Chemical ingredients and molecular mechanisms underlying Yiqi Xingnao oral liquid in alleviating cerebral ischemic stroke based on UHPLC-Q-TOF-MS/MS and integrated network pharmacology.

OBJECTIVES

Ischemic stroke is a worldwide problem associated with substantial morbidity, disability and mortality. Yiqi Xingnao oral liquid (YQXN), derived from Buyanghuanwu Decoction, is clinically effective but mechanistically unclear. This study aimed to identify blood-absorbed components of YQXN and elucidate its therapeutic material basis and potential molecular mechanisms.

METHODS

Middle cerebral artery occlusion rats modelled ischemic stroke, and chemical ingredients of YQXN were analysed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Network pharmacology, molecular docking analyses, enzyme linked immunosorbent assay (ELISA), and western blot were performed to identify potential targets and investigate the mechanisms of action of YQXN. Moreover, cellular thermal shift assay (CETSA) experiment was performed to evaluate the effect of formononetin on the thermal stability of STAT3 and PTGS2.

KEY FINDINGS: A total of 141 chemical compounds were identified, with 23 detected in systemic circulation. Key targets including STAT3, PTGS2, tumour necrosis factor-α, interleukin-6, and interleukin-1β were selected. Enrichment analyses indicated that YQXN may alleviate inflammatory responses by modulating the neuroactive ligand-receptor interaction pathway. Nine key active components (including calycosin, ononin, formononetin, cryptotanshinone, salvianolic acid G, senkyunolide A, ferulic acid, kaempferol, and caffeic acid) showed significant binding affinity for both PTGS2 and STAT3. Western blot and ELISA results confirmed that YQXN modulated the protein levels of these targets. Furthermore, a cell lysate-based CETSA showed that formononetin enhanced the thermal stability of STAT3 and PTGS2, supporting its potential target engagement with these proteins.

CONCLUSIONS

YQXN constituents target STAT3, PTGS2, and inflammatory cytokines to ameliorate ischemic stroke, providing a theoretical basis for its clinical application.