Abstract:Objective To investigate the intervention effect of Tongfu Yangsui prescription on ferroptosis of neural cells in TX mice with Wilsons disease (WD) based on the nuclear factor erythroid 2- related factor (Nrf2) signaling pathway, as well as the protective mechanism of Tongfu Yangsui prescription against neural cell injury in WD. Methods With DL mice as normal control group, TX mice were randomly divided into model group and Tongfu Yangsui prescription group. The mice in the Tongfu Yangsui prescription group were given the traditional Chinese medicine Tongfu Yangsui prescription by gavage for 30 days, and those in the model group and the normal control group were given normal saline by gavage. Brain tissue samples were collected after the last administration by gavage. Inductively coupled plasma mass spectrometry was used to measure the content of the trace elements copper and iron in brain tissue; immunofluorescent staining was used to measure the expression level of transferrin receptor (TfR) in brain tissue; colorimetry was used to measure the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in brain tissue; transmission electron microscopy was used to observe the morphological changes of mitochondria in brain cells; Fluoro-JadeB (FJB) staining was used to observe the degree of neural cell injury in mice; Western blot was used to measure the expression of the proteins associated with ferroptosis and the Nrf2 signaling pathway in brain tissue, i.e., Nrf2, ferritin heavy chain (FTH1), heme oxygenase 1 (HO1), and NADPH quinone oxidoreductase 1 (NQO1). Results Compared with the normal control group, the model group had significant increases in the content of copper and iron, the expression level of TfR, and the content of MDA (P<0.05) and a significant reduction in the activity of SOD in brain tissue (P<0.05); compared with the model group, the Tongfu Yangsui prescription group had significant reductions in the content of iron, the expression level of TfR, and the content of MDA (P<0.05) and a significant increase in the activity of SOD in brain tissue (P<0.05). In the model group, the mitochondria in brain tissue had the morphological changes such as shrinkage of mitochondrial membrane, reduction in the number of cristae or disappearance of cristae, and the formation of vacuoles, while there were significant reductions in the degree of mitochondrial structural damage in the Tongfu Yangsui prescription group. Compared with the normal control group, the model group had a significant increase in the degree of neural cell injury (P<0.05), and compared with the model group, the Tongfu Yangsui prescription group had a significant reduction in the degree of neural cell injury (P<0.05). Compared with the normal control group, the model group had a significant reduction in the expression level of glutathione peroxidase 4 (GPX4) in brain tissue (P <0.05), and compared with the model group, the Tongfu Yangsui prescription group had a significant increase in the expression level of GPX4 (P<0.05). Compared with the normal control group, the model group had significant reductions in the expression levels of Nrf2, FTH1, HO1, and NQO1 in brain tissue (P<0.05), and compared with the model group, the Tongfu Yangsui prescription group had significant increases in the expression levels of Nrf2, FTH1, HO1, and NQO1 (P<0.05). Conclusion By upregulating the expression levels of the ferroptosis-related proteins such as GPX4 and the Nrf2 signaling pathway-related proteins such as Nrf2, FTH1, HO1, and NQO1, Tongfu Yangsui prescription can alleviate oxidative stress caused by copper accumulation and inhibit the ferroptosis of neural cells, thereby exerting an intervention effect on neural cell injury in WD and a protective effect against such injury.