Hfls ra

HFLS and RA‐infected HFLS (RA‐HFLS) were obtained from Cell Applications, Inc. All cells were incubated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37°C. The pcDNA‐BZRAP1‐AS1 vectors, pcDNA‐COL5A2 vectors, BZRAP1‐AS1 siRNA (si‐BZRAP1‐AS1), miR‐1286 mimic/inhibitor, and their corresponding negative controls were delivered into RA‐HFLS using Lipo 3000 transfection reagent (Thermo Fisher Scientific). For BZRAP1‐AS1 or COL5A2 overexpression, the transfected cells underwent a continuous puromycin selection for 7 days and the survival cell clones were kept for next assays.

HFLS-RA (Cell Applications, USA) was used for the in vitro experimental validation in the current study. The cells were cultured in sterile synoviocyte growth medium (Cell Applications, USA) supplemented with 100 U/mL 1 penicillin, 80 U/mL 1 streptomycin, 2 mM Gln-glutamine, and were maintained at 37°C in a humidified 5% CO₂ incubator. HFLS–RA were used at passage numbers 4 to 8 in this study.

Three lots of human fibroblast-like synoviocytes from rheumatoid arthritis (HFLS-RA) patients and three from osteoarthritis (HFLS-OA) (Cell Applications, Inc.) patients were cultured separately in basal medium containing 10% growth supplement and 1% penicillin/streptomycin (Cell Applications, Inc.). Cells were incubated at 37 °C under 5% CO₂. The medium was changed every 2 days. After confluence, the cells were seeded at a density of 3.0 × 10⁵ cells/2 mL/well in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 6-well plates.

Primary HFLS cells from healthy controls (HFLS control), osteoarthritis synovium (HFLS-OA), and RA synovium (HFLS-RA) were purchased from Cell Applications, Inc (San Diego, CA, USA). HFLS cells were grown to confluency in Synoviocyte Growth Medium (Cell Applications). Two independent sets of stimulations were performed on HFLS cells as outlined below.

TCR/DR1 dtg at 12 to 16 wk of age were used as a source of splenic leukocytes. Briefly, mice were killed by cervical dislocation. Single cell suspensions were aseptically prepared from the spleens of the mice. Red blood cells were lysed and splenic leukocytes were isolated after centrifugation over Lympholyte M (Cedarlane Laboratories, Hornby, Canada). Isolated splenic cells were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1.5 mM L-glutamine, 50 mM 2-ME, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂. Human fibroblast-like synoviocytes (HFLS) isolated from synovial tissues obtained from donors without rheumatoid arthritis (HFLS-N; HFLS-Normal) or with rheumatoid arthritis (HFLS-RA) were purchased from Cell Applications, Inc., (San Diego, CA). HFLS were cultured in human synoviocytes growth medium (Cell Applications, Inc.) at 37°C in a humidified incubator with 5% CO₂ and used between passages 5 to 6.

HFLS-RA (Cell Applications, USA) cells were used for in vitro experimental validation. The cells were cultured in sterile synoviocyte growth medium (Cell Applications, USA) supplemented with 100 U/mL 1 penicillin, 80 U/mL 1 streptomycin, and 2 mM glutamine and were maintained at 37 °C in a humidified atmosphere of 5 % CO₂/95 % air. The cells were used between passage 4 and 8 and were incubated with 10 ng/mL IL-1β and different concentrations of GSZD (5.12 × 10⁻⁵, 2.56 × 10⁻⁴ and 1.28 × 10⁻³ μg/mL) for 24 h.

In the current study, HFLS-RA (Cell Applications, San Diego, CA 92121, USA) were used for in vitro experimental validation. The cells were cultured in sterile synoviocyte growth medium (Cell Applications, San Diego, CA 92121, USA) containing 100 U/mL 1 penicillin, 80 U/mL 1 streptomycin, and 2 mM Gln-glutamine in a humidified atmosphere at 37°C in the presence of 5% CO₂.