Network Pharmacology and In Vivo Validation to Explore the Potential Active Ingredients and Mechanisms of Ziyin Buyang Formula in Diminished Ovarian Reserve

The incidence rate of diminished ovarian reserve (DOR) is increasing, which is a difficult problem in the field of reproductive biology. The Ziyin Buyang Formula (ZYBYF), an empirical prescription for traditional Chinese medicine, is often used to improve the ovarian function of women with DOR. However, the underlying mechanism of this phenomenon remains unclear. This study was designed to identify the active components of ZYBYF and to further investigate the mechanism by which ZYBYF treats DOR through network pharmacological analysis and in vivo experiments. The network-based relationships between drug targets and disease-related proteins were screened, predicted, and constructed using a database. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted using R software. AutoDock Vina 1.1.2 was used to perform the molecular docking analysis. In vivo experimental verification was carried out by enzyme-linked immunosorbent assay, histological observation and reverse transcription-polymerase chain reaction. A total of 137 common targets were identified between ZYBYF and DOR. GO and KEGG enrichment analyses suggested that these compounds were associated with the PI3K/AKT signaling pathway and MAPK signaling pathway. Furthermore, the molecular docking analysis revealed that quercetin strongly binds to MAPK1, AKT1, MYC, EGFR, and BCL2; luteolin strongly binds to EGFR, AKT1, and MAPK1; and kaempferol strongly binds to AKT1 and BCL2. In addition, in vivo experiments confirmed that ZYBYF can regulate the serum sex hormone levels, improve the ovarian function of DOR model rats, downregulate the expression of Mapk1 and Mapk14, and increase the levels of Akt1, Egfr, Myc, and Bcl2. ZYBYF improved ovarian function in DOR model rats by regulating the MAPK and PI3K/AKT signaling pathways. This study provides objective evidence that supports the use of ZYBYF in the clinical application of DOR.