This study aimed to develop a novel herbal compound combination to attenuate neuroinflammation with a synergistic approach in vitro. Two novel herbal compound combinations targeting neuroinflammation with a synergistic approach have been identified through a series of studies using network pharmacology, molecular docking, and in vitro experimental verification. The major research findings are shown as follows: 1) AN-SG and BA-SG synergistically inhibited pro-inflammatory mediators, NO, IL-6 and TNF-α in LPS-induced mono-cultured microglial N11 cells; 2) The observed synergy of AN-SG and BA-SG was associated with downregulation of phosphor-MAPKp38/MAPKp38, iNOS and NF-κB p65 nuclear translocation. 3) AN-SG and BA-SG exhibited enhanced anti-neuroinflammatory activities than their individual component in the neuroinflammation tri-culture model and subsequently restored the endothelial tight junction, protected the neuronal survival and reduced p-tau expression. Chapter 3 constructed the compound-gene targets-signaling pathway networks, which highlight the hub gene targets and KEGG pathways for eight selected phytochemicals against neuroinflammation. The top five gene targets included MAPK14, MAPK8, NOS3, EGFR and SRC, and the top KEGG pathway was the MAPK signaling pathway. The molecular docking partly verified the network pharmacology results that demonstrated good bind affinities of all phytochemicals with MAPK14 and NOS3. In Chapter 4, two herbal-compound combinations, AN-SG and BA-SG, were found to exhibit synergistic effects (CI<1) on inhibiting NO, IL-6 and TNF-α productions in LPS-induced N11 cells. Western blot analysis suggested that the observed synergy was associated with downregulation of iNOS and phosphor-MAPKp38/MAPKp38 protein expressions, which were in line with the findings of network pharmacology and molecular docking studies. In Chapter 5, an LPS-induced neuroinflammation tri-culture model was established to further investigate the anti-neuroinflammatory effects of AN-SG and BA-SG. The LPS-induced neuroinflammation tri-culture model provides an efficient and practical cellular model for a wide range of investigations on neuroinflammation mechanisms and for screening potential compounds or drugs candidates to treat neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and schizophrenia (Wolfe, 2012). Our results showed that two combinations significantly reduced the NO, IL-6, and TNF-α in the N11 cell in the tri-culture model. They also protected the endothelial tight junction as evidenced by the honeycomb structure of ZO-1 tight junction protein, reduced permeability and restored TEER values in the MVEC cells. Furthermore, the combinations were found to restore the impaired cell viability and reduced p-tau protein expression in N2A cells in the tri-culture model
