Stroke has a high prevalence of morbidity, mortality, and disability and is the second largest cause of death worldwide. As a result, stroke imposes a significant social and financial impact (GBD 2016 Stroke Collaborators, 2019). 87% of all strokes occur as acute ischemic strokes (AIS), which are defined by an abrupt stoppage of oxygen and blood supply to local brain tissue as a result of arterial blockage (Mozaffarian et al., 2016; Phipps and Cronin, 2020). Tissue plasminogen activator (t-PA) administered intravenously and endovascular therapy are the two treatments that are now readily available to recanalize blood flow in AIS. However, such therapies have the drawback that reperfusion causes the generation of extremely dangerous reactive oxygen species (ROS), leading to oxidative stress, which is the main cause of ischemia-reperfusion injury and therefore causes damage to brain tissue (Orellana-Urzúa et al., 2020). The benefits of AIS treatments are only partially realized despite their high occurrence. Therefore, the creation of obvious mechanisms and efficient therapies is crucial.

With the overproduction of peroxides and the depletion of antioxidants as its main contributing factors, oxidative stress is a major contributor to ischemic stroke and a trigger of neuronal dysfunction and death (Lin and Beal, 2006; Manzanero et al., 2013). As a result of the imbalance between ROS production and consumption caused by excessive ROS synthesis in neuronal cells, AIS, the antioxidant system is depleted. A surplus of ROS leads to lipid peroxidation and the oxidation of DNA, proteins, and RNA, which result in the malfunction and demise of neurons (Ouyang et al., 2015). It has been shown that ferroptosis, which differs from traditional cell death pathways and is linked to oxidative stress-induced lipid peroxidation and glutathione depletion, is essential to the development of ischemic stroke (Alim et al., 2019). According to studies, preventing ferroptosis protects against oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage and improves behavior in stroke models (Liu et al., 2020; Yuan et al., 2021). Research has demonstrated that oxidative stress and ferroptosis share comparable molecular pathways (Fricker et al., 2018). The link between oxidative stress, ferroptosis, and ischemic stroke indicates that these processes are crucial to AIS pathogenesis and that reducing oxidative stress and ferroptosis may help with AIS treatment. Therefore, to guide treatment, it is essential to fully comprehend the molecular processes of oxidative stress and ferroptosis in AIS.

One of the essential isozymes in the metabolism of polyunsaturated fatty acids (PUFAs) is acetyl-CoA synthetase long-chain family member 4 (ACSL4). According to recent research, ACSL4 is essential for ferroptosis, and cells that express more of it are more susceptible to the disease (Doll et al., 2017; H et al., 2016). Animal models of numerous neurological disorders, including ischemic stroke, have indicated that inhibiting ACSL4 prevent ferroptosis (Tuo et al., 2022). In the case of ischemic stroke, for instance, cui et al. found that ACSL4 exacerbates the damage and inflammation to the brain caused by ferroptosis (Cui et al., 2021). Furthermore, according to the research of Li et al., ferroptosis and intestinal ischemia/reperfusion damage are intimately related, and ACSL4 plays a crucial part in this fatal process (Li et al., 2019). Given the important role of ACSL4 in ferroptosis, ACSL4 might be vital in the management of AIS. Together, treatment for AIS may benefit from targeting ACSL4 to prevent ferroptosis and oxidative stress.

Traditional Chinese medicine (TCM) offers thousands of years of clinical expertise in stroke treatment. Because of its capacity to increase blood flow and eliminate stasis, Achyranthes bidentata Blume, a commonly prescribed traditional Chinese herb, is important in traditional Chinese stroke treatment (Gong and Sucher, 2002). Given the critical roles that oxidative stress and ferroptosis play in the pathophysiology of AIS, the development of a new drug to inhibit ferroptosis and oxidative stress is of great significance. Ecdysterone (EDS) is one of the key active compounds in Achyranthes bidentata Blume, and its discovery has improved its medical potential due to its antioxidant and neuroprotective effects (Lin et al., 2019). For instance, it has been proposed that EDS may reduce Aβ-induced behavioral abnormalities by reducing oxidative stress and neuronal degeneration (Gholipour et al., 2022). Additionally, prior research has demonstrated that in models of focal cerebral ischemia, EDS may have a neuronal protective function (Luo et al., 2011). However, it is unclear whether EDS can ameliorate AIS induced oxidative damage by inhibiting ferroptosis in neurons through ACSL4.

Based on the above evidence, we hypothesized that EDS inhibits ferroptosis in neurons through ACSL4, thereby ameliorating oxidative damage induced by AIS. Due to this, we aimed to examine in this work the therapeutic effects of EDS on OGD/R-induced PC12 cells and middle cerebral artery occlusion (MCAO) rats. Simultaneously, we aimed to study the regulatory role of ACSL4 on ferroptosis in a cerebral ischemia stroke model, with a particular focus on whether ACSL4 inhibition mitigates the oxidative damage brought on by AIS in a way that suppresses ferroptosis.