Yulin Yangchao formula improves premature ovarian insufficiency by inhibiting granulosa cell apoptosis through the PI3K/AKT/p53 signaling axis

Background: Abnormal Apoptosis of ovarian granulosa cells (GCs) represents a crucial pathological foundation of premature ovarian insufficiency (POI). Yulin Yangchao Formula (YLYCF) has demonstrated favorable clinical efficacy in the treatment of POI. Nevertheless, its mechanism of action remains elusive.

Purpose: This study aimed to investigate the relevant target sites and potential molecular mechanisms of YLYCF in the treatment of POI.

Methods: Cyclophosphamide (CTX) was employed to stimulate adult female mice and GCs (KGN) to establish in vivo and in vitro models of POI. Following YLYCF treatment, mouse ovarian function was evaluated in vivo by assessing the estrous cycle, hormone levels, follicular development, pregnancy rate, cell Apoptosis, and embryos yield. KGN cell proliferation and Apoptosis were examined in vitro. Besides, active components in YLYCF were identified, and its possible targets for treating POI were identified by network pharmacology, which were related to the PI3K/Akt/p53 axis. Moreover, proteins involved in this axis were analyzed in the CTX-mediated KGN cells and ovarian tissues of POI mice. Moreover, rescue experiments were carried out alongside PI3K/Akt pathway inhibitor MK-2206 and p53 activator Nutlin-3a for validating the specific mechanism through which YLYCF enhances ovarian function.

Results: Following CTX induction, mice with POI exhibited disruptions in the estrous cycle and estrogen secretion, accompanied by a reduced pregnancy rate and litter size, as well as an increased rate of Apoptosis in ovarian tissue. KGN cells exposed to CTX demonstrated decreased proliferation and increased Apoptosis. YLYCF significantly ameliorated the aforementioned POI-related characteristics. Based on the findings of component identification and network pharmacology analysis, the active components glyuranolide, 3'-Methoxyglabridin, glyasperin B, and naringenin in YLYCF exhibit strong binding affinities with Akt1 and TP53. Additionally, YLYCF treatment significantly activated the PI3K/Akt signaling pathway and inhibited p53 expression in both in vivo and in vitro POI models. However, MK-2206 or Nutlin-3a reversed the benefits induced by YLYCF against GC Apoptosis and ovarian function.

Conclusion: YLYCF suppressed GC Apoptosis via PI3K/Akt pathway activation, which then down-regulated p53, thereby enhancing ovarian function in cases of POI.