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Oxymatrine alleviates cerebral ischemia-reperfusion injury by inhibiting microglia ferroptosis via NRF2 pathway
Nanding Wu1, Yutong Wang2, Mengjie Guo2
1Department of Clinical Laboratory Medicine, Affiliated Hospital of Inner Mongolia Minzu University, No. 1742 Huolinhe Street, Horqin District, Tongliao, Inner Mongolia 028000, China; Department of Biochemistry and Molecular Biology, Basic Medical College, Jinzhou Medical University, No.40, Section 3 Songpo Road, Linghe District, Jinzhou, Liaoning 121001, China.
Background:
Cerebral ischemia-reperfusion injury (CIRI) involves secondary damage following blood flow restoration. Ferroptosis, an iron-dependent cell death process, is implicated in CIRI, with mitochondrial dynamics imbalance playing a critical role. Oxymatrine (OMT), a quinolizidine alkaloid extracted from the root of Sophora flavescens, exhibits neuroprotective properties, yet its role in modulating microglia ferroptosis and mitochondrial homeostasis during CIRI remains unclear.
Purpose:
This study aimed to investigate whether OMT attenuates microglia ferroptosis in CIRI by activating the nuclear factor erythroid 2-related factor 2 (NRF2) pathway and restoring mitochondrial dynamic balance.
Methods:
Using a rat middle cerebral artery occlusion/reperfusion model and BV-2 microglia under oxygen-glucose deprivation/reoxygenation, we evaluated the effects of OMT alone or with NRF2 inhibitor ML385 or ferroptosis inducer Erastin. Assessments included neurological scores, infarct volume, Reactive Oxygen Species, ferrous iron, malondialdehyde, glutathione, mitochondrial membrane potential, and related protein expression, including NRF2, kelch-like ECH-associated protein 1 (KEAP1), heme oxygenase-1, solute carrier family 7 member 11, ferritin heavy chain, glutathione peroxidase 4, dynamin-related protein 1, optic atrophy 1. Molecular docking, surface plasmon resonance, and co-immunoprecipitation were used to examine OMT-KEAP1 binding. Finally, in vitro rescue experiments using KEAP1 overexpression and CDDO-ME (a specific KEAP1 inhibitor) confirmed the KEAP1/NRF2 dependency by re-assessing cell viability and ferroptosis markers.
Results:
OMT improved neurological outcomes and suppressed ferroptosis in vivo and in vitro. Mechanistically, OMT disrupted KEAP1-NRF2 binding, promoting NRF2 nuclear translocation and upregulating solute carrier family 7 member 11, heme oxygenase-1, ferritin heavy chain, and glutathione peroxidase 4. This restored mitochondrial homeostasis by balancing optic atrophy 1 and dynamin-related protein 1, thereby reducing lipid peroxidation. Crucially, these effects were abolished by ML385 or Erastin, while KEAP1 overexpression and CDDO-ME respectively mimicked KEAP1-mediated suppression and NRF2-driven protection. These findings confirm OMT acts via a KEAP1/NRF2-dependent anti-ferroptotic axis.
Conclusions:
OMT protects against CIRI by inhibiting microglia ferroptosis through NRF2 pathway activation and improvement of mitochondrial homeostasis, supporting its potential as a therapeutic agent for ischemic stroke.
Insights
Oxymatrine (OMT) protects against cerebral ischemia-reperfusion injury (CIRI) by inhibiting ferroptosis in microglia. This occurs through activating the NRF2 pathway and restoring mitochondrial balance, offering potential as an ischemic stroke therapy.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Cerebral ischemia-reperfusion injury (CIRI) involves secondary damage after blood flow restoration.
- Ferroptosis, an iron-dependent cell death, contributes to CIRI, with impaired mitochondrial dynamics playing a key role.
- Oxymatrine (OMT) shows neuroprotection, but its effect on microglia ferroptosis and mitochondrial homeostasis in CIRI is unknown.
Purpose of the Study:
- To investigate if Oxymatrine (OMT) reduces microglia ferroptosis in CIRI.
- To determine if OMT activates the NRF2 pathway and restores mitochondrial balance.
- To elucidate the molecular mechanisms underlying OMT's effects in CIRI.
Main Methods:
- Utilized rat CIRI models and oxygen-glucose deprived BV-2 microglia.
- Assessed neurological scores, infarct volume, oxidative stress markers, and mitochondrial function.
- Analyzed protein expression of NRF2, KEAP1, and ferroptosis-related molecules.
- Employed molecular docking, SPR, and co-IP to study OMT-KEAP1 interactions.
- Conducted in vitro rescue experiments with KEAP1 overexpression and inhibitors.
Main Results:
- OMT improved neurological outcomes and reduced ferroptosis in vitro and in vivo.
- OMT disrupted KEAP1-NRF2 binding, promoting NRF2 nuclear translocation and upregulating key ferroptosis regulators.
- OMT restored mitochondrial homeostasis by balancing mitochondrial dynamics proteins, reducing lipid peroxidation.
- Inhibiting NRF2 or inducing ferroptosis abolished OMT's protective effects.
- KEAP1 modulation confirmed the KEAP1/NRF2-dependent mechanism of OMT.
Conclusions:
- OMT confers protection against CIRI by inhibiting microglia ferroptosis.
- OMT achieves this via NRF2 pathway activation and improved mitochondrial homeostasis.
- OMT demonstrates potential as a therapeutic agent for ischemic stroke.