基于超高效液相色谱四级杆-飞行时间串联质谱技术（Ultra High Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry/Mass Spectrometry, UHPLC-Q-TOF-MS/MS）分析与网络药理学探究地龙活性成分的抗疲劳作用机制。通过UHPLC-Q-TOF-MS/MS获得地龙化学成分信息，收集成分作用靶点、疲劳和抗疲劳的基因靶点取交集，进行基因本体（Gene Ontology, GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析。利用KEGG Mapper初步阐明抗疲劳机制，设计动物实验验证地龙抗疲劳功效。网络分析表明地龙抗疲劳主要集中在磷酸化的正向调节、核因子-κB（Nuclear Factor kappa-B, NF-κB）信号通路、5-羟色胺能突触、JAK-STAT通路和精氨酸和脯氨酸代谢通路等。动物实验结果表明，与空白组相比，地龙提取物中剂量组小鼠负重游泳实验时间由9.78 min延长至34.07 min（P<0.001），肝糖原含量显著升高，高剂量组为2 591.21 mg/g（P<0.001），高剂量组小鼠尿素氮和血乳酸含量分别降低至3.65 nmol/L和182.21 nmol/L（P<0.01）。综上，地龙通过多成分、多靶点、多途径的协同效应发挥抗疲劳作用，为后续的抗疲劳机制研究提供新的思路与方法。

The anti-fatigue mechanism of the active constituents of earthworm was clarified using ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry/mass spectrometry (UHPLC-Q-TOF-MS/MS and network pharmacology. UHPLC-Q-TOF-MS/MS was used to identify chemical constituents of earthworm extract. The targets of these constituents and fatigue and anti-fatigue associated genes were collected to identify the common targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. KEGG Mapper was utilized to preliminarily elucidate the anti-fatigue mechanism, and animal experiments were performed to verify the antifatigue efficacy of earthworm extract. Network analysis showed that the anti-fatigue effects of earthworm extract mainly function via positive regulation of phosphorylation, NF-κB signaling pathway, serotonergic synapse signaling, JAK-STAT pathway, and arginine and proline metabolic pathways. As shown in the results of animal experiments, the time of weight-bearing swimming in mice in the middle-dose earthworm extract group was 34.07 min, significantly longer than that (9.78 min) of the CK group (P<0.001). Hepatic glycogen content in the high-dose earthworm extract group was 2 591.21 mg/g, which was significantly higher than that in the CK group (P<0.001). Urea nitrogen and blood lactic acid contents in the high-dose earthworm extract group were 3.65 nmol/L and 182.21 nmol/L, respectively, which was significantly lower than those of the CK group (P<0.01). In conclusion, the antifatigue effect of earthworm extract was exerted through synergistic effects of multiple constituents, targets, and pathways, providing valuable insights and methods for further research into anti-fatigue mechanisms.

国家自然科学基金资助项目（22105145）