Gw6471

According to previous reports [53 (link),54 (link),55 (link)], human liver cell line HL7702 and HepG2 were selected for NASH research and purchased from Shanghai Cell bank (Chinese Academy of Science, Shanghai, China), and cultured in DMEM with 25 mM glucose, 5 mM glutamine, 10% fetal bovine serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin at 37 °C under a 5% CO₂ environment. The cell concentrations were adjusted to 1 × 10⁵/mL and 200 μL was added to each cell in a 96-cell plate. GW6471, a PPAR-α antagonist, was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Either 20 μg/mL GW6471 or 10 μg/mL blueberry juice, or both, was added to each cell in a 96-cell plate and further cultured for 3 days. Adherent cells were detached by using 0.1% trypsin and 0.04% EDTA and resuspended in CASYton solution (Roche Applied Science, cat. no. 05651808 001). The debris was removed using a 100-μm nylon filter (Falcon Business USA Inc., Fairfield, CT, USA). Cell concentrations were measured using a glass hemocytometer and coverslip daily.

HCMs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and grown in cardiac myocyte medium supplemented with 5% fetal bovine serum (FBS), 1% cardiac myocyte growth supplement, and 1% penicillin/streptomycin solution. Cells were cultured on a poly-L-lysine-coated dish in a humidified incubator with 5% CO₂ at 37°C. Cells between passages 3–5 were used in all experiments. The Safety Committee of Biological Experiments at Kaohsiung Medical University approved the use of HCMs for in vitro experimental use. The following drugs were used: fenofibrate (a PPARα agonist, Sigma), GW6471 (GW, PPARα antagonist, Santa Cruz), BQ-123 [BQ123, endothelin A receptor (ETA) antagonist, Sigma], BQ788 [BQ788, endothelin B receptor (ETB) antagonist, Sigma], PD98059 (ERK inhibitor, Cell Signaling), and SB203580 (p38 inhibitor, Cell Signaling). None of these drugs significantly influence cell viability (>90%).