Integrating network pharmacology and transcriptomics reveals that Bushen Huoxue recipe ameliorates ferroptosis and apoptosis in granulosa cells by regulating PI3K/Akt signaling pathway in premature ovarian insufficiency mice

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Huoxue recipe (BHR), from records of Jingyue Quanshu, commonly prescribed to treat irregular menstruation, premature ovarian insufficiency (POI), and infertility. BHR has been shown excellent efficacy in improving ovarian function. However, the core mechanisms have yet to be fully clarified. AIM OF THE STUDY: The objective of the study was to investigate the influence of BHR on cyclophosphamide (CTX)-induced POI in mice and to identify the mechanisms involved. MATERIALS AND METHODS: CTX administration induced the establishment of a POI murine model. Different groups of mice were administered distilled water, BHR, and dehydroepiandrosterone for 28 days, respectively. Ovarian function was evaluated based on ovarian index, estrous cyclicity, ovarian morphology, follicular development, and hormone levels. Tunel assays and flow cytometry were used to evaluate apoptosis. Western blotting and q-PCR were employed to detect the molecules related to ferroptosis and apoptosis. An integrated approach combining network pharmacology with transcriptomic analysis elucidated therapeutic targets, subsequently validated through molecular techniques and cellular assays. RESULTS: BHR administration effectively ameliorated estrous cyclicity irregularity, hormonal imbalances, follicular development disorders, increased follicular atresia, and decreased ovarian reserve in POI mice. Transcriptomic analyses illustrated that BHR might enhance ovarian function in POI mice by inhibiting ferroptosis. Significant inhibition of heme oxygenase 1 (Hmox1) was observed concomitant with increased glutathione peroxidase 4 (GPX4) and solute carrier family 7a member 11 (SLC7A11) expression following BHR administration. Meanwhile, BHR administration effectively attenuated oxidative stress disorders in granulosa cells (GCs). Additionally, BHR effectively inhibited apoptosis in GCs. According to network pharmacology and transcriptomic analyses, the beneficial effects of BHR on POI might be due to the PI3K/Akt pathway, which confirms previous results. Furthermore, we verified this conclusion through in vitro experiments. CONCLUSION: BHR effectively ameliorated ovarian function in CTX-induced POI mice by suppressing ferroptosis and apoptosis in GCs. This effect is mechanistically mediated by the activation of the PI3K/Akt signaling pathway. Furthermore, the Hmox1/GPX4 axis may be associated with BHR-mediated inhibition of ferroptosis.