Abstract
Diminished ovarian reserve (DOR), characterized by reduced quantity and diminished quality of oocytes, significantly impairs female fertility. Research indicates that germline stem cells (GSCs) possess the potential to fundamentally replenish the oocyte pool through self-renewal. Clinical studies have confirmed that Yulinzhu (YLZ) can effectively regulate menstrual disorders and hormonal levels, and improve DOR. However, whether YLZ can ameliorate ovarian reserve function by regulating the proliferation of GSCs, as well as its molecular mechanisms of action, remain to be fully elucidated. This study aimed to determine whether YLZ can improve ovarian reserve function by promoting the regeneration of GSCs and to explore the underlying mechanisms involved. The therapeutic efficacy of YLZ against DOR was evaluated in both rat and Drosophila models. This included assessing hormone levels via enzyme-linked immunosorbent assay (ELISA), analyzing ovarian morphology through hematoxylin and eosin (H&E) staining, calculating the ovarian index, examining follicular development, and quantifying GSCs. The LC-MS/MS was then employed to identify the principal constituents of YLZ, and network pharmacology was utilized to predict potential therapeutic targets and signaling pathways relevant to DOR. Component-target interactions were further examined using molecular docking and surface plasmon resonance (SPR). Finally, the predicted targets and signaling pathways were validated through immunofluorescence, reverse transcription-quantitative PCR (RT-qPCR), and western blotting. In vivo experiments showed that YLZ alleviated ovarian atrophy, elevated hormone levels, and promoted follicular development in DOR rats. Furthermore, YLZ increased the expression of Oct4 and MVH markers of ovarian GSCs in DOR rats and raised GSC numbers in aged Drosophila ovaries, supporting GSC regeneration and enhancing ovarian reserve. A total of 93 active components were identified in YLZ, with network analysis highlighting the estrogen signaling pathway and the stem cell pluripotency pathway as central to its therapeutic effect on DOR. ESR1 was identified as the core therapeutic target through component-target network construction and affinity kinetic analysis. Moreover, it was found that kaempferol and quercetin, both belonging to flavonoids, have highly similar parent structures that enable them to recognize and bind to the same binding domains on the ESR1 protein. Further verification indicates that the kaempferol and quercetin in YLZ can promote the self-renewal of GSCs and enhance ovarian reserve capacity by up-regulating ESR1-mediated estrogen signaling and stem cell pluripotency pathways. Kaempferol and quercetin were identified as the core material basis for regulating the core pathway and producing the main pharmacological effects. The results suggest that YLZ regulates estrogen signaling and stem cell pluripotency pathway by targeting ESR1, thereby promoting GSC regeneration to enhance ovarian reserve function. Kaempferol and quercetin were identified as the principal active constituents, underscoring the therapeutic potential of YLZ in the treatment of DOR.
