Toosendanin Alleviates Cerebral Ischemia/Reperfusion Injury via Inhibiting Neural Ferroptosis Through Lipid Metabolic Reprogramming in <scp>MCAO</scp> Mice

BACKGROUND: The crosstalk between ferroptosis and neuroinflammation plays an important role in the pathogenesis of cerebral ischemia-reperfusion injury. Toosendanin (TSN), a triterpenoid compound, exhibits a wide range of pharmacological activities in human diseases. Here we investigated the potential neuroprotective effects of TSN in cerebral ischemia-reperfusion injury. METHODS: In our study, in vivo murine middle cerebral artery occlusion (MCAO) model and in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model were constructed to mimic cerebral ischemia-reperfusion injury. TTC staining, open-field test, Morris water maze test, hanging wire test, rotarod test, foot-fault test, hematoxylin and eosin (H&E) staining, flow cytometry, western blot and reverse transcription-quantitative PCR were conducted to evaluate the potential influence of TSN in cerebral ischemia-reperfusion injury. RESULTS: Our results indicated that post-stroke administration of TSN significantly reduced infarct volume and improved long-term functional recovery of MCAO mice. Mechanistically, TSN alleviated neural oxidative stress, lipid peroxidation, and ferroptosis in MCAO mice or in vitro after OGD/R. In addition, TSN modulated the infiltrating of immune cells in MCAO mice in vivo and T cell differentiation in vitro. Mechanistically, TSN directly interacted with ACSL4 and suppressed its enzymatic activity, thus suppressing the ACSL4/LPCAT3 axis to reduce the incorporation of pro-ferroptotic polyunsaturated fatty acids (PUFA) into phospholipids, and finally inhibiting lipid peroxidation and neural ferroptosis. In addition, the induction of ferroptosis abrogated the influence of TSN on immune infiltration and lipid metabolism in MCAO mice. CONCLUSION: Our findings identify TSN as a promising therapeutic agent for cerebral ischemia-reperfusion injury via inhibiting ACSL4-mediated ferroptosis.