Cardioprotective potential of polyphenols rich Thraatchathi Chooranam against isoproterenol induced myocardial necrosis in experimental rats

Abstract

Background: The present study establishes the cardioprotective role of Thraatchathi Chooranam (TC), a polyherbal traditional Siddha medicine, in terms of membrane stabilizing and antioxidant properties in isoproterenol (ISO) induced myocardial necrosis model in rats.

Methods: Animals were divided into six groups (n = 6), normal (received vehicle 0.5% CMC, p.o.), ISO control (received 0.5% CMC + ISO 120 mg/kg, b.w. s.c. twice at an interval of 48 h), standard control (received Vit-E 100 mg/kg, p.o.) + ISO, TC low and high dose (50 and 100 mg/kg p.o., respectively) + ISO, and drug control (received TC at 100 mg/kg, p.o.). At the end of experimental period, blood samples collected and plasma cardiac troponin-I (CTn-I) was measured by ELISA. Cardiac tissues were isolated, levels of membrane stabilizing enzymes, antioxidants and inflammatory markers were estimated. Gene expression of Bax, Bcl2, Caspase 3, HIF-α, TNF-α, iNOS, TRX1 and TrxR were performed by RT-PCR. Histopathological studies on cardiac tissues were conducted using hematoxylin and eosin (H&E) stain. Statistical analyses were performed by one-way ANOVA followed by Tukey's multiple comparison as post-hoc test.

Results: Administration of ISO resulted in a significant increase in plasma CTn-I, decrease in superoxide dismutase, glutathione and glutathione peroxidase; it also significantly altered membrane stabilizing enzymes like Na⁺/K⁺-ATPase, Mg²⁺-ATPase Ca²⁺-ATPase and Cathepsin D. Pretreatment with TC (50 mg/kg and 100 mg/kg) decreased CTn-I, and improved membrane stabilizing and endogenous antioxidant enzymes and decreased cathespin D level in a dose dependent manner. Histopathological examination revealed that TC improves cellular membrane integrity and decreases inflammatory cell infiltration and necrotic death.

Conclusion: The present study provided a strong evidence on the protective effects of TC against ISO-induced myocardial necrosis in rats.

Keywords: Antioxidants isoproterenol; Apoptosis; Cardioprotective; Inflammation; Membrane enzymes; Oxidative stress; Polyphenols; Thraatchathi Chooranam.

Fig. 1

Effects of TC on plasma cardiac troponin-I in ISO administered rats. Data were expressed as mean ± SEM; (n = 6) and analyzed by one-way ANOVA followed by Tukey’s multiple comparison as post-hoc test. ^###p < 0.001 and vs. Normal control or ^**p < 0.01 and ^*** p < 0.001 vs. ISO control group. A p value < 0.05 was considered as statistically significant

Fig. 2

a-d. Dose dependent effect of TC on mRNA expression pattern of apoptotic markers Bax (2A), Caspase-3 (2B), HIF-α (2C) and Bcl2 (2D) in control and experimental groups. β-actin mRNA was used as housekeeping gene for the normalization of mRNA expressions. Quantification graphs values are expressed as mean ± SEM. ## indicates p < 0.01 vs normal control group; *and **- indicates p < 0.05 and 0.01, respectively, vs ISO group. Mean difference between the groups was analyzed using one-way ANOVA, followed by Tukey’s multiple comparison as post-hoc test. A p value < 0.05 was considered as statistically significant

Fig. 3

a-b. Dose dependent effect of TC on mRNA expression pattern of anti-inflammatory markers iNOS (3A) and TNF-α (3B) in control and experimental groups. β-actin mRNA was used as housekeeping gene for the normalization of mRNA expressions. Quantification graphs values are expressed as mean ± SEM. ## indicates p < 0.01 vs normal control group; *and **- indicates p < 0.05 and 0.01, respectively, vs ISO group. Mean difference between the groups was analyzed using one-way ANOVA, followed by Tukey’s multiple comparison as post-hoc test. A p value < 0.05 was considered as statistically significant

Fig. 4

a-b. Effect of TC on mRNA expression of oxidative stress marker TRX1 (4A) and TrxR (4B) in cardiac muscle in control and experimental groups. β-actin mRNA was used as housekeeping gene for the normalization of mRNA expressions. Quantification graphs values are expressed as mean ± SEM. ## indicates p < 0.01 vs normal control group; *and **- indicates p < 0.05 and 0.01, respectively, vs ISO group. Mean difference between the groups was analyzed using one-way ANOVA, followed by Tukey’s multiple comparison as post-hoc test. A p value < 0.05 was considered as statistically significant

Fig. 5

a-f. Effect of TC on myocardial histopathological changes in rats. Groups: 5A- Normal Control; 5B- ISO (120 mg/kg); 5C- Vit-E (100 mg/kg); 5D- TC (50 mg/kg); 5E- TC (100 mg/kg); 5F- TC alone (100 mg/kg)