Withaferin A protects against epilepsy by promoting LCN2-mediated astrocyte polarization to stopping neuronal ferroptosis.

ABSTRACT

BACKGROUND: Epilepsy is among the most frequent severe brain diseases, with few treatment options available. Neuronal ferroptosis is an important pathogenic mechanism in epilepsy. As a result, addressing ferroptosis appears to be a promising treatment approach for epilepsy. Withaferin A (WFA) is a C28 steroidal lactone that has a broad range of neuroprotective properties. Nonetheless, the antiepileptic action of WFA and the intrinsic mechanism by which it inhibits ferroptosis following epilepsy remain unknown. PURPOSE: This study aimed at investigating to the antiepileptic potential of WFA in epilepsy, as well as to propose a potential therapeutic approach for epilepsy therapy. METHODS: We conducted extensive research to examine the impacts of WFA on epilepsy and ferroptosis, using the kainic acid (KA)-treated primary astrocyte as an in vitro model and KA-induced temporal lobe epilepsy mice as an in vivo model. To analyze the neuroprotective effects of WFA on epileptic mice, electroencephalogram (EEG) recording, Nissl staining, and neurological function assessments such as the Morris water maze (MWM) test, Y-maze test, Elevated-plus maze (O-maze) test, and Open field test were used. Furthermore, the mechanism behind the neuroprotective effect of WFA in epilepsy was investigated using the transcriptomics analysis and verified on epileptic patient and epileptic mouse samples using Western blotting (WB) and immunofluorescence (IF) staining. In addition, WB, IF staining and specific antagonists/agonists were used to investigate astrocyte polarization and the regulatory signaling pathways involved. More critically, ferroptosis was assessed utilizing lipocalin-2 (LCN2) overexpression cell lines, siRNA knockdown, JC-1 staining, WB, IF staining, flow cytometry, electron microscopy (TEM), and ferroptosis-related GSH and MDA indicators. RESULTS: In this study, we observed that WFA treatment reduced the number of recurrent seizures and time in seizure, and the loss of neurons in the hippocampal area in in epileptic mice, and even improved cognitive and anxiety impairment after epilepsy in a dose depend. Furthermore, WFA treatment was proven to enhance to the transformation of post-epileptic astrocytes from neurotoxic-type A1 to A2 astrocytes in both in vivo and in vitro experiments by inhibiting the phosphoinositide 3-kinase /AKT signaling pathway. At last, transcriptomics analysis in combination with functional experimental validation, it was discovered that WFA promoted astrocyte polarity transformation and then LCN2 in astrocytes, which inhibited neuronal ferroptosis to exert neuroprotective effects after epilepsy. In addition, we discovered significant astrocytic LCN2 expression in human TLE patient hippocampal samples. CONCLUSIONS: Taken together, for the first, our findings suggest that WFA has neuroprotective benefits in epilepsy by modulating astrocyte polarization, and that LCN2 may be a novel potential target for the prevention and treatment of ferroptosis after epilepsy.