β-sitosterol in Yijing Hugui decoction prevents cyclophosphamide-induced premature ovarian insufficiency via the AKT1/Nrf2 pathway.

Premature ovarian insufficiency (POI) is a condition marked by premature depletion of ovarian function, affecting a significant portion of women. The objective of this study is to assess the therapeutic efficacy of Yijing Hugui decoction (YJHGD) in the treatment of POI and to elucidate its pharmacological mechanisms. In this study, network pharmacology was used to identify key bioactive compounds in YJHGD, and the components were characterized using LC-MS. In vitro, we used KGN cells treated with cyclophosphamide (CP) to model POI. In vivo, a CP-induced POI mouse model was established. The in vitro therapeutic effects of β-sitosterol on CP-treated KGN cells were evaluated through various parameters. These parameters encompass cell viability, oxidative markers, antioxidant indexes, ATP concentration, intracellular ROS levels, apoptosis rate, and apoptosis-related protein expression. The in vivo therapeutic effects of β-sitosterol in POI mice were assessed through H&E staining, circulating reproductive hormone level detection, reproductive hormone receptor expression measurement, oxidative stress profile, and apoptosis assay. The potential protein target of β-sitosterol was identified utilizing molecular docking in conjunction with drug affinity responsive target stability (DARTS). β-sitosterol was identified as a major active component of YJHGD contributing to its therapeutic effects. In β-sitosterol-treated KGN human granulosa cells, oxidative stress and apoptosis were significantly reduced (P < 0.05). The interaction between β-sitosterol and AKT1 was verified. Furthermore, β-sitosterol significantly activated the AKT1/Nrf2 signaling pathway in vivo and in vitro (P < 0.05). AKT1 activator insulin significantly alleviated CP-induced oxidative stress (P < 0.05). Our results suggest that β-sitosterol inhibits oxidative stress and apoptosis by targeting AKT1 and activating the Keap1/Nrf2/HO-1 signaling. In vivo studies demonstrated that β-sitosterol significantly restored ovarian tissue damage in mice, reduced the circulating levels of reproductive hormones, downregulated the expression of reproductive hormone receptors, alleviated oxidative stress and ROS generation, and improved apoptosis (P < 0.05), which was achieved through the AKT1/Nrf2 pathway. In Conclusion, YJHGD possesses therapeutic potential for the treatment of POI. The active compound, β-sitosterol, demonstrated significant anti-POI effects through its interaction with AKT1, leading to the activation of AKT1/Nrf2 signaling pathway. This interaction contributes to the reduction of oxidative stress and the prevention of cellular apoptosis. Our results suggest that β-sitosterol may represent a novel therapeutic approach. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-025-00740-8.