Network analysis and experimental approach to investigate the potential therapeutic mechanism of zishen yutai pills on premature ovarian insufficiency

BACKGROUND: As society continues to develop, women are more at risk of gonadotoxic substance exposure. Consequently, the incidence of premature ovarian insufficiency (POI) has increased significantly in the past decades. Hormone replacement therapy (HRT) is recommended as the standard treatment to relieve hypoestrogenic symptoms; however, its potential side effects and contraindications have drawn widespread controversy and concern. As such, the Chinese medicine Zishen Yutai Pill (ZSYTP) commonly used for treating miscarriage and menoxenia, is a highly promising alternative drug candidate against POI, however its therapeutic mechanism has not been completely elucidated. OBJECTIVE: To systematically analyze the potential therapeutic targets of ZSYTP on POI, we combined network pharmacology analysis and molecular docking to predict critical target genes, with experimental validation on POI murine models. METHODS: The active compounds of ZSYTP were collected from three online databases, and the candidate targets were predicted based on the chemical structure. The POI-related targets were obtained from four databases. A PPI network was constructed to find the key target genes between ZSYTP and POI, while GO and KEGG enrichment analyses were employed to study the mechanism of ZSYTP against POI. The binding capability of the key co-targets with active components was examined by molecular docking. We used a cyclophosphamide (CTX)-inducible POI mouse model to verify our predictions by histopathological observation, immunohistochemical staining (caspase-3, TUNEL assay), hormone determination (FSH, AMH) and ribonucleic acid sequencing (RNA Seq). Progynova was also used to study the difference between ZSYTP and HRT. RESULT: We identified 21 target genes as the hub between ZSYTP and POI. The GO and KEGG analyses revealed that the molecular mechanism of ZSYTP against POI were mainly based on the regulation of gene and protein expression. A variety of signaling pathways may be involved in the treatment of ZSYTP against POI, especially PI3K-AKT, HIF-1 and the AGE-RAGE cascades. Docking simulation showed that G1, C1, SR5, and F1 had relatively lower binding energy. In vivo, ZSYTP significantly reversed CTX-induced ovarian damage in follicle number, hormone level and apoptosis, with an overall improved therapeutic effect compared to Progynova. Results from RNA-Seq revealed that the PI3K-AKT, Hippo, AGE-RAGE, and Rap1 signaling pathways and regulation of inflammation, immune response, and lipid metabolism may mediate the protective effects of ZSYTP against POI, which is different than Progynova's mechanism of action. CONCLUSIONS: Collectively, this study indicates that ZSYTP could be a highly promising alternative as a non-HRT-based therapy for POI. Its mechanism involves multiple signaling pathways, alleviating ovarian apoptosis and recovering AMH and FSH level. However, the discrepancy between different research techniques highlight the necessity of further experimental verification from other aspects such as translation and posttranslational modification.