Protective Effects of 5-MTP in a Rat Model of Diabetic Cardiomyopathy Through Anti-Inflammatory, Anti-Apoptotic, and Antifibrotic Mechanisms

Figures

• 

  Figure 1. Study design of effects of different dose 5-MTP on rat model of diabetic cardiomyopathy on different treatment duration.

• 

  Figure 2. Representative hematoxylin–eosin staining and myocardial inflammatory score across treatment groups at different treatment durations. (A) Quantitative comparison of myocardial inflammatory scores among treatment groups at day 8 (a), day 16 (b), and day 32 (c). Data are presented as median (minimum–maximum) or mean ± standard deviation, as appropriate. (B) Representative HE-stained myocardial sections (400× magnification; scale bar = 50 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP 100 mg/kg (d); day 16 DCM + vehicle control (e), 5-MTP 25 mg/kg (f), 5-MTP 50 mg/kg (g), and 5-MTP 100 mg/kg (h); day 32 DCM + vehicle control (i), 5-MTP 25 mg/kg (j), 5-MTP 50 mg/kg (k), and 5-MTP 100 mg/kg (l). Histopathological evaluation demonstrated myocardial inflammatory cell infiltration of varying degrees without statistically significant differences among treatment groups at any time point.

• 

  Figure 3. Representative Masson’s trichrome (MT) staining and quantitative myocardial fibrosis area across treatment groups at different treatment durations. (A) Quantitative comparison of myocardial fibrosis (% area) among treatment groups at day 8 (a), day 16 (b), and day 32 (c). At day 16, myocardial fibrosis differed significantly among groups, with all 5-MTP treatment groups demonstrating significantly lower fibrosis area compared with the DCM + vehicle control group (25 mg/kg, p = 0.005; 50 mg/kg, p = 0.008; 100 mg/kg, p = 0.037). No significant differences were observed at day 8 or day 32. * p < 0.05 and ** p < 0.01 vs DCM + vehicle control. (B) Representative MT-stained myocardial sections (400× magnification; scale bar = 60 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP 100 mg/kg (d); day 16 DCM + vehicle control (e), 5-MTP 25 mg/kg (f), 5-MTP 50 mg/kg (g), and 5-MTP 100 mg/kg (h); day 32 DCM + vehicle control (i), 5-MTP 25 mg/kg (j), 5-MTP 50 mg/kg (k), and 5-MTP 100 mg/kg (l). Blue staining represents collagen deposition and myocardial fibrosis. Histopathological evaluation demonstrated reduced…

• 

  Figure 4. Collagen I immunohistochemical expression across treatment groups at different treatment durations. (A) Quantitative comparison of collagen I H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). No significant differences in collagen I expression were observed among groups at any time point. (B) Representative collagen I immunohistochemical-stained myocardial sections (400× magnification; scale bar = 60 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP 100 mg/kg (d); day 16 DCM + vehicle control (e), 5-MTP 25 mg/kg (f), 5-MTP 50 mg/kg (g), and 5-MTP 100 mg/kg (h); day 32 DCM + vehicle control (i), 5-MTP 25 mg/kg (j), 5-MTP 50 mg/kg (k), and 5-MTP 100 mg/kg (l). Brown staining indicates collagen I expression within myocardial tissue. Although early molecular modulation of fibrotic pathways was observed in other markers, collagen I expression did not demonstrate significant between-group differences, suggesting that downstream extracellular matrix deposition may require longer remodeling duration to manifest measurable changes.

• 

  Figure 5. Caspase-3 immunohistochemical expression across treatment groups at different treatment durations. (A) Quantitative comparison of caspase-3 H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). No significant differences were observed at day 8. At day 16, caspase-3 expression differed significantly among groups, with all 5-MTP treatment groups demonstrating significantly lower H-scores compared with the DCM + vehicle control group (25 mg/kg, p = 0.026; 50 mg/kg, p = 0.034; 100 mg/kg, p = 0.005). At day 32, a significant difference was also observed, with only the 100 mg/kg group showing significant reduction compared with the DCM + vehicle control group (p = 0.004). * p < 0.05 and ** p < 0.01 vs DCM + vehicle control. (B) Representative caspase-3 immunohistochemical-stained myocardial sections (400× magnification; scale bar = 60 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP 100 mg/kg (d); day 16 DCM + vehicle control (e), 5-MTP 25 mg/kg (f), 5-MTP 50 mg/kg (g), and 5-MTP 100 mg/kg (h); day 32 DCM + vehicle control (i), 5-MTP 25 mg/kg (j), 5-MTP 50 mg/kg (k), and 5-MTP 100…

• 

  Figure 6. TGF-β immunohistochemical expression across treatment groups at different treatment durations. (A) Quantitative comparison of TGF-β H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). At day 8, TGF-β expression differed significantly among groups, with the 5-MTP 25 mg/kg (p = 0.017) and 50 mg/kg (p = 0.002) groups demonstrating significantly lower H-scores compared with the DCM + vehicle control group, while the 100 mg/kg group did not differ significantly. No significant differences were observed at day 16 or day 32. * p < 0.05 and ** p < 0.01 vs DCM + vehicle control. (B) Representative TGF-β immunohistochemical-stained myocardial sections (400× magnification; scale bar = 60 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP 100 mg/kg (d); day 16 DCM + vehicle control (e), 5-MTP 25 mg/kg (f), 5-MTP 50 mg/kg (g), and 5-MTP 100 mg/kg (h); day 32 DCM + vehicle control (i), 5-MTP 25 mg/kg (j), 5-MTP 50 mg/kg (k), and 5-MTP 100 mg/kg (l). Brown staining indicates TGF-β expression. These findings suggest 5-MTP was associated with early differences in TGF-β expression, particularly…

• 

  Figure 7. SMAD3 immunohistochemical expression across treatment groups at different treatment durations. (A) Quantitative comparison of SMAD3 H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). At day 8, SMAD3 expression did show a significant difference in pairwise 5-MTP 50 mg/kg compared to control. At day 16, SMAD3 expression differed significantly among groups, with the 5-MTP 25 mg/kg (p = 0.017) and 100 mg/kg (p = 0.017) groups demonstrating significantly lower H-scores compared with the DCM + vehicle control group, whereas the 50 mg/kg group did not show a significant difference. No significant differences were observed at day 32. * p < 0.05 vs DCM + vehicle control. (B) Representative SMAD3 immunohistochemical-stained myocardial sections (400× magnification; scale bar = 60 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP 100 mg/kg (d); day 16 DCM + vehicle control (e), 5-MTP 25 mg/kg (f), 5-MTP 50 mg/kg (g), and 5-MTP 100 mg/kg (h); day 32 DCM + vehicle control (i), 5-MTP 25 mg/kg (j), 5-MTP 50 mg/kg (k), and 5-MTP 100 mg/kg (l). Brown staining indicates SMAD3 expression. These…

• 

  Figure 8. Nuclear and cytoplasmic AKT immunohistochemical expression across treatment groups at different treatment durations. (A) Quantitative comparison of AKT nuclear H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). At day 8, no significant overall difference was observed among groups (p = 0.147), although the 100 mg/kg group demonstrated significantly lower nuclear AKT expression compared with the DCM + vehicle control group (p = 0.031). At day 16, AKT nuclear expression differed significantly among groups (p = 0.021), with the 25 mg/kg group demonstrating significantly higher nuclear AKT expression compared with the DCM + vehicle control group (p = 0.003), whereas no significant differences were observed in the 50 mg/kg or 100 mg/kg groups. No significant differences were observed at day 32. * p < 0.05 and ** p < 0.01 vs DCM + vehicle control. (B) Quantitative comparison of AKT cytoplasmic H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). No significant differences were observed at day 8 or day 32. At day 16, AKT cytoplasmic expression differed significantly among groups (p = 0.023), with the 50 mg/kg group demonstrating significantly…

• 

  Figure 9. Nuclear and cytoplasmic NF-κB immunohistochemical expression across treatment groups at different treatment durations. (A) Quantitative comparison of NF-κB nuclear H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). No significant differences in nuclear NF-κB expression were observed among groups at any time point. (B) Quantitative comparison of NF-κB cytoplasmic H-score among treatment groups at day 8 (a), day 16 (b), and day 32 (c). No significant differences were observed at day 8 or day 32. At day 16, cytoplasmic NF-κB expression differed significantly among groups (p = 0.006), with the 5-MTP 25 mg/kg group demonstrating significantly lower expression compared with the DCM + vehicle control group (p < 0.001), and the 100 mg/kg group also demonstrating significantly lower expression (p = 0.045), whereas no significant difference was observed in the 50 mg/kg group. * p < 0.05 and *** p < 0.001 vs DCM + vehicle control. (C) Representative NF-κB immunohistochemical-stained myocardial sections (400× magnification; scale bar = 60 μm) from each treatment group across time points: day 8 DCM + vehicle control (a), 5-MTP 25 mg/kg (b), 5-MTP 50 mg/kg (c), and 5-MTP…

• 

  Figure 10. Proposed temporal framework of observed histopathological and molecular changes following 5-MTP treatment in experimental diabetic cardiomyopathy. Hyperglycemia promotes ROS and AGE formation, activating NF-κB-mediated inflammation, TGF-β/SMAD3-driven fibrosis, and caspase-3-associated apoptosis, which contribute to myocardial remodeling progression. Based on this study, 5-MTP was associated with early changes in TGF-β expression (day 8), broader intermediate changes in NF-κB, SMAD3, AKT, and caspase-3 expression (day 16), and persistent differences in caspase-3 expression at day 32. This figure summarizes the proposed temporal molecular effects of 5-MTP during DCM progression.