7,8-Dihydroxyflavone alleviates ulcerative colitis by binding keap1 to activate an slc7a11-dependent inhibition of NF-κB p65 signaling in macrophages

INTRODUCTION: Ulcerative colitis (UC) is characterized by episodic occurrences of bloody diarrhea and can lead to potentially fatal complications, highlighting the urgent need for effective and safe therapeutic options. OBJECTIVES: This study aims to conduct a mechanistic investigation into the repurposing of 7,8-Dihydroxyflavone (7,8-DHF), a bioactive flavonoid, for ameliorating the clinical symptoms of dextran sulfate sodium (DSS)-induced ulcerative colitis in mice of both sexes. METHOD: To explore the structural-activity relationship of 7,8-DHF, a series of flavone derivatives with different substitution patterns on the A-ring were synthesized. The active 7,8-DHF were evaluated in both LPS-induced RAW 264.7 macrophage and DSS-induced mice UC models. Then we used RNA-sequencing and 16s rRNA sequencing to identify the mechanism underlying of 7,8-DHF and then identified the critical role of slc7a11-mediated macrophage polarization combining with gene silencing and pharmacological intervention. The interaction between slc7a11 and NF-κB p65 in macrophage was verified through immunoprecipitation-mass spectrometry (IP-MS) and Co-Immunoprecipitation (CO-IP). Furthermore, Nrf2/keap1complex dissociation-induced slc7a11 activation was demonstrated using a biotinylated DHF probe in pull-down and molecular dynamic stimulation assays. RESULTS: Anti-colitis capacity of 7,8-DHF was superior to both 7-hydroxyflavone (7-HF) and 5-aminosalicylic acid (5-ASA) across all measured clinical and histological parameters. The protective effects of 7,8-DHF against DSS-induced colitis are attributed to the binding of its A-ring ortho-dihydroxy moiety to keap1, which leads to the activation of Nrf2 and subsequent upregulation of slc7a11 expression. The increased slc7a11 protein binds to NF-κB p65, sequestering it from the nucleus and thereby reducing NF-κB signaling. This modulation of the NF-κB pathway affects macrophage polarization and gut inflammation. Additionally, treatment with 7,8-DHF specifically increased the abundance of beneficial Akkermansia spp. and decreased pathobionts such as Escherichia-Shigella spp. in the DSS-induced colitis model. CONCLUSION: Our study underscores the potential therapeutic application of 7,8-DHF in managing inflammatory conditions, particularly ulcerative colitis.