Adenosine triphosphate (atp)

Synovial tissues removed from five 2-month-old male SD rats were snipped into pieces of ~3 mm³, homogenized in DMEM (Gibco; Thermo Fisher Scientific, Inc.) and incubated for 1 h at 37°C with 1 mg/ml type I collagenase (Sigma-Aldrich; Merck KGaA). The samples were filtered through a 100-µm cell strainer. After dissociation, the FLSs were pelleted via centrifugation at 300 × g at ~25°C for 5 min and plated in DMEM supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Invitrogen; Thermo Fisher Scientific, Inc.). Cells were cultured at 37°C in a humidified atmosphere with 95% air and 5% CO₂, and were identified as described in our previous studies (9 (link),10 (link)). Primary FLSs from passages 3–5 were used for subsequent experiments.
To induce pyroptosis, FLSs were stimulated with LPS (3 µg/ml; Sigma-Aldrich; Merck KGaA) in DMEM for 12 h and then treated with ATP (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C (3 mM) for 4 h. For the control group, FLSs were treated with DMEM for 12 h and then treated with DMEM in combination with 0.9% saline of the same volume as ATP for 4 h. Compounds in the supernatant were detected using specific ELISA kits. FLSs were collected to detect protein and gene expression levels, and caspase-1 activity, as well as to perform immunofluorescence.

PDMs were isolated from 50 male C57BL/6 mice (6-8 weeks old, 20-25 g), purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The C57BL/6 mice were housed under controlled conditions at a temperature of 25±2˚C with a 12-h light-dark cycle and 55±2% relative humidity, as previously described (32 (link)). Mice were and sacrificed by intraperitoneal injection of overdoses of sodium pentobarbital (150 mg/kg). PDMs were cultured in an RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere under 5% CO₂ and 95% O₂ at 37˚C. Cells were randomly separated into three groups: Normal (N), LPS+ATP and AKFPD groups. PDMs in the LPS+ATP group were stimulated with 500 ng/ml lipopolysaccharide (LPS; cat. no. L8880; Beijing Solarbio Science & Technology Co., Ltd.) for 2.5 h, followed by exposure to 5 mM ATP (cat. no. 10519979001; Roche Diagnostics) for 0.5 h at 37˚C. PDMs in the AKFPD group were pre-incubated with 400 µg/ml AKFPD for 24 h at 37˚C and subsequently exposed to LPS plus ATP.