Introduction

Osteoporosis (OP) is a systemic metabolic bone disease characterized by reduced bone mineral density and deterioration of bone microarchitecture, which increases susceptibility to fragility fractures following low-energy trauma1. According to the Seventh National Population Census, China has 264 million individuals aged ≥ 60 years (18.7% of the total population) and over 190 million individuals aged ≥ 65 years (13.5% of the total population), making it the country with the world’s largest elderly population. A national epidemiological survey on osteoporosis reported that the prevalence among individuals aged ≥ 50 years was 19.2%, with 32.1% in women and 6.9% in men. Among those aged ≥ 65 years, the prevalence rose to 32.0%, with 51.6% in women and 10.7% in men2,3. Decreased bone density frequently causes chronic lower back pain and generalized body pain, significantly impairing patients’ quality of life. Osteoporotic fractures are associated with high mortality and disability rates. Furthermore, treatment-related costs impose a substantial financial burden on affected individuals4,5.

At present, the treatment of osteoporosis is mainly achieved through diet regulation, appropriate exercise, and a combination of drugs, aiming to prevent and address the occurrence of osteoporosis and fractures6. In clinical practice, the commonly used drugs for the treatment of OP comprise bisphosphonates, denosumab injection, calcitonin; estrogen, etc7,8. Nevertheless, these drugs can enhance bone mineral density in the short term, but long-term usage will bring about various adverse reactions. Therefore, it is essential to continuously discover new drugs with superior anti-osteoporosis effects and fewer side effects, which are suitable for long-term application. Naringin (Nar) is a natural flavonoid that is not only abundant in vegetables and fruits but also one of the main active ingredients of traditional Chinese medicine (Rhizoma Drynariae)9,10. Studies have revealed that naringin possesses multiple pharmacological activities, including anti-oxidation, anti-inflammation, anti-apoptosis, anti-diabetes, anti-osteoarthritis, anti-arrhythmia, and other systemic diseases, which have attracted significant attention in the research of natural drugs11,12. Recent studies have discovered that naringin can facilitate osteogenic differentiation and inhibit the formation of osteoclasts, and it may exert its effects by regulating BMP-2, Wnt/β-catenin, oxidative stress, etc13,14,15.

The Wnt/β-catenin signaling pathway plays a crucial role in osteogenic differentiation. The activation of this pathway can stabilize β-catenin, facilitate its transfer to the nucleus, and subsequently promote the proliferation and differentiation of osteoblasts, thereby ameliorating osteoporosis16. However, previous studies were mostly conducted in vitro, and in vivo, animal studies were relatively scarce. Therefore, through in vivo and in vitro studies, we unveiled the mechanism of naringin in enhancing bone mineral density in ovariectomized mice, enriched the theoretical basis, and provided a foundation for the clinical development of new drugs.

Materials and methods

Drugs and reagents

Naringin (purity 99.75%) was purchased from MedChemExpress, USA. β-Estradiol (E2) (purity ≥ 98%, Cat# ST1101) was obtained from Beyotime Biotechnology, China. Quantitative PCR Kit was provided by Conway Century Biotechnology, China. BCA Protein Assay Kit was sourced from Biosharp, China. Antibodies against OPN, COL1A1, Wnt3a, β-catenin, and Runx2 were purchased from Beyotime Biotechnology, Shanghai, China. CCK-8 Kit was acquired from Shanghai Yeasen Biotechnology, China. Alizarin Red Staining Solution was obtained from Solarbio Science & Technology, Beijing, China. BCIP/NBT Alkaline Phosphatase Color Development Kit was purchased from Beyotime Biotechnology, China. Mouse PINP, CTX-1, and OCN ELISA Kits were procured from Enzyme-linked Biotechnology, China. Fluorescent secondary antibodies and DAPI Staining Solution were purchased from Servicebio Technology, Wuhan, China.

Cell culture

The acquired MC3T3-E1 cells (cl-0378, Wuhan Prosai Life Technology Co., Ltd., China) were cultivated in a 5% CO2 incubator (Shanghai Yiheng Scientific Instruments Co., Ltd., China) at 37 ℃ in a special medium (CM-0378, Wuhan Punuosai Life Technology Co., Ltd., China) for MC3T3-E1 cells. When the cell density reached over 80%, the cells were rinsed with approximately 2 ml PBS, digested with 1 ml of 0.25% trypsin, collected and centrifuged (1200 rpm, 3 min), the supernatant was discarded, and fresh culture medium was added to re-plate at a ratio of 1:3.

CCK-8 detection of cell viability

MC3T3-E1 cells in the logarithmic growth phase were selected to prepare the cell suspension and inoculated into a 96-well plate for 24 h. After the cells were treated with a special medium containing naringin of different concentrations for 24 h and 48 h, CCK-8 reagent was added to each hole and incubated in the incubator for 2 h. Finally, the absorbance was measured at 450 nm using a microplate reader (SpectraMax190, Molecular Devices (Shanghai) Co., Ltd., China).

Alkaline phosphatase (ALP) staining analysis

MC3T3-E1 cells were inoculated into 6-well plates and incubated for 24 h. The cells were cultivated in a special medium containing naringin of different concentrations. After 14 days, alkaline phosphatase staining was conducted in accordance with the instructions of the kit. Finally, the inverted microscope (CKX41, Olympus, Japan) was utilized to observe and take photos.

Alizarin red staining analysis

Similar to the aforementioned, MC3T3-E1 cells were intervened with naringin of different concentrations for 21 days, and alizarin red staining was conducted by the instructions of the kit. Calcified nodules were observed and photographed using an inverted microscope (CKX41, Olympus, Japan).

Detection of β-catenin nuclear translocation by Immunofluorescence staining

MC3T3-E1 cells were seeded into 12-well plates and incubated for 24 h. Subsequently, the cells were cultured in medium containing different drug concentrations within a 37 °C and 5% CO₂ incubator. After treatment, cells were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.5% Triton X-100 (diluted from 10% stock in PBS) for 15 min, and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibodies were then added and incubated overnight at 4 °C, followed by incubation with fluorescent secondary antibodies for 1 h. Finally, nuclei were stained with DAPI, and samples were visualized using an inverted fluorescence microscope.

OVX mouse model and naringin intervention

Forty 8-week-old female C57BL/6 mice, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., were housed in the experimental animal facility of Jiangxi University of Traditional Chinese Medicine. The temperature in the animal room was set at 22 ± 1 °C, the humidity at 55 ± 5%, and the light-dark cycle was 1:1, both of which were 12 h. All animals had free access to water and food during feeding and were adaptively fed for 1 week. This study has been approved by the Animal Ethics Committee of Jiangxi University of Traditional Chinese Medicine (Approval Number: JZLLSC2022-0595). All experiments were performed in accordance with relevant named guidelines and regulations. The authors complied with the ARRIVE guidelines.

After 1 week of adaptive feeding, the model of osteoporosis in mice was established by referring to Liu et al.17. Firstly, 1.5% Pentobarbital Sodium at a dose of 0.05 ml/10 g was injected intraperitoneally to anesthetize the mice. Then, bilateral oophorectomy was performed. In the sham operation group, only the equal volume of fat around bilateral ovaries was removed. One week after the operation, the mice were randomly divided into five groups, with n = 8 in each group: the sham group, the OVX group, the OVX + E2 0.039 mg/kg group (Subcutaneous Injection), the OVX + naringin 15 mg/kg group, and the OVX + naringin 30 mg/kg group. All mice were treated by gavage daily for 12 weeks, and the sham group and the OVX group were given the same volume of normal saline. The mice were euthanized with excessive anesthesia 24 h after the last administration, and the beating of the heart was observed. (1.5% Pentobarbital Sodium, 0.15 ml/10 g). Blood was collected and centrifuged by a centrifuge (2000 × rpm, 10 min, 4 °C), and serum was collected and stored at -80 °C. Bilateral femurs were soaked in 4% paraformaldehyde or stored at -80 °C for subsequent detection.

Micro-CT

The right femur was scanned by Venus Micro-CT (VNC-102, Pingsheng Medical Technology Company, China), and the bone mineral density (BMD, g/cm³), bone volume fraction (BV/TV), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, 1/mm), and trabecular spacing (Tb. Sp, mm) were analyzed by the correlation analysis software.

Hematoxylin-eosin staining (H&E) analysis

After decalcification, gradient ethanol dehydration, xylene transparency, wax impregnation, and embedding, part of the right femur tissue of mice was made into 4–6 μm tissue sections by paraffin sectioning, and Hematoxylin-Eosin Staining was conducted for microscopic observation.

Immunohistochemical (IHC) staining

Tissue sections were subjected to deparaffinization and rehydration, followed by antigen retrieval. The sections were then incubated with primary antibodies at 4 °C overnight, and subsequently with secondary antibodies for 30 min at room temperature. Color development was performed using 3,3’-diaminobenzidine (DAB) for 20 min, followed by counterstaining with hematoxylin. Finally, the sections were dehydrated, cleared, and mounted with neutral resin. Protein-positive expression was identified as yellow-brown staining under a light microscope.

ELISA detection of serum P1NP, OCN, and CTX-1

Serum levels of bone turnover markers (PINP/CTX-1) and the osteogenic marker OCN were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Real-time fluorescent quantitative PCR

Briefly, the total RNA of MC3T3-E1 cells in each group was extracted using a special RNA Extraction Kit (Trans Company, China). The mRNA was reverse transcribed into cDNA using a mRNA reverse transcription kit, and then the mRNA expression level of the target gene was detected by real-time fluorescent quantitative PCR. The primer sequences were statistically analyzed by the 2−ΔΔCT method as shown in (Table 1).

Table 1 Primer sequences of the gene markers.
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Western blot analysis

Femoral tissue or MC3T3-E1 cells were treated as required and entirely lysed in the 4 °C lysate. The lysed tissues or cells were centrifuged (12000 g, 15 min), and the supernatant was obtained. The total protein in the sample was extracted by the conventional method. A 50 µg sample was loaded and then transferred to the membrane after electrophoresis. The primary antibody (OPN, COL1A1, Wnt3a, β-catenin, Runx2 antibody, with a dilution ratio of 1 ∶ 500) was added and incubated overnight at 4 °C. The secondary antibody was diluted with a 1:10000 dilution of HRP-labeled secondary antibody and incubated with the membrane at 37 °C for 1 h. The ECL chemiluminescence method was used for development. Taking GAPDH as the reference, the gray value of the target protein was quantitatively analyzed by ImageJ software.

Statistical analysis

All data were analyzed by GraphPad Prism (version 10) and expressed as mean ± standard deviation. Statistical significance was determined by one-way analysis of variance (ANOVA). P < 0.05 considered that the difference was statistically significant.

Results

Effect of naringin on osteogenic differentiation of MC3T3-E1 cells

Firstly, we employed the CCK-8 method to detect the activity of naringin at different concentrations (0, 0.05, 0.1, 0.5, 1, 5, 10, 20 µmol/L) after intervention and determined the optimal concentration for subsequent experiments. The results indicated that 1 µmol/L naringin had the most favorable effect on cell proliferation at 24–48 h (Fig. 1A). After 14 days of intervention with naringin at different concentrations, compared with the blank group, the staining of ALP in the treatment group increased with the concentration ranging from 0.01 to 1 µmol/L, suggesting that naringin can enhance ALP activity and promote osteogenic differentiation. High concentrations of the drug inhibited ALP activity (Fig. 1B, C). The alizarin red staining results demonstrated that naringin at the concentration of 0.0 to 1 µmol/L formed more mature calcified nodules with deeper staining and a concentration-dependent increase compared with the control group (Fig. 1D, E). Therefore, we synthesized the above experimental results and selected naringin at 0.1, 0.5 and 1 µmol/L for subsequent experiments.

Fig. 1
figure 1

Effects of naringin on proliferation and osteogenic differentiation of MC3T3-E1 cells. (A) CCK-8 was used to detect the proliferation of MC3T3-E1 cells induced by naringin at different concentrations; (B,C) After naringin at different concentrations intervened MC3T3-E1 cells, ALP activity was observed under the microscope; (D,E) MC3T3-E1 cells were treated with naringin at different concentrations, and calcified nodules were observed under the microscope.

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Naringin affects the expression of OPN, COL1A1, and Runx2 in cells

OPN, COL1A1, and Runx2 are regarded as osteogenic markers. Therefore, we further detected the expression levels of OPN, COL1A1, and Runx2 proteins after naringin intervention in MC3T3-E1 cells. As presented in (Fig. 2A–D), the expressions of OPN, COL1A1, and Runx2 proteins increased after naringin intervention at different concentrations. The higher the drug concentration, the more pronounced the effect. Additionally, the mRNA expressions of OPN, COL1A1, and Runx2 in MC3T3-E1 cells were consistent with the results of Western blot. Naringin at different concentrations could up-regulate the mRNA expressions of OPN, COL1A1, and Runx2 in a dose-dependent manner (Fig. 2E–G). Therefore, we can conclude that naringin can promote osteogenic differentiation of MC3T3-E1 cells.

Fig. 2
figure 2

Naringin on the expression of OPN, COL1A1 and Runx2 (n = 3). (A–D) the expression of OPN, COL1A1, and Runx2 proteins after naringin intervention; (E–G) the expression of OPN, COL1A1, and Runx2 mRNA after naringin intervention. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.

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Naringin increased bone mineral density and improved bone microstructure in OVX mice

To verify that naringin can enhance the bone mineral density of mice, we utilized micro-CT to analyze the bone microstructure of mice in each group. As depicted in (Fig. 3A), in the scanning image of the mouse femur, it was discovered that the number of trabeculae in the sham operation group was large and closely arranged. Compared with the sham operation group, the OVX group exhibited significantly sparse trabecular bone, with the trabecular bone structure being damaged and the bone mineral density reduced. After intervention with E2 or naringin, the bone microstructure and trabecular bone density of mice were ameliorated. Through the data analysis of scanning parameters, the results were consistent with the scanning image. Compared with OVX, the estrogen group could reduce the sparsity of bone trabeculae and improve the bone volume fraction. Naringin in the high-dose group could significantly reverse the reduction and sparsity of bone trabeculae, and enhance bone mineral density and bone volume fraction (Fig. 3B–F).

Fig. 3
figure 3

Bone microstructure of OVX mice. (A) Micro-CT image of the three-dimensional reconstruction image of the femur. (B–F) Group analysis of microscopic CT parameters of interosseous trabeculae (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with OVX.

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Analysis of naringin on bone histopathological changes in OVX mice

HE staining demonstrated that the sham operation group exhibited densely arranged trabecular bone in the distal femur with thickened trabeculae, a high trabecular bone area ratio, and sparse adipocytes in the medullary cavity. In contrast, the OVX group displayed thinned and fragmented trabeculae in the distal femur, accompanied by substantial adipocyte infiltration within the medullary cavity. Following pharmacological intervention, all treatment groups showed improved trabecular bone morphology and reduced adipocyte accumulation, with the most prominent improvements observed in the E2 group and H-Nar treatment group (Fig. 4A, B).

Fig. 4
figure 4

Analysis of bone tissue in OVX mice (n = 6). (A) HE staining and IHC staining; (B) Trabecular bone area in HE staining; (C) OPN protein expression levels in each group; (D) COL1A1 protein expression levels in each group; (E) β-catenin expression levels in each group. *P < 0.05, **P < 0.01, ***P < 0.001, compared with OVX. #P < 0.05, ##P < 0.01, compared with E2.

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Analyze the effects of naringin on the expression of OPN, COL1A1, Runx2, and β-catenin in bone tissues of ovariectomized mice

To validate osteogenic capacity in vivo, we performed IHC staining to detect the expression of osteogenesis-related proteins (Fig. 4A). The expression levels of OPN and COLA1 were lowest in the OVX group, demonstrating a significant difference compared to the sham group. After treatment with E2 and Nar, the expression levels of OPN and COLA1 were markedly increased. Notably, the H-Nar group showed higher expression levels than the E2 group in statistical analysis, although the difference was not statistically significant (Fig. 4C, D). In addition, IHC staining images and quantitative analysis revealed a significant decrease in β-catenin protein expression in the OVX group compared to the sham-operated group. Following E2 and naringin interventions, β-catenin protein levels were notably enhanced, with the H-Nar group demonstrating more pronounced upregulation of β-catenin expression compared to both the E2 and L-Nar groups (Fig. 4E).

Similar to the assessment of osteogenic protein expression in vitro, we conducted a Western blot on the femur. Western blot indicated that the expressions of OPN, COL1A1, and Runx2 proteins in the OVX group were significantly lower than those in the sham operation group. Compared with the OVX group, estrogen, and naringin intervention could significantly increase the expression of OPN, COL1A1, and Runx2 proteins, and the effect of high-dose naringin was more pronounced (Fig. 5A–D).

Fig. 5
figure 5

Expression of osteogenic markers and bone turnover markers in mice (n = 6). (A–D) Western blot was employed to detect the expression of OPN, COL1A1, and Runx2 proteins in MC3T3-E1 cells treated with naringin. (E–G) Expression levels of serum CTX-1, P1NP, and OCN in mice. *P < 0.05, **P < 0.01, ***P < 0.001, compared with OVX. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with E2.

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Changes of serum bone metabolism indexes in each group of mice

The OVX group exhibited significantly higher serum bone resorption marker CTX-1 levels compared to the sham-operated group, indicating enhanced bone resorption activity in OVX mice. Pharmacological interventions notably reduced CTX-1 levels in all treatment groups, with the most pronounced therapeutic effect observed in the E2 group (Fig. 5E). Regarding bone formation markers, serum levels of P1NP and OCN in the OVX group showed marked reductions, reflecting impaired bone formation capacity. Both E2 and naringin treatments significantly elevated P1NP and OCN expression. Among these treatments, H-Nar demonstrated superior efficacy compared to both E2 and L-Nar, though the statistical difference was less pronounced (Fig. 5F, G).

Naringin promotes osteogenesis through Wnt/β-catenin signaling pathway

To investigate whether naringin can promote osteogenesis via the Wnt/β-catenin signaling pathway, we carried out experiments in vivo and in vitro. In Western blot experiments, the expression of Wnt3a and β-catenin protein in the OVX group was lower than that in the sham operation group. Compared with the OVX group, estrogen, and low-dose and high-dose naringin could increase the expression of Wnt3a and β-catenin protein, and naringin at a high concentration had a better effect than the estrogen group (Fig. 6A–C). This finding is consistent with the experimental results observed in our IHC staining.

Fig. 6
figure 6

Effect of Naringin on Wnt/β-catenin Signaling Pathway (n = 3). (A–C) The effect of naringin on Wnt3a and β-catenin protein in vivo; (D–F) The effect of naringin on Wnt3a and β-catenin protein in vitro; (G–J) After the use of inhibitors, naringin has an effect on the osteogenic differentiation of MC3T3-E1 cells. (K,L) The effect of naringin on Wnt3a and β- Catenin mRNA in vivo. *P < 0.05, **P < 0.01, ***P < 0.001, compared with OVX. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with E2.

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In vitro, naringin can increase the expression of Wnt3a and β-catenin in a concentration-dependent manner (Fig. 6D–F), and the mRNA expression levels of Wnt3a and β-catenin are consistent with the results of Western blot (Fig. 6K, L). Subsequently, we added the Wnt/β-catenin signaling pathway inhibitor XAV939 (10 µM) to intervene in MC3T3-E1 cells. Compared with the control group, the inhibitor could reduce the expression of OPN, COL1A1, and Runx2 proteins. Naringin could reverse this phenomenon, and the expression of osteogenic-related proteins increased in a dose-dependent manner (Fig. 6G–J). Therefore, naringin can promote osteogenesis by activating the Wnt/β-catenin signaling pathway.

Naringin promotes nuclear translocation of β-catenin

Nuclear translocation of β-catenin is a critical step in the activation of the Wnt/β-catenin signaling pathway. Immunofluorescence staining revealed that naringin-treated MC3T3-E1 cells exhibited β-catenin accumulation in the nucleus, indicating nuclear translocation, compared to the control group. This phenomenon was markedly suppressed when the Wnt pathway inhibitor XAV939 (10 µM) was co-administered (Fig. 7A). Furthermore, Western blot analysis demonstrated significantly higher nuclear β-catenin levels in naringin-treated cells relative to controls, while XAV939 treatment effectively suppressed nuclear β-catenin expression (Fig. 7B, C). These findings collectively demonstrate that naringin activates the Wnt/β-catenin signaling pathway.

Fig. 7
figure 7

Naringin promotes nuclear translocation of β-catenin (n = 3). (A) Immunofluorescence staining for protein localization analysis. (B,C) Western blot detection of nuclear β-catenin expression. Red arrows indicate nuclear translocation. *P < 0.05, ***P < 0.001, compared with the naringin-treated group.

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Discussion

Naringin is a natural compound. Not only is it one of the effective components of Rhizoma Drynariae, but it also widely exists in various citrus fruits and vegetables. Its various biological activities have drawn increasing attention from scholars18,19,20. Naringin has been intensively studied in the field of bone metabolism. Most studies have revealed that naringin can not only promote osteogenic differentiation but also inhibit the formation of osteoclasts, with multi-target bidirectional regulation. Previous research has shown that it can participate in multiple signaling pathways acting on MC3T3-E1 cells, such as BMP-2 and pi3k/akt, and can boost the expression of pathway-related proteins and osteogenic-related proteins21,22,23. However, as mentioned above, the treatment of osteoporosis with naringin is mostly at the experimental stage in vitro, and no study of naringin through the Wnt/β-catenin signaling pathway in vivo has been found. Therefore, we explored the mechanism of naringin through the Wnt/β-catenin signaling pathway both in vitro and in vivo by constructing the osteoporosis model of OVX mice.

In our study, we discovered that naringin can enhance the expression of Wnt3a and β-catenin proteins in both in vivo and in vitro settings, facilitate osteogenesis, reduce bone loss in OVX mice, and enhance bone mineral density. The dynamic equilibrium between osteoblasts and osteoclasts is a crucial factor for maintaining bone stability, and an imbalance between them will induce osteoporosis. Osteoporotic patients are prone to fractures, which are associated with the malignant transformation of bone microstructure and the decrease in bone trabecular density24,25. Through micro CT analysis, we observed that in the OVX group, bone mineral density declined and the number of trabeculae decreased. After naringin drug treatment, both bone mineral density and the number of trabeculae increased. These findings were also confirmed in femoral tissue staining. This study is consistent with previous ones. Li et al.26 hold that when naringin sustained-release nanomaterials are released into experimental animals, animals in the osteoporosis model group can exhibit a better osteogenic effect and improve bone mineral density. Additionally, Jin et al.27 believed that naringin increased the bone mineral density of femoral tissue, augmented the number of trabeculae, and improved the trabecular structure compared to osteoporosis rats.

COL1A1 is abundant in animal bones and plays a crucial role in maintaining the structural integrity of the human skeleton28. When the formation, structure, and function of COL1A1 change, it can result in a variety of bone disorders, such as osteogenesis imperfecta29,30. Runx2 is a key transcription factor in bone development. It promotes osteogenesis by inducing the expression of col1, including COL1A1, COL1A2, SPP1, etc. It can stimulate the proliferation of osteoblasts and plays a significant role in the maturation of osteoblasts31. OPN is a multifunctional protein that can bind to multiple cell surface receptors to exert a biological effect, including in bone metabolism. It is a key protein for maintaining bone strength and bone mass and plays an important role in bone metabolism32. Our study discovered that naringin increased the expression of OPN, COL1A1, and Runx2 proteins both in vivo and in vitro, and also augmented the mRNA expression of OPN, COL1A1, and Runx2 in MC3T3-E1 cells. Additionally, naringin could enhance the activity of ALP and the formation of calcified nodules in MC3T3-E1 cells as demonstrated by alkaline phosphatase staining and alizarin red staining. Naringin can not only promote osteogenesis in rats but also alleviate the inflammatory reaction, thereby facilitating the healing of rat skull defect33. Previous studies have also revealed that naringin can not only enhance ALP gene expression and mineralization in MC3T3-E1 cells but also increase the viability and osteogenic differentiation of human osteoblasts34. Thus, we can conclude that naringin can promote osteogenesis in OVX mice and osteogenic differentiation of MC3T3-E1 cells.

The Wnt signaling pathway is a highly conserved pathway in the process of species evolution. Its main components include the Wnt family of secreted proteins, the frizzled family of transmembrane receptors, CK1, DVL, GSK3, APC, Axin, β-Catenin, and the TCF/LEF family of transcription factors35. In osteoblast differentiation and bone formation, the β-Catenin/transcription complex factor/Runx2 axis plays a crucial role in the Wnt/β-catenin signaling pathway. Activation of the Wnt signal leads to accumulation of free β-Catenin which then enters the nucleus and combines with transcription factor to initiate Runx2 transcription for controlling bone development and formation36,37. Regulation of the Wnt/β-Catenin pathway can also increase expression of osteogenic-related proteins such as OPN and COL1A1 protein to promote proliferation and osteogenic differentiation of osteoblasts leading to increased bone mineral density and improvement in osteoporosis16,38.

Previous studies have demonstrated that the total flavonoids of Rhizoma Drynariae can partially activate the Wnt/β-catenin signaling pathway to promote bone graft mineralization and osteoblast differentiation in a dose-dependent manner in the bone defect model39. In vivo and in vitro experiments indicated that naringin could activate the Wnt/β-catenin pathway and enhance the expression of Wnt3a and β-catenin proteins in OVX mice, thereby promoting osteogenesis. In vitro, after intervention with different concentrations of naringin in MC3T3-E1 cells, the protein expression of Wnt3a and β-catenin showed a concentration-dependent increase, and naringin promoted the nuclear translocation of β-catenin in MC3T3-E1 cells. In addition, we also employed the Wnt/β-catenin signaling pathway inhibitor XAV939. Naringin can reverse the effect of this pathway inhibitor and promote the expression of osteogenic protein. Therefore, we can conclude that naringin can activate the Wnt/β-catenin pathway and exert its biological activity.

Conclusion

Through comprehensive analysis of the results of this study, it can be concluded that naringin can enhance the bone mineral density of OVX mice and facilitate the osteogenic differentiation of MC3T3-E1 cells via the Wnt/β-catenin signaling pathway. Whether in vivo or in vitro, naringin activates the expression of Wnt3a and β-catenin in the signaling pathway and increases the expression of osteogenic markers such as OPN, COL1A1, and Runx2, thereby promoting osteogenesis and stimulating osteoblast differentiation. Based on previous research results, naringin can be regarded as a potential drug for the treatment of osteoporosis, providing a theoretical basis and reference value for the development of new drugs.