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Abstract Objective To investigate the protective effect and mechanism of hyperin on hydrogen peroxide (H2O2)-induced injury of granulosa cells in mice. Methods Adult female BALB/c mice were randomly divided into control group, POI model group and hyperin treatment group. The primary mice ovarian granulosa cells were extracted, cultured in vitro, and exposed to H 2 O 2 to establish the oxidative damage model of ovarian granulosa cells. The ovarian granulosa cells were added with different concentration hyperin (0µg/l, 5µg/l, 10µg/l, 25µg/l, 50µg/l, 75µg/l respectively); and observed the proliferation under different hyperin concentration. The concentration level with the most significant proliferation would be selected for treatment group. Serum estradiol(E2), follicle-stimulating hormone(FSH), anti-mullerian hormone(AMH), superoxide dismutase(SOD) and catalase(CAT) were measured by ELISA.The mRNA and protein levels of nuclear factor (erythroid-derived 2)-related factor 2(Nrf-2), heme oxygenase-1 (HO-1), Caspase3, Bcl-2 and Bax in ovarian granulosa cells were measured by RT-qPCR and Western blot. The protein levels of phosphorylated phosphatidylinositol 3-hydroxy kinase (p-PI3K) and phosphorylated protein kinase B (p-Akt) were measured by Western blot. The reactive oxygen species (ROS) levels in granulosa cells were determined by H2DCFDA. The apoptosis of granulosa cells was examined by TUNEL assay. Results 50µg/l hyperin has the most significant effect on proliferation of ovarian granulosa cells. Comparing with the model group, the serum levels of E2, SOD and CAT in Hyperin treatment group increased ( P < 0.05).The expression of Nrf-2, HO-1, p-PI3K, p-Akt and Bcl-2 in ovarian granulosa cells increased, while the expression of Caspase3 and Bax decreased ( P < 0.05). ROS level decreased ( P < 0.05). TUNEL assay showed reduced apoptosis of granulosa cells ( P < 0.05). Conclusion Hyperin can improve H2O2-induced ovarian granulosa cell injury through Nrf-2/HO-1 anti-oxidative stress and PI3K/Akt anti-apoptosis.