Human cartilage and chondrocyte culture

The normal human articular cartilages were obtained from the femoral heads of 27 patients with femoral neck fractures undergoing total hip replacement or artificial femoral head replacement surgery, but with no significant clinical and imaging features of OA (18 women, 9 men; 53–88 years old; mean 73.4 years; Kellgren-Lawrence grade, 0). The OA human articular cartilages were obtained from 30 patients undergoing total knee arthroplasty (22 women, 8 men; 49–81 years old; mean 66.2 years; Kellgren-Lawrence grade, III or IV). The cartilages were cut into approximately 1 mm3 pieces, and then they were digested by 0.25% trypsin (Beyotime, Shanghai, China) for 30 min. Next, the samples were incubated with 0.2% collagenase II (Sigma, St. Louis, MO, USA) at 37 °C overnight. The obtained chondrocytes were cultured in the Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Biosharp, Hefei, China) containing 15% fetal bovine serum (FBS; Tianhang, Huzhou, China) in 5% CO2 at 37℃. Finally, the chondrocytes at first passage were selected for the further experiments. Informed consents were obtained from the donors. Our study was conducted based on Declaration of Helsinki. Ethical approval was obtained from Clinical Medical Research Ethics Committee of the First Affiliated Hospital of Anhui Medical University (PJ2019-06-06).

Cell adenovirus infection

Recombinant adenovirus vectors expressing CHMP5 (Ad-CHMP5) were constructed by General Biosystems (Chuzhou, China). Briefly, DNA fragments encoding CHMP5 (NM_016410) were subcloned into the pShuttle-CMV vector (Fenghui, Changsha, China), and recombined with pAdEasy-1 in BJ5183-AD-1 cells (Huayueyang, Beijing, China). The linearized vectors carrying CHMP5 encoding fragments were then transfected into HEK-293A cells (iCell, Shanghai, China) via Lipofectamine 3000 (Invitrogen, USA) to produce adenoviral particles. The empty adenoviral vector (Ad-NC) was used as a negative control. Adenoviruses containing short hairpin RNA (shRNA) targeting CHMP5 (Ad-shCHMP5) or none-targeting shRNA (Ad-shNC) were also generated by General Biosystems (Chuzhou, China). The sequences were as follows: Ad-shCHMP5: 5’-GGATGAAGATGATTTAGAAGC-3’; Ad-shNC: 5’-TTCTCCGAACGTGTCACGT-3’. The titers reached 1.6 × 109 pfu/mL for Ad-CHMP5, 2.8 × 109 pfu/mL for Ad-NC, 2 × 109 pfu/mL for Ad-shCHMP5 and 2.2 × 109 pfu/mL for Ad-shNC, respectively. Chondrocytes were infected with adenoviruses at 100 multiplicity of infection (MOI).

To mimic inflammatory stimulation, chondrocytes were exposed to IL-1β (10 ng/mL; Sinobiological, Beijing, China) for 6 h, 12 h, 24–48 h. To inhibit protein degradation mediated by ubiquitin-proteasome system, chondrocytes were treated with MG132 (10 µM; Aladdin, Shanghai, China) for 4 h.

Experimental mice

OA was induced in the male C57BL/6 mice (10–12 weeks) using destabilization of the medial meniscus (DMM) (Glasson et al. 2007). The mice were anesthetized by isoflurane inhalation (3% for induction and 1.5% for maintenance). The right knee joints of the mice were exposed by incision from the medial side of the patella, and the medial meniscotibial ligaments were transected by micro-surgical scissors. In sham surgery, the ligaments were visualized without further damage. Finally, the incisions were sutured. All mice were euthanized at 4 and 8 weeks after the surgery, and all knee joints were collected to detect CHMP5 expression. For the effect of CHMP5 on OA mice, starting at 10 days post-operation, Ad-CHMP5 was injected into the mouse articular cartilages once a week for the next three weeks. The Ad-NC injection was used as a control. The mice were sacrificed 8 weeks after the operation. The knee joints were isolated and gathered for further histological analysis. Meloxicam (5 mg/kg) was administered subcutaneously about 1 h before the DMM operation and once a day for 3 days after surgery. Ethical approval was obtained from the Experimental Animal Ethics Committee of Anhui Medical University.

Proteomic analysis

The articular cartilages of 3 OA patients (Kellgren-Lawrence grade, III or IV) and 3 normal cartilages (Kellgren-Lawrence grade, 0) were grinded with liquid nitrogen, and then weighed 150 mg. The tissues were lysed using SDT lysis buffer. The supernatant was collected after centrifuging (20,000 g, 4℃). The samples were digested in trypsin and marked using TMT 6plex kit (Thermo Fisher Scientific, Shanghai, China). Then, labeled peptides were fractionated by the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Shanghai, China) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The Easy nLC 1200 chromatographic system was applied to separate the peptides. Data-dependent acquisition (DDA) analysis was performed by a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific, Shanghai, China). Proteome Discoverer 2.4 software was used to retrieve the data and quantify the protein. The false discovery rate (FDR) was set to 1% for protein and peptide spectrum matches.|log2 fold change| > 0.8 and p < 0.05 were considered as the criterions for screening differential proteins.

Histological analysis

The mouse cartilage tissues were decalcified in 10% disodium ethylene diamine tetraacetic acid (Biosharp, Hefei, China), embedded in paraffin and cut into 5-µm-thick sections. All tissues were stained with safranin O-fast green. The articular cartilage destruction was evaluated using the Osteoarthritis Research Society International (OARSI) scoring system by estimating the highest observed scores based on a previous study (Glasson et al. 2010). For immunohistochemistry (IHC), the sections embedded in paraffin were dewaxed, hydrated, and inoculated with 3% hydrogen peroxide. Thereafter, the sections were blocked with 1% bovine serum albumin (Sangon, Shanghai, China) for 15 min at room temperature and incubated with the primary antibodies against CHMP5 (1:50), collagen II (1:50) and matrix metallopeptidase 13 (MMP13; 1:50) purchased from Affinity Biosciences (Changzhou, China) at 4 °C overnight. Then, the slides were inoculated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:500; Thermo Fisher Scientific, Shanghai, China) at 37 °C for 1 h. Next, the 3, 3-diaminobenzidine tetrahydrochloride (Maixin, Fuzhou, China) was used as a chromogenic agent. The images were captured with a microscope (Olympus, Japan).

Real time PCR

Total RNA was extracted from the mouse articular cartilages and the human articular chondrocytes using the TRIpure reagent (BioTeke, Beijing, China). The corresponding cDNA was obtained using BeyoRT II M-MLV Reverse Transcriptase (Beyotime, Shanghai, China). The primer sequences are as follows: mouse-CHMP5 forward primer: 5’-CATTGGGACGGTGGATA-3’, mouse-CHMP5 reverse primer: 5’-GGTTGTCTCGCTGTTGC-3’; human-CHMP5 forward primer: 5’-GGCACGGTGGACAGTAG-3’, human-CHMP5 reverse primer: 5’-ACTCGCAAGGCTTTCTG-3’. Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) were used as the endogenous controls. The relative expression levels were calculated by the 2−ΔΔCt method.

Cell counting kit 8 (CCK-8)

The chondrocytes were seeded in 96-well plates (4 × 103 cells per well) and cultured overnight. After that, IL-1β (10 ng/mL) was applied to the transfected cells for 24 h in 5% CO2 at 37℃. CCK8 reagent (10 µL; Solarbio, Beijing, China) was added into each well. Subsequently, the samples were incubated for 2 h. The OD values were determined at 450 nm by a microplate reader (BioTek, USA).

Flow cytometric

Annexin V-FITC and phycoerythrin (PE) were used to stain the chondrocytes referring to the cell apoptosis detection kit (KeyGene, Nanjing, China). The cells were analyzed through a NovoCyte Flow Cytometer (Aceabio, USA).

Immunofluorescence (IF) analysis

The chondrocytes were fixed with 4% paraformaldehyde (Sinopharm, Shanghai, China) for 15 min, permeabilized with 0.1% Triton X-100 (Beyotime, Shanghai, China) for 30 min, and blocked by 1% bovine serum albumin (Sangon, Shanghai, China) for 15 min. The samples were incubated with collagen II (1:100; Affinity, Changzhou, China) at 4 °C overnight, followed by incubation with Cy3-conjugated goat anti-rabbit IgG (1:200; Invitrogen, USA) for 1 h. Images were taken by a fluorescence microscope (Olympus, Japan).

Co-immunoprecipitation (Co-IP)

The chondrocytes were lysed with lysis buffer (Beyotime, Shanghai, China) on ice for 5 min. The cell lysates were centrifuged at 10,000 g for 5 min. The collected supernatants were incubated with rabbit anti-CHMP5 (1:1000, Affinity, Changzhou, China), anti-NF-κB inhibitor alpha (IκBα1; 1:1000, ABclonal, Wuhan, China), anti-ubiquitin specific peptidase 15 (USP15; 1:500, ABclonal, Wuhan, China) or anti-Ubi (1:1000, ABclonal, Wuhan, China) antibodies at 4 °C overnight, after which they were incubated with protein A agarose beads. Subsequently, western blot was performed as follows.

Western blot

Total articular cartilages and chondrocytes were lysed in radioimmunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). For the examination of NF-κB p65, the cytoplasm and nucleus were separated with cytoplasmic and nuclear extraction reagents according to the Nuclear Protein Extraction Kit (Solarbio, Beijing, China). The proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) afterwards. RIPA, PMSF and SDS-PAGE were purchased from Solarbio Science & Technology (Beijing, China). The membranes were blocked by 5% (M/V) skimmed milk and then incubated with the anti-rabbit primary antibody CHMP5 (1:1000), collagen-II (1:500), cleaved poly (ADP-ribose) polymerases (PARP; 1:500), cleaved caspase-3 (1:1000), aggrecan (1:400) from Affinity Biosciences (Changzhou, China), SOX9 (1:1000), IκBα (1:1000), p-p65 (1:1000), p65 (1:500), MMP13 (1:1000), matrix metallopeptidase 3 (MMP3; 1:2000), ADAMTS5 (1:500) from ABclonal (Wuhan, China), Histone H3 (1:5000) from Gene Tex (USA) as well as anti-mouse control GAPDH (1:10000) from proteintech (Wuhan, China) overnight at 4 °C. The secondary antibody HRP-conjugated goat anti-rabbit IgG (1:3000) and anti-mouse IgG (1:3000) from Solarbio Science & Technology (Beijing, China) were incubated with the membranes at 37 °C for 1 h. The results were imaged using the enhanced chemiluminescence luminous (ECL; Solarbio, Beijing, China).

Statistical analysis

Statistical analysis was performed with GraphPad Prism software, and the data were expressed as mean ± SD. Differences between two groups were evaluated by unpaired t test. Differences among multiple groups were assessed using one-way ANOVA with Tukey’s test. The non-parametric Mann-Whitney test was used to compare the OARSI scores and IHC staining scores between two groups. P < 0.05 was regarded as statistical significance.