Salvia miltiorrhiza extract ameliorated ovarian damage induced by cyclophosphamide in mice by multidimensional mechanisms.

Abstract

Introduction: Cyclophosphamide (CTX) can result in particularly severe ovarian damage by the broad range of damaging mechanisms, yet now single compounds struggle to provide comprehensive protection against CTX-induced ovarian damage. Salviae Miltiorrhizae Radix et Rhizoma (SM), as the traditional Chinese medicines (TCMs) with multi-component nature, offers hope for prevention and treatment against multi-mechanistic drugs causing ovarian damage. In this study, we aimed to investigate the protective effect of SM on ovarian hypofunction induced by CTX and explore its underlying mechanisms in mice.

Methods: The Salvia miltiorrhiza Bunge with a final concentration of 1.33 g/mL was administrated daily by gavage in mice with tumor xenografts and/or CTX intraperitoneal intervention. The subcutaneous xenograft tumor volume of each mouse was recorded and calculated every 3 days by measuring its width and length. After drug administration, estrous cycle was monitored and follicle counting was performed by H&E staining, as well as ELISA assays for serum hormone and mating experiment for fertility assessment. The apoptosis inhibition, antioxidant capacity, vascular protective and anti-fibrotic effects of SM was respectively evaluated by TUNEL staining, ROS, DPPH scavenging and antioxidant enzymes assays, vascular morphological observation, masson staining, as well as immunofluorescence staining and western blotting for the related-protein expression.

Results: SM didn't affect the anti-cancer efficacy of CTX and tumor growth rate in vivo. After SM administration for 41 days, the dropped body weight and decreased ovarian index caused by CTX gained obviously, the disturbed estrus cycle was markedly restored, a significant increase emerged in the levels of AMH and E₂ and a significant decrease appeared synchronously in FSH levels, a significant increase in primordial follicles and a corresponding decrease in atretic follicles was showed with the same increasing trend in the primary follicles, secondary follicles and antral follicles. Our results also showed that CTX could lead to pregnancy rate decreased, especially the average stillbirth rate raised obviously, both of which could be improved by SM, but without effect on the average litter size and body weight of alive pups. In-depth research showed that SM could effectively attenuate CTX-induced granulosa cells apoptosis to a certain extent by blocking the activation of the apoptotic proteins, also alleviate oxidative stress damage caused by CTX through its anti-oxidative stress ability, even reverse CTX-induced ovarian vascular occlusion and alleviate ovarian tissue fibrosis.

Conclusion: Our findings verified that SM could significantly reduce primordial follicle (PMF) loss and follicular atresia, and ameliorate CTX-induced ovarian dysfunction and poor fertility, but didn't affect the anti-tumor effect of CTX and tumor growth. Further compelling evidence was provided that, the protective effect of SM may be related to apoptosis inhibition, low oxidative stress, vascular protection and anti-fibrotic effect, which further confirmed the comprehensive protection of SM on CTX-induced ovarian damage, thereby supporting its potential clinical efficacy in mitigating chemotherapy-induced decline of ovarian function.