Chloroquine exerts antiepileptic and neuroprotective effects through the P62-Keap1-Nrf2-GPX4-ferroptosis pathway in pentatetrazone-induced epileptic rat model

Abstract

Background: Epilepsy is one of the most common brain disease. Increasing studies have revealed that ferroptosis plays a significant role in the development of epilepsy. Choloroquine(CQ) can release P62 by inhibiting the fusion of autophagosomes with lysomes in the process of autophagy. Our study aimed at investigating the antiepileptic and neuroprotective benefits of CQ via the P62-Keap1-Nrf2-GPX4-ferroptosis pathway in epilepic rats and illustrating the possible connection between autophagy and ferroptosis preliminarily. Methods: 45 male SD rats were randomly divided into three groups: control, pentylenetetrazol(PTZ; 35mg/kg), CQ(40mg/kg)+PTZ groups, all the rats were injected 15 times every other day in a total of 29 days. The EEG changes and behavioral manifestations(epileptic score, latency, and number of seizures in 30 minutes) were recorded. Patch clamp technique was applied to evaluating the excitability of hippocampal neurons. Nissl staining was utilized to observe the neurons in hippocampus. Spectrophotometry was used to evaluate the expression levels in hippocampus of Fe2+, glutathione(GSH) and malondialdehyde(MDA). The expression of P62, LC3, Keap1, Nrf2, GPX4 and PTGS2 was determined by Immunohistochemistry and Western-blot. Results: CQ intervention reduced the level and number of seizures, as well as the latency of seizures, in the PTZ-induced epileptic model. Furthermore, as compared to the PTZ group, CQ reduced the excitability of hippocampus neurons according to patch clamp technique. And the number of pathological neurons in the hippocampal CA1 region decreased based on nissl staining. CQ therapy also prevented MDA and iron accumulation while increasing the expression of GSH, according to spectrophotometry..The expression of P62, LC3, Nrf2, GPX4 increased significantly and the level of Keap1 and PTGS2 decreased significantly based on immunohistochemistry and western-blot. Conclusion: CQ could reduce neuronal ferroptosis and seizures via the P62-Keap1-Nrf2-GPX4-ferroptosis pathway, and we preliminary investigated the possible link between autophagy and ferroptosis.