To explore the mechanism of Bushen Tiaojing Formula (BSTJF) underlying the promotion of proliferation and steroid hormone synthesis of ovarian granulosa cells in aged mice.

Twenty 7-8-week-old ICR female mice were assigned to the control group (intragastric administration of distilled water), and eighty 9-10-month-old ICR female mice were equally divided into the model group (intragastric administration of distilled water), the BSTJF low-dose group (intragastric administration of 25.6 g/kg BSTJF), the BSTJF high-dose group (intragastric administration of 51.2 g/kg BSTJF), and the leukemia inhibitory factor (LIF) inhibitor group was treated with intragastric administration of BSTJF (51.2 g/kg) and intraperitoneal injection of EC330 (5.0×10^-4 g/kg) according to random numbers. All groups underwent daily gavage or intraperitoneal administration for 30 days. The estrous cycle changes of mice were observed by a vaginal exfoliated cell smear. The levels of sex hormones were detected by ELISA. The mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), the cell cycle protein D1 (Cyclin D1), the acute steroidogenic regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 19A1 (CYP19A1), LIF, and LIF receptor (LIFR) were detected by quantitative reverse transcription PCR and Western blotting. The expressions of LIF and LIFR were detected by immunohistochemistry.

Compared with the control group, the estrous cycles of the mice in the model group were disordered and prolonged (P<0.05), the levels of estradiol (E₂) and progesterone (P₄) decreased (P<0.05), the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and the FSH/LH ratio increased (P<0.05). The mRNA and protein expressions of PCNA, Cyclin D1, StAR, 3β-HSD, CYP19A1, LIF, and LIFR in granulosa cells decreased (P<0.05). Compared with the model group, the estrous cycles of the low- and high-dose groups of BSTJF were more regular and shorter (P<0.05), the levels of E₂ and P₄ increased (P<0.05), the levels of FSH, LH, and FSH/LH ratio decreased (P<0.05), and the mRNA and protein expressions of PCNA, Cyclin D1, StAR, 3β-HSD, CYP19A1, LIF and LIFR in granulosa cells increased (P<0.05). Compared with the BSTJF high-dose group, the estrous cycle of mice in the LIF inhibitor group was disordered and prolonged (P<0.05), the levels of E₂ and P₄ decreased (P<0.05), FSH and LH levels increased as did the FSH/LH ratio (P<0.05). In addition, the mRNA and protein expressions of PCNA, Cyclin D1, StAR, 3β-HSD, CYP19A1, and LIFR in granulosa cells decreased (P<0.05).

BSTJF may improve ovarian reserve function and promote follicular development by up-regulating LIF and LIFR, mediating the proliferation and steroid hormones synthesis of granulosa cells.