Cardioprotective Effect of Oleanolic Acid on Isoproterenol-Induced Myocardial Ischemia in Rats

Abstract

This study was designed to investigate the protective effect of oleanolic acid (OA) against isoproterenol-induced myocardial ischemia in rat myocardium. Wistar strain rats were pretreated with OA (20, 40, and 60 mg/kg, s.c) for 7 days and then intoxicated with isoproterenol (ISO, 85 mg/kg, sc for 2 consecutive days). Heart were excised from the experimental animals and assessed for the activities of marker enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK)], the levels of lipid peroxide products [thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (HP) and conjugated dienes (CD)], myeloperoxidase (MPO), lipid profiles [total cholesterol (TC), free cholesterol, ester cholesterol, triglycerides (TG), free fatty acids (FFA) and phospholipids (PL)], and membrane-bound ATPase enzymes (total ATPase, Na⁺K⁺ATPase, Ca²⁺ATPase, and Mg²⁺ATPase). Troponin T and I were estimated in plasma. Leakage of cardiac markers, elevated lipid peroxidation with increased lipid profiles and decreased activities of membrane-bound ATPase enzymes were confirmed the severe myocardial damage occurring as a consequence of isoproterenol-induced ischemia, and they also showed the significant improvement effected by oleanolic acid pretreatment. These findings provided evidence that oleanolic acid was found to be protecting rat myocardium against ischemic insult and the protective effect could attribute to its anti-oxidative, anti-hyperlipedemic, and anti-arrhythmic properties as well as its membrane-stabilizing action.

Introduction

Catecholamines are of significance not only in normal cardiovascular regulation but also in certain disease states such as congestive heart failure and ischemic heart disease (Raab, 1960; Harrison and Chidsey, 1962). In normal physiological conditions, the circulatory and interstitial catecholamines concentrations in heart are in the nanomolar range. However, pathological conditions such as ischemia, the levels can increase micromolar range (Lameris, 2000). Excessive endogenous release or exogenous administration of catecholamines depletes the energy reserve of cardiac muscle cells. This leads to complex biochemical and structural changes that cause irreversible cellular damage, which is a prelude to necrosis (Rona, 1959).

The model of isoproterenol-induced myocardial ischemia is considered as one of the most widely used experimental model to study the beneficial effects of many drugs and cardiac function (Grimm et al., 1998). The pathophysiological changes following ISO administration are comparable to those taking place in human myocardial ischemia/infarction (Wexler, 1978). It is also well known to generate free radicals and to stimulate lipid peroxidation, which may be a causative factor for irreversible damage to the myocardial membrane (Chatelain et al., 1987). Increases in the formation of reactive oxygen species during ischemia/reperfusion and the adverse effects of oxyradicals on myocardium have now been well established by both direct and indirect measurements. Thus, increased production of reactive oxygen species (ROS) may be a unifying mechanism in ischemic injury progression, and anti-oxidants may be the therapeutic value in this setting.

Myocardial cell protection and prevention of cell ischemia/necrosis have been therapeutic targets for a long time. New therapies are needed to treat myocardial ischemia because current treatment has only a limited impact on survival and annual costs. Triterpenoids exist widely in nature and are the major components of many traditional medicinal herbs. Oleanolic acid (OA) (Figure 1) is a triterpenoid compound that exists widely in food and herbs (Liu, 1995). It has a variety of biological effects, such as anti-oxidants (Balanehru and Nagarajan, 1991), antifungal, anti-inflammatory, anti-hyperlipdemia, hepatoprotective, tumor prevention, immunomodulatory, (Price et al., 1987; Nishino et al., 1988), anti-HIV (Kashiwada et al., 2000), anti-arrhythmic and cardiotonic (Somova et al., 2004). OA has been used successfully to treat liver disease in humans (Hunan Medical Institute, 1997).

Due to its anti-oxidant, anti-hyperlipedemic, anti-arrhythmic, and cardiotonic effects, it will provide an accessible and cheap traditional medicine source for treatment of myocardial ischemia in developing countries. Previously we have reported the cardioprotective effect of ursolic acid, an isomer of oleanolic acid, against isoproterenol-induced myocardial ischemia (Senthil et al., 2007). This prompted our research on the anti-ischemic effect of OA in isoproterenol-induced myocardial ischemia.

Materials and Methods

Chemicals

Oleanolic acid was a gift from the Active Ingredient Group, Inc., Changsha, China. Isoproterenol hydrochloride was purchased from Sigma Chemical Co., St. Louis, Mo, USA. All other chemicals used were of analytical grade.

Animals

Adult male albino rats of Wistar strain, weighing approximately 120–140 g, were used for the experiment. The animals were housed individually in polypropylene cages under hygienic conditions and maintained at room temperature. They all received pellet diet (Amrut Laboratory Animal Feed, M/S. Pranav Agro Industries Ltd., Bangalore, India) and water ad libitum. The experiment was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approved by Institutional Animal Ethics Committee (IAEC), Annamalai University.

Induction of Myocardial Ischemia

Myocardial ischemia was induced by subcutaneous injection of isoproterenol hydrochloride (ISO, 85 mg kg⁻¹ bw), dissolved in physiological saline, for 2 consecutive days (Rona et al., 1959; Seth et al., 1998).

Experimental Protocols

The rats were randomly divided into 6 groups with 8 rats each. The test compound (OA) was completely dissolved in DMSO and diluted with saline by the volume of 5:100 ratio and then used for the experiments. Group 1 served as control (received 5% DMSO sc, for 7 days). Group 2 rats were administered isoproterenol (ISO, 85 mg kg⁻¹ bw, sc, twice at an interval of 24 hours) on 8th and 9th days. Group 3 rats were administered with OA (60 mg kg⁻¹ bw, sc) for 7 days. Groups 4, 5, and 6 rats received 20, 40, and 60 mg kg⁻¹ b.w., of OA, subcutaneously, for 7 days and received ISO (85 mg kg⁻¹ bw, sc, twice at an interval of 24 hours) on the 8th and 9th days, respectively. Twelve hours after the second dose of ISO injection, the rats were sacrificed by cervical decapitation. The heart tissue was excised immediately, washed with chilled isotonic saline, and used for the estimations.

Biochemical Estimations

The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were estimated by the method of Reitman and Frankel (1957). Lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) were determined by the method of King (1965) and Okinaka et al. (1961), respectively, using commercially available kits. Plasma cardiac troponin T and I (cTnT and cTnI) were quantitatively measured by means of a highly specific enzyme immunoassay using commercially available kits (Katus et al., 1991; Larue et al., 1993). The level of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (HP), conjugated dienes were estimated by the method of Nichans et al. (1968), Jiang et al. (1992), and Rao and Recknagel (1968), respectively. The neutrophils specific MPO activity was determined by the method of Henson et al. (1978). The levels of total cholesterol, ester cholesterol, triglycerides, phospholipids, and free fatty acids were measured by the method of Allain et al. (1974, commercial kit), Varley et al. (1991), McGowan et al. (1983, commercial kit), Stewart (1980) and Falholt et al. (1973), respectively. The activities of total ATPase, Na⁺K⁺ATPase, Ca²⁺ATPase and Mg²⁺ATPase were estimated by the method of Evans (1969), Bonting (1970), Hjerken and Pan (1983), and Ohinishi et al. (1982), respectively. The scavenging effect of oleanolic acid was confirmed by the method of Kaur and Saini (2000).

Statistical Analysis

Results are expressed as means ± SD. The data were statistically analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) using a statistical package program (SPSS 10.0 for windows), taking p < 0.05 as significant.

Results

Table 1 shows the scavenging effect (%) of oleanolic acid on superoxide and hydroxyl radicals generated by alkaline DMSO method and Fenton reaction, respectively. Oleanolic acid strongly and dose-dependently scavenged the superoxide and hydroxyl radicals.

Table 2 shows the activity of AST, ALT, LDH, and CPK in heart and troponin T and I in plasma of control and experimental rats. Activity of AST, ALT, LDH, and CPK were observed to substantially decrease whereas the level of troponin T and I increased in the isoproterenol-treated group as compared with control. Oleanolic acid pre-treatment (20, 40, and 60 mg kg⁻¹ bw) significantly blocked the decrease of AST, ALT, LDH, and CPK and increase of troponin T and I.

Table 3 shows the level of TBARS, HP, CD (markers of lipid peroxidation) and myeloperoxidase (marker of neutrophil infiltration) in the heart tissue of control and experimental rats. TBARS, HP, CD, and MPO elevated significantly in the ISO-treated group. Pretreatment with OA prevented the elevation of lipid peroxidation and neutrophil infiltration when compared to untreated ISO group.

Table 4 shows the activities of total ATPase, Na⁺K⁺ AT-Pase, Ca²⁺ ATPase, and Mg²⁺ ATPase in the heart tissue of control and experimental rats. Total ATPase, Na⁺K⁺ AT-Pase, Ca²⁺ ATPase reduced significantly while decrease in Mg²⁺ ATPase was not significant in the ISO control group. Pretreatment with OA blocked the decrease of heart ATPase activities.

Table 5 shows the level of total cholesterol, free cholesterol, ester cholesterol, triglycerides, free fatty acids, and phospholipids in the heart tissue of control and experimental rats. The level of myocardial lipids except phospholipids showed a significant increase whereas phospholipids decreased significantly in the ISO-treated group. OA pretreatment blocked the amendment of heart lipids to near normal when compared to the untreated ISO group.

In all the parameters studied, OA had the highest activity, at the medium dose of 40 mg kg⁻¹, than the other 2 doses (20 and 60 mg kg⁻¹).

Discussion

Isoproterenol produces relative ischemia or hypoxia due to myocardial hyperactivity and coronary hypotension (Bloom and Davis, 1972), and induce myocardial ischemia due to cytosolic Ca²⁺ overload (Singal et al., 1983). The oxidative stress may be exerted through quinone metabolites of isoproterenol, which reacts with oxygen to produce ROS (Rathore et al., 1998) and interfere with glutathione reductase (Remiao et al., 2000), superoxide dismutase (Rathore et al., 1998), and ATP pumps (Remiao et al., 2000).

When myocardial cells, containing AST, ALT, CPK, and LDH, are damaged or destroyed due to deficient oxygen supply or glucose, the cell membrane becomes permeable or may rupture, which results in the leakage of enzymes. This accounts for the decreased activities of these enzymes in heart of rats with myocardial ischemia-induced by isoproterenol. This might be due to the damage caused to the sarcolemma by the β-agonist that has rendered it leaky (Mathew, 1985). Furthermore, the level of plasma troponin T and I were increased in ISO-treated rats and our finding was in consonance with an earlier report (Acikel et al., 2004). It is more likely that the plasma levels of troponin found in patients with unstable angina pectoris provide information about the severity of myocardial ischemia that caused cellular troponin degradation and release of troponin degradation products in the circulation (Van Der Laarse, 2002).

The OA pretreatment blocked the decrease of marker enzymes and increase of plasma troponins significantly, indicating the cytoprotective activity of OA and effect on recovery. Oleanolic acid is a lipophilic β-blocker in nature. β-Adrenergic blockers have long been useful adjuvants in the management of myocardial ischemic syndromes. The therapeutic benefits of β-blockers in patients with heart failure have been demonstrated in the CIBIS-II (1999) and MERIT-HF (1999) trials. The lipophilic β-blocking drugs intercalate into the lipid matrix and impart stabilization to myocardial cell membranes in relation to the degree of lipophilicity (Cruickshank and Dweyer, 1985). Hence, it is possible that likewise OA may also prolong the viability of myocardial cell membranes from necrotic damage by its membrane-stabilizing action (Han et al., 1997).

The myocardial necrosis observed in the animals receiving isoproterenol can also be attributed to peroxidative damage as it has been previously reported that isoproterenol generates lipid peroxides (Blasig et al., 1984). Our present observations were in keeping with the previous findings indicating the increases of lipid peroxidation. Oleanolic acid pretreatment prevent the elevation of myocardial lipid peroxides by an apparent direct scavenging of superoxide and hydroxyl radicals (Table 1) thereby prevent the initiation and propagation of the lipid peroxidation process (Balanehru and Nagarajan, 1991).

It was noticed earlier that the neutrophils, a major source of free radicals, characteristically invaded the myocardial tissue during ischemia (Abe et al., 1999). The observations of this study showing that oleanolic acid pretreatment blocked the elevation of MPO activity indicated that OA suppressed neutrophils infiltration into the injured myocardium. The inhibition of neutrophils infiltration and its function resulting in reduced generation of oxygen free radicals, during ischemia, may contribute to the protective action of oleanolic acid against myocardial ischemia.

Lipid metabolism plays an important role in myocardial necrosis produced by ischemia (Mathew et al., 1981). Isoproterenol-treated rats showing altered lipid profiles in the heart agrees well with a previous report (Subhash et al., 1978). The significant increase observed in the lipid profiles except phospholipids in the rat treated with ISO alone could be due to enhanced lipid biosynthesis by cardiac cAMP. Changes in membrane cholesterol content affect its fluidity, permeability to ions, activities of membrane-bound enzymes, and increased degradation of phospholipids (Yeagle, 1985). In this study, OA pretreatment was shown to block the alterations of myocardial lipids. This could be due to the ability of oleanolic acid to inhibit cAMP and thereby maintain the normal fluidity and less alteration in the property and function of the myocardial membrane (Wang et al., 1996).

Biological membranes are rich in phospholipids. A significant increase in free fatty acids and a decrease in phospholipid content in ISO-treated rats might have been due to the breakdown of membrane phospholipids. Accelerated phospholipid degradation could produce membrane dysfunction, resulting in cell injury and ultimate cell death. OA pretreatment significantly prevented the degradation of phospholipids thereby increases phospholipids and decrease the ratio of cholesterol/phospholipids (C/P ratio).

Besides lipids, ATPases pay significant role in the contraction and relaxation cycles of the cardiac muscle by maintaining normal ion levels within the myocyte. Several factors are known to alter the levels of ATPase, especially lipid peroxidation and membrane fluidity. It has been reported that ISO treatment resulted in a decrease in the activities of membrane-bound ATPases (Chernysheva et al., 1980). The results obtained in this study also correlates with the preceding reports. The loss of ATPase activity in the ischemic state may be responsible for causing not only functional damage but also reversible necrotic changes in the involved myocardial cell. Hebbel et al. (1986) have correlated the inactivation of membrane-bound enzymes with peroxidation of membrane lipids. Peroxidation of membrane lipids could inactivate Na⁺K⁺ ATPase and Ca²⁺ATPase because of the oxidation of ‘SH’ groups present in its active site leading to the conformational alteration in the enzymes (Kako et al., 1988). Decrease in the activity of Ca²⁺ATPase can increase intracellular concentration of free Ca²⁺ and alter the signal transduction pathways and cellular fluidity (Levy et al., 1986).

The present study shows that the altered activity of the membrane-bound phosphatases by isoproterenol was protected by pretreatment with oleanolic acid. This could be due to the anti-oxidative effect of OA against ROS induced by ISO. The results of this study imply that oleanolic acid pretreatment proved to be effective in reducing the extent of myocardial damage by decreasing lipid peroxidation, prevent the overloading of myocardium with lipids and its β-blocking activity.