The NeuroProtect Formula: A Preventive Approach to AD Targeting the HIF-1/PI3K-AKT Signaling Pathway Evaluated through In Vivo, In Vitro, and Network Pharmacology Approaches

Objective: Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder with limited options for reversing its middle-to-late stages. Early intervention is crucial to slow down disease progression. This study aimed to investigate the potential of the NeuroProtect (NP) formula, a combination of geniposide and Panax notoginseng saponins, in preventing AD. We evaluated the effects of the NP formula on amyloid plaque accumulation, neuronal degeneration, and molecular signaling pathways using in vivo and in vitro models. Methods: To predict functional pathways and potential downstream targets of NP intervention, we employed network pharmacology. The preventative impact of the NP formula was assessed using APP/PS1 mice. We conducted HE staining, ELISA assay, Golgi staining, and immunohistochemistry to detect the protective effect of NP. Additionally, cell experiments were performed to assess cell activity and target protein expression. Results: Network pharmacology analysis revealed 145 drug-disease interactions and identified 5 core active targets associated with AD. Molecular docking results demonstrated strong binding affinity between the components of the NP formula (GP, GN-Rb1, GN-Rg1, NS-R1) and target proteins (STAT3, HIF1A, TLR4, mTOR, VEGFA). Notably, the binding energy between NS-R1 and mTOR was -11.4kcal/mol. Among the top 10 enriched KEGG pathways, the HIF-1 and PI3K-AKT signaling pathways were highlighted. In vivo experiments demonstrated that the NP formula significantly ameliorated pathological changes, decreased the Aβ42/Aβ40 ratio in the hippocampus and cortex, and increased dendritic spine density in the CA1 region during the early stage of AD. In vitro experiments further illustrated the NP formula’s ability to reverse the inhibitory effects of Aβ25-35 on cell viability and regulate the expression of Tlr4, Mtor, Hif1a, Stat3, and Vegfa. Conclusion: Our findings suggest that NP exhibits neuroprotective effects during the early stages of AD, positioning it as a potential candidate for AD prevention. The NP formula may exert its preventive effects through the HIF-1/PI3K-AKT signaling pathway, with mTOR identified as a key target.