Gualou-Xiebai-derived Apigenin-7-O-glucoside inhibits VSMCs foam cell formation and alleviates atherosclerosis by targeting Rab7 to drive SNAP29-dependent lipophagy.

Background

Foam cell formation by vascular smooth muscle cells (VSMCs) is a key event in atherosclerotic plaque development. Lipophagy, a selective autophagy pathway governed by the small GTPase Rab7, helps maintain cellular lipid homeostasis, yet how Rab7-regulated lipophagy controls VSMC lipid balance in atherosclerosis (AS) remains unclear. The traditional formula Gualou-Xiebai herb pair (GLXB) is used clinically for chest-pain conditions now recognized AS and modulates lipid metabolism, but its molecular targets are undefined.

Purpose

To define the role of Rab7 in VSMC lipid homeostasis and lipophagy during AS, and to identify the GLXB constituents that engage Rab7.

Methods

An AS model was established in 6-week-old male apolipoprotein E-deficient (ApoE-/-) mice via a 12-week high-fat diet and confirmed by ultrasonography. Subsequently, the mice were treated with GLXB at doses of 3 or 12 g/kg for an additional 4 weeks. Therapeutic efficacy was evaluated using histological staining and transcriptomic (RNA sequencing [RNA-seq]) profiling, and core downstream targets were pinpointed via adeno-associated virus (AAV)-mediated Rab7 overexpression. Co-immunoprecipitation mass spectrometry (Co-IP-MS) was employed to identify Rab7 downstream targets, and the interaction between Rab7 and SNAP29 was subsequently validated using molecular docking, surface plasmon resonance (SPR), and co-immunoprecipitation (Co-IP) or glutathione S-transferase (GST) pull-down assays. In mouse aortic tissues, Rab7-SNAP29 colocalization, lipophagy, and autophagic flux were assessed via immunofluorescence, Western blotting, and an mCherry-GFP-LC3 tandem fluorescent reporter assay. In vitro, VSMCs challenged with oxidized low-density lipoprotein (ox-LDL; 80 μg/ml) were utilized to examine how GLXB-medicated serum or short hairpin RNA-mediated Rab7 silencing (sh-Rab7) modulated lipophagy. Finally, the direct binding of GLXB active monomers to Rab7 was confirmed by Co-IP, SPR, and drug affinity responsive target stability (DARTS) assays.

Results

GLXB improved dyslipidemia and reduced plaque burden in ApoE-/- mice. RNA‑seq placed Rab7 at the intersection of the enriched "Autophagy" and "Lipid and atherosclerosis" pathways. AAV‑mediated Rab7 overexpression stabilized plaques, improved lipid profiles, and enhanced autophagy in atherosclerotic lesions; conversely, blocking flux with chloroquine largely abrogated the protection afforded by both Rab7 and GLXB. Mechanistically, GTP‑bound Rab7 engaged the SNARE protein SNAP29 in a strictly GTPase‑dependent manner to promote autophagosome-lysosome fusion. Among GLXB constituents, apigenin‑7‑O‑glucoside (Api‑7‑O‑G) was the principal Rab7 ligand, with kaempferol and luteolin‑7‑O‑gentiobioside showing markedly weaker engagement. Api‑7‑O‑G bound to and stabilized Rab7, and mCherry‑GFP‑LC3 imaging confirmed it restored autophagic (lipophagic) flux and limited ox‑LDL‑driven lipid accumulation in VSMCs.

Conclusions

GLXB and its active constituent Api‑7‑O‑G mitigate AS by targeting Rab7 to initiate SNAP29‑dependent lipophagy, supporting Api‑7‑O‑G as a candidate agent for cardiovascular disease.