Breaking the Autophagy-Oxidative Stress Vicious Cycle in Alzheimer's Disease: Lactiflorin Unlocks P62 Dual Phosphorylation via ULK1 Targeting.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Paeonia lactiflora Pall. has a long history of medicinal use in traditional Chinese medicine. Classical materia medica and traditional practice describe Paeonia-related medicinal materials for headache, dizziness, restlessness, and other neurological or behavioral manifestations, particularly in disorders traditionally associated with blood or yin deficiency and liver-yang hyperactivity. Modern pharmacological studies have further demonstrated neuroprotective and antioxidant effects of Paeonia lactiflora and its bioactive monoterpene glycosides. Lactiflorin (LAC) is a naturally occurring monoterpene glycoside reported as a constituent of Paeonia lactiflora and exhibits antioxidant and cytoprotective properties; however, its therapeutic potential and underlying mechanisms in Alzheimer's disease (AD) remain unclear. This study therefore investigated the protective effects of LAC against AD and explored its underlying mechanisms. AIM OF THE STUDY: This study investigated whether LAC could break the self-reinforcing vicious cycle between autophagic dysfunction and oxidative stress in AD by engaging ULK1 to promote P62 phosphorylation at two key residues. MATERIALS AND METHODS: APP/PS1 transgenic mice and Aβ1-42-treated HT22 cells served as in vivo and in vitro AD models, respectively, with cognitive performance assessed through a battery of behavioral tests. Integrated bioinformatics and machine-learning analyses were applied to map AD-related molecular networks and prioritize candidate targets. Aβ deposition, autophagic flux, and oxidative stress were evaluated by immunofluorescence, Western blotting, biochemical assays, and the mCherry-EGFP-LC3 tandem fluorescent reporter system. The interaction between LAC and ULK1 was subsequently evaluated by molecular docking, molecular dynamics simulation, thermal shift assay, DARTS, and SPR. RESULTS: LAC alleviated cognitive deficits in male APP/PS1 mice and reduced hippocampal Aβ deposition. Bioinformatics analysis subsequently suggested that LAC may regulate AD-related pathology mainly through autophagy- and oxidative stress-associated networks. Consistent with these predictions, LAC increased the LC3-II/LC3-I ratio and decreased P62 expression, while also alleviating redox imbalance and lipid peroxidation in brain tissue and HT22 cells, as evidenced by decreased malondialdehyde (MDA) levels and restored superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. Mechanistically, LAC engaged ULK1 via a His24-dependent interaction, thereby enhancing P62 phosphorylation at Ser403/Ser351, activating the Keap1/Nrf2/HO-1 pathway, and restoring the Beclin-1/VPS34-associated autophagy-initiation machinery. CONCLUSIONS: LAC alleviated Aβ deposition, oxidative injury, and cognitive dysfunction by engaging ULK1, suggesting its potential as a promising natural compound for AD treatment.