Isoliquiritigenin Attenuates Neuronal Damage In Epileptogenesis Via NLRP3/CASPASE-1/GSDMD Signaling Pathway

Research backgroundEpilepsy is a common neurological disorder with complex and diverse initiation and triggering factors.Studies have shown that epileptogenic injury to the brain activates neuroinflammation and oxidative stress damage,increases neuronal excitability,lowers the epileptic threshold,and promotes seizures;conversely,seizures also exacerbate neurological inflammation,oxidative stress damage,and induce programmed cell death(PCD)in neuronal cells.Pyroptosis,a PCD dependent on inflammatory cysteinyl aspartate-specific proteinase-1(CASPASE-1)and accompanied by a large release of pro-inflammatory factors,is executed by Gasdermins(GSDMs)transmembrane pore-forming proteins.It can be activated in an NOD-like receptor thermal protein domain associated protein(NLRP3)inflammatory vesicle-dependent manner.Scorch death has been reported in both animal models of epilepsy and patients with epilepsy,but the mechanism of action of scorch death in epilepsy is unclear and still needs to be further explored.Isoliquiritigenin(ISL),a flavonoid isolated from licorice,exerts anti-inflammatory effects mainly by targeting inflammatory bodies and antioxidant functions by activating nuclear factor E2-related factor 2(NRF2).Our group’s previous study confirmed that ISL pretreatment attenuated the neuronal inflammatory damage caused by kainic acid(KA)in the early epileptogenesis,but whether it has some inhibitory effect on neuronal scorching in epileptogenesis still needs to be further explored.Research objectivesThe objective of this study was to investigate the pathogenic mechanism of pyroptosis pathway in epileptic diseases,explore the inhibitory effect of ISL on pyroptosis injury,and determine whether ISL plays an anti-pyroptosis and neuronal protective role by inhibiting the NLRP3/CASPASE-1/GSDMD participating signaling pathway.Research methods1.The epileptic mouse model was induced by the injection of KA into the lateral ventricle using stereostereoscope,and the epileptic mouse model was evaluated by Racine behavioral grading method and open field behavior test.2.Through transcriptome sequencing of hippocampal tissues of epileptic mice and normal mice,the function enrichment of differentially expressed genes and pyroptosis-related pathways was carried out,and disease-causing target genes highly associated with cell pyroptosis were screened.3.The microglia-neuron co-culture model was constructed,and the neuronal scorch damage model was induced by LPS and Nig combined stimulation,and the rescue experiment was performed by different ISL concentrations.The pyroptosis concentration of neurons induced by LPS+Nig and the optimal rescue concentration of ISL were determined by CCK-8 proliferation experiment.Cell damage was evaluated by flow cytometry.The contents and activities of reactive oxygen species(ROS),malondialdehyde(MDA),catalase(CAT)and superoxide dismutase(SOD)in neurons of each group were detected by chemical detection kit,and the oxidative stress damage was determined.The protein expression levels of NLRP3,CASPASE-1,cleaved CASPASE-1,GSDMD and GSDMD-nt were determined by Western Blot.The expression levels of IL-1β and IL-18 in cell culture medium were determined by ELISA.Research results1.KA injection into the lateral ventricle successfully induced the mouse epilepsy model.In the early stage of epilepsy,the mouse activity track was simple,the response was delayed,and the exploration behavior was reduced.2.By sequencing the hippocampal tissue transcriptome of epileptic mice and normal mice,functional enrichment of pyroptosis-related pathway was conducted for differentially expressed genes,and pathogenic target genes highly related to pyroptosis-related cells were screened out:Nlrp3,Caspase-1,Gsdmd.Western Blot further confirmed that NLRP3,CASPASE-1 and GSDMD were overexpressed in the hippocampus of epileptic mice.3.Under the stimulation of LPS 100μg/mL and Nig 2μM,the neuronal cell activity decreased,and some neuronal cell membranes were perforated with foam outflow under microscope.The rate of neuron injury was increased by flow cytometry.The contents of ROS and MDA increased,while the activities of CAT and SOD decreased.NLRP3,CASPASE-1,cleaved-CASPASE-1,GSDMD,GSDMD-nt were detected by Western Blot.Increased levels of Il-1β and IL-18 were detected by ELISA.4.ISL 2μM was administered after LPS and Nig stimulation.After ISL administration,the activity of neuron cells increased and the number of damaged cells decreased under microscope compared with that before administration.The rate of neuron injury was decreased by flow cytometry.The contents of ROS and MDA decreased,while the activities of CAT and SOD increased.NRF2 expression was increased by Western Blot,and NLRP3,CASPASE-1,cleaved-CASPASE-1,GSDMD,GSDMD-nt expression was decreased.The content of Il-1β and IL-18 in culture medium was decreased by ELISA.Research conclusionPyroptosis-related genes(Nlrp3,Caspase-1,Gsdmd)were significantly expressed in early epileptogenesis,and the pyroptosis pathway involved in NLRP3/CASPASE-1/GSDMD was activated in mouse hippocampal neurons.The herbal extract ISL inhibited the NLRP3/CASPASE-1/GSDMD pathway and attenuated neuronal damage,thus exerting a neuronal protective effect in epilepsy.This study provides a new theoretical basis and experimental basis for ISL to be a potential drug candidate to prevent and treat neurological injury.