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目的:本研究运用网络药理学方法结合体内外实验验证，以红藻氨酸(KA)诱导的大鼠癫痫模型和谷氨酸(L-Glu)诱导SH-SY5Y神经元细胞损伤模型阐明癫痫合剂抗癫痫作用及其主要机制。方法:通过数据库筛选癫痫合剂活性成分及潜在治疗靶点，构建蛋白互作(PPI)网络并分析核心靶点，进行GO和KEGG通路富集分析，采用Autodock Vina对核心成分与关键靶点进行分子对接。体内建立红藻氨酸大鼠癫痫模型，设正常对照组、模型对照组、癫痫合剂2、4 g/kg组及阳性对照卡马西平125 mg/kg组，观察癫痫合剂对大鼠脑电(EEG)、认知功能、海马神经元损伤及γ氨基丁酸/谷氨酸(GABA/Glu)等神经递质的影响。体外以谷氨酸9 mmol/L诱导SH-SY5Y细胞损伤进行机制研究，设空白对照组、模型对照组、阳性对照姜黄素2.5μmol/L组及癫痫合剂0.5、1 mg/mL组，采用CCK-8法检测细胞活力；Annexin V-FITC/PI双染法检测细胞凋亡率；JC-1探针检测线粒体膜电位变化；DCFH-DA探针检测细胞内ROS水平；Western blot法检测癫痫合剂对PI3K/AKT/mTOR通路相关蛋白磷酸化表达的影响。结果:网络药理学分析显示，癫痫合剂主要活性成分包括槲皮素、木犀草素、没食子酸、3,4,5-三甲氧基肉桂酸等，关键靶点包括mTOR、AKT1和TP53等。体内实验证实，癫痫合剂能明显抑制红藻氨酸诱导的癫痫发作(P<0.05或P<0.01),改善认知障碍，保护海马神经元，升高GABA、5-HT、DA、NA/NE含量，降低Glu含量(P<0.05或P<0.01)。以谷氨酸诱导神经元细胞损伤模型，癫痫合剂0.5、1 mg/mL组显著提高SH-SY5Y细胞活力，降低细胞凋亡率和ROS水平，降低线粒体膜电位(P<0.05或P<0.01),下调PI3K、AKT、mTOR蛋白磷酸化表达(P<0.05或P<0.01)。结论:癫痫合剂具有明确的抗癫痫及神经保护作用，其机制可能通过调控PI3K/AKT/mTOR信号通路，抑制谷氨酸诱导的神经元氧化应激与凋亡级联反应，并调节脑内GABA/Glu递质平衡，从而发挥整体抗癫痫作用。
Abstract：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. Methods:The active ingredients and potential therapeutic targets of Dianxian Heji were screened through databases to construct a protein-protein interaction(PPI) network and analyze the core targets, gene ontology(GO) and Kyoto Encyclopedia 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),cognitive 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 Dianxian Heji on the phosphorylation expressions of phosphatidylinositide 3-kinase(PI3K)/AKT/mammalian target of rapamycin(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-trimethoxycinnamic 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 impairment, 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 apoptosis 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.
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基本信息:
DOI：10.13412/j.cnki.zyyl.20260123.001
中图分类号:R29
引用信息:
[1]周文文,司丽君,郭君婷,等.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[J].中药药理与临床,2026,42(02):25-35.DOI:10.13412/j.cnki.zyyl.20260123.001.
基金信息:
新疆维吾尔自治区重大科技专项项目(编号：2022A03017-5); 中央级公益性科研院所基本科研业务费专项(编号：2023-RW360-01); 新疆维吾尔自治区重大科技专项项目(编号：2022A03006-1)
2026-01-26
2026-01-26
2026-01-26