Collection of femoral head specimens

In this study, we conducted our research using a cohort of patients with SIONFH who underwent total hip arthroplasty at the Department of Orthopedics of the First Affiliated Hospital of Guangzhou University of Chinese Medicine. The study was conducted following the approval of the Ethical Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (Ethics No. K-2023-181). The selection of patients adhered to specific inclusion criteria outlined as follows: a) Patients were diagnosed with SIONFH based on the Chinese Adult Femoral Head Necrosis Clinical Diagnosis and Treatment Guidelines (2020). b) Early stages of SIONFH such as Association Research Circulation Osseous (ARCO) stages II and IIIA. c) Patients had a clear history of glucocorticoid drug use, with no evidence of hip trauma or long-term alcohol abuse. d) Patients fell within the age range of 18 to 55 years. e) Patients were free from other bone metabolic diseases. f) Prior to participation, patients were fully informed about the study and provided their consent to participate. Nine patients who met these inclusion criteria were recruited for the study. Following total hip arthroplasty, tissue samples of the femoral head were collected. The femoral head tissue was then cut into uniformly sized bone slices, approximately 5 mm thick, along the coronal surface. These bone slices were subsequently stored in an ultra-low temperature refrigerator at −80 °C for future use in the experiments.

Proteomics and bioinformatics analysis

Protein data-independent acquisition (DIA) analysis has proven to be effective and reliable, allowing for a comprehensive exploration of the proteome while preserving relevant data. In this study, we focused on analyzing healthy and necrotic regions within the examined specimens using protein-DIA techniques. Our objective was to uncover unique expression patterns of proteins in different anatomical areas. The raw data obtained were pre-processed and imported into R statistical software for further analysis and visualization. Gene ontology (GO) function and KEGG pathway enrichment analyses of genes corresponding to significantly different proteins among groups were performed using the “clusterProfiler” package in R (v.4.3.1). Additionally, we obtained the corresponding protein structures in PDB format from the Protein Crystal Structure Database (PDB, https://www.rcsb.org). To investigate the interaction between drugs and target proteins, we conducted molecular docking using RDock, a widely used docking software. The binding energy was used as a measure to evaluate the effectiveness of the docking process, with higher binding energy indicating better binding activity of the drug to the receptor protein and a more stable docking state.

Animal experiments

A total of 60 male SD rats, 10 weeks old and of SPF-grade, were randomly divided into six groups: Control group, Experimental group, XJB-5-131 group, Midiv-1 group, SIRT3-agonist group, and Isovitexin group, with 10 rats in each group. Except for the Control group, lipopolysaccharide (2 mg/kg, Sigma) was injected into the tail vein of rats in the other groups. On days 2, 3, and 4, rats in the experimental groups received methylprednisolone injections (30 mg/kg, MedChemExpress) into the gluteal muscle, alternating between the two gluteal muscles. After the completion of the modeling process, the XJB-5-131 (a mitochondria-targeted ROS scavenger, MedChemExpress), Midiv-1 (a mitophagy inhibitor, MedChemExpress), and Isovitexin (Chengdu, Must) groups received intraperitoneal injections of 1 mg/kg,57 15 mg/kg,47 and 15 mg/kg,58 respectively, every two days. The SIRT3-agonist group received a daily oral gavage of 200 mg/kg SIRT3 agonist59 (Nicotinamide riboside chloride, MedChemExpress). The Control group was injected with an equal amount of 0.9% saline. After 4 weeks from the modeling process, all rats were euthanized, and bilateral femurs were collected for further analysis. The animal study was conducted following the approval of the Ethical Review Committee for Animal Experimentation of Guangzhou University of Chinese Medicine (Ethics No. 20230526001).

Micro-computed-tomography scanning

The proximal femur was scanned using a SkyScan micro-CT scanner, with the femoral head as the central area. The scanner was set with basic parameters, including a ray intensity of 1 mm Al, a resolution of 9 μmol/L, a voltage of 65 kV, a current of 385 μA, and a scanning length of 18 mm. Following the scanning process, the original image data were reconstructed using NRecon software. The reconstructed model was then adjusted in three dimensions using Data Viewer software to ensure accurate alignment and positioning. To perform a quantitative analysis of the model, CTAn software was used. This software provided metrics for specific indicators, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) of the bone tissue. These indicators offer insights into the density and microarchitecture of the trabecular bone in the proximal femur. Furthermore, Database Viewer software was utilized to reconstruct two-dimensional (2D) images based on the analyzed data. This allowed for visualizing and examining the bone structure in a simplified format.

Hematoxylin and eosin (HE) staining

The left lower limb femurs of rats from each group were taken and fixed in 4% paraformaldehyde for 48 h and then decalcified using 14% EDTA (PH = 7.4). After the completion of decalcification, the tissues were dehydrated, paraffin-embedded, sectioned (thickness of 5 μm) and baked, and then stained in a series of operations in a fume hood using Harris Hematoxylin Stain and Eosin Stain. The sections were routinely dehydrated, clarified and sealed, then the proximal femoral region was scanned using a tissue section scanner. The number of empty bone lacunae and the ratio of empty bone lacunae versus total bone lacunae were compared within each group.

Immunohistochemistry (IHC) staining

The bone tissue paraffin sections were deparaffinized with distilled water, followed by antigen retrieval and blocking. Subsequently, the RUNX2 (ABways) and GPX4 (Proteintech) antibodies was added dropwise onto the sections, which were then incubated at 4 °C overnight. After washing with water, secondary antibody was added and incubated for 30 min at 37 °C. Finally, SABC was added and incubated for 30 min at 37 °C, and DAB staining was performed for 5–10 min. The sections were routinely dehydrated, clarified, and sealed, then photographed under observation, and the rate of immunohistochemistry positivity was counted in each field of view.

Immunofluorescence (IF) staining and ROS staining

Paraffin sections of bone tissue were dewaxed and subjected to antigen retrieval treatment. Sections were permeabilized with 0.3% TritonX-100, blocked with 5% goat serum for 2 h. Drops were added with the SIRT3 (Proteintech), BNIP3 (ABways), NIX (ABways), MFN1 (Proteintech), MFN2 (Proteintech) and DRP1 (Abcam) antibody or ROS stain solution and incubated overnight at 4 °C. After washing with PBST, the secondary antibody was added, and finally, anti-fluorescence quencher containing DAPI for mounting was used. Fluorescence microscopy was detected to observe the fluorescence signal in the femoral head region and the images were analyzed using lmage J.

Cell culture and cellular interventions

The resuscitated MC3T3-E1 cells were spread on a 100 mm dish containing α-MEM medium with 10% fetal bovine serum by volume, and incubated at 37 °C in an incubator with 5% CO2 by volume. The medium was changed every 2 days, and the cells were passaged when the cell fusion was about 90%. Stably grown MC3T3-E1 cells were differentiated into osteoblasts by culturing in osteogenic induction solution (complete α-MEM medium containing 10 nmol/L dexamethasone, 50 μg/mL vitamin C and 5 mmol/L β-glycerophosphate). Stably grown MC3T3-E1 cells were taken, digested, centrifuged, resuspended, and spread, and the cells were grouped and intervened after the cells were attached to the wall. The cells were divided into the following six groups: Control group, DEX (Dexamethasone, MedChemExpress) group, XJB-5-131 group, Midiv-1 group, SIRT3-agonist group, Isovitexin group. The intervention program was finalized by reviewing the relevant literature and pre-experiments. Control group: α-MEM complete medium containing osteoinductive fluid; DEX group: α-MEM complete medium containing osteoinductive fluid +1 μmol/L DEX; DEX + XJB-5-131 group, DEX+Midiv-1 group, DEX + SIRT3-agonist group, and DEX+Isovitexin group: on the basis of the DEX group, respectively add 800 nmol/L XJB-5-131, 800 μmol/L SIRT3 agonist, 5 μmol/L Midiv-1, 150 μmol/L Isovitexin.

Isolation and culture of rat bone marrow mesenchymal stem cells (BMSCs)

BMSCs were used to validate the experimental results of MC3T3-E1. SD rats were anesthetized and euthanized by decapitation. The tibia and fibula were dissected to expose the bone marrow cavity, which was then flushed with PBS to collect the bone marrow fluid. The collected bone marrow fluid was cultured in α-MEM medium supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin. The cell culture medium was refreshed every 2 days. When the cell growth reached 80%–90% confluence, the cells were passaged. Cells from the third passage were harvested and utilized in the subsequent experiments.

Cell proliferation assay

The MC3T3-E1 cells or BMSCs were grown in 96-well plates at a density of 5 000 cells/well, and after intervention treatment, the medium was discarded and washed with PBS. After reintroducing 100 μL of medium and setting up blank control wells, 10 μL was added to each well, and after incubation for 1–2 h at 37 °C and 5% CO2, the absorbance (OD value) at 450 nm was measured by an enzyme marker.

Alkaline phosphatase (ALP) staining

The above stably grown MC3T3-E1 cells were spread in 48-well plates at a density of 6 × 103 cells/well, and the experimental group was induced to differentiate by adding 800 nmol/L XJB-5-131, 800 μmol/L SIRT3 agonist, 5 μmol/L Midiv-1 and 150 μmol/L Isovitexin in osteogenic induction solution per well, respectively; and the negative control group was cultured using α-MEM complete culture. The medium was changed every 2 days, and the cells were cultured for 7 days until osteoblasts were maturely differentiated. After 7 days, the cells were washed once with PBS and fixed using 4% paraformaldehyde for 30 min. Add the reaction substrate 150 μL/well according to the instructions of Alkaline Phosphatase Assay Kit reagent and incubate at room temperature away from light for 12 h. After washing with PBS for three times, the 48-well plate was placed in a ventilated kitchen to air-dry. Finally, the 48-well plates were scanned using a laser scanner and the staining depth of each group of cells was quantified using Image J software.

Mitochondrial membrane potential (MMP) analysis

Stably grown cells were spread in 6-well plates at a density of 3 × 105 cells/well, and the cells in each group were treated accordingly, rinsed once with PBS, and then 1 mL of cell culture medium was added. Add 1 mL of JC-1 staining solution to each well, and reset to incubate in the incubator for 20 min. Discard the supernatant at the end of staining, and wash the cells with JC-buffer 2 times. Add 2 mL of culture medium and place under fluorescence microscope or laser confocal microscope to observe the results and collect pictures. Observe and detect the fluorescence value by fluorescence microscope and fluorescence spectrophotometer.

Transmission electron microscopy (TEM) observation

Stably grown MC3T3-E1 cells were spread in 6-well plates at a density of 3 × 105 cells/well, and 2.5% room-temperature glutaraldehyde fixative was added after 24 h of cell intervention in each group. The cells were fixed at room temperature for 5 min, scraped off, and centrifuged for 2 min, and a new electron microscopy fixative was added to suspend the cells. After being put at 4 °C for 30 min, fixed dehydrated sections were made, and mitochondria were observed under transmission electron microscope and photographed.

Ferrous ion (Fe2+), malondialdehyde (MDA), glutathione (GSH) and adenosine triphosphate (ATP) detection

Stably grown MC3T3-E1 cells were spread in 6-well plates at a density of 3 × 105 cells/well. After 24 h of cell intervention in each group, the culture medium was discarded, the cells were collected. and the extraction solution was added according to the instructions of the kit, and after ultrasonic crushing, centrifugation, and taking the supernatant. The absorbance of the supernatant was measured by a spectrophotometric assay at 532 nm for MDA, 405 nm for GSH. Determination of RLU values for ATP using a luminometer.

Flow cytometry for ROS detection

Stably grown MC3T3-E1 cells were spread in 12-well plates and the cellular intervention is performed as in “2.8”. Prepare MitoSOX Red (5 mmol/L, MedChemExpress) in the ratio of 1:1 000, incubate at 37 °C for 30 min, wash the cells with PBS and collect them in a centrifuge tube, centrifuge at 1 000 r/min for 5 min, discard the supernatant, and resuspend the cells with PBS. The relative content of ROS in each group of samples was measured by flow cytometry.

Co-immunoprecipitation (Co-IP) assay

After intervention, the cells were lysed, homogenized, and centrifuged and the supernatant was taken, 2.0 ug SIRT3 monoclonal antibody was added and incubated at 4 °C for 1 h. 20 μL protein G agarose was added and incubated inverted at 4 °C for 3–5 h. The cells were then centrifuged at 1 000 g for 5 min at 4 °C. The supernatant was discarded, and the immunization mixture was collected, and the precipitate was suspended in the SDS-PAGE upsampling buffer for Western blot experiments.

Western blotting (WB)

The collected cellular protein samples were separated by SDS-PAGE, transmembrane, closed with TBST blocking solution containing 5% skimmed milk for 1 h. The membrane was washed three times by TBST, and SIRT3, BNIP3 (Cell antibody usage with ABclonal; bone tissue antibody usage with ABways), NIX (Cell antibody usage with Santa Cruz; bone tissue antibody usage with ABways), DRP1, MFN1, MFN2, GPX4(Cell antibody usage with Affinity; bone tissue antibody usage with Proteintech), TFR (Abcam), FTH (Affinity), RUNX2 (Wanleibio), OCN (Wanleibio) and OPG (Wanleibio) antibody was incubated at 4 °C overnight. On the following day, the corresponding secondary antibody was added, incubated at room temperature for 1 h, the membrane was washed by TBST, the color was developed by ECL chemiluminescence, imaging was performed by gel imager, and quantitative analysis was performed using Image J software.

Quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was extracted from bone tissue powder or cells using the Trizol method, RNA concentration was determined by a biophotometer, and reverse transcription was performed using Oligo-dT and other reagents. Finally, the expression of RUNX2 (Forward: 5′-AGTCCCAACTTCCTGTGCT-3′, Rveverse: 5′- GGTGAAACTCTTGCCTCGTC-3′), OCN (Forward: 5′- GAGGGCAATAAGGTAGTGAA-3′, Rveverse: 5′- CATAGATGCGTTTGTAGGC-3′) and OPG (Forward: 5′-AGGGCGTTACCTGGAGAT-3′, Rveverse: 5′- AGGGTGCTTTCGATGAAG-3′) was detected by SYBR Green method, using Hprt as the housekeeping gene, using Bio-Rad real-time fluorescence PCR instrument, in which the RNA reverse transcription program was 42 °C 60 min, 92 °C 10 min, 4 °C, the PCR reaction on-line program was Step1 95 °C 30 s, Step2 (40 Cycles) 95 °C 15 s, Step2 (40 Cycles) 95 °C 15 s, Step3 95 °C 15 s. ΔΔCt method was used to quantitatively analyze the relative expression of target genes.

Statistical analysis

SPSS 21.0 software was used to analyze the results of the measurement data, which conformed to normal distribution. One-way ANOVA was used for comparison between multiple groups. Independent samples t-test was used for comparison between two groups when the variance was uniform, and nonparametric Mann-Whitney test was used when the variance was not uniform. P < 0.05 indicated that the difference was statistically significant.