The effect of hydroxytyrosol on diminished ovarian reserve induced by cyclophosphamide through Nrf2/HO-1 and NF-κB signaling pathways in mice
Objective:To investigate the impact of hydroxytyrosol(HT)on ovarian damage in mice with diminished ovarian reserve(DOR)induced by cyclophosphamide(CTX)through Nrf2/HO-1 and NF-κB signaling pathways as well as the mechanism of ovarian function improvement by HT in DOR mice.Methods:A total of forty female C57BL/6L mice aged 7-8 weeks were randomly divided into four groups:the control group,the model group(CTX group),HT low-dose experimental group(CTX+L-HT group,50 mg·kg-1·d-1)and HT high-dose experimental group(CTX+H-HT group,100 mg·kg-1·d-1).Each group consisted of ten mice.The DOR mouse model was induced through the intraperitoneal administration of CTX(50 mg·kg-1·d-1).Morphological changes in ovarian tissue and the number of follicles at all levels were assessed using HE staining among all experimental groups.The sex hormone,oxidative stress factors and inflammatory factors levels in serum were detected by enzyme-linked immunosorbent assay(ELISA).Western blot was used to evaluate protein expression levels of Nrf2,HO-1,p-NF-κB and NF-κB.Results:Compared with the control group,there was a significant decrease in the number of follicles at all levels,while the presence of atretic follicles significantly increased(P＜0.05),and the granulosa cell layer and corpus luteum were also reduced in the CTX group.Furthermore,the serum concentrations of FSH and LH were significantly higher and the levels of estradiol(E2)and anti-Müllerian hormone(AMH)were significantly lower in the CTX group(P＜0.05).The activities of superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)in ovarian tissue were significantly lower(P＜0.05),and the MDA content and the levels of proinflammatory factors including tumor necrosis factor(TNF)-α,interleukin(IL)-6,and IL-1β in ovarian tissue were significantly higher and the level of anti-inflammatory factor(IL-10)was significantly lower in the CTX group when compared with the control group(P＜0.05).Additionally,the expressions of Nrf2 and HO-1 proteins were significantly lower and the ratio of p-NF-κB/NF-κB protein expression level was significantly higher in the CTX group when compared with the control group(P＜0.05).When compared with the CTX group,the CTX+L-HT group and the CTX+H-HT group exhibited a significant increase in each grade of follicles,along with a significant decrease in atretic follicles(P＜0.05).The serum levels of FSH and LH were significantly lower and the concentrations of E2 and AMH were significantly higher in the CTX+L-HT group and the CTX+H-HT group than those of the CTX group(P＜0.05).In the CTX+L-HT group and the CTX+H-HT group,higher activities of SOD,CAT and GSH-Px in ovarian tissue,lower content of MDA and the levels of proinflammatory factors as well as higher anti-inflammatory factors were observed(P＜0.05).Moreover,the expression levels of Nrf2 and HO-1 proteins in.ovarian tissue were significantly higher,whereas the ratio of p-NF-κB/NF-κB protein expression level exhibited a significant decrease in the CTX+L-HT group and the CTX+H-HT group(P＜0.05).Furthermore,there was significantly higher level of IL-10 in the CTX+H-HT group than that of the CTX+L-HT group(P＜0.05).Conclusions:HT may have a potential protective effect on ovarian damage in mice with diminished ovarian reserve(DOR),likely by activating the Nrf2/HO-1 signaling pathway,inhibiting the NF-κB signaling pathway,and attenuating oxidative stress and inflammation.
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