[Polydatin attenuates high copper induced damage in Atp7b knockout neural stem cells by regulating PPARG mediated...

To investigate the mechanism by which polydatin ameliorates neurological damage in Wilson disease (WD) based on bioinformatics analysis and experimental validation.

The targets of polydatin were predicted using PharmMapper, SEA, and Swiss Target Prediction databases, while targets related to WD and ferroptosis were retrieved from GeneCards, OMIM, DrugBank, Therapeutic Target Database, and FerrDb. Overlapping targets among polydatin, WD, and ferroptosis were identified. A protein-protein interaction (PPI) network was then constructed using the STRING database and Cytoscape software to screen for core targets. Molecular docking between the core targets and polydatin was performed with AutoDock and visualized using PyMOL. A high copper induced Atp7b knockout (Atp7b^-/-)neural stem cellmodel was established. Different drug intervention groups and reverse validation groups (including gene transfection) were set up. Cell viability was measured by CCK 8 assay; apoptosis was assessed by flow cytometry and TUNEL staining; mitochondrial membrane potential (MMP) was detected using JC 1 staining; intracellular malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and ferrous iron (Fe²⁺) levels were determined by colorimetric assays; reactive oxygen species (ROS) levels were measured using the DCFH DA method; mRNA levels of peroxisome proliferator activated receptor gamma (PPARG) and long chain acyl CoA synthetase 4 (ACSL4) were examined by quantitative reverse transcription PCR (qRT PCR); and protein levels of PPARG, ACSL4, nuclear factor erythroid 2 related factor 2 (NRF2), and glutathione peroxidase 4 (GPX4) were analyzed by Western blotting.

Network pharmacology analysis identified 443 polydatin targets, 5,772 WD targets, and 564 ferroptosis related targets. Their intersection generated 37 potential therapeutic targets. Topology analysis using CytoNCA identified the top 10 core targets: ALB, PPARG, EGFR, NFE2L2, PTGS2, HMOX1, GSK3B, PARP1, MAPK8, and PPARA. Molecular docking demonstrated a strong binding affinity between polydatin and PPARG, with a binding energy of -9.6 kcal/mol. Compared with the model control group, polydatin treatment significantly enhanced cell viability (P<0.05) and reduced the apoptosis rate (P<0.01). The levels of MDA, ROS, and Fe²⁺ were decreased (all P<0.05), while SOD activity and MMP were significantly increased (all P<0.01). The protein expression levels of PPARG, NRF2, and GPX4 were significantly upregulated (all P<0.05), and ACSL4 protein expression was notably downregulated (P<0.05). Pretreatment with a ferroptosis activator (Erastin), a PPARG antagonist (GW9662), or siPPARG silencing, which inhibit PPARG function or activate ferroptosis, significantly weakened the protective effects of polydatin on cell viability, oxidative stress status, and iron metabolism (all P<0.05).

Polydatin alleviates WD induced neuronal damage by targeting PPARG to regulate the NRF2/GPX4 and ACSL4 axis, thereby inhibiting cellular ferroptosis.