Ginkgolide B attenuates cerebral ischemia-reperfusion injury via inhibition of ferroptosis through disrupting NCOA4-FTH1 interaction.

Cerebral ischemia/reperfusion (I/R) injury is a major cause of neuronal damage and death. Ginkgolide B (GB) has been shown to exhibit neuroprotective effects in various brain injury models. The aim of study was to investigate the potential role of GB in protecting against cerebral I/R injury and explore the underlying mechanisms. Adult male Sprague-Dawley rats were exposed to transient middle cerebral artery occlusion (tMCAO) followed by reperfusion in order to trigger cerebral I/R injury. The rats were treated with different doses of GB, vehicle control or positive drug. Neurological function, infarct volume, and levels of ferroptosis markers were evaluated. In vitro experiments were performed using OGD/R-induced PC12 cells to further investigate the effects of GB on ferroptosis and its mechanisms. In addition, molecular docking, and microscale thermophoresis (MST) assay were conducted to explore the combination of GB and NCOA4. Reduced infarct volume and enhanced neurological function were signs of dose-dependent protection from cerebral I/R injury by GB therapy. Additionally, GB treatment had an impact on the levels of oxidative stress and ferroptosis markers, including reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and Fe2+ in the cerebral environment during IR injury. Moreover, relevant ferroptosis key factors such as ACSL4, GPX4, FTH1, and NCOA4 can be regulated by GB. In OGD/R-induced PC12 cells, GB protected against ferroptosis by inhibiting autophagy and disrupting the interaction of NCOA4-FTH1. Our findings suggest that GB may protect against cerebral I/R injury by inhibiting ferroptosis through disrupting NCOA4-FTH1 interaction. GB has potential therapeutic applications for cerebral I/R injury, and further investigation of the underlying mechanisms and clinical trials are warranted.

Yang Y ，Wu Q ，Shan X ，Zhou H ，Wang J ，Hu Y ，Chen J ，Lv Z ... -  《-》

Rhein attenuates cerebral ischemia-reperfusion injury via inhibition of ferroptosis through NRF2/SLC7A11/GPX4 pathway.

Liu H ，Zhang TA ，Zhang WY ，Huang SR ，Hu Y ，Sun J ... -  《-》

Tongqiao Huoxue Decoction inhibits ferroptosis by facilitating ACSL4 ubiquitination degradation for neuroprotection against cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear. This study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms. The active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe2+, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR). TQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment. This work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.

Ou Z ，Deng Y ，Wu Y ，Wang Y ，Zhao Y ，Liu C ，Wang Z ，Liu M ，Hu X ，Fang L ，Chen J ... -  《-》

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.

Liu H ，Zhao Z ，Yan M ，Zhang Q ，Jiang T ，Xue J ... -  《-》

Paeoniae Radix Rubra extract attenuates cerebral ischemia injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway.

Paeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, but its effect on cerebral ischemia is still rarely reported. The present study aimed to assess the potential therapeutic possibilities of the extract of PRR (PRRE) on cerebral ischemia, further exploring the underlying mechanism, and preliminary screening of the corresponding active components. The neuroprotective effects of PRRE in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO) injury and mouse hippocampal neuronal cells (HT22 cell line) following oxidative stress were confirmed. The mechanism was investigated using immunohistochemical staining, western blotting, transmission electron microscopy (TEM), and immunofluorescence. The active components of PRRE were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking. The in vivo study showed that PRRE reduced infarct volume and improved neurological deficits in rats, and the expression of GPX4, FTH1, Beclin1, LC3 II, and p-Akt was upregulated in the rat hippocampi. In addition, the vitro research indicated that PRRE can also alleviate H2O2-induced HT22 cell damage by regulating cytokines such as malondialdehyde (MDA), reduced glutathione (GSH) and reactive oxygen species (ROS), and the expressions of GPX4 and Beclin1 were observed to be elevated. The PI3K/Akt signalling pathway was inhibited by LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K). Furthermore, the effective components of PRRE in regulating ferroptosis and autophagy are mainly defined as albiflorin, paeoniflorin, benzoyl paeoniflorin, oleanolic acid, and hederagenin. PRRE exerts neuroprotective effects against cerebral ischaemic injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway. This study provides an experimental basis for the potential application of PRRE as a novel therapeutic drug, and PI3K/Akt-associated ferroptosis and autophagy as therapeutic targets for cerebral ischemia.

Zhao F ，Peng C ，Li H ，Chen H ，Yang Y ，Ai Q ，Chen N ，Liu F ... -  《-》