Protective Effects and Possible Mechanisms of Actions of Bushen Cuyun Recipe on Diminished Ovarian Reserve Induced by Cyclophosphamide in Rats

BACKGROUNDS: Diminished ovarian reserve (DOR) contributes significantly to female infertility. Bushen Cuyun Recipe (BCR, Tradename Yueliang Yin), a product marketed in China, has shown effects in the treatment of female infertility in clinical practices of traditional Chinese medicine (TCM). In this study, we aimed to investigate the chemical compositions of BCR and its efficacy based on scientific evidence and pharmacological mechanisms in DOR treatments. METHODS: The chemical compositions of BCR were determined by the UHPLC-LTQ-Orbitrap MS method. DOR was induced in a rat model by intraperitoneal injection of cyclophosphamide (CTX) 90 mg/kg once. After the CTX treatment for 14 days, rats were intragastrically administrated deionized water, dehydroepiandrosterone (DHEA), or BCR in low, middle, and high doses for 30 days. Ovarian index, ovarian morphology, follicle number, and anti-Müllerian hormone (AMH) in serum were determined to assess the effects of BCR. To investigate possible action mechanisms, network pharmacological analysis was used to predict possible pathways in the effects of BCR on female infertility. In experimental studies, the contents of hormones in the hypothalamic-pituitary-ovarian axis (HPOA, including estradiol (E(2)), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH)) and pyroptosis-related proteins, including gasdermin D (GSDMD), caspase-1, and interleukin-18 (IL-18), in ovarian were detected by ELISA, immunofluorescence and Western blot. RESULTS: Chemical studies revealed a total 84 components in BCR, which included 43 flavonoids, 13 triterpenoids, 11 phenolic acids, 8 alkaloids, 1 coumarin, 1 anthraquinone, and 7 other components. After treatments with BCR, the ovarian morphology, ovarian index, estrous cycle, growing follicles and corpus luteum from last ovulation, and serum AMH in DOR rats were significantly improved. Network pharmacological analysis suggested that the NOD-like receptor signaling pathway ranked No. 1 among the mechanisms by which BCR affects female infertility. Experimental results demonstrated that the content of serum FSH in DOR rats was significantly decreased and the contents of serum GnRH and E(2) were significantly elevated after BCR treatment and that the elevated level of GSDMD, caspase-1, and IL-18 was significantly reversed in BCR-treated rats. CONCLUSIONS: The chemical compositions of BCR were first identified in the present study. BCR was demonstrated to show protective effects on DOR. The possible mechanisms of BCR on DOR might be mediated by regulating gonadal hormones of the HPOA and protecting granulosa cells in ovary against pyroptosis.