Background: Ferroptosis has emerged as a pivotal mechanism in epilepsy development. Kelch-like ECH-associated protein 1 (KEAP1), a critical regulator of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, plays a pivotal role in oxidative stress and is implicated in seizure recurrence. This study aimed to elucidate the mechanistic role of KEAP1 in kainic acid (KA)-induced ferroptosis and neuroinflammation.

Methods: HT22 hippocampal neuronal cells were stimulated with KA to establish the in vitro excitotoxicity model. Cell viability and apoptosis were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Inflammatory responses were evaluated by detecting interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) secretion levels. Ferroptosis was evaluated by measuring Fe²⁺, glutathione (GSH), and reactive oxygen species (ROS) levels. The parkin RBR E3 ubiquitin protein ligase (PRKN)-KEAP1 and histone deacetylase 6 (HDAC6)-PRKN interactions were assessed by co-immunoprecipitation (Co-IP) experiments.

Results: KEAP1 (p = 0.0008) and HDAC6 (p = 0.0004) protein levels were upregulated in the KA-induced HT22 cells, while PRKN expression was downregulated (p = 0.0034). Knockdown of KEAP1 alleviated KA-induced neuroinflammation by reducing the secretion of IL-6 (p = 0.0021) and TNF-α (p = 0.0003). Furthermore, it suppressed ferroptosis, as demonstrated by decreased levels of Fe²⁺ (p = 0.0022) and ROS (p < 0.01), and an increased GSH content (p = 0.0057). In addition, KEAP1 silencing attenuated apoptosis (p = 0.0016) in KA-induced cells. Mechanistically, PRKN mediated KEAP1 ubiquitination and degradation (p = 0.0054); HDAC6 induced PRKN deacetylation (p < 0.001), and HDAC6 modulated KEAP1 expression via PRKN. In KA-treated HT22 cells, KEAP1 reconstitution counteracted PRKN overexpression-mediated anti-inflammatory and ferroptosis-inhibitory effects (p < 0.05), while PRKN knockdown reversed the protective effects conferred by HDAC6 silencing (p < 0.05). Additionally, PRKN regulated the NRF2/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) pathway through KEAP1 (p < 0.05).

Conclusion: Our study uncovers a novel HDAC6-PRKN-KEAP1 cascade that critically modulates KA-induced ferroptosis and neuroinflammation in HT22 neuronal cells.