OBJECTIVE To study the mechanism of Tian-Si-Yin in treating Vascular dementia(VD) by network pharmacology.

METHODS Searched the active ingredients and corresponding targets of Tian-Si-Yin through the TCMSP databases, then obtained the targets of VD through the OMIM and GeneCards database. Constructed protein interaction network map by String online database. At the same time, GO and KEGG enrichment analysis of key targets were carried out by using R language. Modified 2-VO method was used to build the VD rat model. The pathomorphological changes of rat hippocampal neurons by hematoxylin-eosin(HE) staining. Transmission electron microscopy was used to observe the mitochondrial ultrastructure in the hippocampal tissue. Flow cytometry to detect changes in mitochondrial membrane potentialthe and the level of reactive oxygen species(ROS). A biochemical colourimetric assay was used to detect the levels of Fe²⁺, malondialdehyde(MDA), and glutathione(GSH) in hippocampal tissue. Western blotting and q-PCR experiments were used to clarify the protein expression and gene transcription of the relevant targets.

RESULTS A total of 13 active ingredients of Tian-Si-Yin were screened, 103 targets related to VD, The top 20 KEGG related signaling pathways and GO analysis of the top 20 biological processes. Animal experiments showed that Tian-Si-Yin could improved the learning and spatial memory abilities of model rats significantly(P<0.05,P<0.01), alleviated pathological morphology and mitochondrial damage in hippocampal tissue, increased GSH and mitochondrial membrane potential,reduced Fe²⁺, MDA, and ROS levels(P<0.05, P<0.01), Up-regulated the expression of Mfn1, Mfn2, GPX4, SLC7A11, FTH1 protein and mRNA(P<0.05, P<0.01), down-regulated the expression of Drp1, Fis1, ACSL4, COX-2 proteins and mRNA(P<0.05, P<0.01). Tian-Si-Yin treatment increased the phosphorylated levels of AMPK, upregulated the expression of Nrf2, HO-1 protein and mRNA, therefore promoting the activation of the AMPK/Nrf2 pathway(P<0.05,P<0.01). However, the co-treatment of compound C partially reversed the therapeutic effects of Tian-Si-Yin on ferroptosis.

CONCLUSION Tian-Si-Yin improves mitochondrial dynamics imbalance and inhibits ferroptosis by activating the AMPK/Nrf2 pathway, thereby improve cognitive function in VD rats.