Characterization of the bioactive compounds with efficacy against epilepsy from the herb pairs Polygala tenuifolia - Zizyphus jujuba  by modulating CHRNA4/CaMKII signaling pathway: LC-MS/MS combined with network pharmacology analysis and experimental evidence

Background: Epilepsy is a typical nervous system disorders identified by the spontaneous recurrence of seizures which injure periods of electroencephalographic activity and behavior. Traditional Chinese Medicine (TCM) herb pairs Polygala tenuifolia and Zizyphus jujuba have been used in treatment of epilepsy in China, while the mechanism of action still remains unclear. This article aims to disclose the substances and potential mechanisms of the anti-epilepsy activity of P. tenuifolia and Z. jujuba extract ( PZE ) using LC-MS/MS, network pharmacology, ethology and molecular biology methods. Methods: With the help of the self-built components database, identification of the chemical parameters of PZE was possessed through LC-MS/MS method, and the “ingredient-target-pathway” network of PZE was established through online databeses. Molecular docking was performed using Discovery Studio Visualizer. In the setting of the epilepsy model, pentylenetetrazol (PTZ, 10 mg/kg) was administered intraperitoneally injected for a period of 21 days. Mice were assessed for anxiety-like behavior by Elevated plus maze test, open field test, forced swimming test and tail suspension test. HE staining, western blotting, and immunofluorescence staining were used to detect morphological changes and signal pathway. Results: Through network analysis, 37 active ingredients were obtained from PZE , SLC6A4, CHRNA4 and MAOA and were found to play a major role in the PPI network. GO and KEGG analyses that display their anti-epilepsy activity. The"Ingredient-target-pathway"network diagram consists of 99 targets, 24 kinds of constituents, and 20 signaling pathways. The values of M15 and M17 show the largest degree. Molecular docking analysis shows the key components screened by network pharmacology have a good interaction with the predicted targets. Animal experiments results showed that: 1) PZE effectively lengthened the latent time of PTZ-induced epilepsy in mice model. 2) PTZ-induced depression-like behavior was strikingly ameliorated by PZE . 3) Hippocampal neurons are significantly shielded by PZE . 4) PZE was shown to play a key role in modulating the CHRNA4/CaMK II signaling pathway in to show anti-epilepsy potency. Conclusion: This study has successfully identified constituents of PZE through LC-MS/MS methods and predicted the potential targets and CHRNA4/CaMK II as potential signaling pathways of anti-epilepsy effects for PZE , which was proved by animal experiments. The results of this paper are conducive to the systematic elucidating of its mechanism of action and the development of TCM-based anti-epilepsy agents.