Buyang Huanwu Decoction Alleviates Ischemic Stroke Injury by Inhibiting Ferroptosis via the Nrf2/GPX4 Pathway

Hao Huang,1,* Sijie Liu,1,* Jing Wu,2,* Jiayi Zhu,1 Jiaxiang Xu,1 Shuhong Yu,1 Lingna Bei,1 Biao Zhang,1,3 Yi Luo1 1Department of Neurology, Suzhou Hospital of Integrated Traditional Chinese and Western Medicine, Suzhou, People’s Republic of China; 2Department of Traditional Chinese Medicine, Community Health Service Center of the Urban Area, Suzhou, People’s Republic of China; 3Central laboratory, Suzhou Hospital of Integrated Traditional Chinese and Western Medicine, Suzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yi Luo; Biao Zhang, Suzhou Hospital of Integrated Traditional Chinese and Western Medicine, No. 39, Xiashatang, Mudu Town, Wuzhong District, Suzhou City, 215101, People’s Republic of China, Email [email protected]; [email protected]: Acute ischemic stroke poses major challenges due to high disability and mortality rates. Ferroptosis, a form of regulated cell death triggered by iron-induced oxidative stress, plays a key role in stroke injury. Despite its long history in stroke treatment, the mechanism of Buyang Huanwu Decoction (BHD) in ferroptosis remains unclear.Methods: Network pharmacology predicted BHD’s active components and pathways, while Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) confirmed its main ingredients. Middle Cerebral Artery Occlusion (MCAO) was induced in C57 mice, with neurological deficits and infarct size assessed by Longa scoring and TTC staining. Histopathological and ultrastructural changes were assessed by staining and electron microscopy, and biochemical markers (MDA, GSH, SOD, Fe²+) measured by kits. Western blotting and qPCR analyzed ferroptosis-related proteins, Nrf2 localization, and gene expression. In vitro, HT22 cells viability and ROS levels were assessed under Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) conditions. Protein expression, Nrf2 interactions, and nuclear translocation were also investigated.Results: Network pharmacology showed BHD targets key pathways in cerebral infarction, including ferroptosis and antioxidant pathways. BHD improved neurological function and reduced the infarct size in MCAO mice by 10% - 50%, and also significantly decreased the levels of oxidative stress markers (MDA, Fe2+) while increasing the activities of antioxidants (GSH, SOD). Histopathological and ultrastructural analyses demonstrated reduced neuronal damage and improved mitochondrial structure. Western blot and qPCR indicated upregulation of GPX4 and Nrf2, downregulation of Keap1, and Nrf2 nuclear translocation. In vitro, BHD enhanced HT22 cell viability and reduced ROS under stress. Protein analysis confirmed increased Nrf2, GPX4, and HO-1, with decreased Keap1 and enhanced Nrf2 nuclear translocation. Nrf2 inhibitor experiments confirmed BHD’s effects are Nrf2-mediated.Conclusion: In pre-clinical studies, BHD exerts neuroprotective effects in ischemic stroke by inhibiting ferroptosis through the Nrf2/GPX4 pathway. Keywords: ischemic stroke, ferroptosis, Nrf2/GPX4 pathway, buyang huanwu decoction