Network Pharmacology-Guided Discovery of Traditional Chinese Medicine Extracts for Alzheimer’s Disease: Targeting Neuroinflammation and Gut–Brain Axis Dysfunction

Neuroinflammation plays a central role in the pathogenesis of Alzheimer's disease (AD), with amyloid-β (Aβ) deposition and neurofibrillary tangles driving both central and peripheral inflammatory responses. This study investigated the neuroprotective and anti-inflammatory effects of <i>Vitex trifolia</i> (VT), <i>Plantago major</i> (PM), <i>Apocyni Veneti</i> Folium (AVF), and <i>Eucommiae folium</i> (EF) using network pharmacology and a co-culture model of PC12 neuronal and Caco-2 intestinal epithelial cells. Bioactive compounds were identified via high-performance liquid chromatography (HPLC) and screened with network pharmacology analysis, yielding 27 for VT, 10 for PM, 6 for AVF, and 3 for EF. Molecular docking confirmed strong binding affinities between the key bioactive compounds and AD-related targets. A co-culture system of PC12 neuronal and Caco-2 intestinal epithelial cells was established to evaluate the effects of VT, PM, AVF, and EF extracts (at concentrations of 10 µg/mL, 20 µg/mL, and 50 µg/mL) and donepezil hydrochloride (positive-control) on Aβ_25-35-induced neurotoxicity and lipopolysaccharide (LPS)-induced intestinal inflammation, to assess cell viability, and effects on oxidative stress, mitochondrial function, and inflammatory markers. The VT, PM, AVF, and EF extracts activated phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, enhanced phosphorylation of AMP kinase, suggesting inhibition of Aβ accumulation and tau hyperphosphorylation (<i>p</i> < 0.05). However, donepezil hydrochloride only enhanced AMPK phosphorylation. The extracts reduced lipid peroxidation and acetylcholinesterase by about 5-fold. JC-1 staining confirmed preserved mitochondrial membrane potential, while hematoxylin and eosin staining indicated improved intestinal barrier integrity (<i>p</i> < 0.05). PM and AVF reduced the number of mast cells (<i>p</i> < 0.05). In conclusion, these findings highlight the multi-target potential of VT, PM, AVF, and EF in mitigating both neuronal and intestinal inflammation. Their dual regulatory effects on the gut-brain axis suggest promising therapeutic applications in AD through the modulation of central and peripheral immune responses.