Dapagliflozin exerts anti-apoptotic effects by mitigating macrophage polarization via modulation of the phosphoinositide 3-kinase/protein kinase B signaling pathway

Figure 1 Effects of dapagliflozin treatment on blood glucose and liver and kidney functions in diabetic mice. A: Dynamic changes in blood glucose in each group of mice; B-F: Serum concentrations of serum alkaline phosphatase, creatinine, alanine aminotransferase, urea and uric acid in mice. The bars indicate the mean ± SD from three independent experiments (n = 3). ¹P < 0.05 vs normal control group; ²P < 0.05 vs type 2 diabetic mouse group. NC: Normal control group; DM: Type 2 diabetic mouse group; DAPA: Type 2 diabetic mouse group treated with dapagliflozin; STZ: Streptozotocin.

Figure 2 Apoptosis, M1 polarization, and inflammatory factor expression were reduced in peritoneal macrophages from dapagliflozin-treated mice. Relative mRNA expression levels of M1 macrophage surface markers: A: Inducible nitric oxide synthase; B: Cluster of differentiation 86; Relative mRNA expression levels of apoptosis-related factors: C: Proapoptotic factor (Caspase 3); D: Antiapoptotic factor = (BCL2); Relative mRNA expression levels of inflammatory factors: E: Interleukin-6; F: Interleukin-1β; G: Tumor necrosis factor-α. The bars indicate the mean ± SD from three independent experiments (n = 3). ¹P < 0.05 vs normal control group; ²P < 0.05 vs type 2 diabetic mouse group. NC: Normal control group; DM: Type 2 diabetic mouse group; DAPA: Type 2 diabetic mouse group treated with dapagliflozin; IL: Interleukin; TNF: Tumor necrosis factor; CD: Cluster of differentiation; iNOS: Inducible nitric oxide synthase.

Figure 3 Dapagliflozin inhibits the activation of the phosphoinositide 3-kinase/protein kinase B signaling pathway. A: Protein imprints of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and phosphorylated protein kinase B (P-AKT) in mouse peritoneal macrophages after dapagliflozin treatment; B: Quantitative analysis of PI3K; C: Quantitative analysis of AKT; D: Quantitative analysis of P-AKT. The bars indicate the mean ± SD from three independent experiments (n = 3). ¹P < 0.05 vs normal control group; ²P < 0.05 vs type 2 diabetic mouse group. NC: Normal control group; DM: Type 2 diabetic mouse group; DAPA: Type 2 diabetic mouse group treated with dapagliflozin; PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; P-AKT: Phosphorylated protein kinase B.

Figure 4 Effects of dapagliflozin pretreatment on the human monocyte cell line cell viability. Cell viability was measured with a cell counting kit 8. The data are presented as percentages, and the experiment was independently repeated 3 times. ¹P < 0.05 vs control group (100 μM, 24 hours).

Figure 5 Dapagliflozin ameliorated the human monocyte cell line cell apoptosis, which was inhibited by 740Y-P (a phosphoinositide 3-kinase/protein kinase B signaling pathway agonist). Relative mRNA expression levels of the following apoptosis-related factors: A: Proapoptotic factors (Caspase 3) B: Antiapoptotic factors (BCL2); C: Flow cytometry and Annexin V-fluorescein Isothiocyanate/propidium iodide staining were used to observe apoptosis; D: Quantitative analysis of the apoptosis ratio. The bars indicate the mean ± SD from three independent experiments (n = 3). ¹P < 0.05 vs normal control group; ²P < 0.05 vs lipopolysaccharides + interferon-γ; ³P < 0.05 vs lipopolysaccharides + interferon-γ + dapagliflozin. NC: Normal control group; LPS: Lipopolysaccharides; IFN-γ: Interferon-γ; 740Y-P: A PI3K/AKT signaling pathway agonist; PI: Propidium iodide; DAPA: Type 2 diabetic mouse group treated with dapagliflozin.

Figure 6 Dapagliflozin reduces human monocyte cell line cell polarization toward the M1 phenotype and decreases the expression levels of inflammatory factors, effects that are inhibited by 740Y-P (a phosphoinositide 3-kinase/protein kinase B signaling pathway agonist). Relative mRNA expression levels of M1 macrophage surface markers: A: Inducible nitric oxide synthase; B: Cluster of differentiation 86; Expression ratio of M1 macrophages: C: Cluster of differentiation 11b + detected by flow cytometry; D: Cluster of differentiation 86+ detected by flow cytometry; Relative mRNA expression levels of inflammatory factors: E: Interleukin-6; F: Interleukin-1β; G: Tumor necrosis factor-α. The bars indicate the mean ± SD from three independent experiments (n = 3). ¹P < 0.05 vs normal control group; ²P < 0.05 vs lipopolysaccharides + interferon-γ; ³P < 0.05 vs lipopolysaccharides + interferon-γ + dapagliflozin. NC: Normal control group; LPS: Lipopolysaccharides; IFN-γ: Interferon-γ; 740Y-P: A PI3K/AKT signaling pathway agonist; PI: Propidium iodide; DAPA: Type 2 diabetic mouse group treated with dapagliflozin; IL: Interleukin; TNF: Tumor necrosis factor; CD: Cluster of differentiation; iNOS: Inducible nitric oxide synthase.

Figure 7 Dapagliflozin inhibits the activation of the phosphoinositide 3-kinase/protein kinase B signaling pathway. A: Protein imprints of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and phosphorylated protein kinase B (P-AKT) in mouse peritoneal macrophages after dapagliflozin treatment. B: Quantitative analysis of PI3K; C: Quantitative analysis of AKT; D: Quantitative analysis of P-AKT. The bars indicate the mean ± SD from three independent experiments (n = 3). ¹P < 0.05 vs normal control group; ²P < 0.05 vs lipopolysaccharides + interferon-γ; ³P < 0.05 vs lipopolysaccharides + interferon-γ + dapagliflozin. NC: Normal control group; LPS: Lipopolysaccharides; IFN-γ: Interferon-γ; 740Y-P: A PI3K/AKT signaling pathway agonist; DAPA: Type 2 diabetic mouse group treated with dapagliflozin; PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; P-AKT: Phosphorylated protein kinase B.