HepG2 cell culture and treatments

HepG2 cells were cultured in DMEM supplemented with 10% FBS, 100 IU mL⁻¹ penicillin, and 0.1 µg mL⁻¹ streptomycin. The cells were incubated in humidified atmosphere of 5% CO₂ at 37°C, passaged according to the recommended procedures of ATCC, used for experiments from the logarithmic phase of growth, and seeded into 96-well plates (1×10⁴ cells per well, 100 µL). Cells were exposed to d-gal (at 18.75, 37.5, 75, 150, and 300 mM) or Anwulignan (at 0.625, 1.25, 2.5, 5, and 10 µg mL⁻¹) for 24 hours for determining the concentrations of d-gal and Anwulignan. The final concentrations were 75 mM for d-gal to produce the liver injury model, and 0.625, 1.25, and 2.5 µg mL⁻¹ for Anwulignan. All cells were cultured for 24 hours.