Quercetin Prevents Cyclophosphamide-induced Primordial Follicle Loss Via Suppression Of PI3K/AKT/FOXO3a Pathway Activation In The Mouse Ovary

Objective: Premature ovarian failure and infertility are serious side effects of chemotherapy treatments in reproductive age women with malignant tumor,which could also be a severe social problem related to reproductive health.As a common alkylating agent,cyclophosphamide(CY)is highly toxic to the ovaries,previous research has demonstrated that it can promote the activation of primordial follicles and lead to their overall loss.The PI3 K / AKT / FOXO3 a pathway is known to regulate the activation of primordial follicles.Hereby,in this study,rodent model was recruited to to evaluate effect of CY on follicles at various levels and on the expression of the key protein of the PI3 K / AKT / FOXO3 a pathway.Quercetin is a is a natural flavonoid and has been shown to be a potential anti-cancer candidate via inhibiting the PI3 K pathway.Therfore,this study was also designed to investigate whether quercetin can lead to an overall reduction in primordial follicle loss and better fertility in CY-treated mice.Methods:(1)Forty C57BL/6 female mice aged 5-6 weeks were randomly divided into two groups: control group(PBS,ip)and CY group(CY 75mg/kg,ip).Animals were sacrificed 24 h after CY administration,orbital blood was collected to detect AMH,ovaries were removed forhistochemicall analysis of Ki67,cleaved-caspase-3 and TUNEL staining,and Western-blot for the protein expression of the PI3 K / AKT /FOXO3 a pathway were detected by.Animals were also sacrificed 7d after CY administration,and ovaries were taken for HE staining to count the follicles. (2)One hundred and twenty C57BL/6 female mice aged 5-6 weeks were randomly divided into six groups: control group,quercetin of low-dose group(Que1,20mg/kg,ip),quercetin of high-dose group(Que2,40mg/kg,ip),cyclophosphamide group(CY,75mg/kg,ip),cyclophosphamide and quercetin of low-dose co-treatment group(CY +Que1 group),cyclophosphamide and quercetin of high-dose co-treatment group(CY +Que2 group).Animals were sacrificed 24 h after CY administration,and orbital blood was collected to detect the level of AMH,FSH,E2.Similarly,ovaries were removed for histochemical analysis of Ki67 and cleaved-caspase-3,TUNEL staining,and Western-blot for the protein expression of the PI3K/AKT/FOXO3 a pathway.Animals were also sacrificed 7d after CY administration,and ovaries were taken for HE staining to count the follicles.Additionally,the fertility index of two mating cycles was observed respectively at 8 or 14 weeks after CY administration.Results: After CY treatment,the follicles at various levels in mice were significantly reduced in mice,and the phosphorylation of the PI3K/AKT/FOXO3 a pathway protein were significantly increased.The oocytes of the primordial follicles and their surrounding pregranular cells did not undergo apoptosis,but the proliferation of granulosa cells around oocytes of early-growing follicles was increased.The level of AMH and E2 was decreased,while the concentration of FSH was increased.Cotreatment with quercetin significantly inhibited the loss of primordial follicles,supressed the phosphorylation of the PI3K/AKT/FOXO3 a pathway protein and restrained the nuclear export of FOXO3 a,Serum AMH and E2 level was also increased and the FSH level was decreased.Conclusion: Cyclophosphamide(CY)can lead to the overall loss of primordial follicles by activating the PI3K/AKT/FOXO3 a pathway,while quercetin can prevent the loss of primordial follicles induced by CY via inhibiting the PI3K/AKT/FOXO3 a pathway.This finding provides novel insights in alleviating premature ovarian failure and improving fertility for female patients undergoing cancer chemotherapy..