Individual and Combined Cardioprotective Effects of 1,25(OH) ₂ D ₃ and Dimethyl Fumarate in an Isoproterenol‐Induced Myocardial Injury Model

Figures

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  Figure 1. Effects of 1,25(OH)2D3, DMF, and their combination on serum cardiac injury markers in rats with ISO‐induced AMI. Serum levels of alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), lactate dehydrogenase (LDH, U/L), and troponin I (TnI, ng/mL) are presented for Control, 1,25(OH)2D3, DMF, 1,25(OH)2D3 + DMF, ISO, ISO + 1,25(OH)2D3, ISO + DMF, and ISO + 1,25(OH)2D3 + DMF groups. Data are expressed as mean ± SD (n = 7 per group). a p < 0.05 versus Control group; b p < 0.05 versus ISO group. Subscripts indicate levels of significance: 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001. Statistical analyses were performed as described in the Statistical Analysis section.

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  Figure 2. Effects of 1,25(OH)2D3, DMF, and their combination on cardiac oxidative stress markers in rats with ISO‐induced AMI. Cardiac levels of reactive oxygen species (ROS, RFU/mg protein), malondialdehyde (MDA, pmol/mg protein), and advanced oxidation protein products (AOPP, nmol/mg protein) are presented for Control, 1,25(OH)2D3, DMF, 1,25(OH)2D3 + DMF, ISO, ISO + 1,25(OH)2D3, ISO + DMF, and ISO + 1,25(OH)2D3 + DMF groups. Data are expressed as mean ± SD. a p < 0.05 versus Control group; b p < 0.05 versus ISO group. Subscripts indicate levels of significance: 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001. Statistical analyses were performed as described in the Statistical Analysis section.

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  Figure 3. Effects of 1,25(OH)2D3, DMF, and their combination on cardiac oxidative stress parameters in rats with ISO‐induced AMI. Cardiac levels of ferric reducing antioxidant power (FRAP, nmol/mg protein), reduced glutathione (GSH, nmol/mg protein), superoxide dismutase (SOD, U/mg protein), and glutathione peroxidase (GPX, nmol/min/mg protein) are presented for Control, 1,25(OH)2D3, DMF, 1,25(OH)2D3 + DMF, ISO, ISO + 1,25(OH)2D3, ISO + DMF, and ISO + 1,25(OH)2D3 + DMF groups. Data are expressed as mean ± SD (n = 7 per group). a p < 0.05 versus Control group; b p < 0.05 versus ISO group. Subscripts indicate levels of significance: 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001. Statistical analyses were performed as described in the Statistical Analysis section.

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  Figure 4. Effects of 1,25(OH)2D3, DMF, and their combination on cardiac angiotensin II (Ang II) and TNF‐α levels in rats with ISO‐induced AMI. Cardiac Ang II (pg/mg protein) and TNF‐α (ng/mg protein) levels are presented for Control, 1,25(OH)2D3, DMF, 1,25(OH)2D3 + DMF, ISO, ISO + 1,25(OH)2D3, ISO + DMF, and ISO + 1,25(OH)2D3 + DMF groups. Data are expressed as mean ± SD. a p < 0.05 versus Control group; b p < 0.05 versus ISO group. Subscripts indicate levels of significance: 1 p < 0.05,2 p < 0.01, and 3 p < 0.001. DMF, dimethyl fumarate; ISO, isoproterenol; 1,25(OH)2D3, 1,25‐dihydroxyvitamin D3; Ang II, angiotensin II; TNF‐α, tumor necrosis factor‐α. Statistical analyses were performed as described in the Statistical Analysis section.

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  Figure 5. Gene expression levels of Nrf2, NQO1, NF‐κB, AT1R, and ACE in experimental groups. Gene expression levels are presented as fold change relative to the Control group. Data are expressed as mean ± SD. Groups: Control, 1,25D3 + DMF, ISO, 1,25D3 + DMF + ISO, and ISO + 1,25D3 + DMF. a Statistically significant difference compared with the Control group; bstatistically significant difference compared with the ISO group. Subscripts indicate significance levels: 1 p < 0.05, 2 p < 0.01, and 3 p < 0.001. ISO, isoproterenol; DMF, dimethyl fumarate; 1,25D3, 1,25‐dihydroxyvitamin D3; Nrf2, nuclear factor erythroid 2‐related factor 2; NQO1, NAD(P)H quinone oxidoreductase 1; NF‐κB, nuclear factor kappa B; AT1R, angiotensin II type 1 receptor; ACE, angiotensin‐converting enzyme. Statistical analyses were performed using one‐way ANOVA followed by Tukey's post hoc test for normally distributed data and the Kruskal–Wallis test followed by Mann–Whitney U post hoc comparisons for non‐normally distributed data. Statistical analyses were performed as described in the Statistical Analysis section.

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  Figure 6. Histopathological evaluation of myocardial tissue in treated and control groups. (A) Control, DMF, and 1,25(OH)2D3 groups showed normal myocardial architecture with regularly arranged cardiac muscle fibers and intact cardiomyocytes, without evidence of necrosis or inflammatory cell infiltration (H&E, ×100). (B) The ISO group exhibited extensive myocardial necrosis, loss of cardiomyocyte integrity, and dense inflammatory cell infiltration (H&E, ×100). (C) The 1,25(OH)2D3 + ISO group showed focal myocardial necrosis accompanied by marked inflammatory cell infiltration (H&E, ×100). (D) The DMF + ISO group demonstrated largely preserved myocardial architecture with mild‐to‐moderate interstitial inflammatory cell infiltration (H&E, ×100). (E) The DMF + 1,25(OH)2D3 + ISO group revealed interstitial edema with mild inflammatory cell infiltration and predominantly preserved myocardial architecture (H&E, ×100). (F) The ISO + 1,25(OH)2D3 + DMF group showed focal myocardial necrosis associated with moderate inflammatory cell infiltration (H&E, ×100).

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  Figure 7. Immunohistochemical staining for NQO1 (A–E), Nrf2 (F–J), and NF‐κB (K–O) in myocardial tissue. Representative photomicrographs (×100 magnification) of the Control, 1,25(OH)2D3 + DMF, ISO, 1,25(OH)2D3 + DMF + ISO (pretreatment), and ISO + 1,25(OH)2D3 + DMF (post‐treatment) groups. NQO1 and Nrf2 immunostaining were both reduced in the ISO group compared with Control and increased following combination pretreatment and post‐treatment, whereas NF‐κB immunostaining showed the opposite pattern, with marked upregulation in the ISO group and reduced expression in the combination‐treated groups.