Primer sequence information.

Meiju Oral Liquid (MOL), a representative medicinal formula in China, stems from the traditional use of specific Chinese medicinal herbs known for their anti-fatigue properties, including rose, jujube, chicory, and wolfberry. While these individual herbs have been recognized for their benefits, the formulation of MOL itself has not been extensively studied. This study was designed to evaluate the potential anti-fatigue effects of MOL, prepared from these natural herbs, and to explore its underlying mechanisms. In this research, both mouse and zebrafish models were utilized to investigate the anti-fatigue effects of MOL. Chemical characterization of MOL and identification of bioactive compounds in serum were conducted using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The results demonstrated that MOL significantly prolonged the weight-bearing swimming time in mice, increased hepatic and muscle glycogen content, and reduced serum levels of blood urea nitrogen, blood lactate, and inflammatory markers (IL-1β, IL-6, TNF-α, and NO). Furthermore, MOL down-regulated the expression of NOX4 and TNF-α proteins while up-regulating p-PI3K and p-AKT proteins in the liver tissues of fatigued mice. In zebrafish models, MOL exhibited protective effects against sodium sulfite-induced lethality, enhanced high-speed motion trajectories, and increased movement distances in both normal and fatigued zebrafish. Additionally, MOL downregulated IL-1β, IL-6, TNF-α, and TNF-β mRNA levels while up-regulating PI3K and AKT1 mRNA levels in fatigued zebrafish. These findings suggested that the anti-fatigue effects of MOL may be mediated through the activation of the PI3K/AKT signaling pathway as well as the inhibition of TNF-α and NOX4 expression. In addition, a total of ninety-four chemical components were identified in MOL, with twenty-three migration compounds detected in mouse serum. These migration compounds are likely the primary active agents, contributing to the reduction of metabolite accumulation, enhancement of glycogen synthesis, and suppression of inflammatory responses. Taken together, our findings underscore the potential anti-fatigue effects of MOL, warranting further investigation into its therapeutic applications and the specific roles of its bioactive compounds.梅鞠口服液（Meiju Oral Liquid, MOL）是中国代表性药用方剂，源自具有抗疲劳特性的特定中草药的传统应用，包括玫瑰花、大枣、菊苣与枸杞子。尽管上述单味草药的益处已得到广泛认可，但MOL本身的组方尚未得到深入研究。本研究旨在评估由这些天然草药制备的MOL的潜在抗疲劳功效，并探究其潜在作用机制。本研究采用小鼠与斑马鱼两种模型，探究MOL的抗疲劳作用；同时采用超高效液相色谱-四极杆飞行时间质谱（ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, UPLC-Q-TOF/MS）对MOL进行化学表征，并鉴定血清中的生物活性成分。研究结果显示，MOL可显著延长小鼠的负重游泳时间，提升肝脏与肌肉糖原含量，并降低血清中血尿素氮、血乳酸及炎症标志物（IL-1β、IL-6、TNF-α与NO）的水平。此外，MOL可下调疲劳小鼠肝组织中NOX4与TNF-α蛋白的表达，同时上调p-PI3K与p-AKT蛋白的表达。在斑马鱼模型中，MOL对亚硫酸钠诱导的致死效应具有保护作用，可增强正常与疲劳斑马鱼的高速运动轨迹，并增加其运动距离。此外，MOL可下调疲劳斑马鱼体内IL-1β、IL-6、TNF-α与TNF-β的mRNA水平，同时上调PI3K与AKT1的mRNA水平。上述研究结果表明，MOL的抗疲劳作用可能通过激活PI3K/AKT信号通路，以及抑制TNF-α与NOX4的表达来介导。此外，本研究共在MOL中鉴定出94种化学成分，在小鼠血清中检测到23种迁移性化合物。这些迁移性化合物或为主要活性成分，可通过减少代谢产物积累、增强糖原合成并抑制炎症反应发挥抗疲劳功效。综上，本研究结果证实了MOL具有潜在的抗疲劳功效，为其临床治疗应用及其生物活性成分的具体作用机制的进一步研究提供了重要依据。