Patients and samples
Human synovium was obtained from OA patients (n = 4) who underwent artificial total knee arthroplasty at the Second Affiliated Hospital of Shandong First Medical University. Patients had advanced disease and were diagnosed with primary OA, excluding trauma and other structural causes of secondary OA. Control samples from healthy individuals (n = 3) were obtained from patients at the time of knee arthroscopic evaluation. These patients were diagnosed as a result of knee injury caused by trauma to the knee or sport injury, excluding rheumatic diseases and inflammation. This study was approved by the Clinical Research Ethics Committees of the Second Affiliated Hospital of Shandong First Medical University. All patients participating in this study signed informed consent forms.
FLSs were obtained via enzyme digestion and tissue cultivation. In short, after rinsing with phosphate-buffered saline (PBS), synovial tissues were sliced into 1 mm3 sections using sterile ophthalmic scissors and put into 15 ml centrifuge tubes. Next, 1 mg/ml collagen II enzyme (Solarbio, China) was added to the tube, which was four times larger than the tissues. After thorough mixing, the samples were incubated with 5% CO2 in air at 37 °C for 2 h. Then, the tissue digestion fluid was filtered with stainless steel meshes (200) and centrifuged at a rate of 1500 r/min for 5 min. After discarding the supernatant, the samples were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 12% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37 °C, 5% CO2 incubator. The undigested tissue pieces were continuously cultured. All experiments were performed between passage 3 and 5 of cell culture.
Cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After overnight incubation, OA-FLSs were treated with different concentrations (0, 20, 50, 100 and 200 nM) of RvD1 (Cayman Chemical Company, USA) for 48 h. Subsequently, 20 μL MTT solution (0.5 mg/mL) was added to each well, and the plates were incubated for 4 h in a 5% CO2 atmosphere at 37 °C. After a brief centrifugation, the culture medium was removed, and 150 μL (Dimethyl sulfoxide) DMSO was added to each well. The absorbance at 490 nm was measured with a spectrophotometer.
Western blot analysis
Tissue and Cultured cells were collected and lysed in RIPA buffer (Servicebio, China) supplemented with 100 x PMSF (Servicebio, China) and 100 x phosphatase inhibitor (Servicebio, China). The protein concentrations were determined with a BCA protein assay kit (Solarbio, China), and adjust the final protein concentration to 2 μg/μL. The proteins were separated by SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with TBST containing 5% bovine serum albumin for 1 h and then incubated with the antibody overnight at 4 °C. The antibodies used for this assay were cyclin D1 (Santa Cruz, USA, 1:1000), cyclin B1 (Abcam, UK, 1:1000), PCNA (Santa Cruz, USA, 1:1000), p53 (Santa Cruz, USA, 1:1000), MMP-13 (Santa Cruz, USA, 1:1000), YAP (CST, USA, 1:1000), p-YAP (CST, USA, 1:1000), and LATS1 (CST, USA, 1:1000). After washing with TBST, the blots were incubated with HRP-labeled secondary antibodies (Servicebio, China, 1:5000) for 2 h at room temperature. The blots were visualized by using an enhanced chemiluminescence reagent kit.
FLSs were washed in PBS, fixed in 4% paraformaldehyde and treated in 0.5% Triton X-100 for 10 min. After rinsing, the cells were blocked with 5% bovine albumin for 1 h at ambient temperature, rinsed with PBS and incubated with primary antibody for YAP (1:100), diluted in 3% BSA overnight at 4 °C. After washing and incubation with secondary antibodies for 1 h at ambient temperature, the cells were labeled with DAPI for 5 min. A fluorescence microscope (Nikon) was used for observation.
The IL-1β in FLS culture filtrate cytokines were detected using specific ELISA kits (Beyotime Biotechnology, China), according to the manufacturer’s instructions.
Cells were collected and washed twice with ice-cold PBS and then fixed with 75% ethanol at 4 °C for 2 h. After washing 2 times with ice-cold PBS, cells were incubated with 100 μl RNase A for 30 min at 37 °C. Then, (Propidium Iodide) PI (Solarbio, China) was added to each of the tubes to achieve a final concentration of 50 μg/mL, and the tubes were incubated in the dark at 4 °C for 30 min. The samples were then analyzed by flow cytometry.
EdU labeling and analysis
The 5-ethynyl-2-deoxyuridine (EdU) incorporation assay was used to detect the proliferation of FLSs. FLSs were seeded in 96-well plates at a density of 5 × 103 cells/well. After night, RA-FLSs were treated with RvD1 for 48 h. EdU was added to each well and cultured for another 3 h. The KFluor488-EdU cell proliferation detection kit (Beyotime Biotechnology, China) was used to assess proliferation following the manufacturer’s instructions.
The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed by Student’s t-tset for two groups or one-way analysis of variance (ANOVA) or two-way ANOVA with repeated measures. All experiments were performed at least three independent times. NS, not significant, *P < 0.05; ** P < 0.01. GraphPad Prism 8.0 were used to analyze the data.