Human samples collection
Human OA cartilage samples were collected from OA patients who were undergoing knee arthroplasty and healthy non-OA cartilage was excised from the knee joints of donors of trauma patients. The collection of the specimen was approved by the Ethics Committee Board of Fuzhou Second General Hospital (Approval No. 2022140). Written informed consent was obtained from all subjects before knee surgery. The cartilage samples were used for further evaluations using immunohistochemistry and histology.
Animals
Animal experiments were conducted on male C57BL/6J provided by Beijing Vital River Laboratory Animal Technology Company. All mice were housed in pathogen-free barrier facilities at 5 or less per cage and access to food and water. They were maintained at room temperature, with humidity ranging from 30–60%, and a 12-hour light/dark cycle. The mice were allocated to different experimental groups randomly. Animal welfare monitoring and euthanasia practices were implemented throughout the animal research. All animal experiments were approved by the Ethics Committee Board of Fujian Medical University (Approval No. IACUC FJMU 2023 − 0347) and followed the National Research Council and ARRIVE guidelines.
OA mice model construction
For the OA experiment model, the mice were placed under anesthesia using sodium pentobarbital (100 mg/kg) administered intraperitoneally by injection. Then the mice were traversed by the medial collateral ligament and destabilized by the medial meniscus (DMM) in the right knee. The mice were injected with replication-deficient lentivirus (Lenti) alone, Lenti-FBXO7, or saline via intra-articular administration 3 days, 7 days, 14 days, and 21 days after the DMM surgery. Eight weeks after the operation, the mice were sacrificed by the cervical dislocation method, and the knee joints were harvested.
Cell culture and transfection
Femoral articular cartilage was harvested from OA patients and non-OA donors. After conducting the OA model, on the 8th week, the knees of the mice were microdissected by surgically dissecting the OA cartilage under a surgical microscope. The tissue was digested with 0.2% type II collagenase for 40 minutes at 37°C. The isolated cells were washed with D-Hanks solution and suspended in DMEM/F12 (Gibco, USA) containing 10% FBS, 100U/mL penicillin (Gibco, USA), and 100mg/mL streptomycin (Gibco, USA). The chondrocytes were cultured in a high-humidity incubator (37°C; 5% CO2). The media were changed every 2 days until the chondrocytes had grown into sheets and were over 85% confluent(17). After 2 to 3 passages, the chondrocytes were used for future investigations. The FBXO7 overexpression was achieved through pcDNA3.1-FBXO7 transfection, with an empty pcDNA3.1 vector used as a control (Invitrogen, Life Technologies, Carlsbad, CA, USA). Negative control siRNA to knockdown FBXO7 (FBOX7 siRNA) (Sigma-Aldrich, St. Louis, MO, USA) was transfected into chondrocytes at a concentration of 50 nM Lipofectamine® 3000 Transfection Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Senescence-associated β-galactosidase (SA-β-Gal) assay
Using an SA-β-Gal staining kit (#9860, Cell Signaling Technology)as described(18), SA-β-Gal staining was performed. Human chondrocytes were placed into 6-well plates at a density of 1.0×105 cells per well and cultured for about 48 hours at 37°C in an incubator. After culturing the cells for 48 hours, pcDNA3.1-FBXO, pcDNA3.1, FBXO7siRNA, and control siRNA were transfected by Lipofectamine® 3000 Transfection Reagent. Thereafter, cytochemical staining for SA-β-Gal was performed at pH6, and the positive cells were counted.
Edu Assay
Human OA chondrocytes transfected pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA were used to perform Edu assay to analyze the relationship between FBXO7 expression and OA progression. EdU staining was performed according to the manufacturer’s instructions using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime Biotechnology, cat. no. C0075S). Briefly, the cells were incubated for 2 hours at 37°C after EdU was added to the culture media at a final concentration of 10 µM. After labeling, the cells were fixed for 15 minutes with 4% PFA and permeabilized for 10 minutes with 0.2% Triton X-100. The cells were then stained in the dark for 30 minutes with click reaction solution before being counterstained with Hoechst 33342. Under fluorescence microscopy (Olympus, Tokyo, Japan), EdU-positive cells were counted and the percentage of EdU-positive cells was reported.
Flow cytometry Assay
Cells were treated with trypsin and 200 µL Annexin V-FITC, then incubated for 10 minutes in the dark. Cells were rinsed with 200 µL PBS and 10 µL PI added. Cell apoptosis was identified by flow cytometry (Beckman Coulter).
Cell immunofluorescence
Chondrocytes from human donors and mice (after OA completion) were cultured on glass coverslips in 24-well plates at the density of 1×104 cells per well and transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. After 24-hour transfection, the cells on the coverslips were washed twice with PBS, cells were treated with 10ng/mL PA in the absence and presence of Xn for 24 hours for collagen II and MMP13 stain. The glass-seeded chondrocyte monolayers were fixed with 4% paraformaldehyde for 15 minutes at room temperature after they were washed with PBS three times. Then the cells were permeabilized for 10 minutes at room temperature with 0.3% Triton X-100 (in PBS). Then the cells were blocked using 5% BSA for 30 minutes. The chondrocytes were incubated with collagen II and MMP13 primary antibodies overnight at 4°C. The cells were then rinsed three times with PBS before being treated with Alexa Fluor® 647-conjugated goat anti-rabbit IgG (1:500) for 1 hour in the dark at room temperature. Finally, the cells were stained with DAPI to reveal their nuclei. Images were acquired using a fluorescence microscope.
Western blotting analysis
To determine certain protein expressions in transfected human chondrocytes and human SW-1353 chondrosarcoma cells stimulated with IL-1β, human chondrocytes were first transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA. The cells were then lysed with RIPA buffer to extract the whole proteins. The total protein was quantified with a bicinchoninic acid (BCA) protein assay. After being separated and transferred onto polyvinylidenefluoride (PVDF) membranes, the membranes were blocked with 5% skim milk/BSA and incubated with primary antibodies against MMP13 (Cat# Ab39012; Abcam; 1:1000), COL2a1 (Cat# sc52655; Santa Cruz Biotechnology; 1:1000), anti-Fbxo7 antibody (Santa Cruz, sc-271763; 1:1000), JAK1 (Cat# 3332; Cell Signaling Technology; 1:1000), STAT3 (Cat# 9131, Cell Signaling Technology; 1:1000), anti-pSTAT3 (Cat# 9145; Cell Signaling Technology), and anti-IL-6 (Cat# 21865-1-AP; Rosemont, IL, Proteintech).
Histology and immunohistochemistry analyses of OA
Human OA-affected cartilage (OA) and healthy non-OA-affected cartilage (NC), and DMM-induced OA cartilage from mice (transfected with Lenti-FBXO7, Lenti-control, and saline) were fixed with 4% neutral-buffered paraformaldehyde (PFA; Solarbo, Beijing, China), embedded in paraffin, and sectioned into 6 µm thick slices. The cartilage sections were stained with Hematoxylin and eosin, Safranin O using the Safranin O kit (ICH World, Woodstock, NY, USA), and 0.05% Fast green FCF (f7258; Sigma-Aldrich).
For immunohistochemical staining, the paraffin-embedded human articular cartilage sections from OA and NC patients were incubated with 3% H2O2 for 10 minutes to block the endogenous peroxidase activity. This was followed by incubation with 10% goat serum for 1 hour at 21°C. Then the sections were incubated with primary antibodies against FBXO7 (ARP43128_P050; AVIVA, San Diego, USA). Next, the sections were incubated with horseradish peroxidase-linked secondary antibody (1:1, Immunologic, Duiven, The Netherlands) for 1 hour at room temperature. The integrated optical density (IOD) value of positive staining was evaluated using ImageJ software (National Institutes of Health, MD, USA).
RNA-sequencing analysis
The pathway enrichment (KEGG) and GO analyses, including biological process, from different gene lists were carried out by using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/). For GO classification and KEGG, the cutoff conditions were set as FDR = 0.05. The GO graphs of all enriched GO terms and volcano plots were drawn by ggplot2 package in R environment. Heatmaps were generated in R package ‘pheatmap’.
Mass spectrometry analysis
Chondrocytes were transfected with pcDNA3.1-FBXO7, pcDNA3.1, FBXO7siRNA, and control siRNA, then 48 hours later treated with IL-6, and the nuclear fraction was prepared by using a kit (Beyotime, P0028). The nuclear protein was immunoprecipitated with an anti-acetylation antibody and Protein A/G PLUS-agarose (sc-2003, Santa Cruz Biotechnology) overnight at 4°C. immunoprecipitated protein was resolved by 10% SDS-PAGE gels for Coomassie blue staining. The purified protein bands were cut out and digested with trypsin. LC-MS/MS analysis was performed on an EASY-nLC 1000 HPLC system (Thermo Scientific), which was directly interfaced with a Q Exactive mass spectrometer (Thermo Scientific). The analytical column was an AcclaimR PepMap RSLC column (50 µm ID, 15 cm length, C18, 2µm, 100 Å) (Thermo Scientific). The Q Extractive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.2 SPI software and there was a single full-scan mass spectrum in the orbitrap (300–2000 m/z, 70,000 resolution) followed by 20 data-dependent MS/MS scans at 27% normalized collision energy (HCD). The MS/MS spectra from each LC-MS/MS rub were searched against the fasta files using Sequnet HT and phosphoRS 3.0 modules in the proteome Discoverer Software (Version PDL4, Thermo Scientific, USA).
Co-immunoprecipitation
For the co-immunoprecipitation assay, chondrocytes were transfected with Flag-FBXO7 alone, HA-IL-6 alone, or Flag-FBXO7 and HA-IL-6. The cells were then lysed with a cold RIPA lysis buffer. Lysates were centrifuged at 12,000g for 10 minutes, and the supernatants containing proteins were then incubated with 1 µg anti-FBXO7 and anti-HA-IL-6 antibodies at night at 4°C. Protein lysates were subsequently incubated with 20 µL pre-washed protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology, USA) with gentle rotation for 3 hours at 4°C. The immunoprecipitates were analyzed by western blotting as described above using primary antibodies against FBXO7 to detect IL-6 protein or primary antibodies against IL-6 to detect FBXO7 protein.
Ubiquitination Assay
Chondrocytes were transfected with Flag-FBXO7, IL-1β, HA-FBXO7, Myc-k63-ub, and Flag-IL-6 K55R/K82R/K114R/K178R. Following 36 hours of transfection, the cells were treated with 20 µM MG132 for 8 hours and then lysed in RIPA (Low) Lysis Buffer (Meilunbio, China, Cat No: MA0153). For immunoprecipitation, the cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma, USA) or anti-IL-6 Protein A/G immunoprecipitated magnetic beads (Bimake, China, Cat No: B23201) for 4 hours at 4°C. The bound beads were then washed four times with BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. The protein was eluted with FLAG peptide for 4 hours at 4°C. The ubiquitinated form of IL-6 was detected by western blotting with an anti-HA antibody.
Statistical analysis
In this investigation, one-way ANOVA and independent t-tests were employed when the data fit the normal distribution. GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) were utilized for statistical analysis. The data were reported as a mean ± standard deviation (SD). P < 0.05 was determined to be statistically significant.