Ginsenoside Ro attenuates aging-related cognitive impairment and associated neuroinflammatory changes via multi-target modulation of the PI3K-Akt axis: a convergent network pharmacology and experimental validation study.

The increasing global burden of aging-related cognitive impairment (ACI) highlights an urgent need for disease-modifying therapeutics, as current pharmacological options provide only temporary symptomatic relief without affecting underlying pathological trajectories. Ginsenoside Ro, an oleanane-type saponin derived from Panax ginseng, displays anti-inflammatory and cytoprotective properties; however, the target network and signaling mechanisms through which it may alleviate ACI remain inadequately defined. This study aims to thoroughly define the therapeutic efficacy and underlying molecular mechanisms of Ginsenoside Ro against ACI utilizing a convergent analytical approach that integrates network pharmacology, in silico molecular simulation, and empirical validation in aging-model mice and senescent PC12 cells. Putative targets of Ginsenoside Ro were sourced from PubChem, SwissTargetPrediction, STITCH, ChEMBL, and SEA databases. ACI-relevant and aging-associated targets were curated from HAGR, OMIM, and GeneCards databases. A protein-protein interaction (PPI) network was constructed using STRING, and hub targets were identified with the cytoHubba algorithm. Functional enrichment analysis, including GO and KEGG analyses, was performed via DAVID. Binding interactions were evaluated using molecular docking and 100 ns molecular dynamics (MD) simulations. In vivo characterization involved the development of a senescence mouse model via intraperitoneal D-galactose (D-Gal) administration (125 mg/kg/d for 10 weeks), followed by oral treatment with Ginsenoside Ro (5 and 10 mg/kg) or melatonin (100 mg/kg) for 6 weeks. Cognitive performance was assessed using the Morris water maze (MWM) and open field test (OFT). Systemic oxidative status was monitored by measuring serum MDA, SOD, and MAO, while neural tissue integrity was examined through H&E and Nissl staining. In vitro, D-Gal-induced senescent PC12 cells were treated with Ginsenoside Ro (3.125-12.5 µM), followed by evaluations of cell viability, SA-β-galactosidase activity, ROS accumulation, SOD function, and expression levels of TNF-α, IL-6, EGFR, and components of the PI3K-Akt cascade using CCK-8, flow cytometry, RT-qPCR, and Western blot. Target intersection analysis identified 30 overlapping nodes between Ginsenoside Ro and ACI, with TNF-α, IL-6, AKT1, and EGFR recognized as core targets. KEGG enrichment analysis highlighted PI3K-Akt signaling as a key mechanistic pathway. Molecular docking yielded binding energies ranging from - 8.8 to - 4.8 kcal/mol, while MD trajectories demonstrated RMSD/RMSF profiles, hydrogen-bond interactions, and distinct deep free-energy minima consistent with sustained complex formation over the simulation period. In the D-Gal mouse model, Ginsenoside Ro reduced escape latency, increased platform crossings and exploratory behavior, decreased serum MDA, restored SOD and MAO levels, and ameliorated neuronal loss and nuclear pyknosis in the hippocampus and cortex. In PC12 cells, Ginsenoside Ro improved viability, diminished SA-β-gal positivity and ROS accumulation, enhanced SOD activity, restored EGFR expression, suppressed IL-6/TNF-α induction, and normalized the elevated p-PI3K/PI3K and p-AKT/AKT ratios. These findings suggest that Ginsenoside Ro is associated with the modulation of TNF-α, IL-6, AKT1, and EGFR, and that the PI3K-Akt pathway may serve as a central axis correlated with its observed neuroprotective effects. Ginsenoside Ro emerges as a promising candidate for ameliorating aging-associated cognitive dysfunction and related pathological changes, providing a preclinical basis for future translational development.