The blood-brain barrier (BBB), often referred to as the guardian of the brain, plays a fundamental role in establishing and maintaining the microenvironment of the central nervous system (CNS). It comprises specialized endothelial cells that tightly interact with each other, as well as tight junctional complexes, pericytes, extracellular matrix components, and astrocytic endfeet (Schaeffer and Iadecola, 2021; Ali et al., 2023). During the series of events triggered by acute ischemic stroke, BBB integrity is significantly disrupted, leading to a decrease in BBB stability. This disruption damages tight junctions (TJs) and causes BBB leakage (Brouns and De Deyn, 2009; Sifat et al., 2017; Eide and Feng, 2022). As a result, various blood components gain access to the brain parenchyma, resulting in irreversible brain injury (Huang et al., 2013). Tissue-type plasminogen activator (tPA) is currently the only FDA-approved pharmaceutical treatment for ischemic stroke (Yang et al., 2018). However, accumulating evidence suggests that BBB disruption can lead to hemorrhagic transformation (HT), which is a critical limitation to the clinical use of tPA (Wei et al., 2017; Li et al., 2018). Therefore, there is an urgent demand for interventions that can preserve BBB integrity, reduce the risk of HT, and promote functional recovery in stroke patients.

Neutrophils are the first immune cells to infiltrate the CNS after stroke, and their numbers peak at 1–3 days poststroke (Gronberg et al., 2013). The infiltration of neutrophils has been demonstrated to have detrimental effects, as it promotes blood clot formation and contributes to the release of proinflammatory factors (Planas, 2018; Weisenburger-Lile et al., 2019). After activation, a series of events, including chromatin decondensation, histone citrullination, and the release of DNA and histones, occur in neutrophils, indicating the formation of neutrophil extracellular traps (NETs) (Perez-de-Puig et al., 2015). NETs aggravate the death of neurons and promote thrombus formation after stroke, which further contributes to ischemic brain injury (Ducroux et al., 2018; Cai et al., 2020; Denorme et al., 2022; Li et al., 2022). Furthermore, NETs are involved in the BBB disruption after stroke. A previous study suggested that the release of NETs severely damages the BBB during stroke recovery, but inhibiting NET formation can mitigate this damage (Kang et al., 2020). Thus, inhibiting NET formation in stroke lesions is a promising treatment strategy for ischemic stroke.

γ-Glutamylcysteine (γ-GC), the direct precursor of Glutathione (GSH), has been found to have therapeutic potential. γ-GC has the unique ability to cross the BBB and can be absorbed by various cell types (Dringen et al., 1997). Our prior research has shown that γ-GC effectively reduces apoptosis and inhibits ROS-mediated ER stress, thereby mitigating ischemic brain injury (Li et al., 2021). Additionally, γ-GC has been found to protect cerebral endothelial cells from oxidative injury by inhibiting oxidative stress (Nakamura et al., 2012). Furthermore, the esterified form of γ-GC, γ-glutamylcysteine ethyl ester (GCEE), has been shown to attenuate BBB breakdown after traumatic brain injury (Lok et al., 2011). These findings suggest that γ-GC could be a potential drug for treating BBB damage following stroke.

Based on the above factors, this study aimed to investigate the effects of γ-GC on BBB integrity and the formation of NETs in experimental ischemic brain injury. Furthermore, we explored the potential mechanism underlying these effects of γ-GC. We believe that these findings will contribute to a better understanding of γ-GC’s protective influence and provide a potential therapeutic strategy for ischemic stroke.