Sophoridine Counteracts Obesity via Src-Mediated Inhibition of VEGFR Expression and PI3K/AKT Phosphorylation

Figures

• 

  Figure 1. Sophoridine inhibits high-fat diet-induced bodyweight gain and improves glucose tolerance and insulin sensitivity in mice. (a) Schematic diagram of the animal experiment. The dose of SRP fed by gavage is 20 mg/kg. (b) Bodyweight curve during sophoridine treatment. (c) Tissue weight of liver, iWAT, eWAT, and BAT. (d) HE staining images of iWAT (scale bar = 10 μm) and liver (40×, 200×). (e) Plasma levels of triglyceride (TG), total cholesterol (TC), HDL, LDL, GLU, and GSP after 11 weeks treatment. (f) Plasma levels of ALT and AST. (g) Glucose tolerance test (GTT) and AUC. (h) Insulin tolerance test (ITT) and AUC. Data are presented as means ± SEM (n = 6–10). Statistical significance was analyzed by one-way ANOVA followed by Tukey’s post hoc tests. * p < 0.05, ** p < 0.01, **** p < 0.0001, vs. HFD mice; ## p < 0.01, ### p < 0.001, #### p < 0.0001, vs. ND mice. BAT: brown adipose tissue, iWAT: inguinal white adipose tissue, eWAT: epididymal white adipose tissue, TG: triglyceride, TC: total cholesterol, HDL: high density lipoprotein, LDL: low density lipoprotein, GLU: glucose, GSP: glycosylated serum protein.

• 

  Figure 2. Sophoridine inhibits DNA replication and proliferation in 3T3-L1 cells. (a) 3T3-L1 cells were treated with sophoridine (10, 20, 30, 40, 50, 60, 70, 80, and 90 μM) and cell viability was measured. (b) EdU staining after treatment with sophoridine (50 μM) and quantitative analysis (scale bar = 100 μm). (c) The number of cells in different cell cycles was measured by flow cytometry. (d) Relative mRNA expression of proliferation marker genes cyclin B, cyclin D, cyclin E, CDK4, p21, and p27. (e) Western blot detection and quantitation of cell cycle marker gene expression. Data represent mean ± SEM (n = 3). Statistical analysis was performed by Student’s t-test, * p < 0.05, and ** p < 0.01.

• 

  Figure 3. Sophoridine inhibits adipogenesis of 3T3-L1 cells. (a) Protein expression of adipogenesis-related FASN, PPARγ, C/EBPα, C/EBPβ, GLUT4, and FABP4 and lipolytic HSL and ATGL. (b) Quantitative analysis of Western blot. (c) Relative mRNA expression of adipogenesis-related and lipolytic genes. (d) Oil red O staining of 3T3-L1 cells differentiated at 6 days (200×), and determination of TG content. (e) Bodipy staining of 3T3-L1 cells (scale bar = 300 μm). Data represent mean ± SEM (n = 3). Statistical analysis was performed by Student’s t-test, * p < 0.05, and ** p < 0.01.

• 

  Figure 4. Identification of differentially expressed proteins. (a) Flow chart of proteomic sequencing of SRP and control 3T3-L1 cells. (b) Plot of the PCA (principal component analysis) distribution of all samples. (c) Quantitative volcano diagram of differentially expressed proteins. (d) Statistical diagram of the number of differentially expressed proteins. (e) Subcellular location of differentially expressed proteins. (f) KEGG pathway analysis of downregulated proteins.

• 

  Figure 5. Sophoridine targets the Src protein domain and downregulates VEGFR2 expression and phosphorylation of PI3K/AKT. (a) Molecular docking diagram of sophoridine bound to the Src kinase domain (PDB: 1YOJ). Sophoridine stick model showing carbon (gray), hydrogen (white), oxygen (red), and nitrogen (blue) atoms. (b) Hydrogen bonds at receptor surfaces and differential interactions between sophoridine and the Src kinase domain. (c) Protein expression of Src, VEGFR2, and VEGF of 3T3-L1 cells. (d) Quantitative analysis of Western blot. (e) Protein expression of ERK1/2, P-ERK1/2 (T185/Y187), P85α-PI3K, P-P85α-PI3K, AKT1/2/3, P-AKT1/2 (Tyr315/316/312) of 3T3-L1 cells. (f) Quantitative analysis of Western blot. Data represent the mean ± SEM (n = 3). Statistical analysis was performed by Student’s t-test, * p < 0.05, and ** p < 0.01.

• 

  Figure 6. Sophoridine counteracts obesity via Src-mediated inhibition of VEGFR expression and PI3K/AKT phosphorylation.