Shanyao regulates the PI3K/AKT/P21 pathway to promote oogonial stem cell proliferation and stemness restoration to alleviate premature ovarian insufficiency.

Abstract

Ethnopharmacological relevance: Shanyao (SY, yam, Rhizoma Dioscoreae, the dried rhizome of Dioscorea oppositifolia L.) was recorded in the Chinese pharmacopoeia and was often used in the treatment of premature ovarian insufficiency (POI).

Aim of study: To evaluate the efficacy of shanyao in cyclophosphamide (CTX)-induced POI and explore its potential mechanism of action.

Material and methods: We employed network pharmacology, Liquid Chromatograph Mass Spectrometer (LC-MS), and molecular docking methods to identify active compounds and core targets, and predict the mechanism of shanyao for treating POI. The mechanism was subsequently validated through a series of experiments. Female Sprague-Dawley (SD) rats were randomly divided into five groups: control (CON), model, estradiol valerate (EV), low-dose shanyao, and high-dose shanyao. An experimental rat model of POI was established using cyclophosphamide and treated with either shanyao or EV for a duration of two months. We assessed the efficacy of shanyao in vivo through methods such as weighing, Enzyme-linked Immunosorbent Assay (ELISA), and Hematoxylin and Eosin (H&E) staining. Oogonial stem cells (OSCs) were isolated, after modeling, treated them with a serum containing either shanyao or EV. Using methods such as CCK8 assay, immunofluorescence staining, flow cytometry (FCM) analysis, and Western blot analysis to verify the mechanism of shanyao in treating POI.

Results: In this study, we found that after treatment with shanyao, the general condition of POI rats was improved, body weight and the ratio of ovarian weight to body weight were increased, FSH, E2 and AMH levels were improved, primary follicles and preantral follicles were significantly increased, atretic follicles were decreased. However, the number of antral follicles and fresh corpus luteum was no statistical difference. We identified 10 active compounds of shanyao that act on 220 target genes, 176 of which are associated with POI. Denudatin B and Kadsurenone were finally identified as core components. Through topological analysis, 18 key targets were selected, and ultimately PI3K, CCND1, and CDK4 were identified as core targets. Molecular docking results showed that core components had good binding energy with core targets. The results of GO and KEGG enrichment analysis mainly focus on cell cycle regulation and PI3K/AKT signaling pathway. A series of molecular biology experiments confirmed that after shanyao treatment, the phosphorylation level of PI3K and AKT in POI rats were increased, P21 was inhibited, PI3K/AKT/P21 signaling pathway was activated, and the expression levels of CCND1 and CDK4 were increased. At the same time, the expression of Oct4, fragilis and Mvh of ovarian stem cells was up-regulated.

Conclusion: The active compounds of shanyao can regulate the PI3K/AKT/P21 signaling pathway, promote the proliferation of oogonial stem cells, stemness restoration, and delay ovarian aging. This study provides valuable insights into shanyao treatment for POI.