Neuroprotective Potential of Peptide KSVSPKFLTG from Magallana gigas via Activating the ACE2/Ang-(1-7)/Mas Axis in SHSY-5Y Cells

Figures

• 

  Figure 1. Effects of KSVSPKFLTG peptide on the antioxidant defense of SHSY-5Y cells. (A) Effects of different concentrations of KSVSPKFLTG on cell viability. (B) Alleviative effects of KSVSPKFLTG at different concentrations on cellular injury. (C) Fluorescence images of ROS in SH-SY5Y cells under different treatments (scale bar = 50 μm). (D) Quantitative analysis of ROS fluorescence intensity in SH-SY5Y cells under different treatments. (E) The relative MDA content in SHSY-5Y cells. (F) The relative SOD activity in SHSY-5Y cells. All experiments were performed with n = 6 biological replicates. The significance of the ANOVA analysis is expressed as * p < 0.05, ** p < 0.01 and **** p < 0.0001 (ns, not significant).

• 

  Figure 2. Detection of mitochondrial membrane potential upon KSVSPKFLTG peptide treatment using JC-1 staining. (A) Representative JC-1 flow cytometry scatter plots. (B) Quantification of the percentage of cells with high JC-1 aggregate (red, Q2) fluorescence and high JC-1 monomer (green, Q3) fluorescence across six experimental groups. Data are presented as mean ± SD; n = 3 independent biological replicates.

• 

  Figure 3. Cytosolic iron deposition status of SHSY-5Y cells. (A) Prussian blue staining of SHSY-5Y cells (scale bar = 20 μm). (B) Determination of total cytosolic iron content in SHSY-5Y cells. (C) Cytosolic GPX4 activity assay in SHSY-5Y cells. All experiments were performed with n = 3 biological replicates. The significance of the ANOVA analysis is expressed as * p < 0.05, ** p < 0.01 (ns, not significant).

• 

  Figure 4. Western blot analysis of the protein expression of ACE2, MAS1L, and Nrf2. (A) Western blot bands for different proteins. The size of bubbles with the same color corresponds to the relative grayscale values of the target proteins in their respective groups. (B–D) The expression levels of ACE2, MAS1L, and Nrf2 in SHSY-5Y cells. All experiments were performed with n = 3 biological replicates. The significance of the ANOVA analysis is expressed as * p < 0.05 (ns, not significant).

• 

  Figure 5. Western blot analysis of the protein expression of GPX4, BCBP2, SLC7A11, and SLC40A1. (A) Western blot bands for different proteins. The size of bubbles with the same color corresponds to the relative grayscale values of the target proteins in their respective groups. (B–E) The expression levels of GPX4, BCBP2, SLC7A11, and SLC40A1 in SHSY-5Y cells. All experiments were performed with n = 3 biological replicates. The significance of the ANOVA analysis is expressed as * p < 0.05, ** p < 0.01 and *** p < 0.001 (ns, not significant).

• 

  Figure 6. Western blot analysis of the protein expression of ACE2, Nrf2, SLC7A11, MAS, BCBP2, and GPX4. (A) Different Western blot bands. The size of bubbles with the same color corresponds to the relative grayscale values of the target proteins in their respective groups. (B–G) The expression levels of ACE2, Nrf2, SLC7A11, MAS, BCBP2 and GPX4 in SHSY-5Y cells. All experiments were performed with n = 3 biological replicates. The significance of the ANOVA analysis is expressed as * p < 0.05, ** p < 0.01 and *** p < 0.001 (ns, not significant).