Investigation of the mechanism of Yijing Decoction in the treatment of premature ovarian insufficiency: A network pharmacology study

To explore and validate active components in the Yijing Decoction formula that exert therapeutic effects on targets associated with premature ovarian insufficiency (POI) and to identify the underlying mechanisms of action. A rat model of POI was treated with Yijing Decoction. In the model group, cyclophosphamide (CTX; 83.52 mg/kg) and busulfan (BUS; 20.88 mg/kg) were administered by a single intraperitoneal injection given simultaneously. In the control group, an equivalent volume of saline was administered intraperitoneally. After 2 weeks of modeling, 10 rats were randomly selected from each of the model and the control groups and euthanized to evaluate model establishment. After confirming successful model establishment, 20 rats were randomly selected from the model group as the treatment group and received oral gavage. Yijing Decoction (11.79 g/kg raw drug) was administered daily by oral gavage for 21 days; the remaining 20 model-group rats received equivalent volumes of saline gavage for 21 days. At the end of the treatment, 5 rats from each group were randomly selected and housed with sexually mature male rats at a ratio of 3:1 for mating. Efficacy of Yijing Decoction in treating POI was evaluated by: monitoring the estrous cycle; histopathological assessment with hematoxylin and eosin (H&E) staining to determine follicle counts; TUNEL assay to detect apoptotic cells; measurement of serum hormone levels using enzyme-linked immunosorbent assay (ELISA); and counting embryos resulting from mating. Network pharmacology analysis was used to construct a network of "Herb-ingredient-target" network to explore and predict therapeutic mechanisms. Finally, the predictions were validated using polymerase chain reaction, western blotting, and immunohistochemistry. Compared with the model group, Yijing Decoction increased serum hormone levels, elevated counts of primordial, primary and secondary follicles, and increased the number of embryo implantations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that, in terms of molecular function and cellular component categories, treatment of POI with Yijing Decoction was associated with the serine/threonine protein kinase complex; the cyclin-dependent protein kinase holoenzyme complex; membrane-associated compartments; cytokine receptor binding; and scaffold-protein binding. Expression of cyclin-dependent kinase 1 (CDK1), E2F transcription factor 1 (E2F1), cyclin-dependent kinase 4 (CDK4), proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1) and cluster of differentiation 34 (CD34) was upregulated after gavage treatment with Yijing Decoction. Yijing Decoction may primarily act on the CDK1-PCNA-CCND1 axis to upregulate the expression of these genes, which in turn may influence downstream targets such as CDK4, E2F1, and CD34. By reducing apoptosis in ovarian granulosa cells and promoting angiogenesis, Yijing Decoction may help delay chemotherapy-induced ovarian functional decline. These findings may provide a theoretical basis for the clinical use of Yijing Decoction in the treatment of premature ovarian insufficiency caused by chemotherapy.