Mechanism of Gandouling in Ameliorating Brain Injury via Inhibition of Ferroptosis in a Wilson's Disease TX Mouse Model Based on the SIRT1/FoxO3 Pathway

ObjectiveTo investigate the mechanism of action of Gandouling against brain injury in Wilson disease (WD) by regulating the SIRT1/FoxO3 pathway-mediated ferroptosis.MethodsTX mice were randomly divided into 6 groups: WD model control group, Gandouling group, resveratrol group, Gandouling + resveratrol group, SIRT1 inhibitor group, and Gandouling + SIRT1 inhibitor group, with DL mice serving as the blank control group. After acclimatization, a 4-week intervention was conducted. Neurobehavioral function was assessed using the water maze test, wire hanging test, and pole test. Assessment of Apoptosis in Basal Ganglia Tissue by TUNEL assay. ROS levels in the basal ganglia were analyzed using the DCFH-DA method. Fe²⁺ and MDA levels were measured by colorimetric assays. Immunofluorescence was used to determine SIRT1 and FoxO3 fluorescence intensity. Protein expression levels of SIRT1, FoxO3, GPX4, SLC7A11, ACSL4, FTH1, and P53 were detected by western blotting. RT-PCR was used to measure SIRT1 and FoxO3 mRNA expression.ResultsCompared with the blank control group, the model control group showed significant neurofunctional impairment (prolonged escape latency, reduced platform crossings, and significantly increased pole climbing time), obvious neuronal apoptosis, and increased levels of ROS, Fe²⁺, and MDA in the basal ganglia (P < 0.01). The expression of anti-ferroptosis proteins (GPX4, SLC7A11, FTH1) was decreased (P < 0.01), while the expression of pro-ferroptosis proteins (P53, ACSL4) was increased (P < 0.01). Both resveratrol and Gandouling interventions significantly reversed these phenomena, improving neurofunction, inhibiting basal ganglia cell apoptosis, reducing Fe²⁺, ROS, and MDA levels, upregulating anti-ferroptosis protein expression, and downregulating pro-ferroptosis protein expression, with comparable efficacy (all P < 0.01). Furthermore, the combined intervention of Gandouling and resveratrol exhibited a synergistic effect. Conversely, EX527 exacerbated neurofunctional impairment, cell apoptosis, and further increased Fe²⁺, ROS, and MDA levels in the basal ganglia of TX mice (P < 0.01), along with abnormal expression of ferroptosis-related proteins. However, the combination of EX527 and Gandouling counteracted each other's effects.ConclusionGandouling inhibits neuronal ferroptosis in model mice by regulating the SIRT1/FoxO3 signaling pathway, thereby improving their neurofunctional outcomes.