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. 2017 Aug;40(2):389-399.
doi: 10.3892/ijmm.2017.3014. Epub 2017 Jun 8.

(-)-Epigallocatechin-3-gallate attenuates myocardial injury induced by ischemia/reperfusion in diabetic rats and in H9c2 cells under hyperglycemic conditions

Yang Wu  1 , Zhong-Yuan Xia  1 , Bo Zhao  1 , Yan Leng  1 , Juan Dou  2 , Qing-Tao Meng  1 , Shao-Qing Lei  1 , Zhi-Ze Chen  1 , Jie Zhu  3
Affiliations

(-)-Epigallocatechin-3-gallate attenuates myocardial injury induced by ischemia/reperfusion in diabetic rats and in H9c2 cells under hyperglycemic conditions

Yang Wu et al. Int J Mol Med. 2017 Aug.

Abstract

(-)-Epigallocatechin gallate (EGCG) exerts multiple beneficial effects on cardiovascular performance. In this study, we aimed to examine the effects of EGCG on diabetic cardiomyopathy during myocardial ischemia/reperfusion (I/R) injury. EGCG (100 mg/kg/day) was administered at week 6 for 2 weeks to diabetic rats following the induction of type 1 diabetes by streptozotocin (STZ). At the end of week 8, the animals were subjected to myocardial I/R injury. The EGCG-elicited structural and functional effects were analyzed. Additionally, EGCG (20 µM) was administered for 24 h to cultured cardiac H9c2 cells under hyperglycemic conditions (30 mM glucose) prior to hypoxia/reoxygenation (H/R) challenge, and its effects on oxidative stress were compared to H9c2 cells transfecteed with silent information regulator 1 (SIRT1) small interfering RNA (siRNA). In rats with STZ-induced diabetes, EGCG treatment ameliorated post-ischemic cardiac dysfunction, decreased the myocardial infarct size, apoptosis and cardiac fibrosis, and reduced the elevated lactate dehydrogenase (LDH) and malonaldehyde (MDA) levels, and attenuated oxidative stress. Furthermore, EGCG significantly reduced H/R injury in cardiac H9c2 cells exposed to high glucose as evidenced by reduced apoptotic cell death and oxidative stress. The protein expression levels of SIRT1 and manganese superoxide dismutase (MnSOD) were reduced in the diabetic rats and the H9c2 cells under hyperglycemic conditions, compared with the control rats following I/R injury and H9c2 cells under normal glucose conditions. EGCG pre-treatment significantly upregulated the levels of htese proteins in vitro and in vivo. However, treatment with EX527 and SIRT1 siRNA blocked the EGCG-mediated cardioprotective effects. Taken together, our data indicate that SIRT1 plays a critical role in the EGCG-mediated amelioration of I/R injury in diabetic rats, which suggests that EGCG may be a promising dietary supplement for the prevention of diabetic cardiomyopathy.

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Figures

Figure 1
Figure 1
Body weight and serum glucose concentrations in control and diabetic rats. (A) Serum glucose concentrations and (B) body weights in control and streptozotocin-induced diabetic rats that were treated with or without (−)-epigallocatechin gallate (EGCG). Values are the means ± SEM, n=18. *P<0.05 vs. control group.
Figure 2
Figure 2
(-)-Epigallocatechin gallate (EGCG) treatment alleviates cardiac dysfunction following ischemia/reperfusion (I/R) injury in diabetic rats. (A) Left ventricular systolic pressure (LVSP). (B) Maximum speed of pressure development in LV (+dp/dtmax). (C) Maximum speed of pressure decline (−dp/dtmax). Values are the means ± SEM, n=6. *P<0.05 vs. control group; #P<0.05 vs. DM + EGCG group.
Figure 3
Figure 3
(-)-Epigallocatechin gallate (EGCG) treatment decreases infarct size, cardiomyocyte apoptotic index following ischemia/reperfusion (I/R) injury in diabetic rats. (A) Top panel: the blue-stained areas in representative images of heart sections indicate non-ischemic tissue, red-stained areas represent the area at risk and negative-stained areas indicat infarct areas. Bottom panel: images of the apoptotic myocytes are shown by TUNEL staining; green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes. Images are at ×400 magnification; a total of 15 fields/heart were selected. (B) Myocardial infarct size was expressed as a percentage of the area at risk (AAR) (n=6); (C) Apoptotic index was expressed as a percentage of TUNEL-positive cells (n=6). Values are the means ± SEM. *P<0.05 vs. control group; #P<0.05 vs. DM group; $P<0.05 vs. DM + EGCG group.
Figure 4
Figure 4
(-)-Epigallocatechin gallate (EGCG) treatment decreases serum lactate dehydrogenase (LDH), cardiac malonaldehyde (MDA) content and 15-F2t-isoprostane formation following ischemia/reperfusion (I/R) injury in diabetic rats. (A) Serum LDH level, (B) the level of 15-F2t-isoprostane and (C) MDA contents in cardiomyocytes were compared among different groups (n=6). Values are the means ± SEM. *P<0.05 vs. control group; #P<0.05 vs. DM group; $P<0.05 vs. DM + EGCG group.
Figure 5
Figure 5
(-)-Epigallocatechin gallate (EGCG) treatment increases the protein expression of silent information regulator 1 (SIRT1) and manganese superoxide dismutase (MnSOD) in the hearts of diabetic rats after ischemia/reperfusion (I/R) injury. (A) Top panel: representative western blot analysis of SIRT1 protein; Bottom panel: quantification of western blot data from (A) top. (B) Top panel: representative western blots of MnSOD protein; bottom panel: quantification of western blot data from (B) top. Values are the means ± SEM, n=6. *P<0.05 vs. control group; #P<0.05 vs. DM group; $P<0.05 vs. DM + EGCG group.
Figure 6
Figure 6
(-)-Epigallocatechin gallate (EGCG) treatment reduces the extent of cardiac fibrosis in the diabetic rats following ischemia/reperfusion (I/R) injury. (A) Masson trichrome-stained sections of left ventricles. (B) Quantification of cardiac fibrosis area from Masson trichrome-stained sections. Values are the means ± SEM. n=5. *P<0.05 vs. control group; #P<0.05 vs. DM group; $P<0.05 vs. DM + EGCG group.
Figure 7
Figure 7
(-)-Epigallocatechin gallate (EGCG) treatment decreases cellular apoptosis. (A) Evaluation of cardiomyocyte apoptosis by flow cytometric analysis. (B) Quantification of cardiomyocyte apoptosis. Values are the means ± SEM, n=6. *P<0.05 vs. normal glucose (NG) + normaxia group; #P<0.05 vs. high glucose (HG) + normaxia group; $P<0.05 vs. NG + hypoxia/reoxygenation (H/R) group; △P<0.05 vs. HG + H/R group.
Figure 8
Figure 8
(-)-Epigallocatechin gallate (EGCG) treatment decreases lactate dehydrogenase (LDH) activity as well as malonaldehyde (MDA) levels and increases cell viability in hypoxia/reoxygenation (H/R)-injured H9c2 cardiomyocytes. (A) Relative cell viability in cardiomyocytes. (B) LDH activity in culture medium. (C) MDA production in the culture medium. Values are the means ± SEM, n=6. *P<0.05 vs. normal glucose (NG) + H/R group; #P<0.05 vs. high glucose (HG) + H/R group; $P<0.05 vs. HG + EGCG + H/R group.
Figure 9
Figure 9
Silent information regulator 1 (SIRT1) protein expression in high glucose (HG) group and normal glucose (NG) group. (A) Representative western blots of SIRT1 protein in HG group and NG group; (B) Quantification of western blot data from (A). Values are the means ± SEM, n=6. *P<0.05 vs. NG + hypoxia/reoxygenation (H/R) group.
Figure 10
Figure 10
(-)-Epigallocatechin gallate (EGCG) pre-treatment enhances the protein expression of SIRT1 and manganese superoxide dismutase (MnSOD) in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R)-injury. (A) Top panel: representative western blots of SIRT1 protein under the treatment of high glucose (HG), EGCG and/or SIRT1 siRNA. Bottom panel: quantification of western blot data from (A) top. (B) Top panel: representative western blots of MnSOD protein under the treatment of HG, EGCG and/or SIRT1 siRNA. Bottom panel: quantification of western blot data from (B) top. Values are the means ± SEM, n=6. *P<0.05 vs. HG + H/R group; #P<0.05 vs. HG + EGCG + H/R group; $P<0.05 vs. HG + EGCG + H/R + SI RT1 siRNA group.
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