OBJECTIVES: To investigate the mechanism by which the Chinese medicine Gandouling protects against brain injury in hepatolenticular degeneration (Wilson's disease) through regulation of the SIRT1/FoxO3 pathway-mediated ferroptosis.
METHODS: TX mice were randomly divided into six groups: model control, Gandouling, resveratrol (SIRT1 activator), Gandouling+resveratrol, EX-527 (SIRT1 inhibitor), and Gandouling+EX-527 groups, with DL mice serving as the blank control group. After a 4-week intervention, neurological function was assessed using the Morris water maze test, wire hanging test, and pole test. Apoptosis in basal ganglia tissue was detected by TUNEL assay. Reactive oxygen species (ROS) levels in basal ganglia tissue were measured using the DCFH-DA method, while ferrous iron (Fe²⁺) and malondialdehyde (MDA) levels were determined by colorimetric assays. Immunofluorescence was used to evaluate the fluorescence intensity of SIRT1 and FoxO3. The protein expression levels of SIRT1, FoxO3, GPX4, SLC7A11, ACSL4, FTH1, and P53 were assessed by Western blotting, and the mRNA expression levels ofandwere detected by qRT-PCR. Sirt1Foxo3
RESULTS: Compared with the blank control group, the model control group exhibited significant neurological impairment (prolonged escape latency, reduced platform crossing frequency, and prolonged pole-climbing time), increased neuronal apoptosis, decreased transcription and expression of(both<0.01), increased transcription (<0.05) and expression (<0.01) of, and elevated levels of ROS, Fe²⁺, and MDA in basal ganglia tissue (all<0.01). Expression levels of ferroptosis-inhibiting proteins (GPX4, SLC7A11, FTH1) were decreased (all<0.01), while those of ferroptosis-promoting proteins (P53, ACSL4) were increased (all<0.01). Both Gandouling and resveratrol monotherapy significantly reversed the above alterations: neurological function was improved, apoptosis was reduced, ROS, Fe²⁺, and MDA levels were decreased,transcription and expression were upregulated,transcription and expression were downregulated, ferroptosis-inhibiting proteins were increased, and ferroptosis-promoting proteins were decreased (all<0.01), with comparable efficacy between the two treatments (>0.05). Combined Gandouling and resveratrol treatment exhibited synergistic effects. EX-527 exacerbated neurological impairment and apoptosis, further increased ROS, Fe²⁺, and MDA levels, suppressedtranscription and expression, elevatedtranscription and expression, and aggravated the dysregula-tion of ferroptosis-related proteins (all<0.01). However, no significant differences were observed between the Gandouling+EX-527 group and the model control group (all>0.05). Additionally, the transcription levels of Sirt1 andin basal ganglia tissue were negatively correlated (<0.01). Sirt1P P P Foxo3P P P Sirt1Foxo3P P Sirt1Foxo3P P Foxo3P
CONCLUSIONS: Gandouling inhibits neuronal ferroptosis by regulating the SIRT1/FoxO3 signaling pathway, thereby improving neurological function in model mice.
