基于网络药理学与体内外模型探讨癫痫合剂抗癫痫的作用机制OA

Objective:This study aims to elucidate the anti-epileptic effect and primary mechanism of Dianxian Heji(癫痫合剂)in the kainic acid(KA)-induced rat epilepsy model and L-glutamic acid(L-Glu)-induced neuronal cell SH-SY5Y damage model by using network pharmacology combined with in vivo and in vitro experiment validation.Meth-ods:The active ingredients and potential therapeutic targets of Dianxian Heji were screened through databases to con-struct a protein-protein interaction(PPI)network and analyze the core targets,gene ontology(GO)and Kyoto Encyclo-pedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed.The Autodock Vina was used to perform molecular docking between core ingredients and key targets.A KA-induced rat epilepsy model was established in vivo,and blank control group,model control group,Dianxian Heji groups(2 g/kg and 4 g/kg),and carbamazepine group(125 mg/kg,positive control)were set up.The effects of the Dianxian Heji on rats'electroencephalogram(EEG),cog-nitive function,hippocampal neuronal damage,and neurotransmitters such as gamma-aminobutyric acid(GABA)/Glu were observed.The mechanism of 9-mmol/L Glu-induced SH-SY5Y cell damage model was researched in vitro.Models were divided into a blank control group,a model control group,a curcumin group(2.5 μmol/L,positive control),and Dianxian Heji groups(0.5 and 1 mg/mL).Cell viability was assessed by cell counting kit-8(CCK-8)assay,apoptosis rate was detected by Annexin V-FITC/PI double staining,variation of mitochondrial membrane potential was detected by the JC-1 probe.Intracellular reactive oxygen species(ROS)level was detected by DCFH-DA probe,and the effect of Di-anxian Heji on the phosphorylation expressions of phosphatidylinositide 3-kinase(PI3K)/AKT/mammalian target of ra-pamycin(mTOR)pathway-related proteins was detected by Western blot.Results:Network pharmacology analysis showed that the primary active ingredients of the Dianxian Heji included quercetin,luteolin,gallic acid,3,4,5-trime-thoxycinnamic acid,etc.The key targets included mTOR,AKT1,TP53,mTOR,etc.In vivo experiments confirmed that the Dianxian Heji significantly inhibited KA-induced epileptic seizure(P<0.05 or P<0.01),improved cognitive impair-ment,protected hippocampal neurons,and corrected GABA/Glu neurotransmitter imbalance(P<0.05 or P<0.01).In a Glu-induced neuronal cell damage model,the Dianxian Heji significantly increased SH-SY5Y cell viability,reduced apop-tosis rate and ROS level,and improved mitochondrial membrane potential(P<0.05 or P<0.01).Western blot results showed that the Dianxian Heji significantly inhibited Glu-induced overactivation of the PI3K/AKT/mTOR pathway(P<0.05 or P<0.01).Conclusion:The Dianxian Heji has clear anti-epileptic and neuroprotective effects.Its mechanism may be related to regulating the PI3K/AKT/mTOR signaling pathway,inhibiting Glu-induced neuronal oxidative stress and apoptosis cascade,and regulating the GABA/Glu neurotransmitter balance in the brain,thereby exerting an overall anti-epileptic effect.