Fig. 2

HGHF-induced osteocyte ferroptosis in vitro. a Osteocytes were treated with BSA (5.5 mmol·L⁻¹ glucose) or various concentrations of PA (25.5 mmol·L⁻¹ glucose) for different times and evaluated with a CCK‐8 assay. b CCK-8 assay of osteocytes pretreated with DMSO (solvent), Z-VAD-FMK (an apoptosis inhibitor), VitE (a ROS scavenger), Fer-1 (a ferroptosis inhibitor), Nec-1 (a necroptosis inhibitor) or 3-MA (an autophagy inhibitor) and then subjected to HGHF treatment for 24 h. c CCK-8 assay of osteocytes pretreated with various concentrations of Fer-1 and then subjected to BSA or HGHF treatment for 24 h. d Osteocytes treated with BSA, HGHF or HGHF + Fer-1 for 24 h. Representative images of the TUNEL assay (scale bar: 100 μm), C11-BODIPY staining (scale bar: 25 μm), FerroOrange staining (scale bar: 100 μm), and TEM (scale bar: 0.5 μm) images are presented. e Semiquantitative analysis of TUNEL-positive cells. Semiquantitative analysis of the fluorescence intensity of lipid peroxides (f) and ferrous iron (g). h WB analysis of the expression of GPX4 and ACSL4 in osteocytes. i Total iron, ferrous iron, and ferric iron levels in osteocytes were quantitatively determined using an iron assay kit. j MDA levels in osteocytes were quantitatively determined using an MDA assay kit. “*” indicates comparison between the two indicated groups, and “#” indicates comparison with the BSA group at 24 h. *P < 0.05; “**” and ^##P < 0.01. All data are from n = 3 independent experiments