BA, 3-Methyladenin (3-MA), ROS scavenger N-acetyl-L-cysteine (NAC), NLRP3 inhibitor MCC950 and Dorsomorphin (synonyms: Compound c, CC) were purchased from MedChemExpress, and Essential Medium α (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco, Thermo Fisher Scientific, Inc. The primary antibodies against NLRP3 (cat. no. BF8029), Asc (cat. no. DF6304), Caspase-1 (cat. no. AF5418), Cleaved Caspase-1 (cat. no. AF4005), IL-1β (cat. no. BF8021), AMPK (cat. no. AF6423), phosphorylated (p-)AMPK (cat. no. AF3423), mTOR and p-mTOR were obtained from Affinity Biosciences. The primary antibodies for LC3b (cat. no. ab192890), Beclin-1 (cat. no. ab207612), P62 (cat. no. ab109012), OPN (cat. no. ab283656) and RUNX2 (cat. no. ab192256) were purchased from Abcam.

MC3T3-E1 cells, derived from mouse calvarial pre-osteoblasts, were purchased from Procell Life Science & Technology Co., Ltd. (cat. no. CRL-2593). The cells were seeded in 6-well plates containing α-MEM, 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. After 24 h, the old medium was discarded and replaced with the osteogenic induction and differentiation medium (cat. no. CM-0378; Procell Life Science & Technology Co., Ltd.). The medium was then replaced every 3 days to conduct the induction of osteoblasts. After pretreating MC3T3-E1 cells with different concentrations of BA, the cells were treated with 200 μM H₂O₂ to establish an inflammatory injury cell model. The cells were cultured in a humidified incubator with a moist balanced air containing 5% carbon dioxide at 37°C.

MC3T3-E1 cells were seeded into 96-well plates at a density of 3×10³ cells. The cells were then treated with BA at concentrations of 0, 5, 10, 20, 30 and 40 μM, respectively, for 24 and 48 h. Subsequently, the cytotoxicity was evaluated.

The Cell Counting Kit-8 (CCK-8; cat. no. C0037; Beyotime Institute of Biotechnology) detection kit was used to determine cell viability. Briefly, 10 μl CCK-8 was added to each well (100 μl medium), incubated at 37°C for 1 h, and absorbance was measured. The absorbance value, optical density, was accurately measured at a wavelength of 450 nm using a microplate reader (ELx800; Thermo Fisher Scientific, Inc.).

MC3T3-E1 cells were seeded in 6-well plates. Cells were pretreated with 0, 10, and 20 μM of BA for 24 h. The medium was then replaced with osteogenic differentiation medium, treated with H₂O₂ for 24 h, and cultured for 7 days, with the medium changed every 3 days. After fixing the cell samples, they were washed 3-5 times. The BCIP/NBT (cat. no. C3206; Beyotime Institute of Biotechnology) staining working solution was then added for 30 min in the dark, the color development reaction was then terminated. Cell images were obtained using an optical (bright-field) microscope (Leica Microsystems GmbH).

MC3T3-E1 cells were seeded in 6-well plates and pretreated with 0, 10, and 20 μM of BA for 24 h. The medium was then replaced with osteogenic differentiation medium containing H₂O₂ and cultured for 14 days. The medium was changed every 3 days. The samples were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min. The samples were washed three times with PBS and the ARS (cat. no. C0148S; Beyotime Institute of Biotechnology) staining solution was applied for 30 min at room temperature before terminating the reaction. Cell images were obtained using an optical microscope (Leica Microsystems GmbH).

MC3T3-E1 cells were seeded in 96-well plates, treated per experimental protocols, then culture medium was aspirated. 100 μl MDC (cat. no. C3018; Beyotime Institute of Biotechnology) staining solution was added to each well, followed by 30-min light-protected incubation at 37°C. After discarding the stain, cells were rinsed thrice with Assay Buffer, and green fluorescence was visualized via fluorescence microscopy.

MC3T3-E1 cells were plated on coverslips and pretreated with different concentrations of BA. Subsequently, the culture medium was replaced with fresh α-MEM containing 200 μM H₂O₂ and incubated under standard conditions (37°C; 5% CO₂) for 24 h. Initial fixation was performed with 4% PFA for 15 min. Subsequently, the cells were permeabilized with 0.2% Triton X-100 for 10 min. To minimize non-specific binding, specimens were incubated with Rapid Immunofluorescence Blocking Buffer at room temperature for 30 min. Primary antibody incubation was performed using rabbit anti-LC3b (1:50) overnight at 4°C, followed by three PBS washes. Secondary antibody incubation was performed using Cy3-labeled goat anti-rabbit IgG (1:200; cat. no. A0507; Beyotime Institute of Biotechnology) at room temperature for 1 h. Nuclear counterstaining was performed with DAPI (5 μg/ml). Cell images were captured using an inverted fluorescence microscope (Leica Microsystems GmbH). Additionally, ImageJ software (v1.53t; National Institutes of Health) was employed to quantitatively analyze the average fluorescence intensity.

Intracellular ROS generation was assessed using 2',7' dichloro-dihydro-fluorescein diacetate (DCFH-DA; cat. no. S0034; Beyotime Institute of Biotechnology). MC3T3-E1 cells were plated in 96-well culture plates at 3×10³ cells/well. Cells were then pretreated with 0, 10, and 20 μM of BA for 24 h. The medium was then replaced with fresh medium containing H₂O₂ and culture for 24 h. Prior to analysis, cells were loaded with 10 μM DCFH-DA in serum-free medium (37°C; 30 min) and immediately image was captured using an inverted fluorescence microscope (Leica Microsystems GmbH).

After treating the cells under the aforementioned conditions, the cells were lysed using a buffer containing RIPA (cat. no. P0013; Beyotime Institute of Biotechnology), PMSF (cat. no. ST505; Beyotime Institute of Biotechnology) and phosphatase inhibitors (cat. no. P1081; Beyotime Institute of Biotechnology) to extract the total cellular proteins. According to the manufacturer's instructions, a BCA assay kit was used to determine the protein concentration. Subsequently, the proteins in the samples were separated using 10% SDS-polyacrylamide gels (loading: 30 μg per lane) and an electrophoresis apparatus. The proteins then were transferred onto a PVDF membrane. After blocking the membrane with a rapid protein-free blocking solution at room temperature for 30 min, the membrane was probed with the primary antibody overnight at 4°C. After that, the membrane was rinsed three times with Tris-buffered saline containing 0.1% Tween 20 and then incubated with the corresponding secondary antibody (1:1,000; cat. no. A0208 or A0192; Beyotime Institute of Biotechnology) at room temperature for 2 h. Finally, protein expression was detected using an enhanced chemiluminescence reagent (cat. no. MI00607B; Mouse Biotech), and its intensity was analyzed using ImageJ software.

Total RNA was isolated from MC3T3-E1 cells using TRIzol^™ Reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. For RT, RNA (1 μg) was combined with oligo (dT) primers (10 μM) and deoxyribonucleoside triphosphates (dNTPs; 10 mM) in a 20 μl reaction volume. The reverse transcription reaction was carried out at 42°C for 60 min, followed by 85°C for 5 min to inactivate the reverse transcriptase. Primers employed for amplification are shown in Table I. The obtained complementary DNA (cDNA) was used as a template to determine the gene expression levels on the LightCycler^® 96 system using SYBR Green I (Thermo Fisher Scientific, Inc.) as the fluorophore. The reaction conditions of PCR amplification were as follows: Denaturation at 95°C for 10 min and followed by 40 cycles at 95°C for 15 sec, then decreased to 60°C for 15 sec and finally increased to 72°C for 40 sec. Relative gene expression levels were calculated using the 2^−ΔΔCq method (30).

All animal experimental protocols were examined and approved by the Laboratory Animal Management Committee of Xi'an Jiaotong University (approval no. XJTUAE2025-3812). Animals were housed under controlled conditions (22±2°C, 50±10% humidity, 12/12-h light/dark cycle) with free access to food and water and were implemented following the Xi'an Jiaotong University Guide for Animal Experimentation, which conforms to the requirements of the Guide for the Care and Use of Laboratory Animals issued by the US National Institutes of Health. A total of 30 Sprague-Dawley female rats (8-weeks-old; weight, 180±10 g) were acquired from Xi'an Jiaotong University. Based on the formula provided by MedChemExpress official guidelines: Animal A (mg/kg)=Animal B (mg/kg) × (Animal A Km/Animal A Km) and the recommended intraperitoneal administration volume range for rats, the in vivo regimen was rationally designed. The rats were divided into three groups: Sham + Vehicle, OVX + Vehicle, and OVX + BA (15 mg/kg; n=10) randomly. The three groups of rats were acclimated for 1 week. Before all surgical and sampling procedures, the rats were anesthetized with inhaled isoflurane (4% for induction and 2% for maintenance) to ensure adequate anesthesia. OVX + BA group rats were intraperitoneally administered 15 mg/kg/day BA every other day for 8 weeks. Rats in the sham and OVX + Vehicle groups were intraperitoneally administered with equal volumes of the excipient. After 8 weeks of treatment, blood was collected from the heart of the rats. Then, the rats were euthanized by cervical dislocation. The femurs were removed and fixed with 4% PFA for subsequent experiments.

Cardiac blood samples were obtained via cardiac puncture and subjected to two-step centrifugation to isolate serum fractions. Serum concentrations of proinflammatory cytokines were quantified using ELISA kits: IL-1β (cat. no. GER008), IL-6 (cat. no. GER011) and TNF-α (cat. no. GER013; all from Elabscience Biotechnology, Inc.), following the manufacturer's protocols.

The prepared femur samples were placed in a high-resolution Micro-CT scanner (Skyscan 1276; Bruker Corporation). Raw projection data were reconstructed into three-dimensional models through sequential processing with NRecon (v1.7.4.2; Bruker-microCT) and Data Viewer (v1.5.6.2, Bruker-microCT) software packages (Bruker-microCT). Quantitative morphometric analysis within the metaphyseal region of interest included bone mineral density (BMD, mg HA/cm³), bone volume fraction (BV/TV, %), trabecular number (Tb.N, mm⁻¹) and trabecular separation (Tb.Sp, μm).

Femoral specimens were subjected to decalcification in 10% EDTA solution, achieving complete decalcification within 8-week duration. Specimens subsequently underwent graded ethanol dehydration series (70-100%) and paraffin embedding using standard histology protocols. Subsequently, the specimens were cut into thin slices with a thickness of 4 μm. Finally, according to the standard laboratory procedures, the H&E and Masson staining methods were used respectively to stain the slices.

Paraffin sections underwent xylene dewaxing and graded ethanol rehydration. After that, they were subjected to citrate buffer antigen retrieval. Then, 5% bovine serum albumin (cat. no. HY-D0842; MedChemExpress) was used to block the non-specific binding at room temperature for 30 min. Incubation followed successively with the corresponding primary antibody and secondary antibody. Chromogenic development with 3,3'-diaminobenzidine was monitored microscopically until optimal signal intensity, followed by hematoxylin counterstaining (30 sec) and permanent mounting with resinous medium. Brightfield imaging was performed using a microscope. Quantitative histo-morphometric analysis of staining intensity was conducted through ImageJ with IHC Profiler plugin.

Cells were seeded onto chamber slides and infected using the 1/2 small-volume method with HBAD-mRFP-GFP-LC3 adenovirus (cat. no. HB-AP210000; Hanbio Biotechnology Co., Ltd.) at 500 multiplicity of infection at 37°C for 24 h. After transfection, the cells were subjected to the designated treatments and then examined under a confocal microscope at 48 h post-infection. Yellow puncta (RFP⁺GFP⁺) were identified as autophagosomes, while red puncta (RFP⁺GFP⁻) represented autophagolysosomes. Autophagic flux was assessed by manually counting the number of yellow and red puncta in the merged images. Successful transfection efficiency was verified, and representative fluorescence images are shown in Fig. S1.

All experimental data were obtained from at least three independent experiments and were expressed as the mean ± SD. Statistical analyses were performed using SPSS 24.0 software (IBM Corp.). Comparisons between two groups were conducted using unpaired Student's t-test, and Cohen's d was calculated to estimate effect size. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied. When appropriate, 95% confidence intervals (95% CI) were provided for key outcome measures. P<0.05 was considered to indicate a statistically significant difference. In addition, post-hoc power analysis was conducted using G*Power 3.1 based on key outcome measures (effect size f=0.32, α=0.05, n=10 per group). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the in vivo safety of BA, (HC staining was carried out on major organs (liver, heart, lung and kidney) of Sham, OVX and BA groups (Fig. S2). Histopathological analysis revealed intact tissue structures and no obvious abnormalities (such as inflammatory infiltration, necrosis or structural damage) in the major organs of the BA groups. No significant histological differences were observed among the three groups, preliminarily confirming the favorable in vivo safety of BA at the experimental dose in OVX rats. To explore the effect of BA on bone loss in OVX rats, focus was addressed on the rat femurs (Fig. 1A). The micro-CT images and bone parameters of the rat femurs were analyzed. In addition, the changes in the microstructure of the rat bone tissue were evaluated by Masson's trichrome staining and H&E staining. The results of the CT scan showed that, compared with the OVX group, the BMD, BV/TV and Tb.N in the BA group were significantly higher, whereas Tb.Sp was significantly lower. (Fig. 1B-F). The results of Masson's trichrome staining revealed that, compared with the OVX group, the BA group significantly increased the generation of bone collagen fibers (Fig. 1G). The results of H&E staining demonstrated that, compared with the OVX group, the BA group alleviated the manifestations of trabecular bone reduction and fracture (Fig. 1G). Post-hoc power analysis indicated that the current sample size (10 rats per group) achieved 83.6% power for one-way ANOVA, confirming the reliability of these primary outcome measures. These findings indicate that BA can prevent OVX-induced bone loss.

To further investigate the mechanism by which BA prevents bone loss in OVX rats, the expression levels of NLRP3, Asc and Caspase-1 protein in the femoral tissues of the rats were assessed through IHC staining. Additionally, ELISA was used to detect the inflammatory markers IL-1β, IL-6 and TNF-α in the rat serum. IHC staining of the femurs of OVX rats identified that, compared with the sham group, there was a significant increase in the expression levels of NLRP3, Asc and Caspase-1 protein. However, administration of BA significantly reduced the expression of these inflammation-related proteins (Fig. 2A and B). In addition, the results of ELISA showed that BA could significantly reduce the elevated inflammatory indices of IL-1β, IL-6 and TNF-α in the serum of OVX rats (Fig. 2C). Therefore, these data suggest that BA treatment for bone loss in OVX rats may be associated with its inhibition of the NLRP3 inflammatory pathway in the rat bone tissue.

To further explore the potential of BA treatment and prevention of OP, MC3T3-E1 cells were challenged with H₂O₂ (200 μM) and an inflammatory injury cell model was established in vitro. The experimental results showed that, compared with the con group, H₂O₂ intervention significantly increased ROS production in MC3T3-E1 cells (Fig. 3A and B), and the protein expression level of the NLRP3 inflammasome increased (Fig. 3C and D). In addition, as osteogenic differentiation marker factors, both the mRNA and protein expression levels of RUNX2 and OPN were inhibited (Fig. 3E-I). When NLRP3 inhibition experiments were conducted using MCC950 (NLRP3-specific inhibitor) in MC3T3-E1 cells with inflammatory injury, relative to the H₂O₂ group, the expression level of NLRP3 protein decreased significantly (Fig. 3C and D). Moreover, relative to the H₂O₂ group, it significantly improved the expression of RUNX2 and OPN genes and proteins (Fig. 3E-I). Additionally, when NAC (specific ROS inhibitor) was added, in comparison with the H₂O₂ group, the accumulation of ROS was reduced, the expression of NLRP3 protein in the inflammasome was inhibited (Fig. 3A-D), and the inhibition of RUNX2 and OPN gene and protein expression was alleviated (Fig. 3E-I). The aforementioned findings indicate that the effect of H₂O₂ on the osteogenic differentiation of MC3T3-E1 is associated with ROS/NLRP3 inflammasome.

Before evaluating the effect of BA (Fig. 4A) on the differentiation of MC3T3-E1 cells treated with H₂O₂, the CCK-8 activity assay was used to detect whether there were potential cytotoxic effects on MC3T3-E1 cells within 24 and 48 h. When the concentration of BA ≤20 μM, there was no significant change in cell viability (Fig. 4B). Consistent with previous studies reporting that BA shows no cytotoxicity to osteoblast-lineage cells within the range of 0-30 μM (31), 10 and 20 μM were therefore selected for subsequent experiments. MC3T3-E1 cells were treated with BA at different concentrations. The results showed that BA (20 μM) significantly inhibited the activation of the NLRP3 inflammasome pathway induced by H₂O₂ (Fig. 4C-H). In addition, the results of ARS and ALP staining revealed that, BA (20 μM) significantly restored the activity of ALP and the number of mineralized nodules (Fig. 4I). Similarly, by detecting the mRNA expression of RUNX2 and OPN, it was found that when treated with BA (20 μM), the mRNA expression of the two was significantly restored (Fig. 4J-K). These results demonstrate that BA can effectively treat H₂O₂-induced inflammatory damage and significantly alleviate the inhibitory effect of H₂O₂ on osteoblast differentiation.

Based on the previous experimental data, 20 μM was determined as the optimal concentration for BA. To explore the effect of BA on autophagy in MC3T3-E1 cells treated with H₂O₂, the expression of autophagy marker proteins was detected. The results showed that BA significantly increased the expression of LC3b II and Beclin-1 in the cells and inhibited the expression of P62. However, 3-MA could reverse the effects of BA on the expression of LC3b-II, Beclin-1 and P62 (Fig. 5A-D). Immunofluorescence results showed that, compared with the control group, the expression of LC3b in cells increased significantly, and the 3-MA group reversed this result (Fig. 5E and F). MDC can specifically label autophagosomes. MDC staining showed that BA can promote the formation of autophagosomes (Fig. 5G and H). The fluorescent probe DCFH-DA was used to detect intracellular ROS. The BA group significantly inhibited the production of ROS, but the 3-MA group reversed this result (Fig. 5I). Additionally, based on the mRFP-GFP-LC3 puncta assay, the autophagic flux was dynamically evaluated under different conditions. In the BA treatment group, there was a marked increase in both yellow puncta (autophagosomes) and red puncta (autolysosomes), indicating that BA significantly promoted autophagosome formation and enhanced autophagic flux. By contrast, when bafilomycin A1 was added together with BA, yellow puncta remained abundant, but red puncta were markedly reduced. This suggests that although autophagosome formation was still induced, the fusion and degradation steps were blocked, leading to autophagosome accumulation. These findings demonstrate that BA enhances autophagy by promoting autophagic flux (Fig. S3). This indicates that BA can enhance autophagy and inhibit ROS production in H₂O₂-exposed MC3T3-E1 cells.

The regulation of autophagy by BA was investigated. It has been reported that BA can regulate AMPK phosphorylation (32). Western blotting showed that BA effectively promoted the phosphorylation of AMPK and inhibited the phosphorylation of mTOR, while compound C (an AMPK blocker) significantly reversed this effect and inhibited the expression of LC3b-II and Beclin-1, promoting the expression of P62 (Fig. 6A-G). The group treated with compound C also inhibited the expression of LC3b as detected by immunofluorescence and the formation of autophagosomes as shown by MDC staining (Fig. 6H-K). This indicates that BA regulates autophagy in H₂O₂-exposed MC3T3-E1 cells through the AMPK/mTOR pathway.