Calycosin decreases cerebral ischemia/reperfusion injury by suppressing ACSL4-dependent ferroptosis

Ischemic stroke is the second leading cause of death globally. Calycosin is a typical phytoestrogen that protects against cerebral ischemia/reperfusion (I/R) injury. However, the role of ferroptosis in this effect remains unknown. In the present study, we investigated the ferroptosis mechanism of calycosin against cerebral I/R injury using transient middle cerebral artery occlusion/reperfusion (tMCAO/R)-exposed rats and oxygen-glucose deprivation/reperfusion (OGD/R)-stimulated PC12 cells. We found that calycosin treatment significantly improved neurological deficits, brain edema, blood-brain barrier (BBB) breakdown, infarction volume, and neuronal injuries in rats that underwent tMCAO/R; similar to ferrostatin-1 (a ferroptosis inhibitor), calycosin prevented cell viability loss in PC12 cells exposed to OGD/R stimulation. In addition, calycosin intervention decreased ferroptosis, as assessed by iron accumulation, malondialdehyde (MDA), superoxide dismutase (SOD), ceramide, and reactive oxygen species (ROS) levels, as well as ferroptosis-related protein expression (ACSL4, TfR1, FTH1, and GPX4). Furthermore, overexpression of ACSL4 reversed calycosin-induced beneficial efficacy in OGD/R-stimulated PC12 cells. The molecular docking analysis demonstrated that calycosin binds to ACSL4 by forming stable hydrogen bonds at G465, K690, and D573. Collectively, these findings indicate that calycosin ameliorates cerebral I/R injury by depressing ACSL4-dependent ferroptosis.