Nimbolide competitively binds to the 14-3-3 epsilon with TFEB to promote lipophagy and ameliorate atherosclerosis.

BACKGROUND: Atherosclerosis, driven by macrophage foam cell formation and lipid accumulation, remains inadequately addressed by current pharmacotherapies, necessitating novel natural therapeutic strategies. Nimbolide (NIM), a limonoid derived from Azadirachta indica, possesses established anti-inflammatory properties, yet its efficacy against atherosclerosis and underlying mechanisms remain elusive. PURPOSE: The study was designed to assess the impact of NIM on treating atherosclerosis, focusing on cholesterol efflux and lipophagy. STUDY DESIGN: The study combined both in vivo atherosclerotic mouse modeling and in vitro macrophage foam cell studies. METHODS: Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet received NIM to assess therapeutic effect. Oxidized low-density lipoprotein was used to induce macrophage foam cell formation, and effects of NIM on cholesterol efflux and lipophagy were assessed by western blot, immunofluorescence and electron microscopy. Cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and co-immunoprecipitation (Co-IP) were employed to investigate molecular interactions. A TFEB-knockdown mouse model was used to evaluate the mediating role of transcription factor EB (TFEB). RESULTS: NIM administration dose-dependently attenuated aortic plaque burden, improved serum lipid profiles, and reduced vascular inflammation in ApoE-/- mice. In macrophages, NIM enhanced cholesterol efflux via upregulation of ATP-binding cassette transporter A1/G1 and activated lipophagy through TFEB nuclear translocation. Mechanistically, NIM directly bound 14-3-3 epsilon and competitively displaced TFEB, thereby promoting TFEB dephosphorylation and autophagy-lysosomal activation. TFEB knockdown abolished NIM-mediated lipophagy and atheroprotection both in vitro and in vivo. CONCLUSION: NIM exerts anti-atherosclerotic effects by disrupting the 14-3-3 epsilon/TFEB interaction to enhance macrophage cholesterol clearance and lipophagy, identifying this axis as a promising therapeutic target for atherosclerosis.