Modulating Oxidative and ER Stress in Hypoxic Hepatocytes

The mouse hepatocyte cell line AML12 was treated with CoCl₂ (Sigma-Aldrich, St. Louis, USA) to establish the cell hypoxia model as described previously [29 (link)]. AML12 cells were cultured in DMEM/F12 (Hyclone, Logan, USA) containing 10% fetal bovine serum (FBS), ITS liquid media supplement (Sigma-Aldrich, St. Louis, USA), and 40 ng/mL dexamethasone (Solarbio, Beijing, China) at 37°C with 5% CO₂. The cells divided into four treatment groups. These were the control, ORY (240 μg/mL), CoCl₂ (300 μM), and CoCl₂+ORY (240 μg/mL ORY was added at the same time with CoCl₂ (300 μM)) groups [30 (link)]. Levels of ROS in AML12 were determined by DCFH-DA ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer's instruction. The mouse hepatocyte cell line AML12 was treated with tunicamycin (TM) (Solarbio, Beijing, China) as described previously [31 (link)]. These were the control, ORY (240 μg/mL), TM (10 μg/mL), and TM+ORY. The cells were pretreated with γ-oryzanol for 12 h, followed by treatment with 10 μg/mL tunicamycin for an additional 24 h.