Theaflavin attenuates atherosclerosis progression by suppressing pyroptosis-mediated foam cell formation and promoting autophagy through modulation of GSK-3β activity in macrophages.
Key Findings
Results
Theaflavin treatment attenuated aortic plaque formation in ApoE-/- mice fed a high-fat diet.
Twelve male ApoE-/- mice were studied; after 6 weeks of high-fat feeding, mice received TF (10 mg/kg/day, intraperitoneally) or PBS for 6 weeks
Oil Red O staining was used to assess plaque formation
TF treatment reduced markers of pyroptosis including GSDMD-N, cleaved Caspase-1 (C-CASP1), NLRP3, and IL-1β in aortic tissue
The study used unpaired two-tailed t-tests or ANOVA with Dunnett's test for statistical analysis
Results
Theaflavin suppressed lipid uptake in macrophages overexpressing Gsdmd or Caspase-1 but had no effect in Gsdmd-deficient cells.
Macrophages were stimulated with ox-LDL (20 μg/mL) and treated with TF for 48 hours
Gsdmd/Caspase-1-overexpressing and Gsdmd-deficient Ana-1 mouse macrophages were used, generated via CRISPR/Cas9
The absence of effect in Gsdmd-deficient cells indicates TF's anti-foam cell action is GSDMD-dependent
This demonstrates that pyroptosis-mediated mechanisms are required for TF's effect on lipid uptake
Results
Theaflavin directly interacted with GSK-3β and reduced its phosphorylation at Tyr216.
Interaction between TF and GSK-3β was confirmed using molecular docking, cellular thermal shift assay, and co-immunoprecipitation
TF reduced phosphorylated GSK-3β at Tyr216 (p-GSK-3β(Tyr216)), indicating suppression of GSK-3β kinase activity
Phosphorylation at Tyr216 is associated with GSK-3β activation, so its reduction reflects TF-mediated inhibition
GSK-3β was identified as a direct molecular target of theaflavin
Results
Theaflavin promoted autophagy in macrophages through modulation of GSK-3β activity.
TF treatment promoted autophagy in ox-LDL-stimulated Ana-1 macrophages
Western blotting and immunofluorescence were among the methods used to assess autophagy markers
TF enhanced the interaction between GSK-3β and cathepsin B, suggesting a lysosomal/autophagic pathway involvement
Promotion of autophagy is proposed as a complementary mechanism alongside pyroptosis suppression in TF's anti-atherosclerotic effect
Results
Pyroptosis markers including GSDMD-N, NLRP3, cleaved Caspase-1, and IL-1β were elevated in human aortic tissues from atherosclerosis subjects compared to controls.
Human aortic tissues from both control subjects and subjects with atherosclerosis were examined
Immunofluorescence and Western blotting were used to assess pyroptosis-related protein expression
Elevated GSDMD-N, C-CASP1, NLRP3, and IL-1β in AS tissues established the clinical relevance of the pyroptosis pathway
These findings provided the rationale for targeting macrophage pyroptosis as a therapeutic strategy in atherosclerosis
Results
Theaflavin reduced inflammatory cytokine levels consistent with suppression of NLRP3 inflammasome-driven pyroptosis.
ELISA was used to measure cytokine levels including IL-1β
Reduced IL-1β is consistent with inhibition of NLRP3 inflammasome activation and Caspase-1-mediated cytokine processing
Both in vivo and in vitro models showed reductions in pyroptosis-related proteins
The NLRP3-Caspase-1-GSDMD axis was identified as the relevant pyroptotic pathway modulated by TF
What This Means
This research investigates whether theaflavin, a compound found in black tea, can slow the development of atherosclerosis (hardening of the arteries caused by plaque buildup). The researchers studied human artery tissue samples, mice prone to atherosclerosis, and immune cells called macrophages. They found that theaflavin reduced plaque formation in mice and decreased markers of a type of inflammatory cell death called pyroptosis, which contributes to the formation of 'foam cells' — fat-laden macrophages that are key contributors to arterial plaques.
The study identified GSK-3β, a cellular enzyme, as a direct molecular target of theaflavin. By binding to and reducing the activity of GSK-3β, theaflavin both suppressed pyroptosis and promoted autophagy — a cellular 'self-cleaning' process that helps remove lipid buildup. Importantly, when the researchers removed a key pyroptosis protein (GSDMD) from macrophages using gene editing, theaflavin lost its ability to reduce lipid uptake, confirming that its benefits depend on this specific cell death pathway.
This research suggests that theaflavin, a natural compound already present in commonly consumed tea, may have therapeutic potential for atherosclerosis by simultaneously blocking harmful inflammatory cell death and encouraging protective cellular housekeeping processes in immune cells. These findings identify GSK-3β as a possible drug target and support further investigation into theaflavin as a candidate for cardiovascular disease treatment, though studies were conducted in animals and cell models and further research in humans would be needed.
Yang S, Liao L, Yang Q, Xia T, Qin S, Tang Q, et al.. (2026). Theaflavin attenuates atherosclerosis by targeting GSK-3β to suppress pyroptosis and Promote Autophagy in macrophages.. Inflammation research : official journal of the European Histamine Research Society ... [et al.]. https://doi.org/10.1007/s00011-026-02343-1