β-Ecdysterone Attenuates Ang II–Induced Senescence in Human Aortic Smooth Muscle Cells via Autophagy Activation and ROS Suppression Through AKT/mTOR Pathway Inhibition

BACKGROUND: This study aimed to elucidate the protective effects of β-ecdysterone (β-Ecd) against premature senescence in human aortic smooth muscle cells (HASMCs) and to unravel the underlying mechanisms. METHODS: HASMCs' senescence was induced with angiotensin II (Ang II), and cells were then treated with β-Ecd. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cellular senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, cell cycle analysis, and western blotting for the senescence-associated proteins ‌tumor protein p53 (p53) and cyclin-dependent kinase inhibitor 1A (p21). IL-6 and MCP-1 levels in culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA). Autophagy was assessed by microtubule-associated protein 1A/1B light chain 3 (LC3) immunofluorescence, autolysosome staining, and western blotting for LC3 and sequestosome 1 (p62). Intracellular reactive oxygen species (ROS) were quantified by flow cytometry. Transcriptomic profiling using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA), along with analyses and molecular docking, was used to explore potential mechanisms, with key findings validated by western blot. RESULTS: Ang II induced pronounced senescence in HASMCs, characterized by increased SA-β-gal activity, elevated p53 and p21 expression, G0/G1 cell cycle arrest, impaired autophagic flux, increased ROS accumulation, and elevated secretion of IL-6 and MCP-1. CCK-8 assays confirmed that β-Ecd did not affect HASMCs' viability at concentrations up to 200 μM. Treatment with 200 μM β-Ecd effectively attenuated Ang II-induced senescence, restoring cell cycle distribution, reducing p53 and p21 expression, and suppressing IL-6 and MCP-1 secretion. β-Ecd also enhanced autophagic activity, as evidenced by increased LC3II levels, reduced p62 accumulation, and enhanced autophagosome-lysosome fusion, while significantly decreasing intracellular ROS levels. Inhibition of autophagy with bafilomycin A1 abolished the protective effects of β-Ecd. Transcriptomic and bioinformatics analyses revealed enrichment for pathways related to autophagy regulation, with a prominent role for the PI3K/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling axis. Consistently, western blot analysis showed that β-Ecd suppressed Ang II-induced phosphorylation of AKT and mTOR. Modulation of AKT activity further supported its involvement in β-Ecd-mediated protection, as AKT inhibition mimicked this effect. In contrast, AKT activation counteracted the pro-autophagic and anti-senescent effects of β-Ecd. Molecular docking further suggested favorable interactions between β-Ecd and AKT isoforms as well as mTOR. CONCLUSION: β-Ecd attenuates Ang II-induced premature senescence in HASMCs by enhancing autophagy and limiting oxidative stress, a process mediated by suppressed AKT/mTOR signaling.