目的 基于磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）通路探究桂花醇提物抗慢性脑缺血性神经损伤的保护作用与机制。方法 借助文献检索、TCMSP、BATMAN数据库筛选桂花活性成分及靶点；OMIM、GeneCards疾病数据库检索疾病靶点。取交集靶点构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络。借助DAVID数据库进行基因本体（gene ontology，GO）功能和京都基因和基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，并建立“成分-靶点-通路”网络。采用右侧颈总动脉结扎法建立慢性脑缺血小鼠模型，随机分为假手术组、模型组、脑络通（195 mg/kg）组和桂花醇提物低、高剂量（125、375 mg/kg）组。采用旷场实验评价各组小鼠自发能力及探索行为；检测各组小鼠海马组织胆碱乙酰化酶（choline acetylase，ChAC）和胆碱酯酶（cholinesterase，ChE）水平；采用苏木素-伊红（HE）和TUNEL染色观察脑组织病理结构变化及神经元凋亡情况；采用Western blotting检测海马组织PI3K/Akt信号通路及凋亡相关蛋白表达。结果 网络药理学筛选得到12种桂花活性成分及潜在靶点289个，慢性脑缺血疾病靶点3014个，桂花与慢性脑缺血的交集靶点198个，其中AKT1、血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）、半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）等为核心靶点。通过KEGG和GO分析进一步发现，PI3K/Akt信号转导通路可能是桂花发挥慢性脑缺血神经保护的重要途径。采用右侧颈总动脉结扎法复制慢性脑缺血小鼠模型，结果显示，桂花醇提物可显著升高脑组织ChAC水平（P<0.001），降低ChE水平（P<0.001），增强中枢胆碱系统功能；明显提高脑缺血小鼠站立次数及中央区域运动距离（P<0.001），以改善认知等神经功能障碍，并抑制缺血区脑组织的神经元凋亡（P<0.001）。其作用机制与调控PI3K/Akt信号通路，上调p-PI3K、p-Akt、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）蛋白表达（P<0.05、0.01），同时下调促凋亡细胞色素C（cytoehrome C，Cyt-C）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、cleaved Caspase-3蛋白表达相关（P<0.01）。结论 桂花醇提物可有效抑制脑缺血性神经细胞损伤，改善神经功能障碍，其脑神经保护作用与调控PI3K/Akt通路、抑制细胞凋亡有关。

Objective To explore the protective effect and mechanism of Osmanthus fragrans ethanol extract against chronic cerebral ischemic nerve injury based on phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. Methods The active components and targets of O. fragrans were screened by literature, TCMSP and BATMAN database; OMIM and GeneCards disease databases were used to search for disease targets. Protein-protein interaction (PPI) network was constructed by taking intersecting targets. With the help of DAVID database, gene ontology function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were carried out, and “component-target-pathway” network was established. The model of chronic cerebral ischemia in mice was established by ligating the right common carotid artery. The mice were randomly divided into sham group, model group, Naoluotong (脑络通, 195 mg/kg) group and O. fragrans ethanol extract low- and high-dose (125, 375 mg/kg) groups. Open field experiments was used to evaluate the spontaneous ability and exploratory behavior of mice in each group; Levels of choline acetylase (ChAC) and cholinesterase (ChE) in hippocampus of mice in each group were detected; HE and TUNEL staining were used to observe pathological changes in brain tissue and neuronal apoptosis; Western blotting was used to detect PI3K/Akt signaling pathway and apoptosis-related protein expressions in hippocampal tissue. Results A total of 12 active ingredients and 289 potential targets of O. fragrans, 3014 targets of chronic cerebral ischemia disease, and 198 intersection targets of O. fragrans and chronic cerebral ischemia were screened by network pharmacology, among which AKT1, vascular endothelial growth factor A (VEGFA), and cysteine aspartic protease-3 (Caspase-3) were core targets. Further analysis using KEGG and GO revealed that PI3K/Akt signaling pathway may be an important pathway for O. fragrans to exert neuroprotective effects on chronic cerebral ischemia. The mouse model of chronic cerebral ischemia was replicated using the ligation of the right common carotid artery. The results showed that O. fragrans ethanol extract significantly increased ChAC level in brain tissue (P < 0.001), decreased ChE level (P < 0.001), and enhanced the function of the central cholinergic system; Significantly increased the number of standing times and central area movement distance in mice with cerebral ischemia (P < 0.001), improved cognitive and other neurological dysfunction, and inhibited neuronal apoptosis in the ischemic brain tissue (P < 0.001). Its mechanism was related to regulating PI3K/Akt signaling pathway, up-regulating the expressions of p-PI3K, p-Akt and B-cell lymphoma-2 (Bcl-2) proteins (P < 0.05, 0.01), and down-regulating the expressions of cytochrome C (Cyt-C), Bcl-2 associated X protein (Bax) and cleaved Caspase-3 proteins (P < 0.01). Conclusion O. fragrans ethanol extract can effectively inhibit the damage of cerebral ischemic nerve cells and improve the neurological dysfunction. Its protective effect on cranial nerves is related to the regulation of PI3K/Akt pathway and inhibition of cell apoptosis.

R285.5

四川省科技厅重点研发项目（2020YFS0325）；成都医学院研究生创新科研基金项目（YCX2022-01-30）