Dingkun Pill attenuates oxidative stress, autophagy, and apoptosis to alleviate cyclophosphamide-induced premature ovarian failure in rats via activation of the PI3K/AKT/mTOR signaling pathway

Abstract Background Dingkun Pill (DKP) is a traditional Chinese medicine formulation with blood-nourishing and menstruation-regulating properties, has been used clinically for gynecological disorders. However, its effects on Premature ovarian failure (POF) and the underlying mechanisms remain unclear. Methods A cyclophosphamide (CTX)-induced POF model was established in SD rats, and POF rats were allocated into POF group, low-dose (0.45 g/kg), medium-dose (0.9 g/kg), and high-dose (1.8 g/kg) DKP groups, dehydroepiandrosterone (DHEA) positive control group, PI3K inhibitor LY294002 alone treatment group, and DKP (0.9 g/kg, medium-dose)+LY294002 group. Changes in the estrous cycle were monitored via vaginal smear examination for 14 consecutive days starting from the 14th day of drug intervention. After 28 days of treatment, rats were euthanized, and serum sex hormone and oxidative stress marker levels were determined by ELISA kits. Ovarian histopathologic changes were analyzed by hematoxylin-eosin staining. Western blot detected autophagy, apoptosis, proliferation, and PI3K/AKT/mTOR axis-related protein expression, and immunofluorescence was performed to assess the autophagy, proliferation and apoptosis status of granulosa cells (GCs). Results DKP dose-dependently improved the trend of weight loss in CTX-induced POF rats, reversed sex hormone disorders, restored normal estrous cycles, the ovarian protective effect of high-dose DKP was comparable to that of DHEA. In addition, DKP (0.9 g/kg) alleviated ovarian damage, reduced follicular depletion, and promoted follicular development, significantly reduced serum malondialdehyde (from 7.7 to 4.3 µmol/L) and reactive oxygen species (from 215.3 to 142.3 U/mL) levels, and increased superoxide dismutase activity (from 2.3 to 3.2 U/mL). Meanwhile, DKP inhibited excessive autophagy in the ovaries of POF rats, and decreased pro-apoptosis-related protein levels, increased the proliferative activity of GCs and up-regulated function-related proteins of GCs. These effects were associated with PI3K/AKT/mTOR pathway activation, and co-administration of LY294002 partially attenuated DKP-mediated ovarian protection. Conclusion DKP alleviates CTX-induced POF in rats by reducing oxidative stress and regulating autophagy and apoptosis, these effects were associated with the PI3K/AKT/mTOR axis.