目的 基于网络药理学、分子对接技术及动物实验，探讨防己黄芪汤抗疲劳的作用机制。方法 通过中药系统药理学数据库与分析平台（TCMSP）和HERB数据库以及文献检索，获取防己黄芪汤的活性成分；利用TCMSP数据库和SwissTarget Prediction平台确定其作用靶点，并通过Uniprot数据库对靶点进行标准化处理；以Genecards数据库获取疲劳主要靶点；从Venny 2.1.0获取防己黄芪汤与疲劳的交集靶点后，将其导入STRING 11.0平台进行蛋白质-蛋白质相互作用（PPI）分析，并利用CytoScape 3.9.1软件对PPI网络进行可视化处理；利用Metascape平台数据库对交集靶点进行基因本体（GO）和京都基因与基因组百科全书（KEGG）分析；借助CytoScape 3.9.1软件构建“药物-成分-靶点”网络图；使用Auto DockTools 1.5.6软件对活性成分与靶点进行分子对接；构建疲劳大鼠模型，并通过动物实验验证防己黄芪汤抗疲劳的作用机制。结果 获得防己黄芪汤活性成分156个，相应靶点889个；获得疲劳靶点849个；将疾病靶点与活性成分相关靶点取交集，获得防己黄芪汤抗疲劳的潜在作用靶点197个。从防己黄芪汤中筛选出槲皮素、β-谷甾醇、白桦脂酸等主要活性成分，SRC原癌基因（SRC）、信号转导和转录激活因子3（STAT3）、磷脂酰肌醇4,5-二磷酸3-激酶催化亚基α（PIK3CA）、磷脂酰肌醇-3-激酶调节亚基1（PIK3R1）等4个核心靶点。KEGG富集通路主要包括磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K-Akt）、缺氧诱导因子1（HIF-1）、糖尿病并发症中的AGE-RAGE（AGE-RAGE in diabetic complications）等信号通路。分子对接显示关键活性成分与核心靶点具有强烈的结合活性。动物实验显示，防己黄芪汤及阳性对照组可提升疲劳大鼠力竭游泳时间、体质量、脾脏及胸腺指数，改善大鼠尿素（UREA）、乳酸（LA）、肌酸激酶（CK）、血尿素氮（BUN）、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）及肌纤维形态改变、肌细胞损伤及退化情况，并通过上调疲劳大鼠肌肉组织中PI3K、Akt的表达量，下调p-PI3K、p-Akt、SRC、STAT3的表达量从而调控PI3K/Akt信号通路，以防己黄芪汤高剂量组效果最为显著。结论 初步提示防己黄芪汤的核心活性成分槲皮素、β-谷甾醇及白桦脂酸等可能通过调节氧化应激及能量代谢，作用于SRC、STAT3、PIK3CA、PIK3R1等核心靶点及PI3K/Akt信号通路发挥抗疲劳的作用。

Objective To explore the mechanism of Fangji Huangqi Decoction in anti-fatigue based on network pharmacology, molecular docking, and animal experiments. Methods Traditional Chinese Medicine Systems Pharmacology (TCMSP) and HERB databases, as well as literature searches, was used to obtain the active ingredients of Fangji Huangqi Decoction. Acquire the target sites from the TCMSP database and the Swiss Target Prediction platform, and standardize the target sites through the UniProt database. The main targets for fatigue were obtained from the Genecards database. After obtaining the intersection targets of Fangji Huangqi Decoction and fatigue using Venny 2.1.0, they were imported into the STRING 11.0 platform for protein-protein interaction (PPI) analysis. The PPI network was then visualized using Cytoscape 3.9.1 software. Enrichment analysis of GO functions and KEGG pathways for intersecting targets was conducted using the Metascape platform database. Construct the “drug-component-target” network diagram using Cytoscape 3.9.1 software. The active ingredients were docked with the target using AutoDock Tools version 1.5.6 software. Establish a fatigue rat model and validate the anti-fatigue mechanism of Fangji Huangqi Decoction through animal experiments. Results A total of 156 active ingredients were obtained from Fangji Huangqi Decoction, corresponding to 889 targets; A total of 849 targets were also related to fatigue. By intersecting disease targets with targets related to active ingredients, a total of 197 potential anti-fatigue targets of Fangji Huangqi Decoction were identified. The primary active components isolated from Fangji Huangqi Decoction include quercetin, β-sitosterol, and mairin (betula platyphylla fatty acid). The top four core targets identified were the SRC proto-oncogene (SRC), signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol-4,5-bisphosphate 3- kinase catalytic subunit alpha (PIK3CA), and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1). The KEGG enriched pathways mainly include the PI3K-Akt signaling pathway (PI3K-Akt signaling pathway), HIF-1 signaling pathway (HIF-1 signaling pathway), AGE-RAGE signaling pathway in diabetic complications (AGE-RAGE signaling pathway in diabetic complications), and other signaling pathways. Molecular docking reveals that the key active ingredients demonstrate strong binding affinity to the core target. Animal experiments have shown that both Fangji Huangqi Decoction and the positive control group can extend the exhaustive swimming time, increase body weight, and enhance spleen and thymus indices in fatigued rats. Additionally, they improved levels of urea (UREA), lactic acid (LA), creatine kinase (CK), blood urea nitrogen (BUN), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), as well as mitigated morphological changes in muscle fibers, myocyte damage, and degeneration. By upregulating the expression of PI3K and Akt while downregulating p-PI3K, p-Akt, SRC, and STAT3 in the muscle tissues of fatigued rats, they regulated the PI3K/Akt signaling pathway. The high-dose group of Fangji Huangqi Decoction demonstrated the most significant effects. Conclusion In summary, this study initially indicates that the principal active compounds of Fangji Huangqi Decoction, such as quercetin, β-sitosterol, and betula platyphylla fatty acids, may exert anti-fatigue effects by regulating oxidative stress and energy metabolism, targeting core elements including SRC, STAT3, PIK3CA, PIK3R1, and the PI3K/Akt signaling pathway.

R285.5

浙江省自然科学基金探索项目（LBY24H290001）；浙江省医药卫生科技计划项目（2025KY1653）；绍兴市科学技术局（2024A14033）；绍兴市卫生健康科技计划（2022KY007）