The effect and mechanism of HY-021068 on inhibiting ferroptosis and alleviating cerebral ischemia-reperfusion injury in mice

<sec> <p indent="0mm">Ischemic stroke remains one of the leading causes of mortality and long-term disability worldwide, characterized by a complex pathophysiology in which cerebral ischemia-reperfusion injury (CIRI) constitutes a critical aggravating factor. The progression of CIRI involves a cascade of detrimental events, including severe oxidative stress, robust inflammatory responses, and the activation of multiple regulated cell death pathways. Among these, ferroptosis—a recently identified form of iron-dependent regulated cell death driven by uncontrolled lipid peroxidation—has garnered increasing attention for its significant role in CIRI pathogenesis. This process is characterized by glutathione depletion and inactivation of glutathione peroxidase 4 (GPX4), leading to phospholipid hydroperoxide accumulation and subsequent cell membrane damage. The transcription factor nuclear factor E2-related factor 2 (Nrf2), recognized as a master regulator of cellular antioxidant defense mechanisms, has emerged as a promising therapeutic target for neuroprotection. Under physiological conditions, Nrf2 is sequestered in the cytoplasm by its inhibitor KEAP1 and targeted for proteasomal degradation. Upon activation, Nrf2 translocates to the nucleus and initiates the transcription of numerous cytoprotective genes. This study aims to comprehensively evaluate the neuroprotective effects of a novel synthetic compound, HY-021068, against CIRI in mice and to elucidate its detailed molecular mechanisms, with particular emphasis on the KEAP1-Nrf2 signaling axis and its downstream effects on ferroptosis and inflammatory responses. </sec> <sec> A well-established CIRI model was induced in adult male C57BL/6 mice by transient middle cerebral artery occlusion (MCAO). The animals were randomly assigned to six experimental groups: sham-operated, model, ginsenoside Rg1 <sc>(10 mg kg^–1,</sc> serving as a positive control), and three HY-021068 treatment groups (12.5, 25, and <sc>50 mg kg^–1).</sc> Neurological functional recovery was systematically assessed during the reperfusion period using the pole test and contextual fear conditioning paradigm. Histopathological evaluation of neuronal damage was performed through Nissl staining, while immunohistochemistry and Western blotting were employed to quantify the expression of ferroptosis-associated proteins and key inflammatory mediators. Intracellular reactive oxygen species (ROS) accumulation was detected using dihydroethidium (DHE) fluorescence staining. To investigate the direct molecular interaction between HY-021068 and its putative target, comprehensive molecular docking simulations and thermal shift assays were conducted. </sec> <sec> Treatment with medium and high doses of HY-021068 (25 and <sc>50 mg kg^–1)</sc> resulted in significant improvement in neurobehavioral performance and substantial reduction in cerebral infarct volume compared to the model group. Histological examinations demonstrated that HY-021068 administration effectively mitigated neuronal loss in vulnerable brain regions, including the cerebral cortex and hippocampus, while concurrently reducing ROS accumulation. At the molecular level, HY-021068 treatment dose-dependently promoted Nrf2 dissociation from KEAP1 and subsequent nuclear translocation, thereby enhancing the expression of downstream antioxidant proteins, including heme oxygenase-1 (HO-1) and GPX4. Concurrently, HY-021068 significantly suppressed the expression of key ferroptosis-related proteins, including transferrin receptor (TfR) and the divalent metal transporter SLC11A2, both critically involved in cellular iron uptake. Furthermore, HY-021068 effectively inhibited NLRP3 inflammasome activation and subsequent maturation and release of the pro-inflammatory cytokine interleukin-1β (IL-1β). Molecular docking analyses revealed that HY-021068 binds with high affinity to the Kelch domain of KEAP1, while thermal shift assays confirmed that this interaction enhances KEAP1 protein stability, effectively preventing Nrf2 ubiquitination and degradation. </sec> <sec> In conclusion, this study provides compelling evidence that HY-021068 confers significant neuroprotection in a mouse model of CIRI through a multifaceted mechanism. The compound directly targets KEAP1, activating the Nrf2 signaling pathway and subsequently orchestrating a coordinated cellular defense that attenuates oxidative stress, suppresses NLRP3 inflammasome-mediated inflammation, and inhibits ferroptosis execution. These findings not only elucidate a novel pharmacological strategy for mitigating CIRI but also highlight HY-021068 as a promising therapeutic candidate worthy of further preclinical development for ischemic stroke treatment. </sec>