Insights on the synergistic effects of glycyrrhetinic acid and isoliquiritigenin on HMGB1-induced inflammation by multi-spectroscopic and molecular docking studies

Chronic low-grade inflammation, driven by elevated levels of circulating high mobility group box1 (HMGB1), plays a crucial role in the pathogenesis of multiple chronic diseases. Glycyrrhetinic acid (GA) and isoliquiritigenin (ISL) are representative metabolites in licorice, exhibit potent regulatory effects on HMGB1-mediated chronic inflammation. In this research, we investigated the interactions of GA and ISL with HMGB1 using multi-spectroscopy, surface plasmon resonance (SPR), and molecular docking. Fluorescence spectroscopy revealed that GA or ISL binds to HMGB1, leading to static fluorescence quenching of the protein. Additionally, the binding of GA or ISL altered the microenvironment of tryptophan and decreased the α-helix content of HMGB1 to varying extents. Dynamic light scattering (DLS) showed that GA induced aggregation of the HMGB1 complex into large particles, while ISL reduced the particle size of HMGB1. SPR analysis confirmed that GA and ISL bound reversibly to HMGB1 with K d values of 53.0 ± 3.6 and 16.3 ± 0.5 μM, respectively. Molecular docking further elucidated the binding modes and sites of GA and ISL on HMGB1, showing that GA binds to the B-box and ISL binds to the A-box of HMGB1, with hydrogen bonding and hydrophobic interactions as the primary driving forces. Isobologram analysis demonstrated that the combination of GA and ISL exhibited a significant synergistic inhibitory effect on HMGB1-induced inflammation on RAW264.7 cells, with an optimal combination ratio of 1:1. This study reveals that GA and ISL synergistically exert anti-inflammatory effect by modulating HMGB1 conformation, offering new insights into licorice extracts for preventing chronic inflammatory diseases.