Abstract: BACKGROUND: Studies have shown that patients with premature ovarian failure have changes in the structure of intestinal flora and that imbalance of intestinal microbiota may be one of the important mechanisms in the development of premature ovarian failure.
OBJECTIVE: To investigate the effect of Liuwei Dihuang Wan on oxidative stress and intestinal microbiota in premature ovarian failure mice induced by cyclophosphamide. 
METHODS: Forty-five female ICR mice were randomized into three groups: blank group (normal mice), model group (premature ovarian failure mice), and Liuwei Dihuang Wan group. A mouse model of premature ovarian failure was prepared by one-time intraperitoneal injection of cyclophosphamide (120 mg/kg) 
in the latter two groups. After successful modeling, the Liuwei Dihuang Wan group was intragastrically administered for 28 continuous days, and the other two groups were intragastrically administered with the same amount of normal saline for 28 days. Mouse body mass was recorded weekly and ovarian index was calculated. The development of mouse follicles was observed using hematoxylin-eosin staining. ELISA method was used to detect serum levels of anti-Mullerian hormone, estradiol, follicle stimulating hormone, superoxide dismutase, glutathione peroxidase, and malondialdehyde. Meanwhile, the gut microbiome of all mice was detected through 16S rDNA sequencing.
RESULTS AND CONCLUSION: The mice in the model group had loose hair, decreased vigor and grip strength, almost no increase in body mass, and decreased ovarian index. Whereas, the mouse body mass and ovarian index were increased after treatment with Liuwei Dihuang Wan (P < 0.05). The estrous cycle of mice in the model group was disorganized; Liuwei Dihuang Wan could restore the estrous cycle and reduce the number of atretic follicles in mice with premature ovarian failure. The serum levels of follicle stimulating hormone and malondialdehyde in the model group significantly increased (P < 0.01), while the levels of estradiol, anti-Mullerian hormone, superoxide dismutase, and glutathione peroxidase significantly decreased (P < 0.01). Liuwei Dihuang Wan could significantly decrease the serum levels of follicle stimulating hormone and malondialdehyde (P < 0.01), and increase the levels of estradiol, anti-Mullerian hormone, superoxide dismutase, and glutathione peroxidase. According to the 16S rDNA sequencing results, Liuwei Dihuang Wan could regulate the abundance and diversity of intestinal microbiota, and increase the relative abundance of beneficial bacteria. KEGG pathway analysis showed that the intestinal microbiota and metabolic pathways, biosynthesis of secondary metabolites, microbial metabolism in different environments, and biosynthesis of amino acids were regulated by Liuwei Dihuang Wan. To conclude, the changes in the structure of intestinal microbiome may be one of the potential mechanisms of Liuwei Dihuang Wan in treating premature ovarian failure. Liuwei Dihuang Wan can regulate the structure of intestinal microbiome, increase the number of beneficial bacteria, reduce the number of harmful bacteria, and thus improve the balance of intestinal microbiota. This regulatory effect helps to reduce oxidative stress levels and further inhibit ovarian oxidative stress in mice with premature ovarian failure.

Key words: ">Liuwei Dihuang Wan, premature ovarian failure, intestinal microbiome, oxidative stress