Etanercept

Rat cardiomyocytes were stimulated with 100 nM endothelin (Sigma) for 4 days in serum-free DMEM to induce cardiomyocyte hypertrophy. To evaluate the effect of etanercept on cardiomyocyte hypertrophy, cardiomyocytes were then treated with different concentrations of etanercept (0, 1, 10, and 50 μM, Sigma) for 48 h.

Synoviocytes were isolated from synovial tissue by using standard procedures (19 ). Synoviocytes from patients with RA were digested with Liberase TM Research Grade (Sigma–Aldrich) for 90 min at 37°C. The digested synoviocyte slurries were filtered through a 100 μm cell strainer (EASYstrainer™ Cell; Greiner Bio-One, Kremsmünster, Austria). Cells were suspended in the growth medium consisting of Dulbecco's modified Eagle's medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated at 37°C in 5% CO₂. Cells at passages 3 to 6 were used in all experiments.
For cytokine stimulation, cells were treated with or without various concentrations (1, 10, and 100 ng/mL) of PDGF-BB (BioLegend; San Diego, CA, USA), TGF-β (BioLegend), or TNF-α (BioLegend) for 2 days, as per our approved experimental design. The role of therapeutic drugs was determined by treating cells stimulated with cytokines with 10, 25, or 50 μg/mL etanercept (Pfizer, NY, USA) or 7.5 μM or 15 μM palbociclib (Sigma–Aldrich), or a combination of 25 μg/mL etanercept and 7.5 μM palbociclib for 1 day, as per our experimental design. These doses of cytokines and inhibitors were selected based on the findings of previous studies (20 (link)–23 (link)). WST-8 assays were performed to assess cell proliferation by using the Cell Counting Kit-8 (CK04; Dojindo).

Etanercept was purchased from Sigma Chemical Co. (St. Louis, MO, USA). MPO assay kit, CK-MB, cardiac troponin I assay kit and LDH assay kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China). TNF-α ELISA kit was purchased from R&D Corporation, (USA). BCA protein quantification kit was purchased from Bio-Rad (USA). GSH-PX and SOD activity assay kit and MDA content assay kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China).