Zihuai recipe alleviates cyclophosphamide-induced diminished ovarian reserve via suppressing PI3K/AKT-mediated apoptosis

Zihuai recipe (ZHR), a Chinese herbal prescription, is widely used for the clinical treatment of Diminished ovarian reserve (DOR) infertility. However, little is known regarding its underlying mechanisms of DOR treatment. This study aimed to investigate the beneficial effects of ZHR on the treatment of DOR and to reveal the underlying mechanisms. Sixty 8-week-old Sprague–Dawley female rats were randomly divided into the following six groups (n=10 per group): control group, DOR group, low-dose (2.7 g/kg/day) ZHR (L-ZHR) group, medium-dose (5.4 g/kg/day) ZHR (M-ZHR) group, high-dose (10.8 g/kg/day) ZHR (H-ZHR) group, and hormone replacement therapy (HRT) group. The DOR model was established in all the groups, except the control group, by a single intraperitoneal injection of 90 mg/kg cyclophosphamide (CTX). After the induction of the DOR model, rats were weighed and administered either the relevant dose of ZHR or an equal volume of saline solution (in the control and DOR groups). Rats in the HRT group received estradiol valerate tablets (0.16 mg/kg/day) for 4 days, with medroxyprogesterone acetate tablets (0.86 mg/kg/day) added on day 4. After 32 days of treatment, the rats were euthanized and the ovaries were collected for sampling. Ovarian morphology was observed by hematoxylin and eosin staining and the number of follicles was counted under a microscope. The serum levels of anti-Müllerian hormone (AMH), gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were quantified by ELISA. A TUNEL assay was used to analyze the level of apoptosis of the ovarian cells. The protein expressions of p-PI3K, p-AKT, PI3K, AKT, cleaved caspase-3, BAX, and Bcl-2 were measured by western blotting and immunohistochemistry. Data analysis was performed with SPSS 20.0 software. ZHR administration increased the ovarian index and the serum levels of AMH, GnRH, and E2, while it lowered those of FSH and LH. ZHR treatment also increased the numbers of primordial, primary, secondary, and antral follicles, as well as the numbers of corpora lutea, but decreased the number of atretic follicles. Furthermore, ZHR administration decreased the percentage of TUNEL-positive ovarian cells. After treatment with ZHR, the protein expression levels of p-PI3K/PI3K, p-AKT/AKT, cleaved caspase-3 and BAX were decreased, whereas the level of Bcl-2 was increased. ZHR improved the ovarian reserve in CTX-induced DOR rats. The mechanisms of ZHR on DOR may be mediated through the regulation of gonadal hormones of the hypothalamic-pituitary-ovarian axis, and the inhibition of PI3K/AKT-mediated apoptosis in granulosa cells.