Rosmarinic acid mitigates stress-induced gastric ulcers by targeting the ACE2/GPX4 interaction

Figures

• 

  FIGURE 1. RAS-mediated ferroptosis contributes to gastric ulcer occurrence. (A) Volcano plot depicting RNAs that are upregulated (red) and downregulated (green); (B) KEGG enrichment analysis; (C) Venn diagram showing 14 ferroptosis-related DEGs. The yellow circle represents 533 DEGs between groups, the blue circle represents 484 ferroptosis-related genes, and the overlap represents 14 ferroptosis-related DEGs. (D) PPI analysis of the overlapped genes with ACE, ACE2, and AT1R was conducted using STRING. (E) Serum ACE2 concentration in patients by ELISA. Data are means ± SD and analyzed by Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett post hoc test. All tests were two-sided (n = 6). P < 0.05 was considered statistically significant. *P < 0.05, versus the Ctrl group; #P < 0.05, versus the Model group.

• 

  FIGURE 2. Epithelial ACE2/GPX4 interaction. (A) Single-cell sequencing analysis of ACE2 expression levels in the human stomach from the DISCO database. (B) Representative Western blot images and quantitative analysis of ACE, ACE2, and GPX4 in GEC-1 cells. GAPDH was used as an internal standard. Data are means ± SD and analyzed by Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett post hoc test. All tests were two-sided (n = 9). P < 0.05 was considered statistically significant. *P < 0.05, versus the Ctrl group; #P < 0.05, versus the Model group. (C) Molecular docking (GRAMM) among ACE2, GPX4, and SLC7A11. (D) The RMSD, Rg, RMSF, SASA, and H-bonds number of ACE2 complexed with GPX4 and SLC7A11 during the 100 ns molecular dynamics simulation. (E) Gibbs free energy landscape.

• 

  FIGURE 3. Molecular docking and molecular dynamics simulation of RA with ACE2/GPX4 interaction and in vitro experiment. (A–C) Molecular docking (AutoDock Vina v1.2.3) among RA with ACE2/GPX4 interaction. (D) The RMSD, Rg, RMSF, SASA, and H-bonds number of RA with ACE2/GPX4 interaction during the 100 ns molecular dynamics simulation. (E) Gibbs free energy landscape. (F) Representative Western blot images and quantitative analysis of ACE, ACE2, and GPX4 in Fer-1-treated GEC-1 cells. (G) Representative Western blot images and quantitative analysis of ACE, ACE2, and GPX4 in CAP-treated GEC-1 cells. GAPDH was used as an internal standard. Data are means ± SD and analyzed by Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett post hoc test. All tests were two-sided (n = 9). P < 0.05 was considered statistically significant. *P < 0.05, versus the Ctrl group; #P < 0.05, versus the Model group.

• 

  FIGURE 4. Protective effect of RA on CWIR-induced gastric ulcer in mice (A) Gastric tissue images of mice (left panel) and index of ulcer (right panel). (B) Represent HE stain in gastric tissues (scale bars: 50 μm). (C) Pepsin activity. (D) Represent PAS staining in gastric tissues (left panel, scale bars: 50 μm) and PAS-positive area ratio (right panel). Data are means ± SD and analyzed by Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett post hoc test. All tests were two-sided (n = 3). P < 0.05 was considered statistically significant. *P < 0.05, versus the Ctrl group; #P < 0.05, versus the Model group.

• 

  FIGURE 5. Effect of RA on RAS and GPX4 levels in CWIR-induced gastric ulcer mice (A) IHC staining for GPX4 expression in gastric tissues of CWIR-induced gastric ulcer mice (left panel) and the average optical density of GPX4. (B) Representative Western blot images and quantitative analysis of ACE, ACE2, and GPX4 in mice. Data are means ± SD and analyzed by Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett post hoc test. All tests were two-sided (n = 4). P < 0.05 was considered statistically significant. *P < 0.05, versus the Ctrl group; #P < 0.05, versus the Model group.