Liraglutide combined with dapagliflozin treatment improves myocardial disease and endothelial dysfunction in T2DM mice

Figure 1. Liraglutide and dapagliflozin improve systemic metabolism and reduce myocardial injury in a murine model of type 2 diabetic cardiomyopathy. (a) Experimental timeline and group assignment: mice were fed a high‐fat diet (HFD) for 8 weeks followed by low‐dose streptozotocin (STZ) to induce diabetes, then treated with vehicle, liraglutide, dapagliflozin, or their combination for 4 weeks (n = 6 per group). (b) Body weight trajectories during HFD feeding and intervention. (c) Absolute heart weight at endpoint. (d) Intraperitoneal glucose tolerance test (IPGTT; 2 g/kg glucose, i.p.) performed after 4 weeks of treatment; data expressed as blood glucose concentration over time. (e, f) Serum levels of cardiac injury biomarkers measured by ELISA: creatine kinase‐MB (CK‐MB, e), and lactate dehydrogenase (LDH, f). (g) Statistical analysis of left ventricular ejection fraction (EF). (h) Representative M‐mode echocardiographic tracings showing left ventricular systolic function. Data are presented as mean ± SD, n = 6. *P < 0.05, **P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs. model.

Figure 2. Combined liraglutide and dapagliflozin therapy attenuates cardiac fibrosis and suppresses profibrotic signaling in diabetic hearts. (a) Representative hematoxylin and eosin (H&E) staining of left ventricular sections showing myocardial architecture (scale bar: 50 μm). (b) Masson's trichrome staining illustrating interstitial and perivascular collagen deposition (blue); scale bar: 50 μm. (c) Representative images of Picrosirius Red staining viewed under polarized light to visualize collagen deposition (red/yellow birefringence); scale bar: 100 μm. (d–f) mRNA expression levels of Collagen I (d), Collagen III (e), and TGF‐β1 (f) in cardiac tissue. n = 3, Data are presented as mean ± SD, n = 3. **P < 0.01 vs. control; ##P < 0.01 vs. model.

Figure 3. Liraglutide and dapagliflozin alleviate oxidative stress, inflammation, and apoptosis in the diabetic myocardium. (a–c) Biochemical assays of oxidative stress markers in heart homogenates: Superoxide dismutase (SOD) activity (a), reduced glutathione (GSH) content (b), and malondialdehyde (MDA) levels (c), n = 6. (d) Serum levels of pro‐inflammatory cytokines (TNF‐α, IL‐1β, and IL‐6) measured by ELISA, n = 6. (e) Western blot analysis of pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6) and apoptotic proteins (Bax, Bcl‐2, cleaved caspase‐3); GAPDH served as loading control, n = 3. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. model.

Figure 4. Combined therapy inhibits endothelial apoptosis and ameliorates vascular dysfunction in diabetes. (a) Representative H&E staining of thoracic aorta cross‐sections showing intimal‐medial thickness and endothelial integrity (scale bar: 50 μm). (b) TUNEL staining (green) co‐localized with DAPI (blue) to detect apoptotic nuclei in vascular wall; scale bar: 20 μm. Quantification shows percentage of TUNEL+ cells per field. (c) Western blot analysis of Bax, Bcl‐2, and cleaved caspase‐3 in aortic lysates; GAPDH as loading control. Data are presented as mean ± SD, n = 3. **P < 0.01 vs. control; ## P < 0.01 vs. model.

Figure 5. Combination therapy enhances endothelial integrity and activates NRF2/HO‐1 and AMPK/PKA‐eNOS pathways to counteract vascular oxidative stress. (a) Immunofluorescence staining of aortic sections for CD31 (endothelial marker, red) and 3‐nitrotyrosine (3‐NT, oxidative damage marker, green); nuclei stained with DAPI (blue). Scale bar: 50 μm. (b) Quantification of CD31+ area (%) and 3‐NT fluorescence intensity (n = 6 per group). (c) Western blot analysis of NRF2, HO‐1, phospho‐AMPK (Thr172), total AMPK, PKA substrate phosphorylation, eNOS, and phospho‐eNOS (Ser1177) in aortic tissue. β‐Actin served as loading control, n = 3. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs. control; ## P < 0.01 vs. model.