Molecular characterization of herbal medicine formulation Qi-Fu-Yin for the treatment of neuroinflammation in Alzheimer's disease

Abstract

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases and a major cause of progressive dementia. Aberrant activation of microglia is implicated in neuroinflammation and subsequent neurodegeneration in AD. The present thesis aims to examine the anti-neuroinflammatory action of traditional herbal formula Qi-Fu-Yin (QFY) in murine microglial cells.

Firstly, network pharmacology was employed to screen the active compounds of QFY, and to further predict the AD relevant protein targets and the affected pathways. The representative molecular functions and signaling pathways of the targets were analyzed by gene ontology (GO) and pathway enrichment. A total of 145 active compounds were selected from the seven herbal ingredients of QFY. AD-related biological targets (e.g., MAPT, APP, ACHE, iNOS, COX-2) were predicted to interact with the selected active compounds. Indeed, the enrichment analysis revealed that AD was the most significantly enriched pathway for the shortlisted targets.

Secondly, a bioactivity-guided fractionation approach was developed to investigate the anti-neuroinflammatory action of QFY and the principal active compounds. Different fractions of QFY were prepared by aqueous extraction, ethanolic precipitation and HPLC separation. All fractions were assayed for the downregulation of two key pro-inflammatory biomarkers iNOS and COX-2 in lipopolysaccharide(LPS)-stimulated microglial BV-2 cells by Western blot. We found that 90% ethanol solution of QFY downregulated iNOS in LPS-stimulated BV-2 cells but showed no suppressive effect against COX-2 induction. LC-MS/MS technology was employed to profile the chemical composition of the active fraction, suggesting ginsenosides, especially, Rg3, as the potent anti-neuroinflammatory compounds in QFY. This thesis demonstrated that herbal preparation QFY could ameliorate neuroinflammation via downregulating iNOS in microglia.

Thirdly, a chemical biology approach was developed to investigate whether 4-hydroxy-2,4'-dimethoxychalcone (glypallichalcone) could form covalent conjugate with proteins in LPS-activated microglia. We synthesized an azide-tagged glypallichalcone and immobilized it to alkyne agarose resin by “Click chemistry” azido-alkyne cycloaddition reaction. Glypallichalcone-bound proteins were separated from the cellular proteins of LPS-stimulated BV-2 cells by affinity isolation. Proteomic identification and Western blot verification suggested that lactate dehydrogenase A (LDHA), an anaerobic glycolytic enzyme, was the protein target for glypallichalcone. Molecular docking also simulated a high affinity between glypallichalcone and LDHA, as evidenced by the presence of several hydrogen bonds. Presumably, glypallichalcone intervenes with metabolic players of activated microglia, hence modulating their phenotype.

In conclusion, the present thesis employed multiple experimental approaches to characterize the active compounds of herbal formula QFY and protein targets relevant to AD pathology. Specifically, QFY may ameliorate neuroinflammation via selectively inhibiting pro-inflammatory mediator iNOS. In addition, active compound in QFY could covalently conjugate metabolic protein in activated microglia, which may subsequently affect the microglial phenotype in neuroinflammation. The results provide important information to understand the therapeutic effects of QFY towards AD. Ultimately, the active compounds may serve as lead compound for further development of anti-AD therapeutics.