Hydroxysafflor yellow A protects against colitis in mice by suppressing pyroptosis via inhibiting HK1/NLRP3/GSDMD and modulating gut microbiota

Hydroxysafflor yellow A (HSYA), a chalcone glycoside, is a component of Carthamus tinctorius L. and exerts anti-inflammatory and antioxidative effects. However, the therapeutic effect and the underlying mechanism of HSYA on ulcerative colitis is unclear. This study aimed to investigate the unexplored protective effects and underlying mechanisms of HSYA on UC. In vitro analyses showed that HSYA reduced the secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 and inhibited nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3)/gasdermin D (GSDMD)-mediated pyroptosis in lipopolysaccharide/ adenosine-5'-triphosphate (LPS/ATP)-stimulated macrophages. Gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites showed that HSYA reduced the increased levels of glucose, glucose 6-phosphate, and lactic acid, and inhibited the increased hexokinase 1 (HK1) expression caused by LPS/ATP stimulation. HK1 shRNA transfection further confirmed that HSYA inhibited the NLRP3/GSDMD-mediated pyroptosis via HK1 downregulation. In vivo analyses showed that HSYA drastically attenuated UC symptoms by relieving body weight loss, a decline in colon length, and inflammatory infiltration in colonic tissues induced by dextran sulfate sodium (DSS). HSYA also reduced the secretion of pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and IL-18. Moreover, HSYA inhibited HK1/NLRP3/GSDMD-mediated pyroptosis in DSS-induced colitis mice. Finally, 16S rRNA sequencing analyses of gut microbiota revealed that HSYA reversed gut microbiota dysbiosis by reducing the abundance of Proteobacteria and increasing that of Bacteroidetes. This study demonstrated that HSYA not only exerted anti-inflammatory effects by inhibiting HK1/NLRP3/GSDMD and suppressing pyroptosis but also regulated gut microbiota in mice with DSS-induced colitis. Our findings provide new experimental evidence that HSYA might be a potential candidate for treating inflammatory bowel diseases.