Ameliorative Effects of Red-Fleshed Apple Flavonoid Extracts (RAFEs) on High-Fat Diet-Induced Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) in Mice

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease globally, yet effective therapeutic options remain limited. Red-fleshed apples are rich in dietary flavonoids, but their chemical basis and therapeutic potential for MASLD have not been systematically explored. This study integrated LC-MS/MS metabolomics with a high-fat diet (HFD)-induced MASLD mouse model to evaluate the therapeutic effects and mechanisms of 'XJ4' red-fleshed apple flavonoid extracts (RAFEs). Metabolomics identified 120 types of flavonoids in white-fleshed apple 'FJ' and red-fleshed apple 'XJ4', and 57 differentially accumulated metabolites have been both deteceted, among which 39 flavonoids significantly accumulated higher in 'XJ4'.Compared with 'FJ', 'XJ4' predominantly enriched in O-glycosylated flavonols, including isorhamnetin 3-O-glucoside, cacticin, tamarixin, reynoutrin, and guaijaverin. Male ICR mice were randomly divided into nine groups (n = 10): normal control, HFD model control, positive control (simvastatin, 10 mg/kg), and six groups receiving RAFEs or white-fleshed apple flavonoid extracts (WAFEs) at low, medium, or high doses (1, 3, 5 mg/kg). Hepatic parameters were assessed by histopathology, biochemical assays, RT-qPCR, immunofluorescence, and Western blotting; gut microbiota composition was analysed by 16S rRNA gene sequencing. Medium-dose RAFEs (3 mg/kg) conferred optimal efficacy, significantly reducing body weight gain, liver coefficient, and plasma ALT, AST, and ALP levels while restoring hepatic histological architecture. Mechanistically, RAFEs suppressed pro-inflammatory mediators (IL-6, IL-1β, NF-κB, IRF6, TLR4) and the oxidative stress marker CYP2E1, while enhancing antioxidant capacity (SOD, CAT, T-AOC). RAFEs also reduced hepatic TG, TC, and LDL-C, elevated HDL-C, and modulated lipid metabolism via AMPK and PPAR-α upregulation with α-SMA suppression. Furthermore, RAFEs restored gut microbiota diversity, enriched beneficial taxa (Lactobacillus johnsonii, Bifidobacterium pseudolongum, Bacteroides acidifaciens), and suppressed pathogenic Desulfovibrio fairfieldensis. RAFEs consistently outperformed WAFEs, attributable to 'XJ4's unique isorhamnetin-dominated flavonol glycoside profile. These findings support red-fleshed apple flavonoids as promising natural agents for MASLD treatment.