Fabrication of tFNA‑Cur

Firstly, four single-stranded DNA molecules (ssDNAs, Table S1) (Sangon, Shanghai, China) were mixed in equimolar quantities, dissolved in TM buffer (Tris-HCl and MgCl2, pH=8.0), heated at 95 °C for 10 min, and then cooled at 4 °C for 20 min.56,57 Secondly, the product of the first step, tFNA (200 nmol/L), was mixed with various concentrations of curcumin (5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, and 40 μmol/L) and oscillated for 3 h.

Characterization of tFNA‑Cur

The synthesis of tFNA-Cur was assessed using PAGE (polyacrylamide gel electrophoresis). The Zeta potentials and sizes of tFNA-Cur and tFNA were measured using DLS (dynamic light scattering) with the Nano ZS instrument (Malvern, UK). The shapes and sizes of tFNA-Cur were determined using TEM (transmission electron microscopy) with the Libra200 microscope (Zeiss, Oberkochen, Germany) and AFM (atomic force microscopy) with the Cypher VRS microscope (Oxford Instruments, United Kingdom). The complete spectra of tFNA, curcumin, and tFNA-Cur were analyzed using an ultramicrospectrophotometer (UV5 Nano, Mettler Toledo, Switzerland).

Release and stability of tFNA-Cur

Based on previous research,38 we divided the dialysis bag (30 kD; Solarbio, Beijing, China) into an outer liquid (30 mL) and inner fluid (3 mL) using PBS (pH 7.4, 0.01 mol/L) as the medium. Equimolar concentrations of curcumin and tFNA-Cur (20 μmol/L) were dissolved in the inner fluid at 37 °C with constant agitation (150 r/min). The OD value of the released curcumin in the external liquid was determined using an ultra-microspectrophotometer. To assess the stabilities of tFNA and tFNA-Cur, they were separately incubated with 2% or 10% serum for different time intervals (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h). Agarose gel electrophoresis was employed to analyze the stability results. Finally, images of tFNA and tFNA-Cur were captured using an ultraviolet exposure apparatus (Bio-Rad, Hercules, USA).

Isolation and culture of BMSCs

Male C57 mice, aged 4 weeks, were obtained from GemPharmatech (Jiangsu, China). BMSCs were isolated from the mice’s bone marrow, flushed with-MEM (HYCLONE, Pittsburgh, USA), 10% fetal bovine serum, and 100 U/mL penicillin-streptomycin (HYCLONE, Pittsburgh, USA).58,59

BMSCs uptake of curcumin and tFNA-Cur

BMSCs were cultured in a confocal dish with 10 000 cells and treated with curcumin and tFNA-Cur for 6 and 12 h. Images of the cellular uptake of curcumin and tFNA-Cur were captured using a confocal microscope (Olympus, Tokyo, Japan).

AGEs-induced ferroptosis and osteogenic differentiation of BMSCs

The second-passage BMSCs were seeded in a 96-well plate (10 000 cells per well) and treated with different concentrations of tFNA-Cur (with curcumin concentrations of 5, 10, 15, 20, and 25 μmol/L, and tFNA at 200 nmol/L) for 12 h. Additionally, they were exposed to AGEs at various concentrations (25, 50, 100, 150, 200, and 250 µg/mL) for 24 h. Subsequently, the BMSCs were incubated with 10% CCK-8 reagent (KeyGEN Biotech) for 30 min at 37 °C, and their optical density was measured at 450 nm. Based on the results from the CCK-8 assay and ALP staining, the concentration of AGEs (Bioss, Beijing, China) at 150 µg/mL was determined to simulate a diabetic microenvironment. The BMSCs were pre-treated with tFNA, curcumin, and tFNA-Cur for 12 h, and then treated with 150 µg/mL of AGEs for 24 h to investigate the suppression of ferroptosis. Likewise, the same pretreatment was conducted, followed by exposure to an osteogenic differentiation medium (containing 10 nmol/L dexamethasone, 50 µg/mL ascorbic acid, and 5 mmol/L β-glycerophosphate) for 7 days, with continuous exposure to 150 µg/mL AGEs.

ALP and alizarin red staining

After pretreatment with tFNA-Cur for 12 h and subsequent treatment with AGEs (150 µg/mL) for 7 days, BMSCs were fixed with 4% paraformaldehyde (at 4 °C for 20 min) and stained with the ALP Kit (C3250S, Beyotime, China) at 37 °C for 10 min. Similarly, after 14 days, the cells were incubated with Alizarin Red S at room temperature for 5 min. ALP activity and mineralized nodules were observed and photographed under a light microscope.

ROS, MMP level detection assay

Following the aforementioned treatment, BMSCs were first pretreated with tFNA-Cur for 12 h and then exposed to AGEs for 24 h. Subsequently, the cells were incubated with Hoechst 33342 (1X, C1028, Beyotime, China) for 10 min and DCFH-DA (10 μmol/L, S0033S, Beyotime, China) for 20 min to detect the level of ROS. Additionally, they were incubated with Rhodamine 123 (1X, C2008S, Beyotime, China) for 20 min to assess the level of MMP. After washing with PBS, images of ROS and MMP were acquired using a confocal microscope.

FerroOrange staining

The intracellular level of Fe2+ was assessed using the FerroOrange probe (1 μmol/L, MkBio, Mx4559). Following the specified treatments, similar to the procedure for ROS and MMP assessment, the BMSCs were washed with PBS and then incubated with Hoechst 33342 for 10 min and FerroOrange for 20 min. Subsequently, fluorescence images of the BMSCs were captured using a confocal microscope.

TEM

Mitochondrial morphology changes were observed using TEM. Following the aforementioned treatment, BMSCs were fixed with a 3% glutaraldehyde solution at 4 °C for 16 h. Subsequently, the BMSCs were dehydrated using acetone, embedded in Epon812, sectioned (60-90 nm), and stained with uranium acetate and lead citrate. Finally, the mitochondrial morphology of the various treatment groups was examined and captured using a JEM-1400FLASH transmission electron microscope.

Quantitative RT-PCR analysis

The transcriptional levels of Alp, Runx2, Osx, Opn, Gpx4, Acsl4, and Nrf2 were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The primer sequences can be found in Table S2 (Tsingke Biotech). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, MA, USA). cDNA synthesis was performed using the SYBR® Premix Ex Taq II (Perfect Real Time kit; Takara, Dalian, China). The target genes were measured using SYBR® Green I master mix in Q7 (ABI QuantStudio 7, Thermo Fisher, USA). The results were analyzed using the 2−∆∆CT relative quantitative method, with β-Actin (Table S2) serving as the control gene. All experiments were repeated three times.

Western blot analysis

The BMSCs were lysed using a cell protein extraction reagent (KEYGEN Biotech, Nanjing, China), and then mixed with loading buffer at a ratio of 4:1 (v/v) before being boiled for 5 min. The proteins were separated using 10% SDS-PAGE and subsequently transferred onto a PVDF membrane. The membrane was then blocked with blocking buffer (Thermo Fisher Scientific, MA, USA) for 10 min and incubated with the following antibodies (Table S3) overnight at 4 °C. On the second day, the bands were incubated with a secondary anti-rabbit antibody (1:3 000 BEYOTIME, Shanghai, China) for 1 h after washing with TBST. The results were visualized using an ECL chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

IF staining

BMSCs were fixed using a 4% paraformaldehyde solution (4 °C, 25 min), permeabilized with 0.5% Triton X-100 (room temperature, 20 min), blocked with 5% sheep serum (37 °C, 20 min), and washed with PBS at each step, except for the final step. They were then incubated with specific antibodies: anti-ALP (1:200, HUABio, Zhejiang, China), anti-RUNX2 (1:200, HUABio, Zhejiang, China), anti-OSX (1:200, HUABio, Zhejiang, China), anti-GPX4 (1:200, HUABio, Zhejiang, China), anti-ACSL4 (1:200, HUABio, Zhejiang, China), and anti-NRF2 (1:200, HUABio, Zhejiang, China) overnight at 4 °C. The next day, BMSCs were incubated with a secondary anti-rabbit antibody (1:200, Invitrogen, Carlsbad, USA) at 37 °C for 1 h. The cytoskeleton was stained with phalloidin (37 °C, 20 min), and the Nucleus was stained with DAPI (37 °C, 10 min), followed by cleaning with PBS at each step. Images were captured using a confocal microscope (Olympus, Tokyo, Japan).

Animal experiments

All animal experiments were conducted with the approval of the Animal Ethics Committee of West China Hospital of Stomatology, Sichuan University. Four-week-old male C57BL/6J mice were purchased from GemPharmatech (Nanjing, China) and raised in pathogen-free conditions at (55 ± 5)% humidity and (24 ± 2)°C. First, type 2 diabetes mellitus was induced according to previously published studies.52,60 To investigate the effect of diabetes on bone microstructure, mice were randomly divided into two groups: Control, HFD&STZ (n = 6). To assess the therapeutic effects of tFNA, curcumin and tFNA-Cur on DOP, mice were randomly divided into five groups: Control, DOP, DOP+tFNA, DOP+curcumin, and DOP+tFNA-Cur (n = 6). Throughout the experiment, the Control group mice were fed an ordinary diet (10% kcal from fat), while the model and treatment groups were fed a HFD (60% kcal from fat). After four weeks, all groups except the control group were injected intraperitoneally with STZ (35 mg/kg) for seven days to induce diabetes. Mice in the Control group received citrate buffer injections. Subsequently, mice with high plasma glucose levels (11.1 mmol/L) accompanied by polyphagia, polydipsia, and polyuria were considered diabetic mice for subsequent experiments. The treatment groups were injected intraperitoneally with tFNA (1 μmol/L, 200 μL), curcumin (40 μmol/L, 200 μL), or tFNA-Cur (tFNA: 1 μmol/L, curcumin: 40 μmol/L, 200 μL) three times weekly for 8 weeks. Furthermore, the Control and DOP groups were injected with saline (0.9%, 200 µL). Body weight and fasting glucose levels were monitored every 2 weeks. Finally, the bones were harvested and analyzed using micro-CT, H&E staining, masson’s trichrome staining, and IF staining for ALP, GPX4, and NRF2.

Micro-CT analyses

The lower extremities were examined using a SCANCO Medical Micro-Computerized Tomography 50 (70 kV, 200 μA, 300 ms, 10 μm). The regions of interest (ROI) for the femur and tibia were identified at a location 2 mm below the epiphysis.61

AGEs examination in bone and serum

Blood samples were deposited at 4 °C for 30 min and then centrifuged at 3 000 r/min for 10 min to obtain the serum. The levels of AGEs in both the serum and bone samples were determined using a mouse AGEs ELISA kit (YKW-20124, Shanghai, China).

TUNEL assay

Cell death in the bone tissue was assessed using the TUNEL assay (Beyotime, Shanghai, China). Tissue antigens were retrieved using proteinase K (20 μg/mL, 37 °C, 15 min) and then incubated with TUNEL solution (37 °C, 60 min). Subsequently, the Nucleus was stained with DAPI (37 °C, 4 min). Finally, images were captured using a confocal microscope.

Histological analysis

The specimens from various treatment groups were fixed with 4% buffered formalin for 72 h. After one month of decalcification, the bones were dehydrated, embedded in paraffin, and sectioned (3 μm thick). The sections were then stained with H&E and masson’s trichrome to visualize tissue morphology and structural components. Additionally, the bone sections underwent IF staining using anti-ALP, anti-GPX4, and anti-NRF2 antibodies as described above-mentioned.

3D molecular modeling and docking of curcumin to NRF2

The predicted structures of NRF2 were generated using the Alphafold software. Docking grid documents were created using AutoGrid of Sitemap, and the docking simulation was performed using AutoDock Vina (version 1.2.0). The optimal pose was selected for analyzing the interactions. Finally, the protein-compound interaction figure was generated using PyMOL. In the figure, NRF2 is depicted as a slate cartoon model, while curcumin is shown as cyan sticks, and its binding sites are represented by magenta stick structures.

Statistical analysis

All experimental data were analyzed using GraphPad Prism 9.3.1 software (San Diego, CA, USA). Comparisons between two groups were conducted using Student’s t-test, while comparisons among more than two groups were performed using one-way or two-way ANOVA with Sidak’s multiple comparison test. The experimental results are presented as the mean ± SD values, and P < 0.05 was considered statistically significant.