7-Hydroxy Frullanolide Ameliorates Isoproterenol-Induced Myocardial Injury through Modification of iNOS and Nrf2 Genes

Figures

• 

  Figure 1. Research protocol of cardioprotective activity of 7-hydroxy frullanolide against isoproterenol induced myocardial infarction.

• 

  Figure 2. Represents electrocardiogram (ECG) lead II tracing of (A) control showing normal P wave, RS pattern and T wave (B) isoproterenol (ISO)-alone-treated group showed pathological Q wave and marked ST-segment elevation. Representative electrocardiogram (ECG) lead II tracing of 7-HF treated groups (C) 5 mg/kg showed complete disappearance of pathological Q wave and decrease in ST-segment elevation (D) 10 mg/kg further reduced ST elevation (E) 25 mg/kg showed almost normal ECG pattern, (F) shows effect of atenolol on ECG pattern (Recorded at speed of 50 ms/div). ms = millisecond, mv = millivolt, and (G) shows the graphical representation of the effect of 7-HF on ST-segment elevation. Values are expressed as Mean ± SEM (n = 5), analyzed by one way analysis of variance (ANOVA) followed by post hoc comparison with Tukey’s test. Where ** p < 0.05 vs. ISO, *** p < 0.001 vs. ISO, ### p < 0.001 vs. Normal.

• 

  Figure 3. Shows heart tissue stained with TTC. Brick red (dark region) in the normal group indicated mitochondrial aspiration. ISO-alone treated group shows a large white region (arrow) indicating the infarcted tissue (A). Graph shows the effect of 7-HF on infarct size in ISO-induced MI. The infarct size is expressed as % area to the total ventricular area (B). Values are expressed as Mean ± SEM (n = 4), analyzed by one-way analysis of variance (ANOVA) followed by post hoc comparison by Tukey’s test. Where *** p < 0.001 vs. ISO, ### p < 0.001 vs. control.

• 

  Figure 4. Represents the effect of 7-HF and atenolol on cardiac troponin I (cTnI) (A), creatine kinase-MB (CK-MB) (B), lactate dehydrogenase (LDH) (C), alanine transaminase (ALT) (D), and aspartate transaminase (AST) (E) in isoproterenol (ISO)-induced ischemic rats. Values are expressed as Mean ± SEM, analyzed by one-way analysis of variance (ANOVA) followed by post hoc comparison by Tukey’s test. Where ** p < 0.01 vs. ISO, *** p < 0.001 vs. ISO, ### p < 0.001 vs. control.

• 

  Figure 5. H&E staining of heart tissue (40×) (A) Control, shows normal cardiomyocytes with vesicular central oval nuclei (hexagon), acidophilic cytoplasm (pentagon), and the cardiac fibers isolated by interstitial connective tissue with flat nuclei (arrow). (B) Isoproterenol (ISO)-alone treated heart shows degeneration and disorganization in myocardial fibers with separation of myofibrils (thin arrow), pyknotic nuclei (star), cytoplasmic vacuolization of cardiac muscle fibers (circle), edema (diamond), inflammatory cells infiltrations (caret) and hemorrhage (notched arrow). These changes were remarkably reduced by pretreatment with different doses of 7-HF + ISO (5, 10 and 25 mg/kg) (C–E) and the standard drug atenolol (F). 7-HF reduced these changes in a dose-dependent manner with the highest dose 25 mg/kg showing a more significant reduction in ISO-induced myocardial damage. Pretreatment with the standard drug atenolol also considerably reduced myocardial damage.

• 

  Figure 6. Shows the effect of 7-HF and atenolol on the level of malondialdehyde (A), superoxide dismutase (B), catalase (C), and reduced glutathione (D) in heart homogenates. Values are expressed as Mean ± SEM, analyzed by one-way analysis of variance (ANOVA) followed by post hoc comparison by Tukey’s test. Where * p < 0.05 vs. ISO, ** p < 0.01 vs. ISO, *** p < 0.001 vs. ISO, ### p < 0.001 vs. control.

• 

  Figure 7. Shows the images (A) and graphs of PCR analysis of Nrf2 and iNOS in heart tissues. 7-HF significantly decreased the mRNA expression of iNOS (B) and significantly increased the mRNA expression of Nrf2 (C) as compared to the ISO-alone treated group. Values are shown as mean ± SEM. ### p < 0.001 vs. normal, *** p < 0.001 vs. ISO.

• 

  Figure 8. Graph shows the effect of 7-HF on the force and rate of contraction on spontaneous atrium as well as tissue pre-incubated with atenolol (0.5 µM) (A) and atropine (10 µM) (B). Two-way ANOVA followed by Bonferroni post hoc analysis was applied to determine any statistical difference between the variables. (n = 5). Where *** p < 0.001 vs. Atenolol.

• 

  Figure 9. Graphs showing the effect of 7-HF and verapamil against standard vasoconstrictor i.e., phenylephrine (PE), K+ (80 mM) and Ang II (A,B), endothelium-intact and denuded tissue (C), pretreated; L-NAME (10 µM) atropine (1 µM) and indomethacin (1 µM) (D), pretreated with tetraethylammonium (TEA; 1 mM) and Bacl2 (30 µM) (E). The relaxation responses are expressed as mean± SEM for five experiments. Two-way ANOVA followed by Bonferroni post hoc analysis was applied to determine any statistical difference between the variables. Where *** p < 0.001 vs. Control.

• 

  Figure 10. The graph illustrates the effect of 7-HF (A) and verapamil (B) on Ca2+ response curve produced in Ca2+—free/EGTA solution, in isolated rat aorta. The relaxation responses were presented as mean ± SEM for five experiments. Where * p < 0.05 vs. Control, *** p < 0.001 vs. Control, represents the significant difference.

• 

  Figure 11. The graphs show the effects of increasing concentrations of 7-HF (A) and verapamil (B) on peaks of contractions produced by phenylephrine (PE) in Ca2+-free/EGTA solution. The relaxation responses were presented as means ± SEM for five experiments. Where * p < 0.05 vs. Control, ** p < 0.01 vs. Control, *** p < 0.001 vs. Control.