Interaction of bioactive kaempferol with HMGB1: Investigation by multi-spectroscopic and molecular simulation methods

Chronic and persistent inflammation associated with excessive high mobility group protein 1 (HMGB1) is a risk factor for various diseases. Dietary intake of kaempferol has been proven to be effective in reducing HMGB1 levels and the degree of inflammation, but the structural mechanism remains unclear. In this context, we first investigated the interaction between bioactive kaempferol and HMGB1 using multi-spectroscopic and molecular simulation techniques. The surface plasmon resonance (SPR) data indicated that kaempferol binds directly to HMGB1 with a K_d value of 2.89 × 10^-5 M. Binding of kaempferol with HMGB1 led to the intrinsic fluorescence quenching and modest secondary structure change of HMGB1 supported by fluorescence spectrometry and circular dichroism (CD). Using dynamic light scattering (DLS), it was found that kaempferol induced the aggregation of HMGB1 protein complex to form larger particles. On HMGB1-activated RAW264.7 cells, kaempferol co-incubation exhibited a remarkable inhibitory effect on nitric oxide (NO) release with an IC₅₀ value of 5.02 μM, which was lower than that of quercetin. In silico, kaempferol binds to HMGB1 mainly through hydrogen bonds and hydrophobic forces. Collectively, our study showed kaempferol as a potential HMGB1 inhibitor, mainly acting by direct binding to HMGB1 and inducing its conformational changes, which provides clues for the treatment of chronic inflammation by kaempferol.