Glabridin improves cardiac remodeling after myocardial infarction by activating PPARγ to regulate the ubiquitination and degradation of EGFR

Figures

• 

  Fig. 1. Glabridin is effective in the treatment of myocardial infarction, and EGFR is the main target of glabridin’s action. A Venn diagram of 120 Lut targets and 1249 PMOP targets. B PPI network diagram of the intersection targets of myocardial infarction and glabridin. C The chemical structure of glabridin. D GO enrichment analysis of 80 therapeutic targets, including the significant enrichment terms of BP, CC, and MF. E KEGG pathway enrichment bubble plot of overlapping targets between glabridin and myocardial infarction

• 

  Fig. 2. Glabridin improved cardiac function and cardiac fibrosis in mice. A Schematic protocol for glabridin treatment. B The immunohistochemical results showed that the expression of PPARγ in the myocardium of mice with myocardial infarction was decreased. C The statistical analysis diagrams of immunohistochemical staining reflecting PPARγ expression(n = 3). D Western blot showed PPARγ expression in the myocardium of mice with myocardial infarction was decreased. E The statistical analysis diagram of PPARγ protein level relative to GAPDH (n = 3). F-G Echocardiography showed that glabridin enhanced myocardial contractility and improved cardiac function to some extent (n = 3). H-I Masson’s staining showed that glabridin reduced the fibrotic area after myocardial infarction. J The statistical analysis of parameters relative to cardiac function(n = 3). K-L The statistical analysis diagrams of fibrosis area of Masson’s staining (n = 3). M H&E staining showed that glabridin could reduce the infiltration of inflammatory cells and improve the arrangement of myocardial fibers. *:p < 0.05;**:p < 0.01; ****:p < 0.001

• 

  Fig. 3. Glabridin alleviated apoptosis and oxidative stress in cardiac tissue. A Glabridin activated PPARγ and reduced cardiomyocyte size. B The statistical analysis diagrams of WGA reflecting cardiomyocyte size (n = 3). C The statistical analysis diagrams of immunofluorescence staining reflecting PPARγ expression (n = 3). D-E WB showed that glabridin reduced the expression of myocardial fibrosis markers and inhibited the PI3K/Akt signaling pathway(n = 3). F-G TUNEL showed glabridin attenuated cardiomyocyte apoptosis(n = 3). H-I DHE showed that glabridin attenuated ROS production in myocardial tissue (n = 3). *:p < 0.05;**:p < 0.01;***:p < 0.005; ****:p < 0.001

• 

  Fig. 4. Glabridin activated PPARγ and alleviated apoptosis in vitro. A Cell viability at different concentrations of glabridin as measured by CCK8 (n = 5). B Apoptosis measurement by flow cytometry showed that glabridin reduced apoptosis. C Immunofluorescence results showed TGF-β decreased the expression of PPARγ, but glabridin reversed this phenomenon. D The statistical analysis diagrams of cell death rate (n = 3). E The statistical analysis diagrams of immunofluorescence staining reflecting PPARγ expression (n = 3). F-G WB showed that glabridin inhibited the PI3K/Akt signaling pathway (n = 3). *:p < 0.05;**:p < 0.01;***:p < 0.005; ****:p < 0.001

• 

  Fig. 5. Glabridin inhibited the proliferation and migration of fibroblasts in vitro. A Transwell assay results showed glabridin inhibited the migration of fibroblasts. B-C α-SMA immunnofluorescence staining showed that glabridin reduced the expression of α-SMA (n = 3). D-E The scratch test results indicated that glabridin reduced the migration of fibroblasts (n = 3). **:p < 0.01;***:p < 0.005; ****:p < 0.001

• 

  Fig. 6. PPARγ and EGFR interacted with each other, and glabridin promoted the ubiquitination degradation of EGFR. A Representative immunnofluorescence images of the colocalization of PPARγ and EGFR in fibroblasts. B Representative western blots performed with flag or HA antibody after co-immunoprecipitation (IP) of PPARγ and EGFR. C-D The western blotting analysis of ubiquitination assays showed glabridin promoted the ubiquitination of EGFR (n = 3). E-F The docking model of PPARγ and glabridin. ***:p < 0.005; ****:p < 0.001

• 

  Fig. 7. Inhibition of PPARγ or activation of EGFR both intensified the activation of fibroblasts and reduced the ubiquitination degradation of EGFR. A The Transwell assay results indicated that inhibiting PPARγ intensified the migration of fibroblasts. B The western blotting analysis of ubiquitination assays showed the inhibition of PPARγ inhibited the ubiquitination degradation of EGFR. C The α-SMA immunofluorescence staining results showed that inhibiting PPARγ promoted the activation of fibroblasts. D Inhibition of PPARγ promoted the activation of the PI3K/Akt signaling pathway. E The statistical analysis diagrams of immunofluorescence staining reflecting α-SMA expression (n = 3). F The statistical analysis diagram of PPARγ and PI3K/Akt pathway protein level relative to GAPDH (n = 3). G The α-SMA immunofluorescence staining results showed that activating EGFR promoted the activation of fibroblasts. H Activation of EGFR promoted the activation of the PI3K/Akt signaling pathway. I The statistical analysis diagrams of immunofluorescence staining reflecting α-SMA expression (n = 3). J The statistical analysis diagram of EGFR and PI3K/Akt pathway protein level relative to GAPDH (n = 3).…