Xinzhou Wang,1,* Shanshan Liu,1,* Zehao Lei,2 Panxia Cao,2 Weili Shi,1 Shuibo Gao,1 Hong Wu1,3 

1Second Clinical Medical College, Henan University of Chinese Medicine, Zhengzhou, 450002, People’s Republic of China; 2Graduate School, Henan University of Chinese Medicine, Zhengzhou, 450046, People’s Republic of China; 3Institute of Cardiovascular Disease, Henan University of Chinese Medicine, Zhengzhou, 450002, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Hong Wu, Email wuhong@hactcm.edu.cn

Background: Yiqi Huoxue Granule (YQHX), a traditional Chinese medicine (TCM) formulation, is extensively utilized for the treatment of atherosclerotic diseases. However, its active constituents and molecular mechanisms remain unclear. We utilized a systematic methodology to identify bioavailable compounds in vivo and predict and validate the principal targets and pathways responsible for their anti-atherosclerotic actions.
Methods: Serum pharmacochemistry utilizing UPLC-Q-Exactive Orbitrap-MS was employed to identify the bioavailable compounds of YQHX. An integrated methodology combining network pharmacology and molecular docking was implemented to predict its potential targets and mechanisms against atherosclerosis, which were subsequently verified experimentally in apolipoprotein E-deficient (ApoE–/–) mice.
Results: We identified 36 absorbable compounds in the serum of rats following YQHX administration, and 252 potential therapeutic targets were predicted. Protein-protein interaction analysis identified 10 hub targets, which are IL-6, TNF, EGFR, TP53, AKT, STAT3, SRC, CTNNB1, TLR4, and MMP-9. Enrichment analyses indicated that these targets are primarily involved in lipid metabolism and inflammatory responses, with significant enrichment in the PI3K-Akt and SRC signaling pathways. Molecular docking revealed strong binding affinities between the proteins EGFR, SRC, and AKT and their respective compounds. In ApoE–/– mice, YQHX significantly attenuated atherosclerotic plaque progression, enhanced lipid profiles, and inhibited systemic and plaque inflammation (decreased IL-6, IL-1β, sVCAM-1, and macrophage infiltration). Western blotting analysis revealed that these benefits were associated with the inhibition of SRC and AKT phosphorylation within the plaques.
Conclusion: This study systematically identified the bioactive compounds of YQHX and demonstrated its multi-target anti-atherosclerotic effect, which involved the enhancement of lipid metabolism and suppression of inflammation, mediated, at least in part, by the inhibition of the SRC/AKT signaling pathway.

Keywords: Yiqi Huoxue granule, atherosclerosis, UPLC-Q-Exactive Orbitrap-MS, network pharmacology, inflammation