Guilu Erxian Glue combined with Wuzi Yanzong Pill attenuates granulosa cell apoptosis and ameliorates cyclophosphamide-induced diminished ovarian reserve via the lncRNA NEAT1/miR-204-5p/ESR1 axis

ETHNOPHARMACOLOGICAL RELEVANCE: As classical traditional Chinese formulas used to tonify the Kidney and replenish essence, Guilu Erxian Glue and Wuzi Yanzong Pill have long been applied to improve reproductive function. However, their major bioactive components and the underlying mechanisms of action remain poorly defined. AIM OF THE STUDY: This study aimed to investigate whether the combination of Guilu Erxian Glue and Wuzi Yanzong Pill (EWCD) ameliorates cyclophosphamide (CTX)-induced diminished ovarian reserve (DOR) by inhibiting granulosa cell (GC) apoptosis via regulation of the lncRNA NEAT1/miR-204-5p/ESR1 axis, and to elucidate the underlying molecular mechanisms further. MATERIALS AND METHODS: The chemical components of EWCD were identified by liquid chromatography-high-resolution mass spectrometry (LC-HRMS), followed by target prediction based on network pharmacology and molecular docking analyses. A CTX-induced DOR rat model was established in vivo, and animals were treated with different doses of EWCD or estrogen-progesterone. Estrous cycle characteristics, ovarian morphology, GC apoptosis, serum sex hormone levels, and the expression of NEAT1/miR-204-5p/ESR1 axis-related factors, steroidogenic enzymes (CYP19A1 and StAR), and MAPK signaling proteins were evaluated. In vitro, KGN cell injury was induced using 4-hydroperoxycyclophosphamide (4-HC). The effects of EWCD-containing serum and si-NEAT1 transfection on cell proliferation, apoptosis, and target gene expression were subsequently assessed. RESULTS: LC-HRMS identified 51 chemical constituents in EWCD. Network pharmacology and molecular docking analyses suggested that key constituents of EWCD may interact with ESR1 and modulate the MAPK signaling pathway. In vivo, EWCD significantly improved ovarian function and histological architecture in CTX-induced DOR rats and reduced GC apoptosis. These effects were associated with regulation of the NEAT1/miR-204-5p/ESR1 axis, activation of the MAPK pathway, and enhanced estradiol biosynthesis. Consistently, in vitro experiments demonstrated that EWCD-containing serum alleviated 4-HC- and si-NEAT1-induced dysfunction in KGN cells by restoring proliferation capacity and inhibiting apoptosis in a NEAT1/miR-204-5p/ESR1 axis-dependent manner. CONCLUSION: EWCD protects against CTX-induced ovarian damage by activating the MAPK signaling pathway through modulation of the NEAT1/miR-204-5p/ESR1 axis, thereby inhibiting GC apoptosis and promoting estradiol synthesis. These findings provide mechanistic evidence supporting the therapeutic potential of EWCD for the treatment of DOR.