Influência da rutina sobre a ativação in vitro de folículos primordiais ovinos e contra a toxicidade ovariana induzida pela cisplatina e doxorrubicina em camundongos

Abstract

The aims of this study were to analyze the effects of rutin on the follicular development after in vitro culture of ovine ovarian tissue and against the ovarian toxicity induced by cisplatin or doxorubicin in mice, and to verify the possible involvement of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, and its members such as Protein kinase B (AKT), phosphatase and tension homolog (PTEN) and forkhead box O3a (FOXO3a) in the rutin actions in the ovary of these species. For chapter 1 of this thesis, ovine ovarian fragments were cultured in α-minimum essential medium alone (α-MEM+) or in this medium supplemented with 0.1; 1 or 10 μg/mL rutin (chapter 1) for 7 days. Inhibition of PI3K activity was performed in fragments cultured with LY294002. The following endpoints were analysed: follicle survival, activation and growth of primordial follicles, apoptosis and AKT phosphorylation (p-AKT). The results showed that 1 μg/mL rutin has higher percentage of normal follicles, activation and growth (P<0.05) compared to α-MEM+. After PI3K inhibition, there was a reduction (P<0.05) of follicular survival, activation and growth, as well as of p-Akt immunolocalization. For the in vivo experiment with cisplatin (chapter 2), mice were divided in groups: control, which received orally saline solution (0,15 M); the positive control group received N-acetylcysteine (150 mg/kg body weight, p.o.); the cisplatin group received cisplatin (5 mg/kg body weight, i.p.); and rutin groups received rutin (10, 30 e 50 mg/kg, v.o.) once daily for 3 days. In chapter 3, mice received saline solution (control, 0.15 M, i.p.) or doxorubicin (10 mg/kg body weight, i.p.) or they were pre and postreated with rutin (10, 30 or 50 mg/kg body weight, p.o.) before and after doxorubicin (10 mg/kg body weight, i.p.) once daily for 5 days. At the end of the experiments, the ovaries were collected for evaluation of follicular morphology, apoptosis, cell proliferation, PTEN and FOXO3a phosphorylation (p-PTEN; p-FOXO3a), and levels of reactive oxygen species (ROS), glutathione (GSH) and active mitochondria. The results showed that rutin (10 or 30 mg/kg in chapter 2 and 3, respectively) maintained the normal follicles and cell proliferation, reduced apoptosis and increased GSH levels and mitochondrial activity compared to cisplatin or doxorubicin treatments (P<0.05). Moreover, rutin (10 mg/kg) increased the expression of p-FOXO3a and reduced p-PTEN in relation to cisplatin (chapter 2). In conclusion, rutin promotes primordial follicle activation and reduces apoptosis of preantral follicles after in vitro culture of sheep ovarian tissue. In addition, in mouse, rutin can attenuate the ovarian damage caused by cisplatin and doxorubicin treatment through the PI3K pathway and its members. Thus, it is suggested that rutin can act on the survival and development of ovine primordial follicles and can be used as a therapeutic agent before antineoplastic treatment, promoting its biotechnological potential through the redirection of drugs with the intention of preventing ovarian damage.