Hydroxysafflor Yellow A Alleviates Diet-Induced Hepatic Steatosis Through Gut Microbiota Modulation and Suppression of STAT1/CD36 Signaling in Mice

Background: Hydroxysafflor yellow A (HSYA) is the main active monomer of safflower yellow (SY), an adjunct for cardiovascular/cerebrovascular diseases. Our previous studies demonstrated that SY/HSYA improved non-alcoholic fatty liver disease (NAFLD) in obese mice; however, the precise mechanisms remain unclear. This study aimed to investigate the molecular targets and mechanisms by which HSYA ameliorates NAFLD. Methods: Male C57BL/6J mice were fed a high-fat diet for 8 weeks to induce diet-induced obesity (DIO), followed by HSYA treatment (100 mg/kg/d) for 8 weeks and antibiotic or non-antibiotic treatment for another 5 weeks. Body weight, OGTT, IPITT, liver histology, liver transcriptomics, and fecal metagenomics were assessed. In vitro, HepG2 cells were treated with 400 μM of palmitic acid (PA) to induce steatosis, followed by HSYA treatment and/or cluster of differentiation 36 (CD36) overexpression. Results: HSYA significantly reduced body weight and fat mass and improved glucose metabolism in DIO mice. HSYA also alleviated hepatic steatosis, which was partly abolished by antibiotic treatment, indicating partial dependence on gut microbiota. Metagenomics revealed that HSYA increased the abundance of Akkermansia muciniphila, which negatively correlated with NAFLD scores. HSYA significantly downregulated CD36 and its upstream signal transducer and activator of transcription 1 (STAT1) protein expression in liver tissue by 59.5% and 35.4%, respectively. Similarly, HSYA reduced CD36 and phospho-STAT1 expression in PA-treated HepG2 cells, and these effects were abolished by CD36 overexpression. Conclusions: HSYA improved NAFLD by remodeling gut microbiota and by inhibiting the STAT1/CD36 pathway, thereby reducing fatty acid uptake in hepatocytes.