Theaflavin attenuates atherosclerosis by targeting GSK-3β to suppress pyroptosis and Promote Autophagy in macrophages.

OBJECTIVE AND DESIGN: This experimental study investigated theaflavin (TF) as a potential treatment for atherosclerosis (AS), focusing on its regulation of macrophage pyroptosis and autophagy via GSK-3β. MATERIAL OR SUBJECTS: Human aortic tissues from controls and subjects with AS, twelve male ApoE-/- mice, and ox-LDL-induced Ana-1 mouse macrophages, including Gsdmd/Caspase-1-overexpressing and Gsdmd-deficient cells, were studied. TREATMENT: After 6 weeks of high-fat feeding, mice received TF (10 mg/kg/day, intraperitoneally) or PBS for 6 weeks; macrophages were stimulated with ox-LDL (20 μg/mL) and treated with TF for 48 h. METHODS: Oil Red O staining, immunofluorescence, Western blotting, ELISA, CRISPR/Cas9, molecular docking, cellular thermal shift assay, and co-immunoprecipitation were performed. Data were expressed as mean ± SD and analyzed using unpaired two-tailed t-tests or ANOVA with Dunnett's test. RESULTS: TF attenuated aortic plaque formation and reduced GSDMD-N, C-CASP1, NLRP3, and IL-1β. In vitro, TF suppressed lipid uptake in Gsdmd- or Caspase-1-overexpressing macrophages but showed no effect in Gsdmd-deficient cells. TF interacted with GSK-3β, reduced p-GSK-3β(Tyr216), promoted autophagy, and enhanced GSK-3β-cathepsin B interaction. CONCLUSIONS: TF attenuates AS progression by suppressing pyroptosis-mediated foam cell formation and promoting autophagy through modulation of GSK-3β activity.