Naringenin Attenuates Diabetic Cardiac Injury via SIRT1 Activation in Streptozotocin‐Induced Rats

Figure 1. Experimental timeline. Schematic representation of the 70‐day experimental protocol. Day 0: start of 7‐day acclimatization period. Day 7: single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg) to induce diabetes. Days 10–12: diabetes confirmation based on fasting blood glucose (FBG) > 250 mg/dL over three consecutive days. Day 14: commencement of naringenin (NAR) (50 mg/kg/day, oral gavage) treatment for the NAR, DCM + NAR, and DCM + NAR + EX‐527 groups; EX‐527 (5 mg/kg, i.p., every 48 h) coadministration for the DCM + NAR + EX‐527 group. Body weight was monitored at biweekly intervals on Days 14, 28, 42, 56, and 70. Day 70 (Week 8 of treatment): glucose tolerance test (GTT) and insulin tolerance test (ITT) followed by sacrifice and tissue collection. CON, control; DCM, diabetic cardiomyopathy; CMC, carboxymethylcellulose; p.o., per os (oral); i.p., intraperitoneal.

Figure 2. NAR improves physiological and metabolic parameters in STZ‐induced DCM rats. (A) Total body weight, (B) heart‐to‐body (H/B) weight ratio, (C) systolic blood pressure (SBP), and (D) diastolic blood pressure (DBP). Data = mean ± SD; n = 8 rats/group. Statistical analysis was performed using one‐way ANOVA with Tukey′s post hoc test. Groups: CON (untreated control), NAR (naringenin‐only), DCM (STZ‐induced diabetic cardiomyopathy), DCM + NAR (NAR‐treated diabetic), and DCM + NAR + EX‐527 (diabetic animals treated with NAR + SIRT1 inhibitor). ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM; ns, not significant.

Figure 3. NAR improved glucose and insulin tolerance in diabetic animals. (A) Glucose tolerance test (GTT), (B) area under the curve (AUC) analysis of the GTT, (C) insulin tolerance test (ITT), and (D) AUC analysis of the ITT. AUC was calculated using the trapezoidal method with baseline set to zero (Y = 0). Data are presented as mean ± SD; n = 8 rats/group. (A,C) GTT and ITT curves were analyzed using two‐way repeated‐measures ANOVA with Sidak′s multiple comparisons test. (B, D) AUC values were analyzed using one‐way ANOVA with Tukey′s post hoc test. ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM; ns, not significant.

Figure 4. NAR preserves cardiac histoarchitecture in DCM. Representative H&E‐stained myocardial sections (scale bar: 10 μm). Histopathological assessment was performed qualitatively by two independent observers blinded to the experimental groups. (A) CON: organized cardiomyocytes with central nuclei and striations. (B) NAR: normal architecture. (C) DCM: disrupted striations, interstitial edema, and nuclear pyknosis. (D) DCM + NAR: restored striations and reduced edema. (E) DCM + NAR + EX‐527: pathology similar to DCM.

Figure 5. NAR attenuates cardiac apoptosis and fibrosis. (A–C) mRNA expression of Bcl-2, Bax, and CASP-3. (D–F) Protein levels/activity. (G) Bax/Bcl‐2 ratio. Data = mean ± SD; n = 8. Statistical analysis was performed using one‐way ANOVA with Tukey′s post hoc test. ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM.

Figure 6. NAR modulated markers of fibrosis in the cardiac tissue of diabetic animals. (A) TGF‐β levels and (B) Smad2 levels. Data = mean ± SD; n = 8. Statistical analysis was performed using one‐way ANOVA with Tukey′s post hoc test. ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM.

Figure 7. NAR suppresses cardiac inflammatory mediators in diabetic hearts. (A,B) NF‐κB expression (mRNA/protein), (C) TNF‐α levels, and (D) IL‐6 levels. Statistical analysis was performed using one‐way ANOVA with Tukey′s post hoc test. Data = mean ± SD; n = 8. ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM; (p < 0.05).

Figure 8. NAR alleviates oxidative stress in diabetic hearts. (A,B) Nrf2 expression (mRNA/protein), (C) SOD activity, (D) GSH levels, and (E) MDA levels. Data = mean ± SD; n = 8. Statistical analysis was performed using one‐way ANOVA with Tukey′s post hoc test. ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM; (p < 0.05).

Figure 9. NAR restores SIRT1‐FOXO1 signaling in DCM. (A–D) SIRT1 and FOXO1 expression (mRNA/protein). Data = mean ± SD; n = 8. Statistical analysis was performed using one‐way ANOVA with Tukey′s post hoc test. ∗ p < 0.05 vs. CON, # p < 0.05 vs. DCM, and ∗ # p < 0.05 vs. both CON and DCM (p < 0.05).