Isolation and culture of chondrocytes

Chondrocytes were isolated from the knee joints of mice and human patients by finely dicing the cartilage tissue, followed by prolonged incubation at 37 °C with 0.2% Type II Collagenase (Thermo Fisher Scientific, Waltham, MA, USA). After overnight digestion, undigested tissue remnants were filtered out the following day. The resulting cell suspension was then re-suspended and seeded. Primary chondrocytes were cultured in F-12 culture medium (DMEM/F-12, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific), 100 units/mL Penicillin, and 100 units/mL Streptomycin (Thermo Fisher Scientific) to promote cell growth and prevent microbial contamination. Cells were maintained at 37 °C in a 5% CO2 environment, with all subsequent experimental procedures performed using passage P1 chondrocytes.

Cellular interventions

To investigate the impacts of varying concentrations of inflammatory stimuli on chondrocytes, we utilized 1, 5, and 10 ng/mL of human recombinant IL-1β (Thermo Fisher Scientific), alongside establishing a blank control group. To simulate an in vitro arthritic microenvironment, mouse chondrocytes were incubated with 10 ng/mL human recombinant IL-1β. Small interfering RNAs (siRNAs) were sourced from Shanghai GenePharma Co., Ltd., while adeno-associated viruses (AAVs) were obtained from Shanghai OBiO Technology Co., Ltd. Transfection of Small-interfering RNA (siRNA) was facilitated using LipofectamineTM 3000 (Thermo Fisher Scientific), followed by subsequent treatments with specific agents. To elucidate the functions of PINK1 and SIRT3, cells were transfected with either 100 nmol/L siRNA targeting Pink1 or adeno-associated virus-Pink1 (AAV-Pink1) at concentrations ranging from 109 to 1011 genome copies per mL. Similarly, the role of SIRT3 was examined using 100 nmol/L siRNA targeting Sirt3 or adeno-associated virus-Sirt3 (AAV-Sirt3) at equivalent concentrations. Modulation of glycolysis involved transfecting cells with 100 nmol/L siRNA targeting PKM2. To manipulate phosphorylation states at specific sites, cells were transfected with 100 nmol/L of PKM2 mutants: S127A, S287A, and T365A. To examine the impact of deacetylase inhibitors on mitophagy and C-ECM metabolism, chondrocytes were treated with 3-TYP (MCE, HY-108331) at a concentration of 50 μmol/L.

Quantitative real‑time reverse transcription‑polymerase chain reaction (RT‑PCR)

Mouse chondrocytes were seeded in six-well plates and subjected to the described interventions. Total RNA extraction was carried out using TRIzol® reagent (Thermo Fisher Scientific) followed by reverse transcription to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative reverse transcription PCR (RT-PCR) was performed with the ChamQ Blue Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) using the CF X96TM Real-Time PCR System. Transcript levels of the target genes were quantified using the comparative Ct (2−ΔΔCt) method. Primer sequences utilized for RT-qPCR are provided in Supplementary Table 1.

Western blotting

Following the implementation of various interventions outlined previously, cells from the culture dish were harvested and lysed on ice for 30 minutes using NCM RIPA Buffer (NCM Biological Technology Co., Ltd, China), followed by centrifugation. The resulting supernatant was collected to assess total protein content. Protein concentrations of each sample were determined with the BCA Protein Quantification Kit (Vazyme). Subsequently, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Beyotime, Shanghai, China). The membrane was blocked with a blocking buffer for 30 minutes at room temperature to minimize non-specific binding, followed by washing with washing buffer. Next, the membrane was incubated overnight at 4 °C with specific primary antibodies. After three washes with the washing solution, the membrane was incubated with secondary antibodies for 1 hour at room temperature. Following another thorough wash to remove unbound secondary antibodies, immunoreactive bands were visualized using ultra-sensitive Enhanced Chemiluminescent (ECL, NCM) and captured using the SH-523 imager (Shenhua Technology, Hangzhou, China). Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA) for further analysis.

Nuclear and cytoplasmic protein extraction

For the extraction of nuclear and cytoplasmic proteins, the Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime) was employed to effectively fractionate cellular components from samples. Cells were harvested by rinsing with phosphate-buffered saline (PBS, RG-CE-10, KETU) to remove the extracellular culture medium, followed by detachment using a cell scraper. After centrifugation, the supernatant was carefully aspirated to collect the cell pellet, allowing cells to settle. To extract cytoplasmic protein, 200 µL of reagent A, supplemented with 1 mmol/L Phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime), was added per 20 µL of cell pellet. The mixture was incubated on ice for 15 minutes. Subsequently, 1 µL of reagent B was added to initiate cell lysis, followed by a brief vortex at maximum speed for 5 seconds, and further incubation on ice for 1 minute. After vortexing and centrifugation at 4 °C, 12 000 g for 5 minutes, the resulting supernatant containing cytoplasmic proteins was transferred to a precooled tube. For nuclear protein extraction, the residual supernatant was aspirated, and 50 µL of nuclear extraction reagent containing PMSF was added to the pellet. Intense vortexing for 30 seconds suspended the cells, followed by intermittent ice incubation and vortexing for 30 minutes to ensure efficient cell disruption and release of nuclear proteins. The nuclear proteins were pelleted by centrifugation at 4 °C, 12 000 g for 10 minutes, and the supernatant was collected in a precooled tube.

Co-Immunoprecipitation Assay

Following the experimental interventions, chondrocytes underwent analysis using an immunoprecipitation (Co-IP) kit (C600688, BBI Life Science Corporation, Shanghai). Protein A-Agarose was prepared by adding 18 µL per reaction to a spin column, removing the cap, and washing the agarose with 700 µL of PBS, followed by low-speed centrifugation, repeated four times in total. Cells were harvested by centrifugation at 800 r/min for 3 minutes at 4 °C. The supernatant was discarded, and the cell pellet was washed twice with pre-chilled PBS, each time spinning at 800 r/min for 3 minutes and ensuring complete drainage of PBS to retain the cell pellets. To 100 mg of cell pellet, 1 mL of 1X Lysis buffer was added, vortexed, and homogenized using a glass homogenizer for 30 strokes or sonicated for 30-second pulses interspersed with 1-minute intervals, repeated thrice. The lysate was then centrifuged at 12 000 r/min for 5 minutes to collect the supernatant containing cell lysate. In a new microcentrifuge tube, 0.7 mL of cell lysate, 7 µL of PMSF, and 1 µg of purified antibody were combined and incubated overnight at 4 °C on a flat platform. Subsequently, the mixture was added to the washed Protein A beads in the spin column and incubated overnight at 4 °C. Each column was placed into supplied 2 mL microcentrifuge tubes and centrifuged at 12 000 g for 30 seconds at 4 °C. The beads were washed six times with 700 µL of 1X IP buffer, followed by one wash with 0.1X IP buffer, each wash involving centrifugation at 12 000 g for 30 seconds. To the beads, 50 µL of 1X Loading buffer was added and gently mixed (avoiding vortexing). The spin column was securely capped, and the samples were heated at 95 °C for 5 minutes, with excess water around the cap blotted with tissue paper or filter paper. After opening the column cap, it was reinserted into a fresh microcentrifuge tube and centrifuged at 12 000 g for 30 seconds. The eluted immunoprecipitate was subsequently subjected to SDS-PAGE analysis, and the proteins were detected using Western blot techniques as previously described.

Mitochondrial membrane potential detection

Mitochondrial membrane potential was evaluated using fluorescence microscopy with a JC-1 staining kit (Beyotime). Chondrocytes were treated with a 5 μmol/L JC-1 staining solution for 20 minutes at 37 °C in the dark and observed using a Zeiss Axiovert 40CFL microscope. The level of mitochondrial membrane potential (ΔΨm) was quantified by measuring the ratio of red at 590 nm to green fluorescence at 525 nm intensities.

Mitochondrial superoxide detection

Chondrocytes were cultured on coverslips in culture media and subjected to the respective treatments. Mitochondrial superoxide production was assessed using MitoSOX Red (Thermo Fisher Scientific). Cells were washed with PBS, followed by incubation with 1 µmol/L MitoSOX Red for 30 minutes at 37 °C. Following incubation, the coverslips were mounted on glass slides. Fluorescence was visualized under a microscope, and images were analyzed using image analysis software.

NADH level measurement

The cellular NADH content was determined using a Coenzyme I NAD (H) content test kit (Jiancheng, Nanjing, China). Cells were collected and centrifuged to remove the supernatant. The volume of alkaline extraction solution used was adjusted according to cell density, typically 500–1 000 µL per 104 cells, with 1 mL recommended for 5 × 106 cells. Samples underwent ultrasonic disruption at 200 W or 20% power for 1 minute in an ice bath, pulsing 2 seconds on and 1 second off. Boiling with a secure lid for 5 minutes minimized evaporation. After cooling in an ice bath, debris was pelleted by centrifugation at 10 000 g for 10 minutes at 4 °C. The resulting supernatant was transferred to a fresh tube and neutralized by adding an equivalent volume of acidic solution. Additional centrifugation at 10 000 g for 10 minutes at 4 °C clarified the supernatant, which was stored on ice for subsequent analysis. Thorough mixing was crucial to dissolve any purple-black precipitate, with residual white sediment removed by centrifugation at 20 000 g for 5 minutes at room temperature. Absorbance was measured at 570 nm using a 1 cm path length cuvette, with distilled water as the reference blank. The spectrophotometer was preheated for at least 30 minutes before use. Reagents were prepared in appropriate working dilutions for the simultaneous analysis of multiple samples, protected from light to avoid photometric interference. NADH concentration was determined from absorbance values using a validated formula.

ATP level

Intracellular ATP levels were quantified using an ATP detection kit (Beyotime). Chondrocytes were lysed with ATP lysis buffer, followed by centrifugation to separate the supernatant. The supernatant was then mixed with the ATP detection reagent and the absorbance of the resulting mixture was measured using a Varioskan LUX multifunctional plate reader (Thermo Fisher Scientific). ATP concentrations were normalized relative to the total protein content.

Seahorse XF analysis

The Seahorse Extracellular Flux XFe24 Analyzer (Agilent, USA) was employed to investigate extracellular acidification rates (ECARs). Cells were seeded in hippocampal XF-24 plates at a density of 1 × 105 cells per well. Before measurement, cells underwent preconditioning under hypoxic conditions. ECARs were assessed by sequentially administering glucose, oligomycin, and 2-deoxyglucose to the cells, allowing for the evaluation of glycolysis and glycolytic capacity through subsequent calculations and analyses.

Measurements of mitochondrial complex activity

The mitochondrial complex I-V activity assay kit (Elabscience, China) was used to evaluate mitochondrial complex activity. Cells were collected and carefully mixed with the reagent, then sonicated on ice at 200 W (5 seconds on, 10 seconds off, for 15 cycles). After centrifugation at 10 000 g for 3 minutes at a low temperature, the supernatant was extracted for further analysis. The absorbance was measured using a multifunction plate reader (Thermo Fisher Scientific).

Genetic knockout model

The genetic knockout model involved acquiring global Sirt3 knockout (Sirt3−/−) mice from GemPharmatech Co., Ltd. (Nanjing, China) which were subsequently bred with global Pink1 knockout (Pink1−/−) mice (Nanjing, China) to generate Sirt3−/−Pink1−/− double-knockout (DKO) mice. Age-matched group consisted of wild-type (WT) siblings from the C57BL/6 J lineage. Genetic characterization of these mice utilized tail-derived DNA following protocols established by the Jackson Laboratory. The animals were housed under controlled conditions with consistent temperature with 12 h light/dark cycles and humidity and maintained on a standard diet.

Animal model

The animal model involved medial meniscal (DMM) surgery performed on seven-week-old male C57BL/6 J mice, conducted by protocols approved by the Soochow University Ethics Committee (SUDA20240307A07) and adhering to National Institutes of Health guidelines. Mice were obtained from the Soochow University Laboratory Animal Center and anesthetized with sodium pentobarbital before surgery. Following anesthesia, the knee joint area was prepared by shaving, disinfecting, and making an incision from the distal patellar to the proximal tibial region. The joint capsule was then opened, the patellar tendon retracted, and the medial meniscal ligament was exposed and transected. The Sham group underwent a similar incision procedure without transection of the medial meniscotibial ligament. Animals received penicillin to prevent infection and were housed with access to food and water in a designated area for mobility. One week post-surgery, mice, excluding those in the sham operation group (n = 5), were randomly assigned to four groups (n = 5) for respective interventions and group allocation. Joint samples were collected at 4, 8, and 12 weeks before euthanasia for the first group, whereas all remaining groups were assessed at the 8-week time point.

Human OA specimens

Tibial plateau samples were collected from patients with OA who were scheduled for total knee arthroplasty, following their provision of informed consent. Specimens from OA cartilage were classified as relatively healthy cartilage or severely damaged cartilage. Exclusion criteria included the presence of malignant tumors, diabetes mellitus, or other significant chronic conditions. Ethics approval was obtained from The First Affiliated Hospital of Soochow University (Approval No. 2022-520).

Intra-articular injection treatment

Intra-articular injection treatment involved inducing OA in mice through DMM surgery following established protocols. Subsequently, mice were categorized into different groups, consisting of six mice each group. Post-recovery from DMM surgery, saline, AAV-NC, AAV-Pink1, AAV-Sirt3, siNC, or siPink1 was injected into the joint cavity (3 days post-DMM, 1 × 1011 v.g./mL, 5 μL per joint). The animals were euthanized at week 8 for subsequent examination and analysis. The procedure for grouping other intra-articular cavity injections adheres to the same protocol as described.

Gait analysis

Gait analysis was conducted except for the initial group, which underwent experiments at 4, 8, or 12 weeks before euthanasia; all subsequent groups were assessed at 8 weeks. Mice were identified by marking their front paws in red and their rear paws in green, with recording paper positioned within a dark enclosure. Subsequently, mice were placed in the dark enclosure and allowed to move freely from one side to the other without external interference. Each mouse underwent three replicate experiments, and three consecutive footprints from the recording paper were selected for statistical analysis following the experiments.

μCT analysis

Following euthanasia, the knee joints of the mice underwent tissue fixation. Subsequently, high-resolution micro-computed tomography (μCT) was conducted using a Skyscan 1176 system from Kontich, Belgium, operating at a resolution of 9 μm with settings of 50 kV and 200 μA. Two-dimensional image reconstruction was conducted using NRecon v1.6 workstation and CTAn v1.13.8.1 software. A three-dimensional model was reconstructed using Mimics Research software. Regions of interest (ROI) were defined as 30 consecutive layers of subchondral bone on the medial side of the tibia. Statistical analysis of the reconstructed data evaluated parameters including bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm).

Transmission electron microscopy

Isolated mice chondrocytes were fixed for 2 h using Glutaraldehyde (G916053, Macklin, China) at room temperature. The samples were then embedded in epoxy resin and dehydrated using a series of ethanol concentrations. The sections were subsequently visualized using a transmission electron microscope (HT7700, Tokyo, Japan).

Histological assessment

Knee specimens were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 48 hours, and subsequently decalcified in 10% ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich). Following decalcification, specimens were embedded, sectioned into 6-μm-thick sagittal slices, deparaffinized in xylene, and gradually rehydrated through a series of ethanol solutions. Sections were stained with hematoxylin and eosin (H&E) as well as safranin O-fast green (S.O.). Assessment of cartilage degeneration utilized the Osteoarthritis Research Society International (OARSI) guidelines, and the calculation of the hyaline cartilage (HC) to calcified cartilage (CC) ratio.

Immunofluorescence and immunohistochemical staining

Cell cultures were seeded into 24-well plates and treated with chondrocytes as described in the intervention above. Subsequently, cells were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton™ X-100 (Sigma-Aldrich) for 15 min. Mouse knee joint slides and cell cultures were then deparaffinized and blocked with QuickBlock™ Blocking Buffer (Beyotime) for 30 minutes. Samples were incubated overnight at 4 °C with specific primary antibodies. After washing with PBS, cells were treated with Fluor488-conjugated or CY3-conjugated secondary Antibodies at room temperature for 1 h. Nuclei were counterstained with DAPI (Thermo Fisher Scientific) for 1 minute and resulting immunofluorescence images were visualized using a fluorescence microscope. Mitochondrial autophagy staining was performed using the Mitophagy Detection Kit (Dojindo Molecular Technologies, Japan) following the manufacturer’s instructions. Cells treated with Ad-GFP-LC3B (Beyotime) underwent co-staining for lysosomes using Lyso Tracker Red (1:20 000, Beyotime) after a 30-minute incubation at 37 °C. Subsequently, cells were fixed with 3% glutaraldehyde, as previously described. Mitochondria were co-stained with PINK1 using Mito Tracker Red (1:3 000, Beyotime). After blocking, sections were incubated overnight at 4 °C with respective primary antibodies, followed by procedures similar to those used for cell fluorescence. The Intensity and co-localization of immunofluorescence were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA) for further analysis. Immunohistochemical staining of slices was performed using M&R HRP/DAB Detection IHC Kit (Vazyme).

Colocalization analysis

Colocalization analysis of fluorescent signals in chondrocytes was performed using a plugin to compute Pearson’s Correlation Coefficient (PCC), which is well suited for assessing high-intensity signals within cell. Images were captured from multiple coverslips or well plates under each experimental condition, with each chondrocyte imaged once. PCC ranges from +1 indicating perfect positive correlation to −1 indicating perfect negative correlation, providing a quantitative measure of the linear relationship between signal intensities in two channels. Due to its robustness against background noise, PCC serves as an effective tool for colocalization analysis.

Mitochondrial length analysis

Mitochondrial length analysis commenced following the steps of immunofluorescence. Cells were incubated overnight with an anti-TOMM20 antibody, washed with PBS, and incubated with a CY3-conjugated secondary antibody for 1 hour. Following this, cells were co-stained with DAPI for 1 minute. Microscopic observations were conducted and images were captured accordingly. At least 10 cells were randomly selected from each field of view to measure mitochondrial length, with the mean length calculated subsequently. The quantitative analysis involved determining the mean mitochondrial lengths obtained from 5 randomly selected fields of view. Mitochondrial branch length analysis utilized Fiji software through binary and skeletonization processes.

High intelligent and sensitive structured illumination microscopy (HIS-SIM)

Super-resolution imaging of cartilage cells under specific interventions was performed using the HIS-SIM system (CSR Biotech, Guangzhou, China). Following standard procedures, GFP was excited at 488 nm with a 525/20 nm filter, and mCherry at 561 nm with a 610/20 nm filter. Images were captured with a 100x/1.5 NA oil immersion objective from Olympus. Analysis of the 3D-SIM data was conducted using Imaris software, after processing with the HIS-SIM technology, which included Wiener deconvolution of the reconstructed images.

Phosphoproteomics sequencing and bioinformatic analysis

Phosphoproteomics sequencing and bioinformatic analysis involved several systematic steps. Initially, proteins underwent enzymatic digestion to produce peptides. Phosphopeptides were selectively enriched from this peptide mixture using techniques such as metal affinity chromatography, immobilized metal affinity chromatography (IMAC), or titanium dioxide (TiO2) chromatography. This enrichment step enhances the concentration of phosphopeptides while reducing sample complexity. Next, the enriched phosphopeptides were separated using liquid chromatography (LC) and subsequently introduced into a mass spectrometer (MS) for analysis. Tandem mass spectrometry (MS/MS) was employed to fragment the peptides, generating a spectrum of fragment ions. These MS/MS spectra were then compared against protein sequence databases using specialized software tools to identify phosphorylated peptides. The sequencing service was facilitated by Wekemo Tech Group Co., Ltd., ensuring rigorous analytical procedures. Identified differentially expressed proteins underwent further analysis through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assessments using the online platform “OMICSHARE” (www.omicshare.com).

Statistical analysis

Statistical analysis was conducted using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and results are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was employed for multiple comparisons while two-tailed non-paired Student’s t-tests were used for specific pairwise comparisons. Statistical significance was set at P < 0.05.