D glucose

MC3T3-E1 Subclone 14 cells were purchased from Procell company (Wuhan, China) (RRID : CVCL_5437) and were cultured with α-mem medium containing 10% fetal bovine serum, 1% penicillin-streptomycin and a normal glucose concentration (NG) of 5.5 mM. For high-glucose treatment, the culture medium mentioned contained a 33 mM glucose concentration supplied with D-glucose (Procell company, Wuhan, China), and the cells were treated for 7 days prior to performing the experiments. Cells were seeded on 6-well or 12-well plates in complete medium for 12 h. Canagliflozin (Selleck Chemicals, Houston, TX), a p-AMPK inhibitor dorsomorphin (compound C, Selleck Chemicals, Houston, TX), or an AMPK activator, acadesine (AICAR, Selleck Chemicals, Houston, TX) was added and removed after incubation for 12 h. Then, MC3T3-E1 cells were induced with osteogenic inductive medium (Procell company, Wuhan, China) on the indicated days to perform ALP staining, Alizarin red staining, and protein collection.

HCECs (Purchased from BNCC, Beijing, China) were cultured in MEM medium (Basal Media, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and Penicillin–Streptomycin Solution (10 kU/ml Penicillin, 10 mg/ml Streptomycin, Procell, Wuhan, China) in a humidified incubator at 37 °C containing 5% CO₂. According to the groups, D-Glucose (Procell, Wuhan, China) was dilated to different concentration. The whole research procedure was approved by the Ethics Committee of Chongqing Medical University and was performed following the Declaration of Helsinki.