Osthol Ameliorates Alzheimer's Disease-Associated Cognitive Impairment by Targeting the Estrogen-Cholinergic Axis in Age-Stratified Female 3×Tg-AD Mice.

Alzheimer's disease (AD) represents a devastating neurodegenerative disorder globally. It is clinically characterized by cognitive dysfunction, predominantly learning and memory impairments, and is closely associated with cholinergic system damage. Osthol (OST), a bioactive compound with well-documented neuroprotective properties, has been shown to enhance learning and memory functions. However, the precise molecular mechanisms underlying its therapeutic effects on AD-related cognitive impairment remain unclear. This study elucidates the critical interplay between estrogen-cholinergic system imbalance and AD progression across distinct temporal windows in female AD models. By focusing on this sex-specific regulatory axis, we aimed to address a key gap in understanding AD pathogenesis and OST's targeted efficacy. We employed age-stratified 3×Tg-AD mice as an experimental model and utilized comprehensive behavioral paradigms to assess learning and memory functions. An age-dependent gradient of cognitive impairment, accompanied by varying degrees of neuropathological damage, was observed in 3×Tg AD mice across different age groups. OST significantly improved learning and memory performance in 3×Tg AD mice of all age groups. Western blotting, ELISA, and immunofluorescence staining indicated that OST treatment effectively alleviated damage to the estrogen-cholinergic-NGF axis through multiple synergistic mechanisms: upregulation of Acetylcholine (ACh), Choline acetyltransferase (ChAT), Tyrosine kinase-A (TrkA), and Nerve growth factor (NGF) expression; downregulation of Acetylcholinesterase (AChE) activity; and increased expression of Estradiol (E2), Estrogen receptor-α (ERα), and Estrogen receptor-β (ERβ). In addition, OST improved synaptic plasticity (Postsynaptic density protein-95 (PSD95), Synuclein (SYN), and Brain-derived neurotrophic factor (BDNF)), inhibited neuronal apoptosis (B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (BAX)), and enhanced neurotransmitter signaling cascades (γ-aminobutyric acid (GABA), Glutamic acid (Glu), Epinephrine (E), and ACh). These findings provide new insights into the mechanisms of AD and support OST as a promising candidate for targeted AD therapy.