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

Mei Jiang,Yucong Bi,Jingxuan Zhang,Chunguo Wang,Song Yang,Weiling Wang,Ting Wang,Jialin Li,Pin Li,Yan-Tong Xu

doi:10.3389/fphar.2020.00546

Mei Jiang, Yucong Bi + Show 8 more

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https://doi.org/10.3389/fphar.2020.00546

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Abstract

BackgroundsDiminished 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.MethodsThe 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 (E2), 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.ResultsChemical 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 E2 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.ConclusionsThe 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.

Highlights

Diminished ovarian reserve (DOR) refers to reproductive-age women with regular menses whose response to ovarian stimulation or fecundity is reduced compared with healthy women of comparable age (Practice Committee of the American Society for Reproductive, 2015)
The identified 84 chemical components included 43 flavonoids, 13 triterpenoids, 11 phenolic acids, 8 alkaloids, 1 coumarin, 1 anthraquinone, and 7 other components (Figure 1D), among which 5 chemical components were identified by comparison to standard components: kaempferol, quercetin, hesperetin, hesperidin, and caffeic acid
Serum anti-Müllerian hormone (AMH) was increased in H-Bushen Cuyun Recipe (BCR) and DHEA groups compared with the DOR group (p < 0.05) (Figure 2E)

Summary

Introduction

Diminished ovarian reserve (DOR) refers to reproductive-age women with regular menses whose response to ovarian stimulation or fecundity is reduced compared with healthy women of comparable age (Practice Committee of the American Society for Reproductive, 2015). Long-term anxiety could cause dysfunction of the hypothalamic-pituitary-ovarian axis (HPOA), which can lead to DOR or premature ovarian failure (Nicoloro-SantaBarbara et al, 2017) Among these factors, alkylating chemotherapy agents, such as cyclophosphamide (CTX), which is highly gonad-toxic, led to a decrease in ovarian function and in anti-Müllerian hormone (AMH) (BenAharon et al, 2015). It could induce ovarian damage by activation of the PI3K/AKT and mTOR pathways (Goldman et al, 2017), leading to primordial follicle activation and follicular “burnout” (Chang et al, 2015).

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