Necrostatin-1 as a Potential Anticonvulsant: Insights from Zebrafish Larvae Model of PTZ-Induced Seizures.

Abstract

Epilepsy is a common neurological disorder affecting around 70 million people worldwide. Despite significant research and advancements in pharmaceutical therapies, the exact mechanisms underlying epileptogenesis remain unclear. As a result, current antiepileptic drug treatments are ineffective for approximately 30% of patients, providing only symptomatic relief. The epileptic process is influenced by the signaling of tumor necrosis factor alpha (TNFα), which affects neuronal excitability. The TNFα/TNFR1 signaling pathway and the role of RIPK1 in initiating inflammatory cell death pathways are well studied in human disorders. Dysregulation of RIPK1 is linked to inflammation and neurodegenerative diseases. Necrostatin-1 (Nec-1) selectively inhibits RIPK1 in various pathological conditions. The current study aimed to investigate the anticonvulsant properties of Nec-1 (a selective RIPK1 inhibitor) in PTZ-induced seizures in zebrafish larvae. Before the onset of seizures, zebrafish were treated with Nec-1 (15 µM) for 24 h at 6 days post-fertilization (dpf), followed by exposure to 15 mM of PTZ for 30 min. Behavioral assessments were conducted to observe changes in locomotor activity. Additionally, c-Fos expression was measured as an indicator of neuronal activation and analyzed mRNA levels of the astrocyte activation marker (GFAP) along with various inflammatory cytokines. Western blot analysis was conducted to evaluate GABA_A receptor expression. Pretreatment with Nec-1 restored normal behavior and reversed the expression of c-Fos in zebrafish larvae induced by PTZ. Additionally, Nec-1 reduced the elevated mRNA expression of inflammatory cytokines and significantly suppressed TNF/TNFR1 signaling, thereby inhibiting the enhanced internalization of GABA_A receptors. Our findings indicate that Nec-1 reduced the severity of PTZ-induced seizures in zebrafish by regulating behavior changes, suppressing inflammatory mediators, and enhancing the expression of GABA_A receptors, suggesting potential anticonvulsant properties.