| Objective To investigate the effect of Gusong Decoction-containing serum on hydrogen peroxide (H2O2)-induced ferroptosis in mouse embryonic osteoblast precursor cells (MC3T3-E1) and its underlying mechanism. Methods The cell counting kit (CCK-8) was utilized to determine the optimal concentrations of Gusong Decoction-containing serum and Ferrostatin-1 (Fer-1) treatment. Subsequently, appropriate doses of Gusong Decoction-containing serum and Fer-1 were selected. Levels of ferrous ion (Fe2+) and reactive oxygen species (ROS) were measured using fluorescent probes. Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) levels, and alkaline phosphatase (ALP) activity were assessed via enzyme-linked immunosorbent assay (ELISA). Glutathione (GSH) levels were determined using a colorimetric assay. The absorbance of calcium nodules was quantified by alizarin red S staining. Protein expression of glutathione peroxidase 4 (GPX4), long-chain fatty acyl-CoA synthetase 4 (ACSL4), and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) was analyzed by immunoblotting. Additionally, cells overexpressing NOX4 were evaluated for ferroptosis-related indicators (GPX4 and ACSL4 protein expression, GSH levels, MDA content) and osteogenic function (ALP activity and calcium nodule absorbance). Results H2O2 induced ferroptosis in MC3T3-E1 cells. Compared to the H2O2 group, treatment with Gusong Decoction-containing serum and Fer-1 significantly increased cell survival, SOD and CAT activities, GSH levels, and GPX4 protein expression (P<0.05), while decreasing cellular MDA content, Fe2+ and ROS levels, as well as NOX4 and ACSL4 protein expression (P<0.05). Overexpression of NOX4 attenuated the protective effects of Gusong Decoction-containing serum on ferroptosis and osteogenic function in H2O2 treated MC3T3-E1 cells. Conclusion Gusong Decoction-containing serum inhibits H2O2 induced ferroptosis in MC3T3-E1 cells, potentially through the suppression of the NOX4/ROS signaling pathway. |