Niujiaodihuang Detoxify Decoction Inhibits D-GalN/LPS-induced Hepatocyte Ferroptosis by Maintaining Mitochondrial Homeostasis Via OMA1-OPA1 Pathway

Niujiaodihuang Detoxify Decoction (NDD) has been shown to attenuate liver injury during acute liver failure by inhibiting ferroptosis, whereas the specific mechanism regulating ferroptosis remains to be elucidated. The LO2 liver cell injury model was induced with a combination of D-Galactosamine hydrochloride (D-GaIN) and lipopolysaccharide (LPS), followed by treatment with NDD-medicated serum. Hepatocyte injury, ferroptosis, and mitochondrial function homeostasis were assessed by measuring cell viability, inflammatory factors levels, lipid peroxidation, oxidative stress, expression of ferroptosis-related proteins, mitochondrial membrane potential, and expression of mitochondrial injury-related proteins. Ferroptosis inhibitors were used to determine the relationship between ferroptosis and D-GaIN/LPS-induced hepatocellular injury. Ferroptosis inducers and SiRNA-OPA1 were utilized to validate the mechanism of action of NDD. D-GalN/LPS treatment reduced cell viability, increased the release of inflammatory cytokines, and induced ferroptosis. The use of ferroptosis inhibitors attenuated D-GaIN/LPS-induced hepatocyte injury. 10% NDD-medicated serum effectively restored hepatocyte viability and reduced inflammatory cytokine levels. Furthermore, NDD decreased lipid peroxidation and oxidative stress levels in LO2 cells, restored mitochondrial membrane potential, upregulated the expression of SLC7A11, SLC3A2, GPX4, MFN1, MFN2, and L-OPA1, and downregulated the protein expression of ACSL4, DRP1, and OMA1. Ferroptosis inducers weakened the protective effect of NDD on LO2 cells, and interfering with OPA1 inhibited the regulation of mitochondrial function and ferroptosis in LO2 cells by NDD. NDD inhibits ferroptosis in LO2 cells by maintaining mitochondrial functional homeostasis through OMA1-OPA1 pathway, thereby attenuating D-GaIN/LPS-induced LO2 cell injury.