Puerarin alleviates type 2 diabetes by targeting AKT1 to inhibit ferroptosis: Integrating network pharmacology, proteomics and experimental validation.

The progression of type 2 diabetes mellitus (T2DM) is closely linked to oxidative stress-induced damage. Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation. Its underlying mechanism involves an imbalance between reactive oxygen species (ROS) accumulation and cellular antioxidant defense, ultimately resulting in cell death. While puerarin has been shown to exhibit antioxidant properties, its ability to ameliorate T2DM via the ferroptosis pathway remains unclear. In this study, we combined network pharmacology, proteomics, and experimental validation to investigate the regulatory mechanisms of puerarin. Potential targets of puerarin in T2DM were identified using network pharmacology and proteomics. Oxidative stress markers-including ROS, malondialdehyde (MDA), manganese-dependent superoxide dismutase (Mn-SOD), and glutathione (GSH)-as well as insulin levels and ferroptosis-related markers such as glutathione peroxidase 4 (GPX4), cyclooxygenase-2 (COX2), and acyl-CoA synthetase long-chain family member 4 (ACSL4) were measured to assess the effect of puerarin on ferroptosis. Protein kinase B (AKT1) overexpression and knockdown models, together with nuclear factor erythroid 2-related factor 2 (NRF2) inhibitors, were used alongside Western blot analysis to investigate the pathway through which puerarin regulates ferroptosis. Results showed that palmitic acid-induced oxidative stress triggered ferroptosis in mouse pancreatic β-cells (MIN6). Under puerarin intervention, ferroptosis biomarkers including ROS, MDA, Mn-SOD, iron ions, and GSH, as well as mitochondrial morphology, were significantly altered. Mechanistic studies revealed that puerarin upregulates AKT1, leading to enhanced phosphorylation of GSK3β and increased expression of NRF2. Consequently, expression of GPX4, a key enzyme in glutathione metabolism, was elevated, thereby suppressing ferroptosis. This study is the first to identify targets of puerarin in T2DM through an integrated network pharmacology and proteomics approach. It demonstrates that puerarin may upregulate GPX4 via the AKT/GSK3β/NRF2 pathway, thereby mitigating oxidative stress damage and reducing ferroptosis, offering novel mechanistic insight for diabetes treatment.