Fatigue, as a common symptom, affects people’s normal work and life. Long-term fatigue can induce chronic fatigue syndrome (CFS). It is a syndrome accompanied by a variety of symptoms, such as heart disease, aging, depression and other diseases (Natelson et al., 2019; Sharif et al., 2018). Energy expenditure, oxidative damage and immune system dysfunction are regarded as the main causes of CFS (Yang et al., 2023). Currently, most of the studies on anti-fatigue mechanisms have focused on the AMPK signaling pathway. AMPK mediates increased muscle complex I activity, ATP levels, and mitochondrial biogenesis for anti-fatigue effects. Nrf2 is a key transcription factor regulating anti-oxidant stress (Cui et al., 2020). AMPK phosphorylates Nrf2 and promotes its nuclear accumulation, activating AREs to restore cellular anti-oxidant capacity (Cui et al., 2020). It has been shown that the body exerts anti-oxidative stress by activating the Nrf2/HO-1 signaling pathway to enhance the ability of anti-fatigue (Zhang et al., 2019). AMPK is one of several metabolic sensors reported to transmit energy-dependent signals to the clock, AMPK transmits energy by driving phosphorylation of CRY and PER proteins (Jordan and Lamia, 2013). It suggests that the circadian rhythm signaling pathway is also associated with anti-fatigue.

The classic famous formulas are treasure parts of traditional chinese medicine (TCM) culture, the prominent feature of which is the efficient synergistic effects of multi-component, multi-target and multi-pathway (Hao et al., 2017). BYD is one of the first batch of ancient classic formulas published by the State Administration of Traditional Chinese Medicine and originated from the Ming Dynasty’s “Simplified Medical Enough”. It was composed of five well-known medicinal herbs, Astragalus membranaceus (Fisch.) Bunge, Panax ginseng C. A. Mey., Glycyrrhiza uralensis Fisch., Cinnamomum cassia Presl and Zingiberis Rhizoma Recens. In TCM theory, the main function of BYD is suitable to treat yuan-primordial qi weakness, deficiency and laborious timidity, and other related symptoms (Meng et al., 2021). Modern pharmacological research has identified that BYD has anti-fatigue, and anti-oxidant and improved immune function. In clinical research, BYD is treated in patients with qi deficiency and blood stasis syndrome of fatigue and chronic heart failure. Previous researches showed that anti-fatigue mechanism of BYD was elucidated by the metabolic pathways of differential metabolites, such as glycine, serine and threonine were analyzed and enriched mainly by Metabo Analyst (Yang et al., 2018). Modified BYD may alleviated fatigue by the AMPK/SIRT1/PGC-1α signaling pathway in skeletal muscle and heart (Wei et al., 2023). Currently, the mechanism has never been co-elucidated from AMPK, circadian rhythm and Nrf/HO-1 signaling pathways. Hence, targeting the AMPK, circadian rhythm and Nrf/HO-1 signaling pathways may lead to the development of new treatments and intervention strategies to further reveal the mechanism of BYD in relieving fatigue.

Herein, the UHPLC-Q-Exactive Orbitrap/MS was used to detect the main active ingredients in BYD. The anti-fatigue, anti-oxidant and anti-inflammatory effects of BYD were assessed on AB and Tg (lyz: dsRed) transgenic zebrafish. Furthermore, the integrated results of transcriptomic and network pharmacology by visual analysis were used to predict the main targets and signaling pathways by which BYD improves fatigue. Finally, to validate the anti-fatigue mechanism, mRNA expression was further examined by RT-qPCR. This combinatory multidisciplinary approach facilitates a more comprehensive understanding of the potential mechanisms by which BYD improves fatigue, provides more reliable evidence for the development and application of natural medicines.