Study on the Mechanism of Tiansi Yin in Treating Vascular Dementia Based on Network Pharmacology and Experimental Verification

OBJECTIVE To explore the mechanism of Tiansi Yin in the treatment of vascular dementia(VD) based on network pharmacology and animal experiments.

METHODS Searched the effective ingredients and corresponding targets of Tiansi Yin by the TCMSP databases, then obtained the targets of VD through the OMIM and GeneCards database. Constructed protein interaction network map by String online database, and to performed gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis of key targets by using R language. The VD model was established by improved bilateral common carotid artery ligation. The pathomorphological changes of rat hippocampal neurons by hematoxylin-eosin staining. The ultrastructure of mitochondria in hippocampus was observed by transmission electron microscope. The mitochondrial membrane potential and reactive oxygen species(ROS) were detected by flow cytometry. The contents of Fe²⁺, malondialdehyde(MDA) and glutathione(GSH) in hippocampus were detected by biochemical colorimetry assay. Western blotting and RT-qPCR were used to identify the expression of target proteins and gene transcription of the relevant targets.

RESULTS A total of 13 active ingredients of Tiansi 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 Tiansi Yin 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 the level of Fe²⁺, MDA, and ROS(P<0.05, P<0.01), and up-regulated the expression of mitofusion 1(Mfn1), mitofusion 2(Mfn2), glutathione peroxidase 4(GPX4), solute carrier family 7 member 11(SLC7A11), ferritin heavy chain 1(FTH1) protein and mRNA(P<0.05, P<0.01), down-regulated the expression of dynamics related protein 1(Drp1), fission protein 1(Fis1), acyl-CoA synthetase long-chain family member 4(ACSL4), cyclooxygenase-2(COX-2) proteins and mRNA(P<0.05, P<0.01). The treatment of Tiansi Yin increased the phosphorylated levels of AMPK, upregulated the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Heme oxygenase 1(HO-1) protein and mRNA, and promoted the activation of AMPK/Nrf2 pathway(P<0.05, P<0.01). However, the use of Compound C partially reversed the therapeutic effect of Tiansi drink on ferroptosis.

CONCLUSION Tiansi Yin improved the cognitive function of VD rats by activated the AMPK/Nrf2 pathway to improved mitochondrial dynamic imbalance and inhibited ferroptosis.