目的 基于脑和肌肉组织芳香烃受体核转运类似蛋白 1（Brain and Muscle Arnt-Like-1，BMAL1）介导抗 氧化应激探讨葛根素减轻 Aβ25-35诱导海马神经元凋亡的分子机制。方法 （1）采用网络药理学分析筛选葛根素 干预阿尔茨海默病（AD）昼夜节律紊乱的潜在作用靶点，构建蛋白互作（PPI）网络并进行 GO 功能及 KEGG 通路 富集分析。通过分子对接、分子动力学模拟及药物亲和力靶标稳定性（DARTS）测定实验来验证葛根素与昼夜 节律主控因子 BMAL1 蛋白结合的稳定性。（2）提取小鼠原代海马神经元，采用免疫荧光法鉴定原代神经元，并 通过流式细胞术检测原代神经元的细胞纯度。通过 Aβ25-35处理原代海马神经元 47 h，建立 AD 细胞模型。细胞 分组及干预：正常对照组、AD 模型组，以及葛根素低、中、高剂量组（原代细胞先给予葛根素 12、24、 48 µmol·L-1预处理 1 h 后，再给予 Aβ25-35诱导损伤 47 h）。采用 Western Blot 法检测细胞相关蛋白的表达水平； 检测细胞还原型谷胱甘肽（GSH）、总超氧化物歧化酶（T-SOD）及过氧化氢酶（CAT）水平；TMRE 荧光探针法检 测细胞线粒体膜电位变化；流式细胞术检测细胞凋亡率、活性氧水平。结果 （1）共得到葛根素干预 AD 昼夜 节律紊乱的潜在作用靶点 86 个；20 个核心靶点与氧化应激发生、调控密切相关；潜在作用靶点涉及多条昼夜 节律、氧化应激及退行性病变相关信号通路；生物学过程集中于活性氧代谢过程、对氧化应激的反应、昼夜节 律、认知等部分；细胞组分集中于突触膜内、突触后膜、突触前膜等部分；分子功能集中于泛素连接酶、抗氧 化活性、蛋白磷酸酶结合等部分。分子模拟、分子对接、DARTS 实验均显示，葛根素能与 BMAL1 蛋白稳定结 合。（2）与正常对照组比较，AD 模型组细胞 BMAL1 蛋白表达量显著降低（P＜0.01）；活性氧水平显著升高（P＜ 0.01），GSH、T-SOD、CAT 水平及线粒体膜电位显著降低（P＜0.01）；细胞的总凋亡率显著升高（P＜0.01）， Bax 蛋白表达显著上调（P＜0.01），Bcl-2、PSD95、SYN 蛋白表达显著下调（P＜0.01）。与 AD 模型组比较，葛 根素中、高剂量组细胞 BMAL1 蛋白表达量显著升高（P＜0.01），活性氧水平显著降低（P＜0.01），GSH、CAT 水平及线粒体膜电位显著升高（P＜0.01），细胞的总凋亡率显著降低（P＜0.01），Bax 蛋白表达显著下调（P＜ 0.01），Bcl-2、PSD95、SYN 蛋白表达显著上调（P＜0.01），NRF2、HO-1 蛋白表达显著上调（P＜0.05，P＜ 0.01）；葛根素高剂量组细胞的 T-SOD 水平显著升高（P＜0.01）。结论 葛根素可通过 BMAL1 介导抗氧化应激 减轻 Aβ25-35诱导海马神经元凋亡，其作用机制可能与激活 BMAL1-NRF2/HO-1 通路相关。

Objective To investigate the molecular mechanism by which puerarin alleviates Aβ25-35-induced apoptosis in hippocampal neurons， focusing on Brain and Muscle Arnt-Like-1 （BMAL1）-mediated antioxidative stress. Methods （1） Network pharmacology analysis was employed to screen the potential targets of puerarin in ameliorating circadian rhythm disorder in Alzheimer’s disease （AD）. A protein-protein interaction （PPI） network was constructed， followed by GO functional enrichment and KEGG pathway analyses. Molecular docking， molecular dynamics simulation， and Drug Affinity Responsive Target Stability （DARTS） assays were conducted to validate the binding stability of puerarin with the core circadian regulator BMAL1 protein.（2） Primary mouse hippocampal neurons were extracted and identified via immunofluorescence， with purity assessed by flow cytometry. An AD cell model was established by treating primary hippocampal neurons with Aβ25-35 for 47 hours. Cell grouping and interventions were as follows：normal control group，AD model group，and low-，medium-，and high-dose puerarin groups （primary cells were pretreated with 12，24，and 48 µmol·L-1 puerarin for 1 hour，followed by Aβ25-35-induced injury for 47 hours）. Western Blot was used to detect the expression levels of relevant proteins；cellular levels of glutathione （GSH），total superoxide dismutase （T-SOD），and catalase （CAT） were measured；mitochondrial membrane potential was assessed using the TMRE fluorescent probe；apoptosis rate and reactive oxygen species （ROS） levels were determined by flow cytometry. Results （1） A total of 86 potential targets of puerarin for AD-related circadian rhythm disorder were identified；20 core targets were closely associated with the occurrence and regulation of oxidative stress. The potential targets were involved in multiple signaling pathways related to circadian rhythm， oxidative stress， and neurodegeneration. Biological processes were primarily enriched in reactive oxygen species metabolic process，response to oxidative stress，circadian rhythm，and cognition；cellular components were mainly concentrated in the synaptic membrane， postsynaptic membrane， and presynaptic membrane； molecular functions were primarily enriched in ubiquitin-protein ligase binding， antioxidant activity， and protein phosphatase binding. Molecular simulation， molecular docking，and DARTS assays consistently demonstrated stable binding between puerarin and BMAL1 protein. （2） Compared with the normal control group， the AD model group exhibited significantly reduced BMAL1 protein expression （P＜0.01）；significantly elevated ROS levels （P＜0.01）；significantly decreased levels of GSH，T-SOD， CAT，and mitochondrial membrane potential （P＜0.01）；and a significantly increased total apoptosis rate （P＜0.01）， with Bax protein expression significantly upregulated （P＜0.01） and Bcl-2， PSD95， and SYN protein expression significantly downregulated （P＜0.01）. Compared with the AD model group， the medium- and high-dose puerarin groups showed significantly increased BMAL1 protein expression （P＜0.01），significantly decreased ROS levels （P＜ 0.01）， significantly elevated GSH， CAT levels， and mitochondrial membrane potential （P＜0.01）， and a significantly reduced total apoptosis rate （P＜0.01）. Bax expression was significantly downregulated （P＜0.01），while Bcl-2， PSD95， SYN expression were significantly upregulated （P＜0.01）， NRF2 and HO-1 expression were significantly upregulated （P＜0.05， P＜0.01）. T-SOD levels were significantly increased in the high-dose puerarin group （P＜0.01）. Conclusion Puerarin attenuates Aβ25-35-induced apoptosis in hippocampal neurons by mediating antioxidative stress via BMAL1，and its mechanism may involve activation of the BMAL1-NRF2/HO-1 pathway.

R285.5

中国博士后科学基金面上项目（2023M732121）；山东省自然科学基金青年项目（ZR2023QH090）；徐州医科大学附属医院发展基金 优秀人才基金项目（XYFY202343）。