The Protective Effect of Epigallocatechin-3 Gallate on Ischemia/Reperfusion Injury in Isolated Rat Hearts: An ex vivo Approach

Cheng Shi Piao; Do-Sung Kim; Ki-Chan Ha; Hyung-Ryong Kim; Han-Jung Chae; Soo-Wan Chae

doi:10.4196/kjpp.2011.15.5.259

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Piao, Kim, Ha, Kim, Chae, and Chae: The Protective Effect of Epigallocatechin-3 Gallate on Ischemia/Reperfusion Injury in Isolated Rat Hearts: An ex vivo Approach

Original Article

Korean J Physiol Pharmacol 2011;15(5):259-266.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.5.259

The Protective Effect of Epigallocatechin-3 Gallate on Ischemia/Reperfusion Injury in Isolated Rat Hearts: An ex vivo Approach

Cheng Shi Piao¹, Do-Sung Kim¹, Ki-Chan Ha¹, Hyung-Ryong Kim², Han-Jung Chae^1,^∗,, Soo-Wan Chae^1,^∗

¹Department of Pharmacology and Cardiovascular Research Institute, School of Medicine, Chonbuk National University, Jeonju 560-182, Korea

²Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan 570-749, Korea

Corresponding to: Han-Jung Chae, Department of Pharmacology, School of Medicine, Chonbuk National University, San 2-ro, Geumam-dong, Deokjin-gu, Jeonju 560-182, Korea. (Tel) 82-63-270-3092, (Fax) 82-63-275-2855, (E-mail) hjchae@jbnu.ac.kr

^∗ These authors contributed equally to this work.

Received 18 July 2011 Revised 3 September 2011 Accepted 24 September 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The aim of this study was to evaluate the preventive role of epigallocatechin-3 gallate (EGCG, a derivative of green tea) in ischemia/reperfusion (I/R) injury of isolated rat hearts. It has been suggested that EGCG has beneficial health effects, including prevention of cancer and heart disease, and it is also a potent antioxidant. Rat hearts were subjected to 20 min of normoxia, 20 min of zero-flow ischemia and then 50 min of reperfusion. EGCG was perfused 10 min before ischemia and during the whole reperfusion period. EGCG significantly increased left ventricular developed pressure (LVDP) and increased maximum positive and negative dP/dt (+/-dP/dtmax). EGCG also significantly increased the coronary flow (CF) at baseline before ischemia and at the onset of the reperfusion period. Moreover, EGCG decreased left ventricular end diastolic pressure (LVEDP). This study showed that lipid peroxydation was inhibited and Mn-SOD and catalase expressions were increased in the presence of EGCG. In addition, EGCG increased levels of Bcl-2, Mn-superoxide dismutase (SOD), and catalase expression and decreased levels of Bax and increased the ratio of Bcl-2/Bax in isolated rat hearts. Cleaved caspase-3 was decreased after EGCG treatment. EGCG markedly decreased the infarct size while attenuating the increase in lactate dehydrogenase (LDH) levels in the effluent. In summary, we suggest that EGCG has a protective effect on I/R-associated hemodynamic alteration and injury by acting as an antioxidant and anti-apoptotic agent in one.

Keywords: Epigallocatechin-3 gallate, Ischemia/reperfusion injury, Apoptosis, Antioxidant

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Fig. 1.

Experimental protocol. All hearts were perfused for a total of 90 min or 160 min consisting of a 20-min pre-ischemia period followed by 20 min of global ischemia and 50 min or 120 min of reperfusion at 37°C. There was no ischemia period for the sham group. EGCG was perfused 10 min before ischemia and during the whole reperfusion period.

Fig. 2.

Effect of EGCG on LVDP, LVEDP and ±dP/dt in isolated rat heart. EGCG was perfused 10 min before ischemia and during the whole reperfusion period. The percent changes of LVDP (A), LVEDP (B) and ±dP/dt (C, D) during reperfusion. Sham, non-ischemia; I/R, ischemia and reperfusion only; EGCG, ischemia and reperfusion treated with EGCG (5μmol). I/R is square. EGCG is circle. ^∗p <0.01 vs. Sham group, ^#p<0.05 vs. I/R group.

Fig. 3.

Effect of EGCG on coronary flow and LDH in isolated rat hearts. EGCG was perfused 10 min before ischemia and during the whole reperfusion period. The effect of EGCG on coronary flow and LDH before ischemia and during the reperfusion period. Sham, non-ischemia; I/R, ischemia and re-perfusion only; EGCG, ischemia and re-perfusion treated with EGCG (5μmol). ^∗p <0.01 vs. Sham group, ^#p<0.05 vs. I/R group.

Fig. 4.

Effect of EGCG on infarct size in isolated rat hearts. After 120-min reperfusion, hearts were collected to measure the infarct size by TTC staining (A). Infarct size was expressed as percent of the area-at-risk (AAR). Results were representative of five independent experiments. I/R, ischemia and reperfusion only; EGCG, ischemia and reperfusion treated with EGCG (5μmol). ^∗p<0.05 vs. I/R group.

Fig. 5.

Effect of EGCG on Bax and Bcl-2 protein expression levels in isolated rat hearts. After 50-min reperfusion, hearts were collected to measure the protein expression. (A) The expression levels of Bax and Bcl-2 protein were determined by Western blotting. (B) Densitometric analysis of each protein. The ratio of Bcl-2/Bax was shown (lower). Results are representative of three independent experiments. Sham, non-ischemia; I/R, ischemia and reperfusion only; EGCG, ischemia and reperfusion treated with EGCG (5μmol). ^∗p<0.05 vs. Sham group, ^#p<0.05 vs. I/R group.

Fig. 6.

Effect of EGCG on lipid peroxydation and Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase protein expression in isolated rat hearts. After 50-min reperfusion, hearts were collected to measure lipid peroxydation (A), the protein expression. (B) The expression levels of Mn-SOD (C), Cu/Zn-SOD (D) and catalase (E) were determined by Western blotting. Results are representative of three independent experiments. Sham, non-ischemia; I/R, ischemia and reperfusion only; EGCG, ischemia and reperfusion treated with EGCG (5μmol). ^∗p<0.05 vs. Sham group, ^#p<0.05 vs. I/R group.

Fig. 7.

Effect of EGCG on caspase-3. Cleaved caspase-3 was measured by immunohistochemical staining methods (A). The ratio of cleaved caspase-3 to DAPI staining (B). Results are representative of three independent experiments. Sham, non-ischemia; I/R, ischemia and reperfusion only; EGCG, ischemia and re-perfusion treated with EGCG (5μmol). ^∗p<0.05 vs. Sham group, ^#p<0.05 vs. I/R group.

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