Neonatal hypoxic-ischemic brain damage (HIBD) is a serious neurological disease that can result in cerebral palsy, epilepsy, mental retardation, and even death(Lan et al., 2019; Luo et al., 2018; Yin et al., 2013). It significantly impairs the quality of life for affected children, imposing a substantial economic and social burden on families and society(Dai et al., 2017). Currently, subcritical treatment is the only recognized effective approach, although its effectiveness remains relatively limited(Lan et al., 2019; Zhu et al., 2021). The pathogenesis of HIBD involves various factors, including oxidative stress, mitochondrial damage, and inflammatory response(Lai et al., 2016), which may eventually lead to neuronal death. However, the precise underlying process remains unclear, urging the need for further research.

Cell death can be executed via different subroutines. Since the description of ferroptosis as an iron-dependent form of non-apoptotic cell death in 2012, there has been increasing interest in its process and functions(Dixon et al., 2012). Ferroptosis can occur through two main pathways: exogenous or transporter-dependent pathways and endogenous or enzyme-regulated pathways. It is triggered by a redox imbalance resulting from the dysregulation of oxidants and antioxidants. This imbalance arises from the abnormal expression and activity of multiple redox-active enzymes that generate or detoxify free radicals and lipid oxidation products(Stockwell et al., 2020). Thus, ferroptosis is precisely regulated at multiple levels, including epigenetic, transcriptional, post-transcriptional and post-translational levels(Tang et al., 2021).

The signaling pathway of ferroptosis primarily exerts its function by affecting the activity of glutathione peroxidase, thereby reducing the antioxidant capacity of the cell. Currently, it is believed that the process of ferroptosis involves three key regulatory substances. Glutathione Peroxidase 4 (GPX4): This antioxidant enzyme is crucial in the process of ferroptosis, preventing lipid peroxidation within the cell, and thus avoiding cell death. When the function of GPX4 is impaired or its activity is reduced, the risk of cellular lipid peroxidation increases, leading to ferroptosis. System Xc−: A heterodimer formed by solute carrier family 7 member 11 (SLC7A11) and solute carrier family 3 member 2 (SLC3A2), associated with intracellular glutathione (GSH) synthesis. GSH is an important antioxidant. When System Xc− is inhibited, the synthesis of GSH decreases, leading to a reduction in cellular antioxidant capacity and an increase in Reactive Oxygen Species (ROS), further promoting ferroptosis. Iron metabolism-related proteins: such as transferrin and ferritin, play a key role in cellular iron balance and regulation(Stoyanovsky et al., 2019). During the process of ferroptosis, the imbalance of cellular iron levels leads to an excess of free iron, promoting lipid peroxidation and ROS production, triggering ferroptosis.

The dysregulation of ferroptosis has been implicated in the development of various neurological disorders, including Traumatic Brain Injury (TBI), neurodegenerative diseases, strokes and gliomas(Alim et al., 2019; Do Van et al., 2016; Yee et al., 2020). Iron overload has also been observed in neurological conditions like Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), epilepsy, and neurological diseases. Previous studies have provided initial evidence of the involvement of ferroptosis in neonatal HIBD in rats(Belaidi and Bush, 2016; Chen et al., 2020; Domínguez et al., 2016; Tuo et al., 2022; Xie et al., 2019; Yan and Zhang, 2019). However, the specific mechanisms by which ferroptosis contributes to the HIBD process remain unclear. In this study, we aimed to investigate the role of ferroptosis in HIBD by establishing a neonatal rat model. We examined pathological structures and protein expression to shed light on the mechanism underlying ferroptosis in HIBD.