Dynorphin/KOR inhibits neuronal autophagy by activating mTOR signaling pathway to prevent acute seizure epilepsy

Abstract

In previous studies, we found that dynorphin exerts antiepileptic effect by activating the kappa opioid receptor (KOR). However, the role of neuronal autophagy in dynorphin/KOR-mediated antiepileptic is still unclear. This study aimed to investigate the molecular mechanism of dynorphin's antiepileptic effect by inhibiting autophagy and reducing neuronal apoptosis. Here, a pilocarpine-induced rat model of epilepsy was established and hippocampal neurons were treated with Mg²⁺ -free exposed for epileptiform activity induction. The real-time polymerase chain reaction and Western blot analysis were used to evaluate messenger RNA and protein expression. The TdT-mediated dUTP-biotin nick end labeling staining and flow cytometry were used to analyze cell apoptosis in vivo and in vitro. Neuron cells viability was detected by Cell Counting Kit-8 assay. Immunofluorescent staining and green fluorescent protein-light chain 3 immunofluorescence were used to measure autophagy in vivo and in vitro. Results showed that overexpression of prodynorphin alleviated neuronal apoptosis, activated the mammalian target of rapamycin (mTOR) signaling pathway, and inhibited neuronal autophagy in epileptic rats. Dynorphin inhibited Mg²⁺ -free-induced seizure-like neuron apoptosis, partially reversing the effect of Mg²⁺ -free on the mTOR signaling pathway and seizure-like neuron autophagy. Further, using rapamycin, we found that dynorphin inhibited Mg²⁺ -free-induced seizure-like neuron autophagy and apoptosis by activating the mTOR signaling pathway. In conclusion, dynorphin inhibits autophagy by activating the mTOR signaling pathway and has a protective effect on epilepsy acute seizure and epilepsy-induced brain injury.

Keywords: autophagy; dynorphin; epilepsy; mTOR signaling pathway.