School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 102488, China.
Dong'e Ejiao Co., Ltd., Liaocheng, 252200, China; Shandong Key Laboratory of Gelatine TCM Research and Development, Liaocheng, 252200, China; Shandong Technology Innovation Center of Gelatin-based Traditional Chinese Medicine, Liaocheng, 252200, China; National Engineering Technology Research Center for Gelatin-based Traditional Chinese Medicine, Liaocheng, 252200, China.
J Ethnopharmacol. 2024 Jun 28;328:118058. doi: 10.1016/j.jep.2024.118058. Epub 2024 Mar 20.
Baoyuan Decoction (BYD) was initially recorded in the classic of "Bo Ai Xin Jian" in the Ming dynasty. It is traditionally used for treating weakness and cowardice, and deficiency of vital energy. In researches related to anti-fatigue effects, the reciprocal regulation of AMPK and circadian clocks likely plays an important role in anti-fatigue mechanism, while it has not yet been revealed. Therefore, we elucidated the anti-fatigue mechanism of BYD through AMPK/CRY2/PER1 pathway.
To investigate the effect and mechanism of BYD in reducing fatigue, using pharmacodynamics, network pharmacology and transcriptomics through the AMPK/CRY2/PER1 signaling pathway.
Firstly, the chemical constituents of BYD were qualitatively identified by UHPLC-Q-Exactive Orbitrap/MS, establishing a comprehensive strategy with an in-house library, Xcalibur software and Pubchem combined. Secondly, a NaSO-induced fatigue model and 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH)-induced oxidative stress model were developed to evaluate the anti-fatigue and anti-oxidant activities of BYD using AB zebrafish. The anti-inflammatory activity of BYD was evaluated using CuSO-induced and tail cutting-induced Tg (lyz: dsRed) transgenic zebrafish inflammation models. Then, target screening was performed by Swiss ADME, GeneCards, OMIM and DrugBank databases, the network was constructed using Cytoscape 3.9.0. Transcriptome and network pharmacology technology were used to investigate the related signaling pathways and potential mechanisms after treatment with BYD, which were verified by real-time quantitative PCR (RT-qPCR).
In total, 114 compounds from the water extract of BYD were identified as major compounds. Na₂SO₃-induced fatigue model and AAPH-induced oxidative stress model indicated that BYD has significant anti-fatigue and antioxidant effects. Meanwhile, BYD showed significant anti-inflammatory effects on CuSO-induced and tail cutting-induced zebrafish inflammation models. The KEGG result of network pharmacology showed that the anti-fatigue function of BYD was mainly effected through AMPK signaling pathway. Besides, transcriptome analysis indicated that the circadian rhythm, AMPK and IL-17 signaling pathways were recommended as the main pathways related to the anti-fatigue effect of BYD. The RT-qPCR results showed that compared with a model control group, the treatment of BYD significantly elevated the expression mRNA of AMPK, CRY2 and PER1.
Herein, we identified 114 chemical constituents of BYD, performed zebrafish activity validation, while demonstrated that BYD can relieve fatigue by AMPK/CRY2/PER1 signaling pathway through network pharmacology and transcriptome.
保元汤（BYD）最初记录在明朝的《抱朴子》经典中。它传统上用于治疗虚弱和胆怯，以及气虚。在与抗疲劳效果相关的研究中，AMPK 和生物钟的相互调节可能在抗疲劳机制中发挥重要作用，但尚未揭示。因此，我们通过 AMPK/CRY2/PER1 通路阐明了 BYD 的抗疲劳机制。
通过 AMPK/CRY2/PER1 信号通路，利用药效学、网络药理学和转录组学研究 BYD 减轻疲劳的作用和机制。
首先，通过 UHPLC-Q-Exactive Orbitrap/MS 对 BYD 的化学成分进行定性鉴定，建立了一种综合策略，该策略使用内部库、Xcalibur 软件和 Pubchem 相结合。其次，建立 Na₂SO₃诱导的疲劳模型和 2,2'-偶氮双（2-甲基丙脒）二盐酸盐（AAPH）诱导的氧化应激模型，使用 AB 斑马鱼评估 BYD 的抗疲劳和抗氧化活性。使用 CuSO₄诱导和尾巴切割诱导的 Tg（lyz：dsRed）转基因斑马鱼炎症模型评估 BYD 的抗炎活性。然后，通过 Swiss ADME、GeneCards、OMIM 和 DrugBank 数据库进行靶标筛选，使用 Cytoscape 3.9.0 构建网络。利用转录组学和网络药理学技术，研究 BYD 治疗后的相关信号通路和潜在机制，并通过实时定量 PCR（RT-qPCR）进行验证。
共鉴定出水提 BYD 的 114 种主要化合物。Na₂SO₃诱导的疲劳模型和 AAPH 诱导的氧化应激模型表明，BYD 具有显著的抗疲劳和抗氧化作用。同时，BYD 对 CuSO₄诱导和尾巴切割诱导的斑马鱼炎症模型表现出显著的抗炎作用。网络药理学的 KEGG 结果表明，BYD 的抗疲劳功能主要通过 AMPK 信号通路发挥作用。此外，转录组分析表明，昼夜节律、AMPK 和 IL-17 信号通路被推荐为与 BYD 抗疲劳作用相关的主要通路。RT-qPCR 结果表明，与模型对照组相比，BYD 处理组显著上调了 AMPK、CRY2 和 PER1 的 mRNA 表达。
本文鉴定了 BYD 的 114 种化学成分，通过斑马鱼活性验证，通过网络药理学和转录组学证明 BYD 可以通过 AMPK/CRY2/PER1 信号通路缓解疲劳。
