Eupalinolide B alleviates ulcerative colitis by targeting HMGB1 through modulation of the HMGB1/TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND

Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease with limited therapeutic options. Eupalinolide B (EB), a guaianolide sesquiterpene lactone derived from Eupatorium lindleyanum DC., exhibits anti-inflammatory and immunomodulatory activities. This study investigated the therapeutic potential and mechanism of EB against UC.

METHODS

In vitro anti-inflammatory effects of EB were assessed in LPS-induced RAW264.7 and IEC-6 cells by measuring NO production and inflammatory cytokine mRNA levels (IL-6, IL-1β, TNF-α). In vivo, a DSS-induced UC mouse model was used to evaluate EB's efficacy through body weight, DAI score, immunohistochemistry, and inflammatory factor levels. Target identification employed activity-based protein profiling (ABPP) with an EB-based probe, followed by validation via pull-down, CETSA, SIP, and immunofluorescence assays. Binding affinity and site were characterized using isothermal titration calorimetry (ITC), LC-MS/MS, molecular docking, and mutagenesis. Molecular mechanism was explored by analyzing HMGB1/TLR4/MyD88/NF-κB pathway by WB, qPCR and co-precipitation.

RESULTS

EB exerted anti UC effects by alleviating inflammation and enhancing intestinal barrier integrity in vitro and in vivo. Using ABPP, high mobility group box-1 protein (HMGB1) was established as a high-confidence target of EB, characterized by the covalent binding of Cys106 residue. Mechanistically, EB suppressed the TLR4/MyD88/NF κB pathway and disrupted HMGB1 TLR4 interaction.

CONCLUSION

EB ameliorates UC and exerts anti-inflammatory effects by targeting HMGB1 at Cys106 residue. By downregulating HMGB1 protein expression and reducing the binding affinity between HMGB1 and TLR4, EB inhibits the TLR4/MyD88/NF-κB signaling pathway. This study provides theoretical support for the development of EB as a potential anti-UC candidate drug.