BACKGROUND: Disruption of the blood-brain barrier (BBB) is a critical pathological event in Alzheimer's disease (AD) progression. The ZeXieYin Formula (ZXYF), as described in the ancient Chinese medical text Huangdi Neijing, has shown multitarget neuroprotective effects and promising pharmacokinetics in preclinical studies with transgenic AD rodent models. Despite these findings, the exact molecular mechanisms by which its bioactive components interact with BBB regulatory pathways are not fully understood, necessitating comprehensive analysis through integrated systems pharmacology approaches.
PURPOSE: The primary objective of this study is to explore the protective properties of ZXYF against BBB disruption in the context of AD. Additionally, the investigation seeks to elucidate the molecular pathways underlying the therapeutic effects of ZXYF in this scenario.
METHODS: In vivo, APP/PS1 mice modeled AD. Y-maze and MWM tests assessed ZXYF's effects on cognition. Transmission electron microscopy (TEM) evaluated ZXYF's impact on BBB ultrastructure, while immunohistochemistry (IHC) and western blotting (WB) quantified tight junction (TJ) protein expression. Immunofluorescence detected GFAP (astrocytic marker), and ELISA measured hippocampal neuroinflammatory cytokines. Bioactive ZXYF components in systemic circulation were identified via UPLC-Q-TOF-MS/MS, followed by compound-target network construction and computational prioritization of AD pathways via multiplex network analysis. In vitro, ZXYF (2/6 mg/ml, 24 h) was applied to two BBB models: (1) LPS+TNF-α+IL-1α-stimulated bEnd.3 monolayers and (2) bEnd.3/C8-D1A astrocyte co-cultures. qPCR and WB assessed A1-specific astrocyte gene and protein expression. Molecular docking, Molecular dynamics (MD), Cellular thermal shift assay (CETSA) and Surface plasmon resonance (SPR) binding analysis simulations characterized ZXYF constituent binding to JAK2. Furthermore, siRNA was applied to knockdown JAK2 in the C8-D1A cell to further verify the role of JAK2/STAT3 pathway in ZXYF inhibition of A1 astrocyte activation.
RESULTS: Our findings demonstrated that ZXYF preserved BBB integrity and improved cognitive function in AD mice. Mechanistically, ZXYF restored TJ protein expression (ZO-1, occludin, claudin-5), attenuated astrocyte activation (GFAP↓), and reduced neuroinflammation. Systemic component analysis identified 13 major bioactive constituents of ZXYF. Network pharmacology and GO/KEGG enrichment revealed the JAK2/STAT3 pathway as the core mechanism underlying ZXYF's anti-AD effects. In vitro, ZXYF protected endothelial cells in co-cultured BBB models against LPS/cytokine-induced injury by suppressing A1 astrocyte polarization. Crucially, molecular docking/dynamics confirmed strong binding affinity of key ZXYF components to JAK2, while WB validated ZXYF-mediated inhibition of JAK2/STAT3 phosphorylation (p-JAK2↓, p-STAT3↓) in AD mice.
CONCLUSIONS: Our study shows that ZXYF significantly improved cognitive impairments and maintained BBB integrity in an AD mouse model. Compositional analysis revealed 13 major bioactive components of ZXYF. Mechanistically, ZXYF inhibited A1 astrocyte activation by suppressing the JAK2/STAT3 signaling pathway, enhancing endothelial barrier function. These results provide a mechanistic foundation for further investigation of ZXYF's therapeutic potential for AD and other cognitive disorders involving BBB dysfunction.
