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. 2025 May 22;73(22):13891–13901. doi: 10.1021/acs.jafc.5c01124

Sodium Benzoate Inhibits Osteoblast Differentiation and Accelerates Bone Loss by Regulating the FGF2/p38/RUNX2 Pathway

Zhonghao Wang b, Yexin Wang a, Yu Tang c, Xiaoyan Guo a, Qize Gao b, Yiming Shao b, Jingxuan Wang b, Ronghua Tian a, Yingxu Shi a,*
PMCID: PMC12147136  PMID: 40404584

Abstract

Sodium benzoate (NaB) is a commonly used food ingredient that is also found in cosmetics and medicines. Previous studies have demonstrated that long-term NaB intake has detrimental effects on human health, while its effects on bone mass remain unknown. In the present study, intragastric NaB administration was found to decrease bone mass and deteriorate bone microstructure in vivo, while prolonged NaB gavage further accelerated bone loss. The in vitro study revealed that NaB inhibited osteoblast differentiation of bone marrow mesenchymal stem cells and MC3T3-E1 cells. Mechanistically, RNA sequencing analysis elucidated that NaB greatly suppressed fibroblast growth factor 2 (FGF2) expression. Further studies revealed that NaB inhibited p38/RUNX2 signaling transduction, which was downstream of FGF2 for modulating osteoblast differentiation. The rescue studies suggested that NaB inhibited RUNX2 expression and osteoblast differentiation through the p38/MAPK signaling pathway. Collectively, NaB accelerated bone loss by inhibiting osteoblast differentiation through downregulating FGF2/p38/RUNX2 signaling pathway. The present study revealed that the long-term intake of NaB-containing food increased the risk of bone loss and osteoporosis (OP). Therefore, a reasonable oral intake of NaB-containing food is an important but convenient initiative for preventing OP.

Keywords: osteoporosis, p38/MAPK pathway, food additives, bone loss, osteogenic differentiation

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1. Introduction

Osteoporosis (OP) is a prevalent skeletal disorder affecting ∼ 200 million people globally, and is characterized by decreased bone mass and deterioration of bone microstructure, resulting in bone fragility and fracture susceptibility. Accordingly, an osteoporotic fracture occurs globally every 3 s, with approximately 50% of women and 20% of men experiencing a primary osteoporotic fracture after the age of 50. Osteoporotic fractures can lead to mortality, particularly osteoporotic hip fractures with a mortality rate of approximately 20% within 6 months of fracture occurrence. Therefore, osteoporotic fractures impose significant clinical and economic burdens. Approximately 2 million osteoporotic fractures occur annually in the U.S. costing $17 billion, the costs in China being approximately $19.92 billion annually, while in Europe, the economic burden was predicted to be €37·5 billion annually in 2017, with an estimated 27% increase by 2030 (€47·4 billion). With the accelerating aging process globally, the incidence of OP is gradually increasing and the number of fractures is estimated to increase by 310% from 1990 to 2050. Currently, OP is globally a heavy economic, social, and clinical burden, therefore, its prevention is a urgent and critical public health challenge.

Dietary interventions could be primary and effective strategies in preventing OP. Among dietary factors, the precise effect of the ingredients on OP remains largely unknown and needs further study. Sodium benzoate (NaB) is a commonly used food ingredient that is found in foods such as carbonated beverages, sauces, and canned foods. , In addition to food, NaB is used in medicines, with it reported to be beneficial for the treatment of several diseases, including clozapine-resistant schizophrenia and later-phase dementia. Consequently, significant amounts of NaB are consumed or encountered frequently, resulting in adverse effects. Beezhold et al. reported that a high intake of NaB-rich beverages may contribute to attention deficit hyperactivity disorder-related symptoms in college students. Additionally, studies have shown that even when the NaB concentration is lower than the highest allowable concentration, its long-term intake can harm male reproductive health and contribute to the pathogenesis of diabetes by inducing β cell apoptosis partially via benzoylation. These studies indicated that long-term or excessive NaB exposure is harmful for human health. Currently, the effect and mechanism of NaB in OP remains unknown both in vivo and in vitro.

The generation and development of osteoporosis are modulated by various regulatory factors, such as fibroblast growth factor 2 (FGF2), bone morphogenetic protein (BMP), and transforming growth factor β (TGF-β), through multiple pathways, including the mitogen-activated protein kinase (MAPK) signaling pathway, Wnt signaling pathway, and Notch signaling pathway. Among these regulatory factors, FGF2 is implicated in osteogenic differentiation. , FGF2, also known as basic fibroblast growth factor, is a well-studied member of the fibroblast growth factor superfamily. To date, the role of FGF2 in osteogenic differentiation has been conflicting and widely debated. Ogura et al. reported that recombinant human FGF2 (20 ng/mL) stimulates the differentiation of mesenchymal stem cells (MSCs) only when applied during the proliferation phase of preosteoblast cells, and not during the differentiation stage. Lee et al. obtained a different conclusion. In their experiment, mesenchymal lineage C3H10T1/2 cells were precultured with 10 ng/mL recombinant mouse FGF2 for 4 days and then incubated with osteogenic medium. As a result, the osteogenic differentiation of the FGF2-primed cells was significantly decreased. So the various roles of FGF2 in osteoblast differentiation depend on several factors, including the stages of osteoblast differentiation when FGF2 is applied and the concentrations used in an experiment. ,,

In the present study, we found that NaB inhibits FGF2 expression both in vivo and in vitro, subsequently suppressing FGF2/p38/runt-related protein 2 (RUNX2) signaling transduction, thereby inhibiting osteoblast differentiation and accelerating bone loss. The mechanism described above indicates that a reasonable oral intake of NaB-containing foods is an important and effective approach for preventing OP.

2. Materials and Methods

2.1. Reagents

The cell culture reagents such as α-minimal Eagle’s medium (α-MEM), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), penicillin/streptomycin solution, phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Anisomycin, l-ascorbic acid, and dexamethasone were purchased from MedChem Express (Monmouth Junction, NJ, USA), NaB was acquired from Merck (Billerica, MA, USA), and bone marrow mesenchymal stem cells (BMSCs), the osteogenic induction differentiation kit, and the adipogenic induction differentiation kit were obtained from OriCell (Guangzhou, China). Alizarin Red S (ARS) Staining Solution, BCIP/NBT Alkaline Phosphatase Color Development Kit, Oil Red O (ORO) Staining Kit, 4% paraformaldehyde (PFA), and antifluorescence quenching mounting solution were purchased from Beyotime (Shanghai, China). β-glycerophosphate and cetylpyridinium chloride (CPC) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Isolation and Identification of BMSCs

BMSCs were isolated and cultured following a previously reported protocol. The femur from specific pathogen free (SPF) SPF-grade Sprague–Dawley rats (3–4-weeks-old) were aseptically isolated, and the surrounding muscles were excised. Subsequently, the bones were immersed in a PBS solution with 100 U/mL penicillin and 100 μg/mL streptomycin. The bilateral epiphysis was cut away to expose the bone marrow cavity, which was thoroughly washed in DMEM/F12 medium. The bone marrow fluid was filtered through a 70 μm cell sieve and centrifuged at 1500 rpm for 3 min at room temperature (RT). The supernatant was discarded, and the cells were resuspended in growth medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Subsequently, the cells were inoculated in a culture bottle and maintained at 37 °C under 5% CO2. The culture medium was replaced for the first time after 24 h, and subsequent medium changes were made every 3 days.

The BMSCs cells were identified by morphological observation, adipogenic ability, osteogenic ability, and flow cytometry. Following induction, the ORO Staining Kit was used to stain lipid droplets, while alkaline phosphatase (ALP) activity was determined using the BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitro Blue Tetrazolium) Alkaline Phosphatase Color Development Kit and the calcium nodules were stained using the ARS kit. Flow cytometry experiments were conducted to investigate cell surface markers, including CD29, CD45, and CD90. Anti-CD29, anti-CD45, and anti-CD90 antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

2.3. Cell Culture and Osteoblastic Differentiation

MC3T3-E1 preosteoblast cells were obtained from Procell Life Science & Technology (Wuhan, China) and cultured in α-MEM with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, maintained at 37 °C in a 5% CO2 atmosphere. When MC3T3-E1 cells attained confluency and BMSCs reached 80%, osteogenic differentiation was induced in these cells in osteoblast osteogenic induction medium (OIM), which contained culture medium supplemented with 100 μg/mL l-ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone. The medium was changed every 3 days. MC3T3-E1 cells and BMSCs were treated with different NaB concentrations as indicated.

2.4. ALP, ARS, and ORO Staining

MC3T3-E1 cells and BMSCs were seeded onto 12-well plates at a density of 1 × 105 cells/well. After maintaining the cells in OIM containing different NaB concentrations for 7 days, they were washed with PBS and fixed with 4% PFA. ALP staining was conducted using the BCIP/NBT Alkaline Phosphatase Color Development Kit following the manufacturer’s instructions. Subsequently, the cells were lysed with 0.2% Triton X-100 buffer and centrifuged. The cell lysate was incubated with p-nitrophenyl phosphate solution for 1 h at RT, followed by treating with stop solution. The absorbance was determined at 405 nm using a microplate reader.

For ARS staining, MC3T3-E1 cells and BMSCs were induced in OIM for 14 and 21 days, respectively. The cells were washed twice with PBS and fixed with 4% PFA. The level of mineralization (calcium accumulation) was determined by ARS staining. For quantitative analysis, cells were destained with CPC solution and transferred to a 96-well plate to determine the optical density (OD) values at 562 nm. For ORO staining, BMSCs were maintained in lipid induction medium for 21 days, washed with PBS, fixed with 4% PFA, and stained with ORO staining solution to determine the new lipid droplets.

2.5. Western Blotting

Western blotting was performed as described previously. In brief, after the cells were treated under the indicated conditions, the proteins were extracted from each sample with RIPA buffer, separated on sodium dodecyl sulfate-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Merck). After blocking with 5% skimmed milk, the membranes were incubated with the primary antibodies (1:1000) and secondary antibodies (1:10,000) accordingly. The target protein was visualized using an enhanced chemiluminescence kit (Affinity, Changzhou, Jiangsu, China). Anti-RUNX2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), antiosteocalcin (OCN), anti-ALP, and anti-α-tubulin antibodies were obtained from Proteintech (Wuhan, China), anti-p38, anti-p-p38, antiextracellular signal regulated kinases 1 and 2 (ERK1/2), anti-p-ERK, anti- c-Jun N-terminal kinases (JNK), anti-p-JNK, and anti-GAPDH antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA), and anti-FGF2 antibody was purchased from Abcam (Cambridge, UK). Goat antirabbit and goat antimouse antibodies were purchased from Affinity (China). The bands were quantified using ImageJ software (NIH, Bethesda, MD, USA).

2.6. RNA Extraction and qRT-PCR

Total RNA was extracted from MC3T3-E1 cells with TRIzol reagent (Invitrogen). After determining RNA purity and concentration using the NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), cDNA was obtained by reverse transcription using a reverse transcription kit (BioSharp, Hefei, China). The UltraSYBR Mixture (Cwbio, Beijing, China) was used to perform RT-PCR in the CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). The whole procedure was performed as follows: predenaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s, 65 °C for 30 s and 72 °C for 50 s. GAPDH served as the loading control, and the qPCR results were quantified by the 2–ΔΔCT method. The sequences of all primers are as follows: ALP (Forward, 5′-AACCCAGACACAAGCATTCC-3′, Reverse, 5′-GCCTTTGAGGTTTTTGGTCA-3′); FGF2 (Forward, 5′-GTCACGGAAATACTCCAGTTGGT-3′, Reverse, 5′-CCCGTTTTGGATCCGAGTTT-3′); OCN (Forward, 5′-CTGACAAAGCCTTCATGTCCAA-3′, Reverse, 5′-GCGCCGGAGTCTGTTCACTA-3′); RUNX2 (Forward, 5′ -CGGCCCTCCCTGAACTCT-3′, Reverse, 5′- TGCCTGCCTGGGATCTGTA-3′); GAPDH (Forward, 5′-GCATCTCCCTCACAATTTCCA-3′, Reverse, 5′-TGCAGCGAACTTTATTGATGGT-3′).

2.7. Immunofluorescence (IF) Assay

MC3T3-E1 cells at a density of 4 × 104 cells per well were seeded on glass coverslips in 24-well plates. The glass coverslips were washed with PBS and the cells were fixed with 4% PFA at RT for 15 min, permeabilized with 0.3% Triton X-100 on ice for 10 min, and blocked with 3% goat serum for 1 h at RT. The glass coverslips were placed in a wet box and incubated overnight at 4 °C with a primary antibody against RUNX2 (Santa Cruz, 1:200), OCN (Proteintech, 1:200), ALP (Proteintech, 1:200), FGF2 (Abcam, 1:200), and p-p38 (Cell Signaling Technology, 1:200). Subsequently, the cells were washed and incubated with Alexa Fluor-conjugated secondary antibody (1:500) for 1 h at RT. The nuclei were stained with DAPI (1:1000) and the slides kept in the dark. The slides were viewed and images obtained using a fluorescence microscope (IX73, Olympus, Tokyo, Japan).

2.8. RNA-Sequencing (RNA-seq)

MC3T3-E1 cells and BMSCs were both divided into two groups. The NaB group underwent exposure to OIM containing 10 mM NaB and the control group received OIM containing the same volume of DMSO as the NaB volume. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s instructions and sequenced on the NovaSeq 6000 platform (Illumina) by Beijing Qinglian Biotech Co., Ltd. (Beijing, China).

2.9. Animal Study

The animal study protocol was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University (Approval Number: 2023–02-B002). Thirty female C57BL/6J mice (8-weeks-old, weight: 19 ± 1 g, Ji’nan Pengyue Laboratory Animal Breeding Co., Ji’nan, China) were maintained under standard conditions in an animal house with a conventional temperature of 22–25 °C, a constant humidity of 50–60%, and on a 12-h light/dark cycle. After 7 days of adaptation, the mice were randomly assigned to five groups: (1) The control group (n = 6) was administered 0.2 mL PBS intragastrically every 2 days. (2) The ovariectomy (OVX) group (n = 6) underwent bilateral ovariectomy and was subsequently administered PBS intragastrically once every 2 days. (3) The NaB gavage group (n = 6), received 400 mg/kg NaB dissolved in 0.2 mL PBS by gavage every 2 days. Six weeks later, the mice of these three groups were euthanized and the femurs were obtained for micro-CT and histological detection. (4) The NaB-PBS group (n = 6) received 400 mg/kg NaB dissolved in 0.2 mL PBS by gavage every 2 days, and after 6 weeks, 0.2 mL PBS were administered intragastrically every 2 days for a further 3 weeks. (5) The NaB-NaB group (n = 6) received 400 mg/kg NaB dissolved in 0.2 mL PBS by gavage every 2 days for 9 weeks. After 9 weeks of treatment, the mice in groups (4) and (5) were euthanized and the femurs obtained for micro-CT analysis. The NaB dosage was selected based on previously reported studies. ,

2.10. Micro-CT Analysis

The distal femoral epiphyses were scanned using the SkyScan 1176 micro-CT system (Bruker, Karlsruhe, Germany) with the following settings: 18 μm thickness, 65 kV of energy, 385 mA of intensity, and an integration time of 340 ms. Three-dimensional images were constructed using CTvox. Trabecular bone parameters, including bone mineral density (BMD, g/cm3), bone volume/total volume (BV/TV, %), trabecular bone number (Tb. N, 1/mm), trabecular separation (Tb. Sp, mm), and trabecular thickness (Tb. Th, mm), were analyzed, and the mean ± standard deviation (SD) value for the parameters in each group were obtained.

2.11. Immunohistochemistry (IHC)

The femoral samples were fixed in 4% PFA and decalcified in 10% EDTA for 30 days. Subsequently, the samples were embedded in paraffin. IHC was conducted using the primary antibody against FGF2 (1:100, ABclonal, Wuhan, China) and a horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (H+L) secondary antibody (1:400, ABclonal, Wuhan, China). The signals were visualized with a 0.05% solution of 3,3′-diaminobenzidine tetrahydrochloride before counterstaining with hematoxylin. Images were acquired under a light microscope.

2.12. Statistical Analysis

Statistical analysis was performed using GraphPad Prism9 (La Jolla, CA, USA). All data are expressed as mean ± SD. Comparisons between two groups were analyzed using Student’s t-tests. P < 0.05 was regarded to be statistically significant (*P < 0.05, ** P < 0.01). All experiments were conducted independently a minimum of three times.

3. Results

3.1. Characterization and Identification of BMSCs

BMSCs were reported to grow in a long spindle shape and have multiple differentiation potentials. The cells we isolated were identified as to whether they possessed the characteristics of BMSCs. The results showed that the isolated cells were spindle-shaped and displayed polar growth and stable morphology (Figure A). Additionally, ORO staining showed the appearance of lipid droplets in the cells, which suggested that isolated BMSCs had the ability of adipogenic differentiation (Figure B). As shown in Figure C,D, the ALP and ARS staining suggested ALP accumulation and calcium nodule formation in cells, respectively, indicating that the isolated cells had osteogenic differentiation potency. Moreover, cell surface markers were determined by flow cytometry. The isolated BMSCs expressed the mesenchymal stem cell-positive markers CD90 and CD29, and the negative marker CD45 (Figure E). The above results were consistent with the properties of mesenchymal stem cells, suggesting the BMSCs were isolated and cultured successfully.

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Characterization and identification of BMSCs. (A) Cell morphology of P3-generation BMSCs. Scale bar = 200 μm. (B) Adipogenic differentiation of BMSCs was determined by Oil Red O staining after 21 days of culture in adipogenic induction medium. Scale bar = 200 μm. Osteogenic differentiation was induced in isolated BMSCs for 7 days followed by ALP staining (C) and for 21 days followed by ARS staining (D). Scale bar = 200 μm. (E) Flow cytometry detection of BMSC surface markers, including CD45, CD29, and CD90.

3.2. NaB Inhibits BMSC Osteogenic Differentiation

The in vitro concentration of NaB was chosen based on previously reported studies , and the CCK8 results (Figure S1). Ultimately, the 0 mM, 5 mM, and 10 mM concentrations of NaB were chosen. The effect of NaB on BMSC osteogenic differentiation was investigated by treating BMSCs with different NaB concentrations (0, 5, and 10 mM). ALP, an early bone formation marker secreted by osteoblasts, was selected as the osteoblast differentiation marker. As shown in Figure A, the ALP activity decreased on NaB treatment in a dose-dependent manner. Similarly, the number and quantification of calcium nodules stained with ARS were also decreased following NaB exposure (Figure B). These results confirmed that NaB inhibited BMSC osteogenic differentiation.

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NaB inhibits BMSC osteogenic differentiation. (A) ALP staining of BMSCs after treatment with NaB (0, 5, or 10 mM) in OIM for 7 days. The right panel shows the quantification of ALP staining, scale bar = 500 μm. (B) After treatment with different NaB concentrations in OIM for 14 days, the calcium nodules were stained with ARS. After ARS staining, BMSCs were destained with 10% CPC solution in a shaker and transferred into a 96-cell plate to determine the OD values at 562 nm. The right panel shows the quantitative analysis of ARS staining, scale bar = 500 μm. Data are presented as the mean ± SD *P < 0.05; **P < 0.01 compared with the 0 mM group.

3.3. NaB Inhibits MC3T3-E1 Cell Osteogenic Differentiation

To further verify the negative effect of NaB on osteogenic differentiation, MC3T3-E1 cells were used in the following experiment. Figure A,B show that NaB inhibited ALP activity and calcium nodule accumulation of MC3T3-E1 cells, indicating that NaB inhibits the osteogenic differentiation of MC3T3-E1 cells. Osteogenic differentiation is an orchestrated cellular process triggered by several transcription factors, including RUNX2. , RUNX2 binds to the promoter regions of its downstream target genes, including OCN and ALP, to regulate osteoblastic transcription, , thus we investigated the expression of RUNX2, OCN, and ALP by IF experiments. As shown in Figure C–E, treatment with 10 mM NaB significantly downregulated the fluorescence intensity for RUNX2, OCN, and ALP in MC3T3-E1 cells. Comparable results were obtained from the qRT-PCR and Western blotting assays after exposure to NaB, and the mRNA (Figure F) and protein levels (Figure G,H) of osteogenic markers RUNX2, OCN, and ALP were significantly reduced. These results demonstrated that NaB inhibited MC3T3-E1 cell osteogenic differentiation.

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NaB inhibits MC3T3-E1 cell osteogenic differentiation. (A) ALP staining of MC3T3-E1 cells after induction in OIM containing different NaB concentrations (0, 5, and 10 mM) for 7 days. The right panel shows the quantification of ALP staining, scale bar = 500 μm. (B) ARS staining of MC3T3-E1 cells after treatment with NaB (0, 5, or 10 mM) in OIM for 21 days. The right panel shows the relative OD values of the different groups, scale bar = 500 μm. (C–E) After MC3T3-E1 cells were cultured in OIM for 7 days, images were obtained to show the fluorescence intensity for RUNX2, OCN, and ALP, scale bar = 100 μm. (F) qRT-PCR was performed to evaluate the RUNX2, OCN, and ALP mRNA levels in MC3T3-E1 cells treated with NaB (0, 5, or 10 mM) for 7 days, GAPDH was used as the loading control. (G) The RUNX2, OCN, and ALP protein levels in MC3T3-E1 cells were determined by Western blotting. (H) Quantification and normalization of the indicated protein levels shown in (G); GAPDH was used as the loading control. Data are presented as the mean ± SD *P < 0.05; **P < 0.01 compared with the 0 mM group.

3.4. NaB Induces Bone Loss in Mice

The above in vitro experiments indicated that NaB inhibited osteoblast differentiation. Considering the crucial role of osteoblast differentiation during the process of bone loss and OP, we considered whether NaB treatment affects bone mass. The experimental design for NaB gavage is illustrated in Figure A. The OVX group was considered a positive control for OP. At the indicated end point, the mice were euthanized by cervical dislocation after anesthesia and their femurs were collected for subsequent processing. As shown in Figure B, compared with the control group, the NaB gavage (NaB group) displayed decreased bone trabecular density and bone microstructure destruction. The structural parameters, including BMD, BV/TV, Tb.Th, and Tb.N, were statistically decreased after NaB gavage, whereas Tb.Sp increased upon NaB treatment (Figure C–G).

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NaB induces bone loss in mice. (A) Schematic diagram illustrating the experimental design for the OVX and NaB gavage treatment. (B) Representative micro-CT images of the three-dimensional reconstruction of mouse femurs in the different groups. The quantitative analysis results of BMD (C), BV/TV (D), Tb.N (E), Tb.Th (F), and Tb.Sp (G) are presented for the indicated groups (n = 6). Data are presented as the mean ± SD ns, no significant difference; *P < 0.05; **P < 0.01 compared with the control group.

Interestingly, there was no statistically significant difference of bone trabecular density or structural parameters between the NaB and NaB-PBS groups (P > 0.05). This indicated that on stopping NaB oral administration, bone mass loss did not progress, however, the bone mass loss did not reverse, which may be attributed to the limited observation time. Moreover, compared with the NaB and NaB-PBS groups, the additional 3 weeks of NaB gavage in the NaB-NaB group further decreased bone trabecular density, accompanied by bone microstructure destruction (Figure B–G). These in vivo findings demonstrated that both the treatment and prolonged treatment duration of NaB gavage led to bone loss and the destruction of its microstructure in mice. Therefore, we concluded that there was a strong causal relationship between NaB oral intake and bone loss in mice.

3.5. NaB Inhibits FGF2 Expression In Vitro and In Vivo

To investigate the molecular mechanism of NaB inhibition of osteogenic differentiation, MC3T3-E1 cells and BMSCs were differentiated in OIM with 10 mM NaB for 4 days, the cells subsequently undergoing RNA-seq analysis. The heatmap displayed the overall changes in the expression pattern, with upregulated genes indicated in red and downregulated genes in blue (Figure A). As shown in Figure B, under this treatment with 10 mM NaB, FGF2 was one of the most significantly downregulated differentially expressed genes (DEGs) in MC3T3-E1 cells, with similar results obtained for BMSCs (Figure C). The top five DEGs between the Ctrl group and the NaB group in MC3T3-E1 cells were FGF2, GM44250, MESD, EGR1, and GOLM1. In BMSCs, the top five DEGs were EMP1, GM55521, APBB2, FGF2, and PCYT1A. Thus, FGF2 was one of the overlapping DEGs that were significantly expressed in both MC3T3-E1 cells and BMSCs; therefore, we closely focused on FGF2. Further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the most altered genes in MC3T3-E1 cells were enriched in pathways including the regulation of the actin cytoskeleton, the PI3K-AKT signaling pathway, EGFR tyrosine kinase inhibitor resistance, and the MAPK signaling pathway, all of which were regulated by FGF2 (Figure S2A). In BMSCs, the enriched pathways included signaling pathways regulating the pluripotency of stem cells and the MAPK signaling pathway, both of which are closely related to FGF2 (Figure S2B).

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NaB inhibits FGF2 expression in vitro and in vivo. (A) MC3T3-E1 cells and BMSCs were treated with or without 10 mM NaB. The cells were then subjected to RNA sequencing analysis, and a heatmap was generated to show differentially expressed genes (DEGs). Volcano plot of DEGs analyzed by RNA-seq of MC3T3-E1 cells (B) and BMSCs (C) treated with 0 or 10 mM NaB. FGF2 mRNA (D) and protein levels (E) in MC3T3-E1 cells treated with NaB (0, 5, or 10 mM) in OIM for 4 days. GAPDH was used as a loading control. (F) Quantification of the FGF2 protein level as shown in (E). (G) IF staining for FGF2 in MC3T3-E1 cells cultured in OIM for 4 days, scale bar = 50 μm. (H) IHC staining for FGF2 in the distal femur of mice, scale bar = 100 μm. Data are presented as the mean ± SD *P < 0.05; **P < 0.01 compared with the 0 mM group.

Because of the important regulatory role of FGF2 on osteoblast differentiation, ,,, we hypothesized that NaB inhibited osteoblast differentiation by downregulating FGF2. To verify this, we investigated FGF2 expression in MC3T3-E1 cells. Treatment with 10 mM NaB significantly reduced FGF2 at both the mRNA and protein levels (Figure D–F). Comparable results were obtained from the IF staining (Figure G), whereby, NaB decreased the fluorescence intensity for FGF2 in MC3T3-E1 cells. In addition to the in vitro experiments, the IHC of the femur also indicated that, compared with PBS gavage for 6 weeks, the FGF2 expression was decreased in the mice that underwent 400 mg/kg NaB gavage for 6 weeks (Figure H). These results revealed that NaB inhibited FGF2 expression in vitro and in vivo.

3.6. NaB Inhibits the p38/MAPK Signaling Pathway

Accordingly, the FGF2/MAPK signaling pathway is one of the major mechanisms involved in osteoblast differentiation and bone metabolism. , To further investigate the downstream mechanism of FGF2 in inhibiting osteoblast differentiation, we determined the MAPK pathways, including ERK1/2, p38, and JNK. As shown in Figure A, NaB treatment diminished the relative p38 phosphorylation level (p-p38/total p38) in a dose-dependent manner (P < 0.01). By contrast, the relative index of the phosphorylation level of ERK1/2/ERK and p-JNK/JNK remained constant. Consistent with the results of Western blotting experiments, the fluorescence dots of p-p38 were significantly diminished compared with the control (0 mM NaB) group (Figure B). These data clarified that NaB specifically inhibited the p38/MAPK pathway, rather than the ERK/MAPK and JNK/MAPK pathways.

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NaB inhibits the p38/MAPK signaling pathway. (A) MC3T3-E1 cells were lysed after treatment with indicated NaB concentration in OIM for 4 days. The whole cell extracts were immunoblotted with the indicated antibodies. The GAPDH was used as the loading control. (B) IF staining for p-p38 in MC3T3-E1 cells treated with 0 or 10 mM NaB in OIM for 4 days. Scale bar = 50 μm. Data are presented as the mean ± SD (n = 3), ns, no significant difference; *P < 0.05; **P < 0.01 compared with the 0 mM group.

3.7. NaB Inhibition of Osteogenic Differentiation Is Dependent on p38/MAPK Activity

To further reveal whether NaB inhibition of osteogenic differentiation is dependent on p38/MAPK activity, anisomycin, a p38/MAPK activator, was used. , After treating MC3T3-E1 cells with 10 mM NaB with or without 1 nM anisomycin in OIM for 4 days, anisomycin reversed the inhibitory effect of NaB on the p38 phosphorylation level, with similar results obtained for osteoblast differentiation markers, including RUNX2, OCN, and ALP (Figure A–E). To further verify the role of p38/MAPK activity in the regulatory role of NaB on osteogenic differentiation, ALP and ARS staining were also performed. As showed in Figure F,G, anisomycin attenuated the negative role of NaB on osteoblast differentiation. Collectively, these results revealed that NaB inhibition of osteogenic differentiation was dependent on p38/MAPK activity.

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NaB inhibits osteogenic differentiation dependent on p38/MAPK activity. (A) After MC3T3-E1 cells were treated with NaB (10 mM) with or without anisomycin (1 nM) in OIM for 4 days, the p-p38, p38, RUNX2, OCN, and ALP expression levels were determined by Western blotting. (B–E) Quantification and normalization of indicated protein levels shown in (A). (F) ALP staining of MC3T3-E1 cells after treatment with NaB (10 mM) with or without anisomycin (1 nM) in OIM for 7 days. The right panel shows the quantification of ALP staining, scale bar = 500 μm. (G) ARS staining of MC3T3-E1 cells after treatment with NaB (10 mM) with or without anisomycin (1 nM) in OIM for 21 days. The right panel shows the quantification of ALP staining, scale bar = 500 μm. Data are expressed as the mean ± SD (n = 3). *P < 0.05; **P < 0.01 compared with the control group.

4. Discussion

In this study, NaB was found to inhibit osteoblast differentiation by suppressing the FGF2/p38/RUNX2 signaling pathway in vitro. In vivo, NaB gavage decreased bone trabecular density and resulted in bone microstructure destruction in mice, and prolonged NaB gavage further accelerated bone loss (Figure ), suggesting that NaB intake leads to bone loss. More importantly, the additional 3 weeks of PBS gavage not only did not exacerbate the bone loss, but also did not reverse the bone loss in mice (Figure ). This demonstrated that there was a causal relationship between NaB gavage and bone loss, thus indicating that an appropriate NaB intake was a crucial but convenient initiative for OP prevention.

To investigate the mechanism of NaB regulation of osteoblast differentiation, we performed RNA-seq analysis and found that FGF2 was one of the most significantly downregulated molecules, particularly in MC3T3-E1 cells (Figure B,C). Considering the tight relationship between FGF2 and osteoblast differentiation, we focused on FGF2 for further study. FGF2 serves as a regulatory molecule that interacts and activates FGF receptors (FGFRs). FGF2 is able to activate five of the seven FGFR receptor variants: FGFR 1b, FGFR 1c, FGFR 2c, FGFR 3c, and FGFR 4, while it is unable to activate FGFR 2b and FGFR 3b. , Once FGF2 binds with a target FGFR, the tyrosine protein kinase activity domain in the cell first undergoes dimerization followed by autophosphorylation, subsequently, the target protein of the FGFR is trans-phosphorylated. Murahashi et al. reported that under the treatment of recombinant human FGF2, FGFR1 was activated at 4 weeks on trilayered nanosheets and eventually led to osteoblast differentiation. In the present study, we found that NaB decreased the FGF2 mRNA and protein levels in vivo and in vitro (Figure D–H). However, which FGFR was bound and activated by FGF2 for the subsequent signaling transduction remains unknown and awaits clarification.

FGF2 regulates osteoblast differentiation and bone mass by stimulating several signaling pathways, including the MAPK pathway. , The mammalian MAPK pathway primarily encompasses ERK1/2, p38, JNK1–3, and ERK5. As reported, FGF2 facilitates osteoblast differentiation through stimulating ERK activation in C3H10T1/2 cells. In the present study, we found that NaB inhibits FGF2/p38 activation for modulating osteoblast differentiation in MC3T3-E1 cells, whereas it had no effect on the phosphorylation of ERK1/2 or JNK (Figure ). The difference in mechanisms could be attributed to cell-context dependence. To further confirm whether NaB inhibited p38-dependent osteoblast differentiation, we treated preosteoblasts with a classic p38 activator, anisomycin. , As shown in Figure , p38 activation reversed the inhibitory effect of NaB on osteoblast differentiation, which indicated that NaB inhibited osteoblast differentiation dependent on the p38 pathway. It was also reported that anisomycin increased new bone formation in distraction osteogenesis, while the inhibition of p38 activity by SB203580 suppressed osteoblast differentiation. Our findings further indicate that p38 activation, such as through the application of anisomycin, may be a potential approach for the treatment of bone diseases, including nonhealing fractures and bone defects.

Activation of the p38/MAPK pathway is important in determining RUNX2 expression and activity, a master transcription factor involved in osteoblast differentiation. , Accordingly, the regulatory effect of p38/MAPK on RUNX2 is strongly discussed and primarily has two aspects. On the one hand, p38/MAPK are involved in RUNX2 protein activation but have no effect on its expression. For example, blocking of p38 activity by SB203580 increased RUNX2 transcriptional activity but did not affect the RUNX2 protein level in COS7 cells or MC3T3-E1 preosteoblast cells. On the other hand, inhibition of p38 phosphorylation by SB203580 decreased the protein levels of RUNX2 and other osteoblast differentiation markers, including OCN, ALP, and collagen I, in MC3T3-E1 cells and human dental pulp stem cells. Moreover, p38/AMPK activation by anisomycin significantly increased Runx2 expression in myoblast C2C12 cells. Similarly, the results of the present study indicate that anisomycin stimulated the phosphorylation levels of p-p38 and the expression of RUNX2, moreover, the OCN and ALP expression levels were both increased accordingly (Figure A–E). More importantly, anisomycin abolished the inhibitory effect of NaB on the expression of osteoblast differentiation markers, including RUNX2, OCN, and ALP (Figure A–E). Anisomycin also diminished the inhibitory effect of NaB on osteoblast differentiation (Figure F–G). These results suggested that NaB inhibition of RUNX2 expression and osteoblast differentiation were dependent on p38/MAPK activity.

The present study has several limitations. First, how NaB regulates the expression of FGF2 is still unknown. According to our results, NaB inhibits the mRNA and protein levels of FGF2 (Figure ), indicating that NaB mostly regulates FGF2 at the pretranscriptional or transcriptional level. Therefore, identifying the transcription factors regulated by NaB is one of the future research objectives. Interestingly, we hypothesized that RUNX2 may serve as a transcription factor that triggers FGF2 transcription, thus constructed an FGF2/p38/RUNX2/FGF2 regulatory feedback loop. However, the FGF2 mRNA did not change following RUNX2 overexpression in MC3T3-E1 cells (data not shown). Second, the specific FGF2 receptor involved in the regulation of NaB, as well as whether the interaction between FGF2 and its receptors is affected by NaB, has not been elucidated by the present study. Further research is needed to investigate the regulatory mechanism of NaB on FGF2.

Supplementary Material

jf5c01124_si_001.pdf (278.6KB, pdf)

Acknowledgments

This work was supported by the Key R&D project of Jining City (2023YXNS084) and the high level scientific research program cultivation project of Jining Medical College (JYGC2021FKJ007).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.5c01124.

  • Effects of NaB on the cell viability of BMSCs and MC3T3-E1 cells (Supplementary Figure 1); KEGG analysis of the differentially expressed genes under NaB treatment (Supplementary Figure 2) (PDF)

‡.

Z.W., Y.W., and Y.T. contributed equally to this work. Z.W. performed methodology, investigation, formal analysis, validation, and writing–original draft; Y.W. performed methodology, investigation, data curation, and supervision; Y.T. was in charge of investigation, validation, and software; X.G., Q.G., and Y.S. performed investigation and validation; J.W. and R.T. performed investigation; Y.S. performed visualization, supervision, project administration, data curation, writing–review and editing, and funding acquisition.

The authors declare no competing financial interest.

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