Cardioprotective effects of Schisantherin A against isoproterenol-induced acute myocardial infarction through amelioration of oxidative stress and inflammation via modulation of PI3K-AKT/Nrf2/ARE and TLR4/MAPK/NF-κB pathways in rats

Figures

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  Fig. 1. (A): Schematic diagram of experimental design. Effect of SCA treatment on (B): Myocardial infarct size (C): Heart-to-body weight ratio in ISO-injected rats. (D): The myocardial infarct size was assessed through a histological TTC staining assay. The myocardial infarct area is indicated by the black arrow. C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05. S.C: Subcutaneous; ISO: isoproterenol

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  Fig. 2. Effect of SCA treatment on ISO-induced changes in cardiac biochemical markers in rats. (A): Creatine kinase-MB (CK-MB); (B): Cardiac Troponin I (cTnI); (C): Brain natriuretic peptide (BNP); (D) Histopathological changes through hematoxylin and eosin (H&E) staining in heart. ISO or SCA treated group shows myocardial damage with fragmented and separated cardiac muscle fibers (black arrow). Vascular leakage of RBCs is indicated by blue arrows. The ISO group also exhibits necrosis (*) and inflammatory cell infiltration. Fibrosis is shown by the green arrow. C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05. Scale bar = 50 μm. Magnification: X400

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  Fig. 3. Effect of SCA treatment on ISO-induced changes in rats’ cardiac oxidative stress and antioxidant defense markers. (A): Malondialdehyde (MDA); (B): Nitric oxide (NO) (C): superoxide dismutase (SOD); (D): Catalase (CAT); (E): Glutathione peroxidase (GPx); (F): Glutathione (GSH). C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05

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  Fig. 4. Effect of SCA treatment on ISO-induced changes in PI3K/Akt/Nrf2/ARE signaling in cardiac tissue of rats. Relative mRNA expression of (A): PI3K; (B): Akt; (C): Nrf2; (D): Keap1; (E): Nqo1;(F): Ho-1. C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05

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  Fig. 5. Immunohistochemical staining for myocardial (A) Nrf2 and (B) Keap 1 distribution. (C) Nrf2 and (D) Keap 1 brown staining indicate the cells with positive staining. C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05. The black arrow indicates the protein expression specifically in cardiomyocytes. (Scale bar = 50 μm; magnification, x400)

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  Fig. 6. Effect of SCA treatment on ISO-induced changes in TLR4/MAPK/NF-kB signaling in cardiac tissue of rats. Relative mRNA expression of (A): Tlr4; (C): IKKβ;(D): ERK; (E): JNK; (F): P38; ELISA reading for (B) NF-kB-p65 levels; C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. Data are Mean ± SEM (n = 10). *p < 0.05 versus control, and ^p < 0.05 versus ISO. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05

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  Fig. 7. Immunofluorescent results of myocardial (A) NF-κΒ p65 and immunohistochemical staining (B) P38 MAPK; (C) Fluorescence intensity (red color) of NF-κΒ p65 and (D) p38 MAPK (% of brown staining). C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/ kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05. The black and white arrow indicates the protein expression specifically in cardiomyocytes. (Scale bar = 50 μm; magnification, x400)

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  Fig. 8. ELISA assay for cardiac (A) TNF-α, (B) IL-1β, (C) IL-6; C: Control rats; MI: Isoproterenol (ISO) induced myocardial infarction (MI) in rats; 5 SCA: Treatment of ISO-induced rats with 5 mg SCA/ kg body weight; 10 SCA: Treatment of ISO-induced rats with 10 mg SCA/kg body weight. The data is presented as mean ± SD (n = 10). Statistical analysis was performed using One-Way ANOVA followed by Tukey’s post hoc analysis to compare differences between groups. Mean values with different superscripts indicate significant differences at a significance level of p < 0.05