/mL) and similar results were found with BGLAP and RUNX2 (Figure 6A,B).

The inhibition of BMP-2 and p-SMAD 1/5/9 was reversed to a certain extent following the addition of 0.05 μM CB, with the osteogenic differentiation-related genes showing the similar recovery results (Figure 6A,B). The immunofluorescence cytochemistry assay supported these results, namely, Noggin down-regulated the levels of ALP and RUNX2 fluorescence intensity, which improved after the addition of 0.05 μM CB (Figure 6C). Consistent with these results, the osteogenic differentiation ability and number of mineralized nodules followed the same pattern; they were depressed by Noggin treatment, but CB alleviated these changes (Figure 8). Therefore, we can conclude that CB reversed the suppressive effect of Noggin-mediated osteogenic differentiation, and the effect is likely realized through BMP/SMAD signaling.

Cinobufotalin also reversed the suppression of osteogenic differentiation induced by the Wnt/β-catenin inhibitor Dikkopf-1 (DKK1) in hBMMSCs

The Wnt/β-catenin inhibitor Dikkopf-1 (DKK1; 0.5 μg/mL) was also tested in hBMMSCs. At the protein level, 0.05 μM CB reversed DKK1-mediated GSK3-β and β-Catenin inhibition to a certain degree (Figure 7A,B). The rescue conditions revealed the expression of osteogenic factor RUNX2, but not BGLAP (Figure 7A,B). Immunofluorescence cytochemistry showed the same results for ALP and RUNX2. Finally, ALP and ARS staining (Figure 8) clearly showed that DKK1 inhibited osteogenic differentiation, decreased the ALP activity of hBMMSCs, and reduced the number of mineralized nodules; the addition of CB alleviated these inhibitions. Therefore, Wnt/β-Catenin signaling is also engaged in the regulatory mechanism by which CB promotes osteogenic differentiation in hBMMSCs.

DISCUSSION

In this study, the DLEPS system was utilized to screen a series of natural compounds for osteoporosis treatment. Candidates with clearly reported efficacy were excluded through a literature search, and CB was finally selected for a follow-up study. The protective effect of CB on bone loss was first evaluated in a mouse model of OVX-induced osteoporosis. We also investigated possible signal regulatory mechanisms of CB in hBMMSCs in vitro, which indicated that the BMP/SMAD and Wnt/β-Catenin signaling pathways likely mediated CB-induced osteogenic differentiation and functional promotion.

Osteoporosis leads to poor bone quality and increased risk of bone fracture, resulting in clinical and dental treatment challenges.^31,44,45 Therefore, there is great clinical value in exploiting drugs that promote bone formation and that can be used for a long time with minimal adverse drug reactions. With the advancement of artificial intelligence (AI), information technology (IT) and biomedical science (BS), numerous analytical tools and software based on existing big data and information are being developed and used.^46,47 In the context of drug discovery, VS and DLEPS provide a new avenue for drug selection.¹⁶ The retrieved results consist of a series of highly correlated compounds with gene expression patterns associated with disease phenotypes, sequenced according to genomic enrichment scores.¹⁶ A recent study using DLEPS in a drug candidate examination revealed that perillen, chikusetsusaponin IV, and trametinib had an impact on obesity, hyperuricemia, and nonalcoholic steatohepatitis, respectively.¹⁶ A further investigation by Hajjo et al. algorithmically identified Raloxifene as a 5-HT₆R antagonist with potential utility in Alzheimer's disease.⁴⁸ Using a deep neural network for predicting molecules with antibacterial activity, Stokes et al. discovered ‘Halicin’, which displays bactericidal activity against a wide phylogenetic spectrum of pathogens.⁴⁹ In this study, based on the ArrayExpress database, the gene expression profiles of human MSCs were inspected for primary osteoporosis (E-GEOD-35959). DLEPS was used to subsequently screen out FDA-approved TCM compounds with potential anti-osteoporosis effects.

CB is derived from bufadienolides or toad venom extracted from the skin secretions of giant toads like Bufo gargarizans. Historically, CB has been ingested as a diuretic, cardiotonic, and hemostatic drug.⁵⁰ Moreover, CB alleviates the adverse effects associated with chemotherapy and improves patient quality of life.^51,52 The recommended concentration of CB in vivo (below 4 mg/kg) and in vitro (0.025–10 mM or 20–2000 ng/mL) are considered as safe doses.^53–55 In our present study, two different concentrations (low dose, 0.25 mg/kg; high dose, 0.5 mg/kg) were established to validate in vivo osteogenesis and no toxic effects were identified (Figure 2A). Based on the in vitro cell culture and CCK-8 assay, and it was determined that CB below 1.25 μM was a safe concentration for cell culture and induction (Figure 2B,C).

In our study, we successfully constructed a C57BL/6 animal model of bone loss induced by an estrogen deficiency (Figure 3; Figures S2 and S3). We explored the protective effect of CB on bone mass in an osteoporosis model using Micro CT and histomorphological examination. It was confirmed by double-labeled MAR detection that treatment with 0.25 mg/kg CB in vivo could significantly promote new bone formation (Figure 3). Considering their capacity for multi-lineage differentiation, and based on the requirements of specific biomedical applications, MSCs have achieved satisfactory therapeutic results in the repair of human and animal models of tissue damage.⁵⁶ We therefore also verified the osteogenesis promoting effect of CB in vitro based on the osteogenic differentiation activity and functional detection of hBMMSCs. It was found that 0.05 μM CB could significantly and efficiently promote osteogenic differentiation and the formation of mineralized nodules (Figure 4). Moreover, CB up-regulated the level of BGLAP, ALPL, and RUNX2 gene expression, and enhanced the protein activity of ALP and RUNX2 (Figures 4 and 5). Therefore, these results confirmed that CB could promote bone formation both in vitro and in vivo.

While the molecular regulatory mechanism behind CB-induced osteogenic differentiation and function of hBMMSCs is still unclear, our results suggest that BMPs/SMAD and Wnt/β-catenin signaling are important signal regulation cascades in osteogenesis. In hBMMSCs, the canonical BMP signaling pathway is the main regulatory mechanism by which BMPs fulfill their biological functions. BMPs bind and activate the corresponding receptors (mainly I and II).⁵⁷ Next, activated BMP receptors further phosphorylate cytoplasmic SMAD 1/5/8 to promote the combination and formation of molecular complexes with SMAD 4, finally entering the nucleus for translocation of the complexes to regulate the transcription of other target genes.⁵⁸ In addition, Wnt/β-catenin signaling, in which GSK3-β and β-catenin are the core regulators, is equally crucial.⁵⁹ Evidence has demonstrated that β-catenin is degraded when the Wnt/β-catenin signaling pathway is not activated (with the degradation complex activated by APC protein, GSK3-β, etc.).⁶⁰ The phosphorylation activity of GSK3-β increases when the signaling pathway is activated after the degradation complex is inactivated.⁶⁰ At this time, β-catenin is not degraded; however, it accumulates in the cytoplasm, enters the nucleus, and activates the expression of downstream genes including RUNX2 and OSX.⁶¹ Thus, the transcription of related target genes is initiated to promote osteoblast proliferation and differentiation.⁶¹ Here, we discovered that CB can specifically enhance the expression level of BMP-2, phosphorylated SMAD 1/5/9, and Wnt signal-related factors GSK3-β and β-catenin, thereby enhancing the protein expression of osteogenic-specific genes (BGLAP and RUNX2) in hBMMSCs. Therefore, it is reasonable to conclude that BMPs/SMAD and Wnt/β-catenin signals are involved in the promotion of hBMMSC osteogenic differentiation mediated by CB.

In this study, DLEPS indicated that CB may be an anti-osteoporosis drug candidate. We first demonstrated the osteogenesis-promoting function of CB both in vivo and in vitro, then determined the appropriate dosage (0.25 mg/kg and 0.05 μM). We also elucidated that the BMPs/SMAD and Wnt/β-catenin signaling cascades were all involved in the promotion of CB-induced bone formation. Our study showed that DLEPS can provide novel insights into potential drug investigations for translational medicine. Our results also provide a theoretical basis for the application of CB as an osteoporosis treatment strategy.

CONCLUSIONS

Collectively, our results suggest that CB screened by DLEPS could rescue estrogen deficiency-induced bone loss, and further promote the osteogenic differentiation and function of hBMMSCs. Mechanistically, both the BMPs/SMAD and Wnt/β-catenin signaling pathways were shown to be involved in the CB-induced osteogenic differentiation of hBMMCs. Thus, the expression of osteogenic factors BGLAP and Runx2 were regulated by CB, ultimately promoting osteogenesis.

AUTHOR CONTRIBUTIONS

Da-zhuang Lu and Hao Liu: conceptualization, data curation, funding acquisition, methodology, supervision, validation, writing, review & editing. Li-jun Zeng and Ran-li Gu: resources, formal analysis, investigation, methodology. Yang Li and Meng-long Hu: molecular biology, data curation, visualization. Ping Zhang, Xiao Zhang, Peng Yu and Zheng-wei Xie: conceptualization, project administration, resources, visualization, supervision. Yongsheng Zhou: funding acquisition, supervision, review & editing. All the authors read and approved the final manuscript. All the authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.

ACKNOWLEDGMENTS

The authors thank ELIXIGEN Co. for the editorial and English language revisions, and all the staff at the Stomatology Research Center & Hospital of Peking University Health Science Center.

FUNDING INFORMATION

This study was supported by grants of Beijing Natural Science Foundation (L222145 & L222030), Peking University Medicine Fund of Fostering Young Scholars' Scientific & Technological Innovation (BMU2022PY010), Emerging Engineering Interdisciplinary Project and the Fundamental Research Funds for the Central Universities (Peking University, PKU2022XGK008).

CONFLICT OF INTEREST STATEMENT

Authors declare that there are no conflicts of interest regarding the publication of this article.

DATA AVAILABILITY STATEMENT

Authors confirm that all data in this study are fully available.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Authorization of all animal experiments was awarded by the Peking University School of Medicine Institutional Committee for Animal Care and Use (LA2021040). All operations are performed under anesthesia and efforts were made to minimize suffering to the animals.

CONSENT FOR PUBLICATION

Written informed consent for publication was obtained from all participants.