Abstract

The crosstalk between hepatocytes and macrophages is a key mechanism driving the pathogenesis of cholestatic liver injury (CLI). Glycyrrhetinic acid (GA), a bioactive compound derived from licorice, offers protection against CLI, but its effect on hepatocyte-macrophage crosstalk is not fully understood. This study aims to clarify the anti-inflammatory mechanisms of GA in blocking hepatocyte-macrophage interactions and identify its potential therapeutic targets. Wild-type mice was administrated α-naphthyl isothiocyanate (ANIT) intragastrically to induce CLI. The anti-inflammatory effects of GA were assessed by hepatic pathology, serum biochemistry, inflammatory cytokines expression, and modulation of acHMGB1/TLR2 axis. To recapitulate hepatocyte-macrophage crosstalk, primary mouse hepatocyte and macrophage was separately challenged with taurocholic acid (TCA) or HMGB1, co-treated with GA. Lastly, the contribution of TLR2 to the therapeutic action of GA was confirmed in TLR2-silenced RAW264.7 cells and Tlr2 -/- cholestasis model mice. The results showed that GA dose-dependently attenuated CLI and alleviated inflammatory responses, as corroborated by suppressed expression of hepatic pro-inflammatory cytokines and diminished inflammatory cells infiltration. Concurrently, GA lowered circulating HMGB1 and blunted hepatic TLR2/NF-κB pathway. Moreover, GA inhibited TCA-induced acHMGB1 release in hepatocytes, and reduced inflammatory responses in Raw264.7 cells stimulated by HMGB1. Silencing or pharmacologically inhibiting TLR2 abolished the anti-inflammatory activity of GA in RAW264.7 cells, and Tlr2 -/- mice displayed an attenuated responses to GA, confirming that TLR2 is an essential mediator of GA’s hepatoprotective effect. In conclusion, this study reveals that GA alleviates CLI by blocking acHMGB1/TLR2 axis, thereby interrupting crosstalk between hepatocytes and macrophages.