2.1 Ethics approval
The study protocol was approved by the Institutional Review Board of Xiangya Hospital Central South University, and each participant or participant’s authorized principal were informed about the objectives and agreed to the study.
2.2 Cultures of chondrocytes
Samples of osteoarthritic cartilage were isolated from 10 knee OA patients who underwent total knee replacement, and the knee OA diagnosis was performed according to the guidelines for the diagnosis and treatment of osteoarthritis in China (2019 edition)[24]. Normal samples were obtained from the knees of 3 age-matched traumatic amputation patients, without any history of OA or other joint diseases, including rheumatoid arthritis and septic arthritis. Normal cartilage and OA cartilage were confirmed using the Modified Outerbridge Classification[25, 26]. The cartilage was first washed with phosphate-buffered saline (PBS) twice, and then the cartilage tissue was chopped into 1-5 mm3 slices by a scalpel. The small cartilage pieces were added to a test tube containing 5-8 ml 0.2% collagenase II (Sigma-Aldrich, St. Louis, MO, USA), and the tube was then placed in a constant temperature shaker for digestion for 12-16 h at 37 ℃. The progress of digestion was terminated by the addition of 8-10 ml of Dulbecco's modified Eagle's medium/F12 (DMEM/F12; HyClone, Logan, UT, USA). Then, the released cell pellet was aspirated from the bottom of the test tube, centrifuged at 1000 rpm for 5 min, and transferred to a culture flask. Each culture well was contained with 5 ml DMEM/F12 with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (Gibco) and was incubated for 24 h at 37 ℃ with 5% CO2. Subsequently, the DMEM/F12 was changed every 3 days before trypsinization to the next experiment, and the nonadherent cells were washed out with PBS when the growth medium was changed.
2.3 Cell treatment
Chondrocytes were plated in 6-well culture plates at 5×105/well and serum-starved for 24 h in DMEM/F12 containing 1% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) for 24 h. First, to determine whether OPN had an effect on autophagy, the human OPN (rhOPN) group was treated with 100 ng/ml rhOPN (1433-OP; R&D Systems, Minneapolis, MN, USA) for 48 h, and the control group was unstimulated and untreated. To induce autophagy activity, the cells were treated with rapamycin (10 μmol/L; Cell Signaling Technology, Boston, MA, USA). Next , blocking experiments were performed to determine the possible role of CD44 and αvβ3 integrin. Chondrocytes were treated with mouse anti-CD44 monoclonal antibody (20 µg/ml; BD Biosciences, USA) or isotype control IgG2 (20 µg/ml BD Biosciences, USA) and anti-CD51/61 monoclonal antibody (20 µg/ml; BD Biosciences, USA) to block the binding of OPN-αvβ3 integrin or isotype control IgG1 (20 µg/ml BD Biosciences, USA) 1 h prior to the rhOPN treatment, which was performed for 48h.
Subsequently, to investigate the role of MAPK in the process, special inhibitors of ERK MAPK (FR180204 10 µmol/L , Beyotime,China) were used 1 h prior to rhOPN treatment for 48 h according to previous experimental results.
2.4 mRFP-GFP-LC3 lentivirus transfer and laser confocal imaging
Chondrocytes were seeded into glass bottom cell culture dishes (nest, No. 801002) at 2×105/ dish. The next day, the mRFP-GFP-LC3 lentivirus (HanBio, shanghai, China) was transferred at a multiplicity of infection (MOI) of 100. After 8 h, the virus-containing medium was changed to DMEM/F12 containing 10% fetal bovine serum, and the cells were treated as previously described. After 48 h, the mRFP-GFP-LC3 puncta were visualized using a Leica laser confocal microscope. mRFP was used to label and track LC3. The weakening of GFP can indicate the fusion of lysosomes and autophagosomes to form autophagolysosomes because GFP fluorescent protein was sensitive to acid. When autophagosomes and lysosomes were fused, GFP fluorescence was quenched, and only red fluorescence could be detected at this time. Under microscope imaging, the strength of the autophagy flux can be clearly observed by counting the different color spots. The red spots (RFP) represented autophagolysosomes, and the yellow spots (merge) represented autophagosomes (RFP+GFP).
2.5 Western blot
The proteins extracted from cartilage tissue and cultured chondrocytes were tested. The protein extracted from cartilage tissue in liquid nitrogen immediately after the cartilage tissue was washed and ground into small sections. For the western blot analysis of cultured chondrocytes, 100μl/well SDS containing protease inhibitor (100:1) was used to lyse chondrocytes, and a BCA protein assay kit (Thermo Fisher Scientific, Boston, MA, USA) was used to measure the protein concentration of the lysate. Forty micrograms of protein was used for subsequent experiments, such as electrophoresis. Twelve percent SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Genescript, Piscataway, USA) was performed to separate the proteins at 80 v for 30 min and then 120 v for 60 min, and they were then transferred to polyvinylidenedifluoride (PVDF) membranes with 300mA ,75min. The PVDF membrane was sealed in skimmed milk powder for 1 h and incubated overnight at 4 ℃ with beclin1 (1:1000, Cell Signaling Technology, Boston, MA, USA), LC3 (1:1000, Cell Signaling Technology, Boston, MA, USA), p38(1:1000,Abcam, Cambridge, UK) , p-p38(1:1000,Abcam, Cambridge, UK), extracellular signal-regulated kinase (ERK) (1:1000,Abcam, Cambridge, UK), p-ERK(1:1000,Abcam, Cambridge, UK), Jun Nterminal kinase (JNK) (1:1000,Abcam, Cambridge, UK), p-JNK(1:1000,Abcam, Cambridge, UK), and primary antibody dilution buffer and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2000) for 1 h at room temperature. β-actin and GAPDH were used as housekeeping genes. After that, enhanced chemiluminescence (ECL) (NCM Biotech,China) was performed to develop the membranes , Bio-Rad ChemiDoc-XRS with Image Lab software (BioRad, Richmond, CA, USA) was used to expose and qualify the membranes, and western blot experiments were repeated 3 times.
2.6 Cell proliferation assay
Chondrocytes were seeded at 104/well in a 96-well plate, and five wells were measured for each same treatment. After 48 h of treatment, a Cell Counting Kit-8 (CCK-8) assay (Beyotime,China) was used to determine cell proliferation. Then, 90 μl DMEM/F12 culture medium whit 10 μl CCK-8 solution was added to each well and incubated for an additional 2 h. Absorbance was detected at a wavelength of 450 nm by a microplate reader (BioRad, Richmond, CA, USA). For each intervention, only the data from middle three wells were taken for analysis.
2.7 Statistical analysis
All statistical calculations were performed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data are expressed as the mean ± standard error of the mean. The statistical analysis of the differences between two experimental groups was performed using Student's t test. Differences among groups were determined by the one-way ANOVA; to compare the difference of each group, the Bonferroni test was used. A P value less than 0.05 was considered as statistically significant.