Cell culture

HFLSs were obtained from Cell Applications (San Diego, CA) in February 2024 and seeded in 100-mm dishes (Corning, Corning, NY) at a density of 1 × 104 cells/cm2. The cells were incubated in a humidified environment at 37 °C with 5% CO2 in 10 mL α-modified Eagle’s medium (Sigma Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Sigma Aldrich), 50 µg/mL penicillin (Meiji Seika, Tokyo, Japan), and 60 µg/mL kanamycin (Meiji Seika).

The expression of signaling proteins was maximally elevated 10 min following IL-1β (Fujifilm, Osaka, Japan) treatment for HFLS, while RNA expression of MMP-3 and MMP-13 reached its peak at 12 h, and their protein levels were most significantly increased at 24 h. Therefore, the cells were treated with 1 ng/mL recombinant human IL-1β for 12 h for molecular analysis. The cells were treated with IL-1β for 10 min for expression analysis of signaling pathway proteins, and the cells were treated for 24 h for IL-1β and MMP protein analysis.

The cells were then treated with RV-D1 (Cat#10,012,554, Cayman, MI) and or 10 μM WRW4 (Cat#S9818, Selleck, TX, USA), an antagonist of ALX/ FPR2, for 2 h prior to treatment with IL-1β.

All cells used in the experiment were mycoplasma-free cells, and mycoplasma contamination was examined by PCR every month. Cells were last tested at Hiroshima University in July 2024.

Immunocytochemical staining

HFLSs were fixed with 4% paraformaldehyde in 0.1% Triton/PBS for 15 min and blocked with 1% bovine serum albumin (BSA)/PBS in phosphate-buffered saline for 60 min. Next, a 1:100 dilution of anti-ALX/FPR2 (Cat# ab203129; Abcam, Cambridge, United Kingdom) and anti-GPR32 (Cat# ab79516, Abcam) antibodies in 1% BSA/PBS were added and incubated at 4 °C overnight. After washing with PBS, we used Alexa Fluor 594 Goat anti- Rat IgG Secondary antibodies (Cat # A-11007; Thermo Fisher Scientific, Waltham, MA) with 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature.

Fluorescence intensities of SYTOX green-stained apoptotic cells

HFLSs were seeded in 24-well cell culture dishes and cultured until they reached 80% confluency, after which RV-D1 was added at concentrations of 0, 0.1, 1, 10, and 100 nM. Subsequently, SYTOX green (Cat#S7020, Thermo Fisher Scientific) was added and incubated for 24 h in a CELLCYTE X Live Cell Analyzer (Echo, San Diego, CA) equipped with a fluorescence detection device. Apoptosis was assessed by measuring the area fluorescing at 523 nm in an area of 3.72 mm2 at two random locations from each well.

Real-time RT-PCR

Total RNA was extracted from HFLSs using a TRIzol RNA Isolation Reagents (Invitrogen, Carlsbad, MA). Total RNA concentration was measured using a NanoDrop (NanoDrop, ThermoScientific) and cDNA was synthesized from 1 µg of total RNA using ReverTra Ace® (Toyobo, Osaka, Japan) and random primers (Toyobo). Then, according to the specific primers (Table 1), the expression levels of each gene were analyzed by real-time RT-PCR using the LightCycler®480 real-time PCR system (Roche Diagnostic, Basel, Switzerland). Relative gene expression levels in each sample were calculated as the ratio of target gene expression to that of β-actin. All experiments were carried out in triplicate to ensure accuracy, and the results were averaged.

Table 1. Primer sequences for real-time reverse transcription-polymerase chain reaction Western blot analysis

Proteins were extracted from HFLSs using Triton buffer (50 mM Tris, 250 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, and 50 mM NaF) with the protease inhibitor (Sigma Aldrich, St. Louis, MO). Twenty micrograms of protein was loaded into each well and further separated based on molecular weight by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (ATTO, Tokyo, Japan). After electrophoresis, the proteins were electro-transferred for 60 min to PVDF membranes. The membranes were blocked with 3% nonfat dry milk (Cell Signaling Technology, Danvers, MA) and incubated with primary antibodies: anti-IL-1β (Cat# 9211, Cell Signaling Technology), anti-MMP-3 (Cat# 8690, Cell Signaling Technology), anti-MMP-13 (Cat# 4370, Cell Signaling Technology), anti-phosphorylated p-38 (Cat# 9211, Cell Signaling Technology), anti-total p-38 (Cat# 8690, Cell Signaling Technology), anti-phosphorylated extracellular signal-regulated kinase (ERK1/2; Cat# 4370, Cell Signaling Technology), anti-total ERK1/2 (Cat# 4695, Cell Signaling Technology), anti-phosphorylated c-Jun N-terminal kinase (JNK; Cat# 4668S, Cell Signaling Technology), anti-total JNK (Cat# 9252, Cell Signaling Technology), anti-phosphorylated AKT (Cat# 4051S, Cell Signaling Technology), anti-total Akt (Cat# 9272S, Cell Signaling Technology), anti-phosphorylated nuclear factor kappa B (NF-κB) p65 (Cat# 3033S, Cell Signaling Technology), anti-total NF-κB p65 (Cat# 8242, Cell Signaling Technology), and anti-β-actin (Cat# 4970, Cell Signaling Technology). The protein bands were scanned using Image Studio software (LI-COR, Lincoln, NE) and normalized by β-actin as a control.

Statistical analysis

All experiments were conducted in triplicate. All data were presented as the mean ± standard deviation, and values of p < 0.05 were considered significant. For multiple comparisons, significance was determined using ANOVA followed by Tukey–Kramer multiple comparisons post-hoc analysis (Statcel version 3 (OMS publishing, Saitama, Japan).