Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats

Abstract

Epigallocatechin-3-gallate (EGCG) is the most prominent catechin in green tea. EGCG has been shown to modulate numerous molecular targets in the setting of inflammation and cancer. These molecular targets have also been demonstrated to be important participants in reperfusion injury, hence this study examines the effects of EGCG in myocardial reperfusion injury. Male Wistar rats were subjected to myocardial ischemia (30 min) and reperfusion (up to 2 h). Rats were treated with EGCG (10 mg/kg intravenously) or with vehicle at the end of the ischemia period followed by a continuous infusion (EGCG 10 mg/kg/h) during the reperfusion period. In vehicle-treated rats, extensive myocardial injury was associated with tissue neutrophil infiltration as evaluated by myeloperoxidase activity, and elevated levels of plasma creatine phosphokinase. Vehicle-treated rats also demonstrated increased plasma levels of interleukin-6. These events were associated with cytosol degradation of inhibitor kappaB-alpha, activation of IkappaB kinase, phosphorylation of c-Jun, and subsequent activation of nuclear factor-kappaB and activator protein-1 in the infarcted heart. In vivo treatment with EGCG reduced myocardial damage and myeloperoxidase activity. Plasma IL-6 and creatine phosphokinase levels were decreased after EGCG administration. This beneficial effect of EGCG was associated with reduction of nuclear factor-kB and activator protein-1 DNA binding. The results of this study suggest that EGCG is beneficial for the treatment of reperfusion-induced myocardial damage by inhibition of the NF-kappaB and AP-1 pathway.

Figures

Figure 1

Effect of in vivo treatment with EGCG on myocardial histology. A: Representative cardiac section from a sham-operated rat displays normal tissue structure. B: After 30 min occlusion and 120 min reperfusion of the left coronary artery (I-R), a marked disruption of the myocardial structure characterized by appearance of extensive necrosis, contraction bands (arrow), and thinning of myofibrils (arrow head) was demonstrated in cardiac sections of a vehicle-treated rat. C: In vivo treatment with EGCG ameliorated myocardial damage. EGCG-treated rats received a bolus dose of EGCG 10 mg/kg at the end of the ischemia period followed by 10 mg/kg/h intravenous infusion for the entire period of reperfusion. Magnification × 100. A similar pattern was seen in 6 to 8 different tissue sections in each experimental group.

Figure 2

Effect of in vivo treatment with EGCG on plasma levels of creatine phosphokinase in rats subjected to myocardial ischemia and reperfusion (I-R). Each data point represents the mean ± SEM of 6 animals for each group. EGCG-treated rats received a bolus dose of EGCG 10 mg/kg at the end of the ischemia period followed by 10 mg/kg/h intravenous infusion for the entire period of reperfusion. *P < 0.05 compared with sham operated rats control); #P < 0.05 versus vehicle-treated rats subjected to myocardial ischemia and reperfusion (I-R + vehicle).

Figure 3

Effect of in vivo treatment with EGCG on plasma IL-6 levels in rats subjected to myocardial ischemia and reperfusion (I-R). Each data point represents the mean ± SEM of 6 animals for each group. EGCG-treated rats received a bolus dose of EGCG 10 mg/kg at the end of the ischemia period followed by 10 mg/kg/h intravenous infusion for the entire period of reperfusion. *P < 0.05 compared with control; #P < 0.05 compared with vehicle-treated rats subjected to myocardial ischemia and reperfusion (I-R + vehicle).

Figure 4

Effect of in vivo treatment with EGCG on MPO activity in rats subjected to myocardial ischemia and reperfusion (I-R). Tissue MPO activity was measured in myocardial tissue in vehicle-treated and EGCG-treated rats and compared with control rats. Each data point represents the mean ± SEM of 6 animals for each group. EGCG-treated rats received a bolus dose of EGCG 10 mg/kg at the end of the ischemia period followed by 10 mg/kg/h intravenous infusion for the entire period of reperfusion.*P < 0.05 compared with control; #P < 0.05 compared with vehicle-treated rats subjected to myocardial ischemia and reperfusion (I-R + vehicle).

Figure 5

Effect of in vivo treatment with EGCG on pressure-rate index in rats subjected to myocardial ischemia and reperfusion. Data represent pressure-rate index as recorded before occlusion of the left main coronary artery (basal) after 30 min of ischemia (end of ischemia) and after 120 min of reperfusion (end of reperfusion). Pressure rate index is calculated by the following formula (mean blood pressure [mmHg] × beats/min/1000). Values are means ± SEM of 5 to 6 rats per group.

Figure 6

Effect of in vivo treatment with EGCG on the activation of NF-κB, IKK activation, and IκB-α degradation in rats subjected to myocardial ischemia and reperfusion. EGCG-treated rats received a bolus dose of EGCG 10 mg/kg at the end of the ischemia period followed by 10 mg/kg/h intravenous infusion for the entire period of reperfusion. A: Representative autoradiograph of electrophoretic mobility shift assay for NF-κB binding hearts of vehicle-treated rats at 0 (sham value), 30, 60, and 120 min after reperfusion. B: Representative Western blot analysis demonstrating the effect of vehicle or EGCG on IκB-α degradation at 0 (sham value), 30, 60, and 120 min after reperfusion. Western blot analysis is representative of 3 separate experiments performed at different times. Fold increase was calculated compared with respective sham value (time 0) set to 1.0. Results are representative of 3 separate time-course experiments. *P < 0.05 compared with respective sham value (time 0); #P < 0.05 compared with vehicle-treated rats subjected to myocardial ischemia and reperfusion. C: Representative of autoradiograph of kinase assay demonstrating the effect of EGCG or vehicle treatment on activation of the IKK complex at 0 (sham value), 30, 60, and 120 min after reperfusion. The autoradiograph is representative of 3 experiments with similar results. Fold increase was calculated compared with respective sham value (time 0) set to 1.0. Results are representative of 3 separate time-course experiments. *P < 0.05 compared with respective sham value (time 0); #P < 0.05 versus vehicle-treated rats subjected to myocardial ischemia and reperfusion.

Figure 7

Effect of in vivo treatment with EGCG on activation of AP-1 and phosphorylation of c-Jun in rats subjected to myocardial ischemia and reperfusion. EGCG-treated rats received a bolus dose of EGCG 10 mg/kg at the end of the ischemia period followed by 10 mg/kg/h intravenous infusion for the entire period of reperfusion. A: Representative autoradiograph of electrophoretic mobility shift assay for AP-1 in vehicle-treated rats and EGCG-treated rats at 0 (sham value), 30, 60, and 120 min after reperfusion. The autoradiograph is representative of 3 separate experiments performed at different times. B: Western blot analysis demonstrating the effect of vehicle or EGCG on treatment phosphorylation of c-Jun at 0 (sham value), 30, 60, and 120 min after reperfusion. Western blot analysis is representative of separate experiments performed at different times. Fold increase was calculated compared with respective sham value (time 0) set to 1.0. Results are representative of 3 separate time-course experiments. *P < 0.05 compared with respective sham value (time 0); #P < 0.05 compared with vehicle-treated rats subjected to myocardial ischemia and reperfusion.