目的 基于转录组学探讨蜂毒肽对小鼠癫痫发作的改善效果及分子机制。方法 （1）采用腹腔注射匹罗 卡品诱导小鼠急性癫痫模型。将 20 只癫痫小鼠随机分为模型组、阴性对照组（人工脑脊液）及蜂毒肽低、中、 高剂量组（1 μmol·L -1 、2 μmol·L -1 、3 μmol·L -1 ），每组 4 只。采用脑电图检测痫样波频率和振幅确定蜂毒肽改 善急性癫痫发作的最佳剂量。（2）在确定蜂毒肽最佳干预浓度后，将 24 只癫痫小鼠随机分为模型组、阴性对照 组（人工脑脊液）和蜂毒肽组（2 μmol·L -1 ），每组 8 只。癫痫发作前向小鼠皮层注射相应浓度药物，脑电图检测 小鼠癫痫急性发作潜伏期。（3）选择 6 只小鼠，采用标准局部场电位（LFPs）记录法观察蜂毒肽急性灌流对小鼠 海马 CA1 区 θ 振荡发作持续时长、发作间期持续时长、簇放电频率、振幅的影响。（4）将 9 只雄性 C57BL/6 小鼠分为空白组（生理盐水）、阴性对照组（人工脑脊液）和蜂毒肽组（2 μmol·L -1 ），每组 3 只。各组小鼠皮层注 射相应药物 1 h 后提取小鼠脑皮层组织总 RNA 进行转录组学测序，筛选差异表达基因；利用 STRING 数据库 构建潜在作用靶点蛋白互作（PPI）网络，筛选出核心靶点，并进行 GO 功能和 KEGG 通路富集分析。 结果 （1）小鼠注射匹罗卡品后，小鼠出现Ⅱ-Ⅲ级 Racine 分级的癫痫发作症状，脑电图数据从正常背景活动 逐渐发展为离散及连续的癫痫样放电，且典型癫痫信号稳定持续超 1.5 h，成功构建了小鼠癫痫模型。与模型 组比较，蜂毒肽中、高剂量组小鼠的脑电图痫样波频率明显降低（P＜0.05，P＜0.01），阴性对照组、蜂毒肽低 剂量组小鼠的脑电图痫样波频率有降低的趋势，但差异无统计学意义（P＞0.05）。各组小鼠脑电图痫样波振幅 比较，差异无统计学意义（P＞0.05）。最终确定 2 μmol·L -1 为蜂毒肽的最佳给药浓度。（2）与模型组比较，蜂毒 肽组小鼠的癫痫急性发作潜伏期显著延长（P＜0.05）。（3）经蜂毒肽灌流后，卡巴胆碱小鼠海马区 θ 振荡的发作 持续时长缩短（P＜0.01），θ 振荡簇放电频率减小（P＜0.01），振幅降低（P＜0.05）；θ 振荡的发作持续时长和 θ 振荡簇放电频率在 50 μmol·L -1 卡巴胆碱冲洗后显著升高（P＜0.05）。但是蜂毒肽治疗对 θ 振荡发作间期持续 时长无影响（P＞0.05）。（4）与空白组比较，阴性对照组脑皮层组织中有 666 个差异表达基因，蜂毒肽治疗可逆 转其中 76 个差异表达基因，包括下调基因 20 个和上调基因 56 个。核心靶点为：BATF3、BST2、KLRB1C、 GABRA5、GRM5、NLGN、MAPK1、ATP1B1 等，潜在作用靶点主要参与调控 mTOR 信号通路、相关的炎症通 路和突触传递。结论 蜂毒肽可能通过调控 mTOR 信号通路，抑制神经炎症，恢复突触抑制功能，降低癫痫 发作频率并抑制神经网络同步化，从而发挥抗癫痫作用。

Objective To investigate the ameliorative effect and molecular mechanism of melittin on epileptic seizure in mice based on transcriptomics. Methods （1） An acute seizure model was induced in mice by intraperitoneal injection of pilocarpine. Twenty epileptic mice were randomly divided into five groups （n=4 per group）： the model group， a negative control group （artificial cerebrospinal fluid，aCSF），and low- （1 μmol·L -1 ），medium- （2 μmol·L -1 ）， and high-dose （3 μmol·L -1 ） melittin groups. The optimal dose of melittin for ameliorating acute seizure was determined by electroencephalogram detection of epileptic wave frequency and amplitude. （2） After determining the optimal concentration of melittin， 24 epileptic mice were randomly divided into three groups （n=8 per group）：the model group， the negative control group （aCSF）， and the melittin group （2 μmol·L -1 ）. The corresponding drugs were injected intracortically into mice before seizure induction. The seizure latency was detected by electroencephalogram. （3） Six mice were used to observe the effects of acute melittin perfusion on theta oscillation （θ oscillation） in the hippocampal CA1 region using standard local field potential （LFP） recordings，specifically measuring seizure duration， inter-cluster interval duration，cluster discharge frequency，and amplitude. （4） Nine male C57BL/6 mice were divided into three groups （n=3 per group）：a blank control group （saline），the negative control group （aCSF），and the melittin group （2 μmol·L -1 ）. Total RNA was extracted from the cerebral cortex tissues of mice 1 hour after intracortical injection for transcriptome sequencing to identify differentially expressed genes （DEGs）. A protein-protein interaction （PPI） network of potential targets was constructed using the STRING database to identify core targets，followed by Gene Ontology （GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. Results （1） After pilocarpine injection，mice exhibited seizure symptoms corresponding to Racine stages Ⅱ- Ⅲ . electroencephalogram data showed a progression from normal background activity to discrete and continuous epileptiform discharges，with typical seizure signals stably persisting for over 1.5 hours，indicating successful model establishment. Compared with the model group， the frequency of epileptiform waves in the EEG was significantly reduced in the medium-and high-dose melittin groups （P＜0.05， P＜0.01）. There was a trend towards reduced frequency in the negative control and low-dose melittin groups，but the difference was not statistically significant （P＞ 0.05）. No statistically significant differences were observed in the amplitude of epileptiform waves among the groups （P＞0.05）. Thus，2 μmol·L -1 was determined as the optimal concentration of melittin. （2） Compared with the model group，the melittin group showed a significantly prolonged seizure latency （P＜0.05）. （3） After melittin perfusion，the seizure duration of carbachol-induced θ oscillations in the hippocampal region was shortened （P＜0.01），the cluster discharge frequency was reduced （P＜0.01），and the amplitude was decreased （P＜0.05）. The seizure duration and cluster discharge frequency of θ oscillations significantly increased after 50 μmol·L -1 carbachol flushing （P＜0.05）. However，melittin treatment had no effect on the inter-cluster interval duration of θ oscillations （P＞0.05）. （4） Compared with the blank control group，666 DEGs were identified in the cortical tissues of the negative control group. Melittin treatment reversed 76 of these DEGs，including 20 downregulated and 56 upregulated genes. Core targets included BATF3，BST2，KLRB1C，GABRA5，GRM5，NLGN，MAPK1，ATP1B1，etc. The potential targets were primarily involved in regulating the mTOR signaling pathway， related inflammatory pathways， and synaptic transmission. Conclusion Melittin may exert its anti-seizure effects by regulating the mTOR signaling pathway， inhibiting neuroinflammation， restoring synaptic inhibitory function， reducing seizure frequency， and suppressing neural network synchronization.

R285.5

汕头市科技项目（200622165260881）；国家自然科学基金青年基金项目（82201629）；汕头大学医学院人才科研基金项目（510858055）。