Figures & data
Figure 1. Chemical structure of astragaloside-IV (AS-IV) obtained from Astragalus membranaceus.
Figure 2. High glucose promotes ferroptosis in retinal pigment endothelial (RPE) cells. (a) Cell viability determined by calcein-AM/propidium iodide (PI) staining. (b) Intracellular lipid oxidation rate determined using the C11-BODIPY™ 581/591 probe. C) Total intracellular content of reactive oxygen species (ROS) detected by the CM-H2DCFDA probe. (d) Glutathione (GSH) and oxidized glutathione (GSSG) assay. (e) Cell substructure determined by transmission electron microscopy (TEM); scale bar: 1.0 μm. (f) Cell death rate analyzed by calcein-AM/PI staining. * indicates p values < 0.05; ** indicates p values < 0.01.
Figure 3. Astragaloside-IV (AS-IV) inhibits ferroptosis induced by high glucose in retinal pigment endothelial (RPE) cells. (a) Intracellular lipid oxidation level measured by the C11-C11-BODIPY™ 581/591 probe. (b) Total intracellular content of reactive oxygen species (ROS) detected with the CM-H2DCFDA probe. (c) The cell substructure observed by TEM; D. The level of cell death analyzed by calcein-AM/propidium iodide (PI) staining. * indicates p values < 0.05; ** indicates p values < 0.01.
Figure 4. Astragaloside-IV (AS-IV) alleviates the damage caused by high glucose by reducing Sirt1/Nrf2 signaling activity in retinal pigment endothelial (RPE) cells. (a, d) Detection of Nrf2 by Western blot analysis. (b, c) Detection of Sirt1 by Western blot analysis. (e) Levels of glutathione peroxidase 4 (GPX4), glutamate cysteine ligase catalytic subunit (GCLC), and glutamate cysteine ligase modifier subunit (GCLM) proteins detected by Western blot analysis. ** indicates p values < 0.01.
Figure 5. High glucose inhibits the Sirt1/Nrf2 signaling cascade activity by increasing expression of miR-138-5p in retinal pigment endothelial (RPE) cells. (a) Expression of miRNAs by quantitative reverse-transcription PCR (qRT-PCR). (b, d) Expression levels of miR-138-5p in RPE cells determined by qRT-PCR. (c, e) Expression levels of miR-211-5p in RPE cells detected by qRT-PCR. (f) Protein levels of Sirt1 detected by Western blot analysis. (g) Protein levels of Nrf2 in RPE cells detected by Western blot analysis. * indicates p values < 0.05; ** indicates p values < 0.01.
Figure 6. Astragaloside-IV (AS-IV) enhances Sirt1/Nrf2 signaling activity in retinal pigment endothelial (RPE) cells under high glucose conditions by inhibiting expression of miR-138-5p. (a, b) Expression levels of miR-138-5p in RPE cells detected by qRT-PCR. (c) Protein level of Sirt1 in RPE cells detected by Western blot analysis. (d) Protein level of Nrf2 in RPE nucleus detected by Western blot analysis and grayscale map. (e) Protein levels of glutathione peroxidase 4 (GPX4), glutamate cysteine ligase modifier subunit (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC) in RPE cells detected by Western blot analysis. ** indicates p values < 0.01.
Figure 7. Astragaloside-IV (AS-IV) decreases ferroptosis in retinal pigment endothelial (RPE) cells under high glucose conditions by inhibiting expression of miR-138-5p. (a) Lipid oxidation level in cells detected by the C11-BODIPY™ 581/591 probe. (b) Total content of reactive oxygen species (ROS) detected by the CM-H2DCFDA probe. (c) Glutathione (GSH), oxidized glutathione (GSSG), and GSH:GSSG ratio analyses. (d) Cell substructure as determined by transmission electron microscopy (TEM); scale bar: 1.0 μm. (e) Cell death rate measured by flow cytometry. ** indicates p values < 0.01.
