Hyodeoxycholic acid in Arisaema Cum Bile can alleviate pentylenetetrazol-induced epilepsy by activating p53/Nrf2/GPX4 to inhibit ferroptosis.

BACKGROUND: Epilepsy is a prevalent neurological disorder characterized by recurrent seizures and has limited effectiveness of available clinical treatments. Ferroptosis of neurons is one of the main mechanisms of epilepsy. Arisaema Cum Bile (ACB) is a traditional Chinese medicine that is prepared by fermenting animals bile with Rhizoma Arisaematis rhizomes, has demonstrated therapeutic effects on epilepsy and other nervous system diseases. Furthermore, the active components responsible for the anti-epileptic effects of ACB and the intrinsic mechanisms are still unclear.

PURPOSE: This study aims to clarify the principal bioactive constituents of ACB and their mechanisms in the treatment of epilepsy. It also seeks to provide data support for its clinical application in antiepileptic combination therapy.

METHOD: Pentylenetetrazol injections were used to induce epilepsy in rats. ACB and brain metabolites were identified via UPLC-Q-Exactive Orbitrap MS/MS, and active components were predicted through bioinformatics analysis. In vivo, ACB and hyodeoxycholic acid (HDCA) were administered by gavage, and the effects on oxidative stress, inflammation, and cognitive impairment were evaluated. Additionally, ferroptosis-related protein and gene expression were analyzed using western blot, PCR, immunofluorescence, and flow cytometry. In vitro, using primary neurons and HT22 cells, with electrophysiology employed to assess neuronal activity and glutamate used to induce ferroptosis in HT22 cells, in order to explore how HDCA alleviates epilepsy by inhibiting neuronal ferroptosis.

RESULT: A total of 37 chemical components were identified from the ACB, of which 10 active constituents can cross the blood-brain barrier, including steroids and terpenoids. Bioinformatics analysis suggests that HDCA may be one of the key active components responsible for ACB anti-epileptic effects and ferroptosis may represent a crucial underlying therapeutic mechanism. Studies in animals have demonstrated that treatment with ACB and HDCA reduced the frequency and severity of seizures in epileptic rats, mitigated hippocampal neuronal damage, improved post-epileptic cognitive learning capacity, alleviated neuroinflammatory injury and decreased markers of ferroptosis. This neuroprotective effect likely occurs through activation of the p53/Nrf2/GPX4 signaling pathway. Further in vitro cell experiments demonstrated that HDCA can inhibit the generation of action potentials in primary hippocampal neurons, protects HT22 neurons from glutamate-induced cell death, an effect that is reduced by Nrf2 inhibitors. These findings corroborate earlier bioinformatics analyses, confirming that the neuroprotective effects of HDCA are closely linked to Nrf2-mediated ferroptosis pathways.

CONCLUSION: HDCA is a key component of ACB anti-epileptic effects, potentially inhibiting ferroptosis through the p53/Nrf2/GPX4 pathway to reduce epilepsy.