A progressive discovery strategy for anti-Alzheimer’s drug identification: In vitro screening with an LPS-induced neuroinflammatory cell model and in vivo validation in 5xFAD mice

Background: Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. Cellular drug screening represents a key approach in the preclinical discovery of AD therapeutics. Moreover, selecting an appropriate cellular model is essential for exploring the mechanisms underlying AD pathogenesis. Objective: This study aimed to establish a progressive strategy integrating cell model construction, monomer evaluation, and effective component group-based drug discovery for the development of novel therapeutics targeting AD. Methods: A novel AD12 cell line stably expressing the double-transgenic genes amyloid precursor protein (APP)695/presenilin 1 (PS1)-ΔE9 was first constructed and subjected to lipopolysaccharide (LPS) stimulation. The resulting LPS-induced neuroinflammatory response was characterized using metabolomic analysis. Subsequently, 16 alkaloids derived from traditional Chinese medicine (TCM) were screened with the LPS-AD12 cell model. Molecular docking was then performed to assess the binding affinity of benzoylaconitine (BAC) with APP and PS1 proteins. A targeted metabolomics approach focusing on amino acids and the tricarboxylic acid (TCA) cycle was applied to BAC-treated LPS-AD12 cells. Finally, aconite extract was administered to five-familial-AD-mutations (5xFAD) transgenic mice, and therapeutic efficacy was evaluated through phenotypic and metabolomic analyses. Results: LPS stimulation induced neuroinflammation in AD12 cells, characterized by elevated levels of interleukin-6, amyloid-β 1-42 (Aβ 1-42 ), and PS1. The primary metabolic pathways affected in AD12 cells included alanine, aspartate, and glutamate metabolism, as well as arginine biosynthesis. Notably, BAC significantly reduced the levels of Aβ 1-42 and interleukin-6 in LPS-AD12 cells. Furthermore, molecular docking analyses revealed a strong binding affinity of BAC with APP and PS1 proteins. Targeted metabolomics analysis indicated that the pharmacological activity of BAC was primarily associated with its anti-inflammatory effects, modulation of the cholinergic signaling pathway, and regulation of energy metabolism. Behavioral experiments in 5xFAD mice, together with immunofluorescence assessments of brain tissues [amyloid-β (Aβ), glial fibrillary acidic protein, and ionized calcium-binding adapter molecule 1], demonstrated that aconite extract treatment significantly alleviated AD-related symptoms. In addition, metabolomics-based mechanistic analyses revealed that serine, acetylcholine, ornithine, and gamma-aminobutyric acid exhibited consistent trends in both brain tissue and serum samples from treated mice. Conclusion: This study established a novel AD12 cell model and an effective, reliable LPS-induced cell-based screening system. Additionally, we proposed a new drug discovery strategy integrating cell model construction, monomer evaluation, and effective component group-based drug discovery. This screening system has the potential to accelerate the identification of anti-AD agents and provides a feasible framework for subsequent in-depth investigations into new drug development from TCM, thereby contributing to the modernization of TCM-derived therapeutics.