Ferroptosis inhibition and AMPK activation: key mechanisms of Soy Isoflavones against cerebral injury

Cailian Wu¹Tiantian Luo²Jinfeng Huang³Ruikang Mo^4*

• ¹Department of Emergency, The Second Affiliated Hospital of Guangxi Medical University, Nanning, 530007, Guangxi Province, China, Nanning, China
• ²Mental Health Center, The Second Affiliated Hospital of Guangxi Medical University,Nanning, 530007, Guangxi Province, China, Nanning, China
• ³Department of Rehabilitation, The First People’s Hospital of Nanning, Nanning, 530022, Guangxi Province, China, Nanning, China
• ⁴Department of Neurology, The First People’s Hospital of Nanning, Nanning, 530022, Guangxi Province, China, Nanning, China

Background: Cerebral ischemia–reperfusion injury (CIRI) remains a major cause of neurological disability and lacks effective neuroprotective interventions. Soy isoflavones (SI), phytoestrogens with antioxidant and anti-inflammatory properties, have shown neuroprotective potential. Given that ferroptosis contributes to CIRI pathogenesis and AMP-activated protein kinase (AMPK) regulates redox homeostasis, iron metabolism, and lipid peroxidation, we investigated whether SI protect against CIRI by activating AMPK and inhibiting ferroptosis. Methods: Male Sprague–Dawley rats subjected to middle cerebral artery occlusion (MCAO) were pretreated with SI (120 mg/kg, gavage) for 21 days. Neurological outcomes were assessed by infarct volume (TTC), brain water content, and behavioral scoring (Longa, mNSS). Ferroptosis was evaluated via cerebral Fe²⁺/total iron levels, oxidative stress indices (MDA, GSH, SOD, ROS), and protein expression of GPX4, ACSL4/3, xCT, and ferritin (FTH/FTMT). AMPK activation (p-AMPK/AMPK) was determined by Western blotting. Mechanistic validation employed RSL3 (ferroptosis inducer) and compound C (AMPK inhibitor). Results: SI pretreatment significantly reduced infarct size, alleviated edema, and improved neurological function. SI attenuated cerebral iron accumulation, suppressed ROS and MDA production, and enhanced GSH and SOD activities. Western blot analysis revealed downregulation of ACSL4 and upregulation of GPX4, ACSL3, xCT, and ferritin, consistent with ferroptosis suppression. The protective effects of SI were abolished by RSL3, confirming ferroptosis dependence. Furthermore, MCAO suppressed AMPK activation, which was restored by SI; inhibition of AMPK with compound C intensified ferroptotic injury and negated SI’s benefits, whereas co-treatment with SI reversed these detrimental effects. Conclusion: SI confer neuroprotection against CIRI by activating AMPK signaling and inhibiting ferroptosis. These findings highlight the AMPK–ferroptosis axis as a promising therapeutic target for ischemic stroke.