Integrating serum pharmacology, network pharmacology, and molecular biology analysis to reveal the mechanisms of Baihe Dihuang decoction in treating Alzheimer’s disease

Aim: This study aimed to evaluate the potential neuroprotecive effects of Baihe Dihuang Decoction (BDD) in APP/PS1 double-transgenic (TG) mice and to investigate the role of the gut-brain axis (GBA) using an integrated approach combining serum pharmacology, network pharmacology, and molecular biology. Materials and methods: The blood-borne bioactive components of BDD were initially identified using UPLC-Q-Orbitrap HRMS. Subsequently, network pharmacology was employed to prioritize key therapeutic targets and elucidate the primary pathways underlying the anti-Alzheimer's disease (AD) effects of BDD. The neuroprotective efficacy of BDD in TG mice was systematically evaluated using the morris water maze (MWM) test, histopathological observation (HE staining), transmission electron microscope (TEM) test, and ELISA-based inflammatory cytokine assays. The potential mechanisms were further elucidated by integrating network pharmacology with 16S ribosomal RNA (16S rRNA) sequencing. Finally, molecular docking and Western blotting (WB) were performed to validate the interactions within the identified pathways. Results: A total of 49 BDD-derived compounds were identified in serum samples. Network pharmacology revealed 116 common targets of BDD against AD. Remarkably, KEGG analysis highlighted 57 signaling pathways potentially involved in the anti-AD effects of BDD. Pharmacodynamic analysis showed that BDD ameliorated cognitive impairment in TG mice, mitigated pathological damage, and suppressed the release of IL-6, IL-1β, and TNF-α in the colon, brain, and serum. Moreover, 16S rRNA sequencing indicated that BDD modulated gut microbiota (GM) structure and restored intestinal flora imbalance in TG mice. Integrative analysis of network pharmacology and GM analysis identified several Key pathways (FoxO, MAPK, PI3K-Akt, HIF-1, Th17, IL-17, and Toll/Imd) and core anti-AD targets (TLR4, PTGS2, SIRT1, BDNF, NF-κB, STAT3, JAK2, EGFR, GSK3β, and CD86). Molecular docking results showed that the five complexes with the lowest docking scores (TLR4-salidroside, NF-κB-glabranin, TRKB-alantolactone, PTGS2-3'_4'_dihydroxyflavone, and SIRTI-abietic acid) exhibited strong binding affinity. QSAR and WB analyses further demonstrated the modulatory effects of BDD on these five core targets. Conclusion: This study demonstrated that BDD effectively restored GM structure and ameliorated cognitive impairment in TG mice, thereby exerting therapeutic effects against AD. These findings support BDD as a potential traditional Chinese medicine (TCM) strategy for AD treatment.