Senkyunolide A interrupts TRAF6-HDAC3 interaction to epigenetically suppress c-MYC and attenuate cholestatic liver injury

• SenA alleviated cholangiocyte proliferation by inhibiting c-MYC-related H19-Let-7a axis activation in cholestasis. • SenA occupied binding site on TRAF6 that interacted with HDAC3 and rescuing HDAC3 from c-MYC promoters. • SenA promoted H3K9 deacetylation and subsequently inhibited c-MYC transcription. • This study identified SenA as a potential candidate drug for cholangiopathy-associated liver fibrosis. • This study unveiled a novel approach for discovering epigenetic regulators by targeting protein-protein interactions. Introduction Cholestatic liver diseases are highly prevalent and lack effective treatment, ultimately progressing to end-stage liver diseases. Our recent study indicates that the interplay between c-MYC and lncRNA H19 exacerbates the ductular reaction during cholestasis. Objective: This study aims to unveil the underlying mechanisms of the protective effects of senkyunolide A (SenA) on cholangiocyte overproliferation in cholestatic liver diseases. Methods : Through comprehensive characterization using RNA sequencing, CHIP analysis, protein truncation, amino acid mutation or deletion, and the development of SenA derivatives, we explored the effects and mechanisms of SenA in vivo in bile duct ligation mice and in vitro in primary cholangiocytes. Results : We demonstrated that SenA effectively mitigates cholangiocyte hyperproliferation by epigenetically suppressing c-MYC expression and disrupting the downstream H19, Let-7a and Lin28a. Mechanically, we identified a potential interaction between the carbonyl group in SenA and Arg483 in TRAF6, disrupting the TRAF6-HDAC3 complex. This dissociation facilitates the binding of HDAC3 to the MYC promoter region, resulting in enhanced histone deacetylation and transcriptional suppression. Conclusion : We highlight the therapeutic potential of SenA in cholestatic liver diseases by elucidating its role in epigenetic regulation.