Identifying Baicalein as a Key Bioactive Compound in XueBiJing Targeting KEAP1: Implications for Antioxidant Effects

XueBiJing injection (XBJ) is renowned for its multi-target pharmacological effects, including immunomodulatory, antithrombotic, and antioxidant activities, offering potential therapeutic benefits for patients with severe infections such as sepsis and Coronavirus disease 2019 (COVID-19). Despite its clinical effectiveness, the molecular targets and mechanisms of XBJ remain unclear, warranting further investigation. This study aimed to identify the key bioactive compounds in XBJ and elucidate their molecular targets and mechanisms. The zebrafish model was first used to evaluate the anti-inflammatory and antioxidant effects of XBJ, and the differentially expressed genes (DEGs) were identified by RNA sequencing and network analysis. Network pharmacology was used to analyze the relationship between bioactive compounds and molecular targets, and molecular docking and kinetic simulation were used to explore the target binding ability of key compounds. Cellular Thermal Shift Assay-Western Blot (CETSA-WB) and Surface Plasmon Resonance (SPR) further verified the interaction between compounds and targets; finally, the key pathways were confirmed by gene silencing experiments. The zebrafish model results reveal that XBJ significantly reduced neutrophil and macrophage counts in a dose-dependent manner, emphasizing its potent anti-inflammatory effects. A transcriptomic analysis highlighted the differential expression of key genes in the KEAP1/NRF2 pathway, including HMOX1, SLC7A11, NQO1, and TXNRD1. A network analysis further pinpointed KEAP1 as a central molecular target, with tanshinone IIA, baicalein, and luteolin identified as key active compounds modulating this pathway. Among these, tanshinone IIA and baicalein exhibited strong binding interactions with KEAP1, which were confirmed through molecular docking and kinetic simulations. Further validation showed that baicalein directly targets KEAP1, as demonstrated by CETSA-WB and SPR analysis. Additionally, the gene silencing experiments of KEAP1 and NRF2 reinforced their crucial roles in activating the KEAP1/NRF2 pathway. These findings collectively establish baicalein as a critical bioactive compound in XBJ, driving its antioxidant and anti-inflammatory effects via KEAP1/NRF2 pathway activation through direct binding to KEAP1, providing new insights into the mechanism of action of XBJ.