Fig 2: E2supplementation alleviated ferroptosis of the hippocampus in female rats after OVX. (A) Measurement of MDA levels (nmol/g prot) in the hippocampus (n = 6). (B) Measurement of GSH levels (µmol/g prot) in the hippocampus (n = 6). (C) and (D) Representative images of 4-HNE immunohistochemical staining (scale bar = 20 µm, upper panel and scale bar = 10 µm, down panel) and quantitative analysis. (G) Representative TEM images of the hippocampus. Yellow triangles indicate mitochondria (scale bar = 500 nm). (H) Representative images of western blots and quantitative analysis showing the expression levels of DHODH, GPX4, TfR, ferritin, FPN1, PCBP1 and GAPDH in rat hippocampi (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: The ferroptosis resistance to FAC induced ferroptosis mediated by E2can be blocked by BQR in primary cultured hippocampal neurons. (A) Cell viability of primary cultured hippocampal neurons treated with or without FAC (0.5 mg/L), E2 (30 µM) and BQR (5 µM) for 12 h (n = 3). (B) Relative RNA level of DHODH (n = 3). (C) Representative images of western blots and quantitative analysis showing the expression levels of DHODH, GPX4, TfR, ferritin, FPN1, PCBP1 and GAPDH in primary cultured hippocampal neurons (n = 3). (D) and (G) Representative images of BODIPY C11 581/591 staining (scale bar = 50 µm) and quantitative analysis of lipid peroxidation in hippocampal neurons by the green/red fluorescence ratio. The cell nuclei were stained with DAPI (blue). (E) and (H) Representative images of MitoSOX staining (scale bar = 50 µm) and quantitative analysis of the red/blue fluorescence ratio. (F) and (I) MMP changes were measured by FCM analysis of JC-1 using annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: The ferroptosis resistance to erastin induced ferroptosis mediated by E2 can be blocked by BQR in primary cultured hippocampal neurons. (A) Cell viability of primary cultured hippocampal neurons treated with or without erastin (20 µM), E2 (30 µM) and BQR (5 µM) for 12 h (n = 3). (B) Relative RNA level of DHODH (n = 3). (C) Representative images of western blots and quantitative analysis showing the expression levels of DHODH, GPX4, TfR, ferritin, FPN1, PCBP1 and GAPDH in primary cultured hippocampal neurons (n = 3). (D) and (F) Representative images of BODIPY C11 581/591 staining (scale bar = 50 µm) and quantitative analysis of lipid peroxidation in hippocampal neurons by the green/red fluorescence ratio. The cell nuclei were stained with DAPI (blue). (E) and (G) Representative images of MitoSOX staining (scale bar = 50 µm) and quantitative analysis of the red/blue fluorescence ratio. (H) and (I) MMP changes were measured by FCM analysis of JC-1 using annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Female rats showed decreased memory ability and increased ferroptosis in the hippocampus after OVX. (A) Experimental diagram of oestrous cycle confirmation, grouping, OVX modelling and behavioural tests in female SD rats. (B) Weight gain of rats after modelling (n = 6). (C) Levels of reproduction-related serum hormones, including LH, FSH and E2 (n = 6). (D) Relative hippocampal volumes from brain MRI (cm3, n = 6). The hippocampal volume was the area times the layer thickness. (E) Representative MRI images and division of the hippocampus in various scanning layers from MRI images. White dotted lines are edges of the hippocampal region. (F) Representative traces of rats recorded in the MWM. (G) Swimming speed (cm/s, n = 6) in the MWM test. (H) Escape latency during the learning period in the MWM test (s, n = 6). (I) Platform crossover times during the probe test in the MWM (n = 6). (J) Percentage of the time or distance travelled in the platform quadrant or in the quadrant opposite the platform (n = 6). (K) Representative images of Prussian blue iron staining (enhanced with DAB) in the hippocampal region in female rats (n = 3, scale bar = 50 µm). (L) Measurement of iron content in hippocampal tissue (µg/mg, n = 6). (M) Representative images of western blots and quantitative analyses showing the expression levels of DHODH at 2, 4 and 10 weeks after modelling (n = 3). (N) Representative images of western blots and quantitative analysis showing the expression levels of DHODH, GPX4, TfR, ferritin, FPN1, PCBP1 and GAPDH in rat hippocampi at 10 weeks after modelling (n = 3). (O) Representative TEM images of the hippocampus in female rats (left, scale bar = 2 µm); yellow triangles indicate mitochondria (right, scale bar = 500 nm). *P < 0.05, OVX vs. sham, E2 and Lip-1; #P < 0.05, OVX vs. sham. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)