Zuogui Wan alleviates intergenerational premature ovarian insufficiency through C9-mediated complement-coagulation cascade.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Zuogui Wan (ZGW), a classical kidney-nourishing and yin-tonifying TCM formula composed of multiple herbs, is clinically used to improve premature ovarian insufficiency (POI) and perimenopausal symptoms. Compared to hormonal therapy, ZGW exhibits efficacy with fewer adverse effects, however, its impact on the intergenerational transmission of POI remains unclear. AIM OF THE STUDY: This study investigates whether maternal ZGW administration mitigates POI transmission to offspring and elucidates its underlying molecular mechanism. MATERIALS AND METHODS: A chronic unpredictable mild stress (CUMS) rat model of POI received ZGW intervention. We assessed estrous cycles, ovarian morphology, and sex hormones (E2, AMH, FSH) in F1/F2 offspring. Network pharmacology, limited proteolysis-mass spectrometry (LiP-MS), proteomics, and metabolomics identified key targets. Molecular docking, dynamics simulations (100 ns), and surface plasmon resonance (SPR) validated compound-target interactions. Ovarian tissues from three generations underwent Western blotting, RT-qPCR, and immunofluorescence. A 4-HC-induced KGN cell model was treated with C9 inhibitor, siRNA-C9, and generational sera for functional validation. RESULTS: ZGW improved estrous cycles and ovarian morphology, and tended to normalize serum levels of E2, AMH, and FSH. Multi-omics integration revealed C9-driven complement signaling (C9/ITGAM/KNG1). Quercetin (ZGW's key component) bound C9 with high affinity (KD = 506 nM) via SPR. Ovarian tissues showed generationally altered C9/ITGAM/KNG1 expression. In vitro, C9 inhibition or knockdown mitigated ZGW serum's protective effects on AMH/E2 levels. CONCLUSIONS: ZGW can alleviate the intergenerational transmission of POI via the C9-mediated complement-coagulation cascade pathway, providing strategic insights for managing hereditary ovarian disorders.