【摘要】 目的 采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法（UHPLC-Q-TOF-MS/MS）、网络药理学和实验验证探究读书丸（DSP）防治阿尔茨海默病（AD）的作用机制和药效物质基础。方法 利用UHPLC-Q-TOF-MS/MS技术对读书丸的成分进行分析及鉴定，利用网络药理学及分子对接技术预测读书丸防治AD的关键靶点及通路。使用Aβ_25-35构建AD细胞模型，随机分为空白组、模型组、盐酸多奈哌齐组及读书丸各剂量组，Annexin V-FITC/Mito-Tracker Red双荧光染色法检测细胞凋亡及线粒体功能；通过活性氧（ROS）荧光探针检测读书丸对ROS生成的影响；而后选取高剂量组，采用Annexin V-FITC/PI双染色流式细胞术检测细胞凋亡；Western blot检测JAK2、p-JAK2、STAT3、p-STAT3蛋白表达。结果 UHPLC-Q-TOF-MS/MS共鉴定出读书丸化学成分162个，经网络药理学技术筛选后得到58个活性成分，成分与疾病交集靶点226个，筛选出核心靶点为STAT3、SRC、HSP90AA1、PIK3R1、PIK3CA。KEGG富集分析结果显示，读书丸可能通过癌症的途径、PI3K-Akt信号通路、JAK2/STAT3信号通路对AD起作用。细胞实验研究表明，读书丸能降低细胞凋亡率，有效抑制细胞内ROS的异常增加，降低p-STAT3与p-JAK2的蛋白表达。结论 读书丸可以通过多成分、多靶点改善AD，其作用机制可能与调控JAK2/STAT3信号通路，抑制细胞凋亡有关。更多还原

【Abstract】 Objective To determine the mechanism and pharmacodynamic material basis of Dushu pills（DSP） in preventing and treating Alzheimer’s disease（AD） by ultra-high performance liquid chromatography-quadrupole-orbitrap tandem mass spectrometry（UHPLC-Q-TOF-MS/MS）, network pharmacology, and experiment verification. Methods The components of DSP lyophilized powder were analyzed and characterized by UHPLC-Q-TOF-MS/MS. Network pharmacology and molecular docking were used to predict the key targets and pathways of DSP in AD prevention and treatment. The AD cell models were established with Aβ_25-35 and randomly divided into a blank group, a model group, a donepezil hydrochloride group, and DSP groups of different doses. Annexin V-FITC/Mito-Tracker Red double fluorescent staining was used to detect cell apoptosis and mitochondrial function; reactive oxygen species（ROS） fluorescent probe was applied to determine the effect of DSP on ROS production; fand Annexin V-FITC/PI double staining combined with flow cytometry was performed to detect cell apoptosis; Western blot was performed to measure the protein expressions of JAK2, p-JAK2, STAT3, and p-STAT3. Results UHPLC-Q-TOF-MS/MS identified 162 chemical components in DSP. After screening via network pharmacology, 58 active components were obtained. Network pharmacology revealed 226 overlapping targets among these components and AD, with STAT3, SRC, HSP90 AA1, PIK3R1, and PIK3 CA being identified as the core targets. KEGG enrichment analysis indicated that DSP exerted therapeutic effect on AD through the cancer pathway, PI3K-Akt signaling pathway, and JAK2/STAT3 signaling pathway. Cell experiments demonstrated that DSP reduced the apoptosis rate, effectively inhibited the abnormal increase of intracellular ROS, and downregulated the protein expressions of p-STAT3 and p-JAK2. Conclusion DSP improves AD by multi-component and multi-target regulatory effects, whose mechanism may be associated with regulating the JAK2/STAT3 signaling pathway to inhibit the cell apoptosis.更多还原