Study On The Mechanism Of Fer-1 Regulation Of Ferroptosis Affecting Ovarian Function In Chemotherapy-induced Premature Failure

ObjectiveIn this study,we investigated the promoting effect of ferroptosis on premature ovarian failure(POF)by Cis-dichlorodiammine platinum(Ⅱ)(CDDP),and then explored the effect of ferrostatin-1(FER-1)inhibition of ferroptosis on ovarian function and related mechanisms.To provide new ideas for the treatment of chemotherapy-induced POF.MethodsIn the first part,rat ovarian granulosa cells(GCs)model was constructed and divided into 6 groups: control group(CON group),model group(CDDP group),FER-1 pretreatment group(FER-1 group),GSH peroxidase 4 inhibitor(1S,3R-RSL3,RSL3)pretreatment group(RSL3 group),treatment group(CDDP+FER-1 group)and the over injury group(CDDP+RSL3 group).The cell viability under different influencing factors was detected by CCK8 to determine the drug concentration for subsequent experiments.1.Ferro Orange probe was chosen to detect intracellular Fe2+ levels,and Mito-Ferrogreen probe was taken to analyze intracellular Fe2+ levels in mitochondria.2.DCFH-DA was used to detect intracellular Reactive oxygen species(ROS)levels,and Mito SOX Red probe was taken to observe mitochondrial ROS levels.3.Mitochondrial membrane potential levels were detected by the Rhodamine123 probe.In the second part,POF rat model was established and divided into three groups: control group(CON group),model group(CDDP group)and treatment group(CDDP+FER-1 group).The vaginal smear was used to monitor the motility cycle of the rats,and serum follicle stimulating hormone(FSH)and estradiol(E2)levels were analyzed by enzyme-linked immunosorbent assay(ELISA).Body weight,bilateral ovarian and uterine wet weight were measured to calculate ovarian index and uterine index,respectively.1.The level of oxidative stress in rats were assessed by malondialdehyde(MDA),superoxide dismutase(SOD),and total glutathione(GSH).2.The Fe2+ content in the tissues was detected using the ferrous zine method.3.Western blotting was used to analyze the relative protein expression levels of Keclch like ECH associated protein 1(KEAP-1),Nuclear factor erythroid 2 related factor 2(NRF2),Haemoxygenase-1(HO-1)and Glutathione peroxidase 4(GPX4).ResultsPart I rat GCs model: 30 μmol/L CDDP,30 μmol/L FER-1 and 3 μmol/L RSL3 were used as experimental drug concentrations.1.Compared with the CON group,there was no significant change in the FER-1 group(P>0.05)and the difference was not statistically significant.There was Fe2+ accumulation in intracellular and mitochondria in the CDDP and RSL3 groups compared with the CON group(P<0.01),and the difference was statistically significant.Compared with the CDDP group,the intracellular and mitochondrial Fe2+ levels could continue to increase in the CDDP+RSL3 group(P<0.01);the CDDP+FER-1 group could improve the intracellular and mitochondrial excess Fe2+(P<0.01),the difference were all statistically significant.2.There was no significant change in the FER-1 group compared with the CON group(P>0.05),the difference was not statistically significant.Compared with the CON group,both the CDDP and RSL3 groups produced excessive intracellular and mitochondrial ROS(P<0.01),the difference was statistically significant.Compared with the CDDP group,the intracellular and mitochondrial ROS levels would be further increased in the CDDP+RSL3 group(P<0.01);the intracellular and mitochondrial ROS levels were significantly reduced in the CDDP+FER-1 group(P<0.01),the difference were all statistically significant.3.Compared with the CON group,no significant changes in mitochondrial membrane potential were seen in the FER-1 group(P>0.05),the difference was not statistically significant.Compared with the CON group,both the CDDP and RSL3 groups showed partial loss of mitochondrial membrane potential(P<0.01),the difference was statistically significant.Compared with the CDDP group,most of the mitochondrial membrane potential was lost in the CDDP+RSL3 group(P<0.01);however,the CDDP+FER-1 group could significantly restore the mitochondrial membrane potential(P<0.01),the differences were all statistically significant.Part Ⅱ POF rat model: Compared with the CON group,the CDDP group showed disturbed motile cycle,increased FSH level(P<0.01),decreased E2level(P<0.01),decreased ovarian and uterine indices(P<0.01),these differences were all statistically significant.Compared with the CDDP group,the CDDP+FER-1 group improved the disturbed motile cycle,decreased FSH level(P<0.01),increased E2 level(P<0.05),increased ovarian index and uterine index after two weeks of treatment(P<0.01),the differences were all statistically significant.1.Compared to the CON group,the CDDP group had increased serum MDA levels(P<0.01),decreased serum SOD levels(P<0.01),and decreased total tissue GSH levels(P<0.01),the differences were all statistically significant.Compared with the CDDP group,the CDDP+FER-1 group had decreased serum MDA levels(P<0.01),increased serum SOD levels(P<0.01),and increased total tissue GSH levels(P<0.05),the difference were all statistically significant.2.Increased ovarian tissue iron levels within the CDDP group compared to the CON group(P<0.01),the difference was statistically significant.Ovarian tissue iron content was decreased in the CDDP+FER-1 group compared to the CDDP group(P<0.01),and the difference was statistically significant.3.Compared with the CON group,KEAP-1 protein expression was increased(P<0.01),however,the NRF2(P<0.05),HO-1(P<0.01)and GPX4(P<0.01)protein expression was decreased in the CDDP group,the differences were all statistically significant.Compared with CDDP group,KEAP-1 protein expression was decreased(P<0.01),NRF2(P<0.05),HO-1(P<0.01)and GPX4(P<0.01)protein expression was increased in the CDDP+FER-1 group,the differences were all statistically significant.Conclusions1.Ferroptosis was present in CDDP-induced ovarian dysfunction and GCs injury.2.RSL3 promotes ferroptosis to aggravate GCs injury,FER-1 inhibits ferroptosis to reduce GCs injury.3.FER-1 can regulate serum FSH and E2 hormone levels and improve ovarian oxidative stress status.4.FER-1 inhibits ferroptosis through KEAP-1/NRF2/HO-1 pathway and improves CDDP-induced ovarian injury.