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. 2026 May 20;40(8):5104–5118. doi: 10.1002/ptr.70384

Bakuchiol Ameliorates Glucocorticoid‐Induced Osteoporosis by Enhancing Osteoblast Differentiation via Targeting FAT4 to Activate YAP1

Hengjun Wang 1,2,3, Tingting Zhou 1,2,3, Yunchao Zhao 1,2,3, Zhengxin Meng 1,2,3, Zhiqiang Sun 1,2,3, Hao Zhou 1,2,3, Huan Liu 4, Changyu Du 5, Shuquan Lv 2,6, Jianyong Zhao 1,2,3,✉, Zhongyong Zhang 2,6,✉, Huantian Cui 7,✉
PMCID: PMC13436232  PMID: 42157729

ABSTRACT

Psoralea corylifolia Linn. exhibits osteogenic effects; however, the mechanism by which its active component Bakuchiol (BAK) alleviates glucocorticoid‐induced osteoporosis (GIOP) remains unclear. We aim to investigate BAK's therapeutic effects and potential mechanisms in GIOP. We treated GIOP mice with BAK. We evaluated BAK's therapeutic efficacy using micro‐CT and histopathological staining. We performed transcriptomic analysis and identified significantly enriched GO pathways. We then used biochemical assays and Western blotting to examine the effects of BAK on osteogenic differentiation‐related signaling factors in GIOP mice. We conducted in vitro experiments using osteoblasts. We employed RT‐qPCR, Western blot, ALP & ARS staining, immunofluorescence and dual‐luciferase reporter assays to assess BAK's influence on signaling pathways related to osteoblast differentiation. We used molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) to confirm the direct binding interaction between BAK and FAT4. Finally, we silenced Fat4 to validate that BAK exerts its anti‐GIOP effects by activating YAP1 through FAT4. BAK treatment significantly enhanced bone quality and strength in GIOP mice, and mitigated femoral pathological damage. BAK upregulated key osteoblast differentiation‐related transcription factors and enhanced serum alkaline phosphatase activity. Western blot confirmed that BAK increased protein levels of YAP1 while reducing levels of FAT4 and DCHS1. In vitro, BAK similarly promoted osteoblast differentiation and activated YAP1 expression and Runx2 transcription. Molecular docking, CETSA and DARTS analyses demonstrated that BAK directly binds to FAT4. Silencing Fat4 abolished the pro‐osteogenic effects of BAK. BAK likely exerts its therapeutic effects in GIOP by targeting FAT4, activating YAP1 and promoting osteoblast differentiation.

Keywords: bakuchiol, FAT4, glucocorticoid‐induced osteoporosis, osteoblasts, transcriptomics, YAP1

1. Introduction

Glucocorticoid‐induced osteoporosis (GIOP) is a form of secondary osteoporosis marked by progressive bone loss, decreased bone mass, and impaired bone microarchitecture. It commonly occurs in patients receiving long‐term or high‐dose glucocorticoid (GC) therapy (Xing et al. 2021). Epidemiological studies report that up to one‐third of individuals undergoing prolonged GC treatment develop osteoporosis (Zavatta and Clarke 2021). Current clinical interventions for GIOP primarily involve pharmacologic agents that either inhibit bone resorption or stimulate bone formation, such as bisphosphonates and parathyroid hormone analogs (Laurent et al. 2022). However, these treatments frequently cause adverse effects, including abnormal serum calcium levels and muscle pain (Rizzoli et al. 2011). This underscores the urgent need to develop safer and more effective therapeutic alternatives for GIOP.

GIOP arises from an imbalance between osteoblast‐mediated bone formation and osteoclast‐mediated bone resorption (Lane 2019). Increasing evidence indicates that GC exposure suppresses osteoblast proliferation, differentiation, and function, while also inducing apoptosis in mature osteoblasts (Jiang et al. 2025). Transcriptomic analyses in GIOP mouse models have revealed significant downregulation of the osteogenic transcription factor Runx2, which is partially restored by pharmacological interventions (Wang, Wang, et al. 2024). Clinical studies further support these findings, showing impaired osteogenic differentiation and compromised bone quality in GIOP patients (Yuan et al. 2025; Zhang et al. 2025). These data highlight the pivotal role of osteogenesis in GIOP pathogenesis and suggest that restoring osteoblast function and differentiation capacity may offer a promising therapeutic approach.

Psoralea corylifolia Linn., derived from the seeds of the leguminous plant P. corylifolia , possesses a wide range of pharmacological properties, including antioxidant, anticancer, and osteogenic effects, making it a promising candidate for osteoporosis treatment (Xin et al. 2019; Chai et al. 2018). Experimental studies have demonstrated that P. corylifolia Linn. significantly improves bone mineral density and prevents bone loss in rat models (Lim et al. 2009). Despite this, the pharmacological mechanisms by which its active component, Bakuchiol (BAK), alleviates GIOP remain poorly understood. This study builds on previous research to evaluate the therapeutic efficacy of BAK in GIOP and to uncover its underlying molecular mechanisms. We first established a GIOP mouse model and assessed BAK's pharmacological effects through a series of systematic interventions. Transcriptomic analysis of bone tissue following BAK treatment revealed significant alterations in gene expression, with initial findings suggesting that BAK modulates YAP1 within the Hippo signaling pathway. Further in vitro experiments confirmed that BAK promotes osteoblast differentiation by activating YAP1 and identified its direct target. These findings support the potential of BAK as a novel therapeutic agent for GIOP.

2. Materials and Methods

2.1. Drugs and Reagents

BAK (Catalog No.: SMB00604) was purchased from Shanghai Merck Technology Co. Ltd. And dexamethasone (DEX, Catalog No.: S17003) was obtained from Shanghai yuanye Bio‐Technology Co. Ltd. Additional details regarding experimental drugs and reagents are provided in the Supporting Information S1.

2.2. Animals Feeding, Modeling, Grouping and Drug Administration

We used specific pathogen‐free (SPF) female Balb/c mice, purchased from Beijing Huafukang Bioscience Co. Ltd. (Production License No.: SYXK(Jing)2024‐0015). Mice were 2 months old and weighed 23 ± 2 g at the start of the experiment. The protocol for the study was reviewed by the Ethical Review Committee of Animal Experiments in Yunnan University of Chinese Medicine (Ethics approval number: YUCM‐AE‐2024‐042) and adhered to animal ethics guidelines. And all experimental procedures adhered to institutional and national guidelines for animal welfare. Mice were housed under controlled conditions at 24°C ± 2°C with 45% ± 5% relative humidity and a 12 h light/dark cycle. Food and water were available ad libitum.

We randomly divided 60 female Balb/c mice into six groups (n = 10 per group): normal control group (Control), model group (GIOP), positive control group (Alendronate sodium, ALN), low‐dose BAK group (BAK‐L), medium‐dose BAK group (BAK‐M), and high‐dose BAK group (BAK‐H). To induce GIOP, we administered subcutaneous injection of 25 mg/kg DEX once daily for 4 weeks to the remaining five groups except the Control group (Wang, Zhao, et al. 2024), following established protocols. Starting on the first day of model induction, the ALN group received daily oral gavage of 0.9 mg/kg ALN (Luthfiana et al. 2023), while the BAK‐L, BAK‐M, and BAK‐H groups received daily oral gavage of 5, 10, 20 mg/kg BAK at respective doses (Xu et al. 2025). The Control group and model group received daily subcutaneous injections of 0.1 mL saline. All treatments were continued for four weeks. During this period, the body weight of mice was continuously observed and recorded.

2.3. Micro‐CT

We scanned femurs using a micro‐CT system and analyzed the reconstructed images with VGStudio Max 2.2 software. Quantitative parameters included bone mineral density (BMD), bone volume fraction (bone volume/total volume, BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th).

2.4. Biomechanical Testing

We subjected the left femurs from each group to a three‐point bending test, using a support span of 6 mm and a loading speed of 1 mm/min until fracture occurred. We continuously recorded load–displacement curves and calculated biomechanical properties, including ultimate load, elastic modulus, energy to failure, and ultimate stress.

2.5. Serum Biochemical Analysis

Mice were anesthetized deeply with pentobarbital sodium i.p. We collected blood from the abdominal aorta and centrifuged for 15 min (3000 rpm) to isolate the serum. We measured the activities of serum calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP) following the manufacturer's protocols. Remaining serum was kept at −80°C until analyses.

2.6. Pathological Staining

After micro‐CT scanning, we decalcified the femurs in 10% EDTA, followed by dehydration in graded ethanol solutions and drying. Samples were cleared in xylene and embedded in paraffin. Sections (5‐μm thick) were prepared for Hematoxylin and Eosin (HE) and Masson's trichrome staining. We examined the stained sections under a light microscope. We performed quantitative analysis using Image‐Pro Plus 6.2 to assess trabecular bone content and collagen fiber deposition.

2.7. Transcriptomics Analysis

We performed transcriptomic analysis based on established protocols from previous studies (Cui et al. 2024). Bone tissues were collected from the Control, GIOP, and BAK‐H groups for total RNA extraction. We evaluated RNA purity, concentration, and integrity to ensure sample quality. High‐quality RNA was then used for library preparation and sequencing on the Illumina platform. We identified differentially expressed genes (DEGs) between the GIOP and Control groups, as well as between the BAK‐H and GIOP groups, using DESeq2 software. DEGs met the following criteria: |Log2(Fold Change)| > 1 and p‐value < 0.05. We subsequently performed GO pathway enrichment analysis on the DEGs and considered pathways with p‐value < 0.05 statistically significant.

2.8. Osteoblast Culture and Induction

We obtained the mouse osteoblast cell line MC3T3‐E1 from Shanghai Fuheng Biotechnology Co. Ltd. and cultured the cells in 10% fetal bovine serum‐supplemented α‐MEM at 37°C in an incubator with 5% CO2. After attachment of cells, we replaced the medium with osteogenic induction medium (OM) containing 50 mg/L ascorbic acid, 10 mM β‐glycerophosphate, and 10 nM DEX to induce osteogenic differentiation for 7 days. In parallel, we treated cells with 10 μM DEX or 20 μM BAK (Xu et al. 2025) to evaluate BAK's effects on osteoblast differentiation.

2.9. FAT4 Knockdown in Osteoblasts

We seeded MC3T3‐E1 cells into 12‐well plates at a density of 4 × 104 cells/well and induced differentiation using OM. We then transfected the cells with either shFAT4 or shNC (negative control) using Lipo3000 transfection reagent for 6 h. Following transfection, we replaced the medium without transfection reagent and continued with subsequent treatments. The SS sequence of shFAT4 was: GGAAGTTTAAGTTAGACAA, the AS sequence of shFAT4 was: TTGTCTAACTTAAACTTCC.

2.10. MTT Assay

We seeded cells into 96‐well plates and cultured them in OM containing varying concentrations of DEX or BAK. After treatment, we added 20 μL of 5 mg/mL MTT solution to each well and incubated the plates for 4 h at 37°C with 5% CO2. Next, we added 150 μL of dimethyl sulfoxide (DMSO) per well and gently shook the plates for 10 min. Using a microplate reader, we measured the absorbance at 490 nm.

2.11. RT‐qPCR Analysis

We extracted total RNA from osteoblasts using a commercial RNA extraction kit, followed by reverse transcription to synthesize complementary DNA (cDNA). We quantified mRNA expression levels of target genes by qPCR using the 2−ΔΔCT method, with Actb as the reference gene. Primer sequences are provided in Table S1.

2.12. Cell Alkaline Phosphatase (ALP) Activity Assay

We harvested cells from each group and prepared cell homogenates through ultrasonic disruption. We measured ALP activity according to the manufacturer's instructions and assessed total protein concentration using the BCA method.

2.13. ALP Staining and Alizarin Red (ARS) Staining

After 14 days of induction in MC3T3‐E1 cells, an ALP staining kit was used to detect ALP expression. On the 21st day of osteogenic induction, alizarin red S solution was used to observe bone mineralization level. The specific methods were referenced from literature (Shang et al. 2024; Chen et al. 2025; Wu et al. 2025). In brief, the old culture medium was removed, and the cells were washed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 20 min. After washing the cells twice with PBS, they were stained. The staining was observed under a microscope, and the stained area was quantified using Image Pro Plus 6.2 software.

2.14. Immunofluorescence

We fixed cells at room temperature with 4% paraformaldehyde for 15 min and permeabilized them with 0.5% Triton X‐100. After goat serum blocking for 30 min, we incubated the samples overnight at 4°C with primary YAP1 antibody. With three washes, we incubated the cells with a fluorescent secondary antibody for 1 h at room temperature. We counterstained the nuclei with DAPI and applied an anti‐fade mounting medium. We captured fluorescent images using confocal microscopy, and YAP1 nuclear translocation was quantified using Image‐Pro Plus 6.2.

2.15. Dual‐Luciferase Reporter Assay

We assessed RUNX2 transcriptional activity in osteoblasts using a dual‐luciferase reporter assay, following established protocols (Crespo‐Enriquez et al. 2019; Zaidi et al. 2004; Zhang et al. 2024; Liao et al. 2024). MC3T3‐E1 cells were seeded in 12‐well plates at a density of 1.5 × 104 cells per well and induced with osteogenic medium. Once cells adhered, we replaced the medium with Lipofectamine 2000 transfection reagent and transfected the cells with 250 ng of the Runx2 luciferase reporter plasmid and 50 ng of Renilla plasmid. After a 6‐h incubation, the cells were treated with DEX or BAK for 48 h. Luciferase activity was measured using a Berthold LB 960 luminometer.

2.16. Molecular Docking

We conducted molecular docking using methods described in previous studies (Duan et al. 2024). Briefly, we obtained the 2D chemical structure of BAK from PubChem (https://pubchem.ncbi.nlm.nih.gov/) in SDF format. We downloaded the FAT4 3D structure from the Protein Data Bank (PDB https://www.rcsb.org/). Using PyMOL software, we removed water molecules and small molecule ligands and added hydrogen atoms using AutoDockTools. We performed molecular docking between BAK (the ligand) and FAT4 (the receptor) to evaluate their binding affinity. A binding score (S value) below −5.0 kcal/mol indicated a strong binding interaction (Wu et al. 2024; Xie et al. 2024; Deng et al. 2024). The docking results were visualized to confirm the interaction.

2.17. Cellular Thermal Shift Assay (CETSA)

We performed CETSA following established protocols (Tu et al. 2023; Friman 2020; Wang et al. 2023). Briefly, MC3T3‐E1 cells, induced to differentiate with OM, were harvested and resuspended in PBS containing 1% protease and phosphatase inhibitors at a density of 5 × 105 cells/mL. The cell suspension was subjected to a freeze–thaw cycle: 30 s in liquid nitrogen followed by 30 s at 37°C. Next, lysates were divided into 14 aliquots (7 treated with 20 μM BAK and 7 with DMSO) and incubated at 37°C for 1 h. Samples were heated at increasing temperatures (37°C and 40°C to 65°C in 5°C increments) for 5 min, then cooled on ice. After centrifugation (12,000 × g, 25 min, 4°C), the supernatants were collected, and FAT4 levels were analyzed by Western blot. Thermal denaturation curves were generated to assess the thermal stability of FAT4.

2.18. Drug Affinity Responsive Target Stability (DARTS)

We used DARTS assay to confirm BAK's targeted binding to FAT4, based on previous methods (Shi et al. 2022). The protein supernatant from repeated freeze‐thawing (following the CETSA procedure) was mixed with TNC solution (10×) at a 9:1 ratio. After protein quantification by BCA, the samples were divided into six tubes. Three tubes received 20 μM BAK, and three received an equivalent volume of DMSO. The samples were incubated at 37°C for 1 h. Different concentrations of pronase E were added to the DMSO and BAK‐treated groups, with a pronase E to protein mass ratio of 1/500 and 1/2000. A control group without pronase E was included. Digestion occurred at room temperature for 30 min. FAT4 degradation was assessed via Western blot to evaluate binding stability.

2.19. Western Blot

We quantified total protein extracts from bone tissues and cells using the BCA assay. Equal amounts of protein from each group were separated by SDS‐PAGE and transferred using a wet transfer method to PVDF membranes. After 5% skim milk blocking for 2 h, the membranes were incubated overnight at 4°C with primary antibodies targeting RUNX2, OSTERIX, FAT4, DCHS1, YAP1, and p‐YAP1. We applied HRP‐conjugated secondary antibodies at room temperature for 2 h. We visualized protein bands using ECL and analyzed them with Image‐Pro Plus 6.2, with β‐actin serving as the loading control.

2.20. Statistical Analysis

Statistical analysis was performed using SPSS Pro software. Data are presented as mean ± standard deviation (SD). Intergroup comparisons were conducted using one‐way ANOVA, with p < 0.05 considered statistically significant.

3. Results

3.1. BAK Ameliorates Bone Loss and Improves Bone Strength in GIOP Mice

After 4 weeks of treatment, micro‐CT analysis showed that GIOP mice experienced significant reductions in key bone mass parameters, such as bone mineral density (BMD), bone volume/tissue volume ratio (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) when compared to the Control group. Intervention with either ALN or BAK partially restored these parameters (Figure 1A–E). Biomechanical testing showed that GIOP mice had significantly reduced bone strength indices, such as ultimate load, elastic modulus, energy to failure, and ultimate stress, relative to the Control group. Treatment with either ALN or BAK significantly reversed these deficits (Figure 1F–I). These results demonstrate that bone loss is effectively mitigated by BAK in GIOP mice, which can also preserve bone strength.

FIGURE 1.

FIGURE 1

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BAK intervention significantly improves bone loss and strength in GIOP mice. GIOP models were established via daily injection of 25 mg/kg (i.h.) DEX in mice, and concurrent BAK intervention at 5, 10, 20 mg/kg (i.g.) for 4 weeks. Effects of BAK on bone microstructure and strength in GIOP mice were assessed through femoral micro‐CT scanning and biomechanical testing. (A–E) BAK intervention significantly increases BMD (A, B), BV/TV (A, C), Tb.N (A, D), and Tb.Th (A, E) in GIOP mice. (F–I) BAK intervention significantly enhances ultimate load (F), elastic modulus (G), energy to failure (H), and ultimate stress (I) in GIOP mice. n = 6 per group for A–I. ## p < 0.01 compared to the Control group; *p < 0.05, **p < 0.01, ns indicates a nonsignificant difference as compared to the GIOP group.

3.2. BAK Restores Calcium‐Phosphorus Homeostasis and Alleviates Pathological Damage in GIOP Mice

Serum biochemical analysis revealed significantly lower levels of Ca and P in GIOP mice compared to the Control group. Both ALN and BAK treatments elevated serum Ca and P concentrations, with the most pronounced effects observed in the ALN and BAK‐H groups (Figure 2A,B). Histopathological analysis, using HE and Masson's trichrome staining, showed that GIOP mice had thin, fragmented trabeculae, enlarged marrow cavities, and reduced collagen fiber content, indicating osteopenia and compromised bone quality. In contrast, ALN and BAK treatments significantly improved trabecular structure and collagen deposition (Figure 2C–F). Among all treatment groups, ALN and BAK‐H exhibited the most substantial therapeutic effects. Based on these findings, the Control, GIOP, and BAK‐H groups were selected for further analysis.

FIGURE 2.

FIGURE 2

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BAK intervention alleviates calcium‐phosphorus imbalance and pathological damage in GIOP mice. (A, B) BAK intervention increases serum calcium (Ca) (A) phosphorus (P) (B) in GIOP mice. (C–F) Pathological staining results showing BAK treatment improves trabecular thinning and fracture (C, D), as well as significantly reduces collagen fibers (E, F). n = 10 per group for A, B; n = 6 per group for C–F. ## p < 0.01 compared to the Control group; *p < 0.05, **p < 0.01, ns indicates a nonsignificant difference as compared to the GIOP group.

3.3. BAK Treatment Promotes Transcriptional Expression of Osteoblast Differentiation in GIOP Mice

Transcriptomic analysis of bone tissues from the Control, GIOP, and BAK‐H groups identified DEGs using the criteria |Log2(Fold Change)| > 1 and p‐value < 0.05. As shown in Figure 3A,B, osteoblast‐specific transcription factors Runx2 and Osterix (Wang, Zhao, et al. 2024) were downregulated in the GIOP group but were upregulated following BAK treatment. Western blot analysis confirmed these results, demonstrating that BAK significantly increased the protein levels of RUNX2 and OSTERIX in GIOP mice (Figure 3C–E). Consistent with this, serum ALP activity was significantly reduced in the GIOP group compared to Controls but was restored with BAK treatment (Figure 3F). These results indicate that BAK enhances osteoblast activity and improves bone metabolism.

FIGURE 3.

FIGURE 3

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BAK intervention promotes osteoblast differentiation in GIOP mice. Transcriptome analysis was performed on bone tissues from mice in the Control, GIOP, and BAK‐H groups. DEGs from GIOP vs. Control and BAK‐H vs. GIOP were screened using |Log2(Fold Change)| > 1 and p‐value < 0.05. (A, B) Runx2 and Osterix gene expression is upregulated after BAK intervention. (C–E) Western blot results showing BAK treatment significantly upregulates RUNX2 (C, D) and OSTERIX (C, E) proteins. (F) BAK enhances serum ALP activity in GIOP mice. n = 6 per group for A, B; n = 3 per group for C–E; n = 10 per group for F. ## p < 0.01 compared to the Control group; **p < 0.01 compared to the GIOP group.

3.4. BAK Promotes Osteogenesis by Regulating the Hippo Signaling Pathway

GO enrichment analysis of the DEGs (Tables S2 and S3) identified significant pathways based on a p‐value threshold of < 0.05. Among the pathways enriched in both the GIOP vs. Control and BAK‐H vs. GIOP comparisons, we focused on the Hippo signaling pathway (Figure 4A,B). A heatmap of Hippo signaling‐related genes is shown in Figure 4C, revealing that, in the GIOP group, Dchs1 and Fat4 were upregulated, while Sox11, Yap1, Amotl2, and Amot were downregulated. BAK treatment reversed these alterations. The Hippo signaling pathway plays a critical role in the phosphorylation regulation of YAP1. Inhibition of this pathway reduces YAP1 phosphorylation, allowing dephosphorylated YAP1 to translocate into the nucleus, where it activates Runx2 transcription (Collins et al. 2024; Chuang and Ito 2021). DCHS1 and FAT4, as upstream regulators of the Hippo signaling pathway, suppress YAP1 nuclear translocation by activating the pathway (Crespo‐Enriquez et al. 2019) (Figure 4D schematic). Western blot analysis confirmed that BAK treatment increased YAP1 protein levels while decreasing p‐YAP1, FAT4, and DCHS1 expression in GIOP mice (Figure 4E–I). These findings suggest that BAK may activate YAP1 by inhibiting the Hippo signaling pathway, thereby promoting osteoblast differentiation. Consequently, we conducted further in vitro validation.

FIGURE 4.

FIGURE 4

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BAK intervention regulates the Hippo signaling pathway to promote osteogenic growth. GO enrichment analysis was performed on DEGs from the Control, GIOP, and BAK‐H groups. Significant pathways from GIOP vs. Control and BAK‐H vs. GIOP were screened using p‐value < 0.05. (A, B) Hippo signaling pathway is significantly enriched in both GIOP vs. Control and BAK‐H vs. GIOP. (C) BAK intervention significantly downregulates Dchs1 and Fat4 expressions and upregulates Sox11, Yap1, Amotl2, and Amot expressions. (D) Schematic diagram of molecular relationships related to the Hippo signaling pathway. (E–I) Western blot results showing BAK intervention increases YAP1 (E, F) expression, while decreasing p‐YAP1 (E, G), FAT4 (E, H), and DCHS1 (E, I) expressions. n = 6 per group for A–C; n = 3 per group for E–H. ## p < 0.01 compared to the Control group; *p < 0.05, **p < 0.01 compared to the GIOP group.

3.5. BAK Enhances Osteoblast Differentiation via Activation of YAP1

We initially assessed the impact of DEX and BAK on the viability of OM‐induced MC3T3‐E1 cells. MTT assays revealed that DEX reduced osteoblast viability in a dose‐dependent manner (Figure 5A), while BAK at less than 20 μM had no significant effect on cell viability (Figure 5B). Based on a prior study (Huang et al. 2024), we selected 10 μM DEX for the intervention in OM‐induced MC3T3‐E1 cells to establish an in vitro osteoblast injury model, while 5, 10, and 20 μM BAK were used for intervention. MTT assays indicated that BAK enhanced osteoblast viability in a dose‐dependent manner (Figure 5C). Subsequently, we examined the expression levels of key osteoblast differentiation markers using RT‐qPCR and Western blot. DEX treatment downregulated the expression of RUNX2 and OSTERIX, whereas BAK significantly upregulated these markers in a dose‐dependent manner (Figure 5D–H). Additionally, cell ALP activity assays showed a similar trend, with BAK increasing ALP activity in osteoblasts (Figure 5I). Consistently, the staining results for ALP&ARS revealed that upon the addition of BAK, the formation of ALP (Figure 5J,K) and the calcium deposits (Figure 5J,L) in osteoblasts increased in a dose‐dependent manner compared to the group treated with DEX alone. Collectively, these results suggest that BAK promotes osteoblast differentiation.

FIGURE 5.

FIGURE 5

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BAK promotes osteoblast differentiation in vitro. In vitro experiments were conducted using MC3T3‐E1 cells induced to differentiate into osteoblasts with OM. Different concentrations of DEX were used to screen required DEX doses for the in vitro GIOP model. Different concentrations of BAK were used to screen nontoxic doses. Subsequently, MC3T3‐E1 osteoblasts were treated with 10 μM DEX for 7 days to establish the in vitro GIOP model. BAK intervention at 5, 10, 20 μM was applied to assess effects of BAK on osteoblast differentiation. (A, B) MTT results showing DEX dose‐dependently decrease viability of MC3T3‐E1 osteoblasts (A), and BAK exhibits no cytotoxicity on MC3T3‐E1 osteoblasts under 20 μM (B). (C) BAK dose‐dependently increases osteoblast viability. (D, E) RT‐qPCR detection showing BAK dose‐dependently upregulates Runx2 (D) and Osterix (E) gene expressions. (F–H) Western blot results showing BAK intervention increases RUNX2 (F, G) and OSTERIX (F, H) protein expressions. (I) BAK intervention enhances cell ALP activity of the osteoblast injury model in vitro. (J–L) ALP&ARS staining reveals that BAK intervention increases the ALP formation (J, K) and the calcium deposits (J, L) in osteoblasts in a dose‐dependent manner. n = 6 per group for A‐C, *p < 0.05, **p < 0.01, ns indicates a nonsignificant difference as compared to the DEX (0 μM) group. n = 3 per group for D–L, ## p < 0.01 compared to the Control group; *p < 0.05, **p < 0.01, ns indicates a nonsignificant difference as compared to the DEX‐treated alone group.

To investigate the mechanism underlying BAK's effect on YAP1‐mediated osteoblast differentiation, we continued our analysis using the aforementioned cell model. We again applied 10 μM DEX and 20 μM BAK for intervention. Western blot analysis demonstrated that BAK significantly increased the protein expression of YAP1 and reduced p‐YAP1 protein expression in osteoblasts (Figure 6A–C). Immunofluorescence staining further revealed that BAK treatment increased both the overall expression of YAP1 and its nuclear localization (Figure 6D,E). As previously discussed, nuclear YAP1 enhances the transcriptional activity of Runx2. To confirm BAK's effect on the transcriptional activity of nuclear RUNX2 in osteoblasts, we employed a dual‐luciferase reporter gene assay. The results showed that DEX decreased Runx2 transcriptional activity, while BAK enhanced its activity (Figure 6F). This finding supports the hypothesis that BAK promotes osteoblast differentiation through the activation of YAP1.

FIGURE 6.

FIGURE 6

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BAK increases Runx2 transcription levels by promoting YAP1 nuclear translocation. We treated OM‐induced MC3T3‐E1 cells with 10 μM DEX and 20 μM BAK to assess the effects of BAK on YAP1 nuclear translocation and Runx2 transcriptional activity in osteoblasts. (A–C) Western blot results showing BAK upregulated YAP1 (A, B) and downregulated p‐YAP1 (A, C) protein expression in osteoblasts. (D, E) YAP1 nuclear translocation levels increase after BAK intervention as observed by immunofluorescence staining. (F) Dual‐luciferase reporter gene assay indicating BAK intervention enhances Runx2 transcriptional activity. n = 3 per group. ## p < 0.01 compared to the Control group; *p < 0.05, **p < 0.01 compared to the DEX‐treated alone group.

3.6. BAK Promotes Osteoblast Differentiation by Targeting FAT4 to Activate YAP1

As shown in Figure 4C, FAT4 exhibited a significant difference among the genes in the Hippo signaling pathway. Previous studies have demonstrated that the binding of the ligand DCHS1 to the receptor FAT4 inhibits osteoblast proliferation (Crespo‐Enriquez et al. 2019). Based on these findings, we hypothesized that BAK might promote osteogenic differentiation by directly interacting with and binding to FAT4. To test this hypothesis, we performed molecular docking, CETSA, and DARTS experiments (Figure 7A). The molecular docking results revealed a binding energy of −6 kcal/mol (< −5 kcal/mol) (Wu et al. 2024; Xie et al. 2024; Deng et al. 2024), indicating a strong interaction between BAK and FAT4 (Figure 7B). CETSA results showed that the intensity of the specific band for FAT4 decreased progressively with increasing temperature. Moreover, after BAK treatment, the melting curve of FAT4 shifted to the right (Figure 7C), suggesting that BAK enhances the thermal stability of FAT4. DARTS analysis revealed that FAT4 exhibited increased resistance to proteolytic digestion following BAK intervention (Figure 7D). Together, these data preliminarily confirm that BAK promotes osteoblast differentiation by directly targeting FAT4.

FIGURE 7.

FIGURE 7

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BAK activates YAP1 by targeting FAT4 and thereby promoting osteoblast differentiation. We first used molecular docking, CETSA, and DARTS to detect targeting effects of BAK on FAT4. Subsequently, Fat4 was silenced to assess effects of BAK on osteoblast differentiation. (A) Schematic diagram of CETSA and DARTS experiments. (B) Molecular docking results showing BAK had strong binding ability to FAT4. (C) CETSA results showing BAK significantly increases FAT4 thermal stability. (D) DARTS results showing BAK intervention improves FAT4 resistance to proteolysis. (E) Schematic diagram of the in vitro Fat4 silencing experiment. (F–K) Silencing Fat4 significantly increases osteoblast viability (F), upregulates YAP1 (G, H), RUNX2 (G, J), and OSTERIX (G, K) protein expressions in osteoblasts, and enhances ALP activity in the supernatant (L), while decreasing p‐YAP1 (G, I). (M–O) ALP&ARS staining shows that Fat4 silencing increases the ALP formation (M, N) and the calcium deposits (M, O) in osteoblasts. n = 3 per group. *p < 0.05, **p < 0.01, ns indicates a nonsignificant difference.

Next, we explored the role of FAT4 in osteogenic differentiation by silencing Fat4 in OM‐induced MC3T3‐E1 cells, followed by treatment with DEX and BAK (Figure 7E). The results indicated that Fat4 silencing led to enhanced osteoblast differentiation (Figure 7F). Furthermore, the protein levels of YAP1, RUNX2, and OSTERIX were upregulated and p‐YAP1 was downregulated in osteoblasts (Figure 7G–K), and ALP activity was significantly increased (Figure 7L). Meanwhile, ALP&ARS staining revealed that the formation of ALP (Figure 7M,N) and the calcium deposits (Figure 7M,O) in osteoblasts were increased after Fat4 silencing. However, the pro‐differentiation effect of BAK was abolished after Fat4 silencing. These findings demonstrate that BAK promotes osteoblast differentiation by targeting FAT4 and thereby activating YAP1.

4. Discussion

In recent years, the incidence of GIOP has steadily increased. As the first‐line agents for treating osteoporosis, bisphosphonates reduce bone resorption by inhibiting osteoclast activity. However, the side effects of bisphosphonates have been reported in various aspects, such as causing gastrointestinal reactions (Yang et al. 2025), inducing severe osteonecrosis of the jaw and atypical femoral fractures (Reyes et al. 2016), as well as ocular diseases (Qian et al. 2024). This highlights the urgent need for effective prevention and treatment strategies. P. corylifolia Linn. has traditionally been used to treat osteoporosis (Wei et al. 2024), but research on its primary active component, BAK, in alleviating osteoporosis remains limited (Hu et al. 2025). To address this gap, we evaluated the anti‐GIOP effects of BAK using a DEX‐induced GIOP mouse model, which is widely utilized in GIOP research (Ma et al. 2023). As a positive control, we selected alendronate (ALN), a commonly used therapeutic bisphosphonate drug for GIOP (Liu et al. 2022; Squadrito et al. 2023). Our results revealed that DEX‐induced GIOP mice showed reductions in bone quality parameters, including bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as well as bone strength measures such as ultimate load, elastic modulus, energy to failure, and ultimate stress. Additionally, we observed decreased serum Ca and P levels, along with diminished trabecular bone and collagen fibers. These findings are consistent with previous studies (Wang, Zhao, et al. 2024; Ochiai et al. 2024; Rong et al. 2022), confirming the successful modeling of GIOP in our experiment. Importantly, BAK treatment dose‐dependently improved these symptoms, with the high dose of BAK showing efficacy comparable to ALN, suggesting BAK's potential as an alternative therapeutic agent for GIOP. Nevertheless, further research is required to evaluate the therapeutic safety advantages of BAK, in order to provide more detailed experimental evidences for the usage of BAK in clinic.

Transcriptomic analysis revealed that BAK intervention upregulated the expression of the Runx2 and Osterix genes in the bone tissue of GIOP mice. Runx2 and Osterix are critical transcription factors involved in osteoblast differentiation and are commonly used to evaluate osteogenic maturity (Yamashita et al. 2025). In GIOP patients, reduced expression of Runx2 and Osterix is associated with impaired osteoblast function and decreased differentiation capacity (Yamashita et al. 2025). Consequently, RUNX2 and OSTERIX serve as key markers for osteogenic differentiation (Lee et al. 2015). In our study, BAK intervention increased the protein expression levels of RUNX2 and OSTERIX. Additionally, we observed enhanced ALP activity in the serum of GIOP mice. Elevated ALP levels reflect improved bone metabolic balance and indicate increased bone formation in treated GIOP patients (Ponzano et al. 2023), suggesting that BAK enhances the osteogenic differentiation capacity of osteoblasts in GIOP mice.

Transcriptomic analysis revealed significant enrichment of the Hippo signaling pathway following BAK treatment. Specifically, BAK downregulated Dchs1 and Fat4 while upregulating Yap1, Amotl2, Amot, and Sox11. The Hippo pathway, a key regulator of cell proliferation, differentiation, and survival, plays an essential role in bone development and homeostasis through its downstream effector YAP1 (Li et al. 2024). Inhibition of Hippo signaling reduces YAP1 phosphorylation in the cytoplasm, thereby enhancing its nuclear translocation. Once in the nucleus, YAP1 upregulates RUNX2 expression and promotes osteogenic activity (Fu et al. 2022). AMOT and AMOTL2, members of the angiomotin protein family, are critical for maintaining cell tight junctions and facilitating YAP1 nuclear translocation (Ragni et al. 2017). SOX11, a transcription factor, promotes osteogenic differentiation by regulating osteogenesis‐related genes Runx2 and Osterix; silencing Sox11 impairs osteogenic progression (Gadi et al. 2013; Yu et al. 2020). FAT4, a single‐pass transmembrane receptor, interacts with DCHS1 to modulate Hippo signaling through YAP1. Loss of FAT4 function leads to increased YAP1 activity (Van Hateren et al. 2011; Sadaf et al. 2024; Che et al. 2019), thereby enhancing osteoblast proliferation and differentiation. Thus, activating YAP1 within the Hippo signaling pathway represents a promising strategy for osteoporosis treatment. Our results showed that BAK increased YAP1 protein expression, while it decreased FAT4 and DCHS1 protein levels. These findings suggest that BAK may promote osteoblast differentiation by activating YAP1 signaling in the Hippo pathway. In this study, we preliminarily explored the role of SOX11 in osteogenic differentiation, but a limitation is that we did not thoroughly elucidate its underlying mechanisms. In future research, we will focus on further investigating the function of SOX11 in GIOP treatment, with an emphasis on deciphering its mediated molecular signaling pathways and clarifying its regulatory mechanisms in bone metabolism.

To validate our hypothesis, we established an in vitro osteoblast injury model (Jin et al. 2024) using DEX and treated it with BAK. The results demonstrated that BAK promoted osteoblast differentiation and increased both YAP1 protein expression and its nuclear translocation. Previous studies have shown that nuclear YAP1 can enhance RUNX2's transcriptional activity (Collins et al. 2024). Consistent with this, our dual‐luciferase reporter assay revealed that BAK significantly enhanced Runx2 transcriptional activity, confirming our hypothesis.

We further investigated the molecular mechanism underlying this effect and identified FAT4 as the direct target of BAK in activating YAP1. As noted earlier, FAT4 acts as a positive upstream regulator of the Hippo signaling pathway and typically suppresses osteoblast proliferation when bound to DCHS1 (Crespo‐Enriquez et al. 2019). Thus, FAT4 has been recognized as a potential therapeutic target for GIOP and a key biomarker for osteoporosis prevention and treatment (Crespo‐Enriquez et al. 2019; Sadaf et al. 2024). Our findings showed that BAK improved the protein stability of FAT4 while simultaneously reducing its interaction with DCHS1. Silencing Fat4 significantly enhanced osteoblast differentiation, increased the protein expression of YAP1, RUNX2, and OSTERIX in osteoblasts, and elevated serum ALP activity. Notably, BAK treatment produced similar effects to Fat4 silencing, suggesting that BAK promotes osteoblast differentiation by targeting FAT4 and activating YAP1.

Although our study has elucidated the mechanism by which BAK enhances osteogenic differentiation in GIOP, there are still areas that require improvement. In this study, a termination time point was set for observing the therapeutic effects of BAK intervention in mice, which may prevent us from evaluating the dynamic therapeutic effects of BAK on mice and the timeframe over which the drug exerts its effects. Moreover, this study is currently at the basic research stage and has not yet conducted in vivo validation experiments involving Fat4 knockdown and experiments with human osteoblasts. However, these experiments would provide more reliable experimental support for the clinical application of BAK. These limitations will be taken into account in our ongoing and future research endeavors. We aim to employ conditional Fat4‐knockout mice and use human osteoblasts or hMSCs to verify the translational applicability of our findings.

5. Conclusion

In summary, BAK significantly reduces bone loss and enhances bone strength in GIOP mice, while also alleviating DEX‐induced pathological damage. Through both in vivo and in vitro experiments, we demonstrate that BAK exerts its therapeutic effects by targeting and binding to FAT4, which activates YAP1 (Figure 8). This activation promotes osteoblast differentiation and counteracts the progression of GIOP. Our findings not only confirm the therapeutic potential of BAK for GIOP but also clarify its underlying molecular mechanism, thereby providing a theoretical foundation for the development of novel GIOP treatments.

FIGURE 8.

FIGURE 8

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BAK activates YAP1 by targeting FAT4, promoting osteoblast differentiation and ameliorating GIOP (by FigDraw).

Author Contributions

Hengjun Wang: writing – original draft, investigation. Tingting Zhou: investigation, data curation. Yunchao Zhao: investigation, data curation. Zhengxin Meng: investigation, data curation. Zhiqiang Sun: data curation, validation. Hao Zhou: validation, data curation. Huan Liu: investigation, validation. Changyu Du: investigation, validation. Shuquan Lv: data curation. Jianyong Zhao: writing – review and editing. Zhongyong Zhang: writing – review and editing. Huantian Cui: conceptualization, writing – review and editing.

Funding

This work was supported by Research Project of the Science and Technology Planning Program of the Department of Traditional Chinese Medicine of Hebei Province (2025164).

Ethics Statement

Approval for all animal experiments was obtained from the Ethical Review Committee of Animal Experiments in Yunnan University of Chinese Medicine (Ethics approval number: YUCM‐AE‐2024‐042). All animal experiments were conducted in accordance with the 1986 British “Animal (Scientific Procedures) Act” and relevant guidelines.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Primer sequence.

Figure S1: Statistical results of body weight of mice during the experiment period. n = 10 per group. **p < 0.01.

Figure S2: shFAT4 knockdown resulted in a significant reduction of FAT4 expression in the osteoblast cells. n = 3 per group. **p < 0.01.

PTR-40-5104-s002.docx (82KB, docx)

Table S2: Significant GO pathways from GIOP vs. Control.

PTR-40-5104-s003.pdf (454.4KB, pdf)

Table S3: Significant GO pathways from BAK‐H vs. GIOP.

PTR-40-5104-s001.pdf (723KB, pdf)

Contributor Information

Jianyong Zhao, Email: zhaojyvip@126.com.

Zhongyong Zhang, Email: zzyhappy666@126.com.

Huantian Cui, Email: 1762316411@qq.com.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Primer sequence.

Figure S1: Statistical results of body weight of mice during the experiment period. n = 10 per group. **p < 0.01.

Figure S2: shFAT4 knockdown resulted in a significant reduction of FAT4 expression in the osteoblast cells. n = 3 per group. **p < 0.01.

PTR-40-5104-s002.docx (82KB, docx)

Table S2: Significant GO pathways from GIOP vs. Control.

PTR-40-5104-s003.pdf (454.4KB, pdf)

Table S3: Significant GO pathways from BAK‐H vs. GIOP.

PTR-40-5104-s001.pdf (723KB, pdf)

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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