Objective To evaluate the effects of the traditional Chinese herbal formula YA3D3 on cognitive impairment and cerebral β-amyloid (Aβ) deposition in APP/PS1 transgenic mice and to determine whether these effects are mediated by up-regulation of the autophagy-related gene 14 (ATG14). Methods Network pharmacology was employed to predict YA3D3 targets.Total antioxidant capacity and hydroxyl free radical scavenging activity were measured in vitro.BV2 microglial cells were infected with short hairpin RNA (shRNA) lentivirus targeting ATG14 (designated as sh-ATG14) and then treated with 0.5 mg/mL of either water-extract (YA3D3-HF) or fermented (YA3D3-MHF) preparation for 24 h.The expression levels of ATG14 and downstream ATG16L1 were assessed by qPCR and Western blot.Sixty-four 3-month-old male APP/PS1 mice were randomly allocated to eight groups (n=8):APP/PS1 model,donepezil,low-,medium-,and high-dose YA3D3-HF,and low-,medium-,and high-dose YA3D3-MHF.All the drugs were administered ad libitum in drinking water for 90 days.Spatial learning and memory were evaluated through the Morris water maze.Cerebral Aβ deposition was quantified by immunofluorescence.Autophagy-related gene expression in the brain tissue was analyzed by qPCR. Results Network pharmacology indicated significant enrichment of YA3D3 targets in autophagy and phosphatidylinositol 3-kinase/protein kinase B signaling pathways.YA3D3-MHF showed stronger antioxidant activity than YA3D3-HF in vitro (P<0.001).In BV2 cells,both preparations reversed ATG14 knock-down-induced down-regulation in the expression of ATG14 and ATG16L1,with YA3D3-MHF being more potent (P=0.003).The animal experiment showed that all the YA3D3 groups exhibited shorter escape latency and more platform crossings than the APP/PS1 model group (all P<0.05),along with reduced Aβ fluorescence intensity (all P<0.001).The efficacy of high-dose YA3D3-MHF was similar to that of donepezil.qPCR confirmed that YA3D3 up-regulated the expression of ATG14 and modulated the expression of downstream autophagy-related genes in the brain tissue (all P<0.05). Conclusions The traditional Chinese herbal formula YA3D3 may up-regulate the expression of ATG14 to slow down Aβ deposition and ameliorate cognitive impairment in APP/PS1 mice,serving as a potential candidate for Alzheimer’s disease intervention.It should be noted that this study has not directly verified the necessity of ATG14 in vivo,and the causal relationship between the in vivo therapeutic efficacy of YA3D3 and the up-regulation of ATG14 requires further confirmation.In addition,this study only verifies the expression regulation of autophagy-related molecules and does not confirm the enhancement of autophagy flux.