Combinational effect of resveratrol and atorvastatin on isoproterenol-induced cardiac hypertrophy in rats.

INTRODUCTION: Resveratrol is a natural polyphenol present mainly in grapes. It has been shown to offer strong cardio protection in animal models due to its ability to correct lipid peroxidation and maintain antioxidants level. Atorvastatin, a HMG-CoA reductase inhibitor, lowers cholesterol level and is commonly prescribed to heart patients. Our aim in this study was to see the combination effect of these two drugs against Isoproterenol-induced cardiac hypertrophy in rats.
MATERIALS AND METHODS: Wister Albino rats were treated with resveratrol (20 mg/kg/day, p.o), atorvastatin (20 mg/kg/day, p.o) and in combination (resveratrol [10 mg/kg/day, p.o] + atorvastatin [10 mg/kg/day, p.o]) for a period of 25 days and from 15(th) till 25(th) day Isoproterenol (5 mg/kg/day, s.c) was co-administered to rats to induce cardiac hypertrophy.
RESULTS: A significant increase in creatine kinase, lactate dehydrogenase, aspartate transaminase and lipid peroxidation with the significant decrease in reduced glutathione, superoxide dismutase and catalase were observed in Isoproterenol treated rats. Resveratrol, atorvastatin and their combination significantly reversed the effect. The histopathological studies and myocardial infarct size evaluation also confirmed the protection.
CONCLUSION: Comparing the data we came to this conclusion that atorvastatin although showed the protection along all the parameters, the extent of protection offered by resveratrol alone and in combination were more effective. Hence, it can be concluded that resveratrol, an herbal nutritional supplement, alone and in combination is better against cardiac hypertrophy.

Cardiac myocytes enlarges in response to various pathological stimuli like hypertension, valvular dysfunction, and myocardial infarction leading to cardiac remodeling called cardiac hypertrophy.[123] It is a compensatory response initially but often leads to heart failure when hypertrophic stimuli persist. Therefore, it is an independent and major risk factor for cardiovascular morbidity and mortality.[4] Excess production of reactive oxygen species (ROS) under pathophysiological conditions; prevail over the antioxidant defenses which lead to oxidative stress. Under oxidative stress, the cellular components get injured considerably.[5678] Although we see revolution in the field of cardiovascular disease (CVD) therapy in the last many years due to the development of a large number of drugs, yet the deaths due to CVD still accounts for 25%.[9] The increasing risk factors, non-compliance to drug therapy and unabated disease pathology may be the reason behind it. A worldwide attempt is continuously being made to look for newer effective drugs in terms of cost, safety, and effectiveness. The success however achieved is still limited. Traditional medicinal herbs are enthusiastically being explored now-a-days by the researchers and information on their preclinical and clinical efficacy is being worked out. Thus, herbal medicines are greatly emerging as effective alternative or adjuncts to modern medicine. In this connection, we also tried to look for an herbal drug which can prove to be a better cardioprotective agent.

Resveratrol (3, 4, 5-trihydroxy-trans-stilbene) is a natural polyphenol, found in grapes, mulberries etc., is traditionally used as medicinal herb by Asians and proved to increase the life span of yeast,[10] mice, insulin sensitivity,[11] prevented cancer,[12] and shown antiatherosclerotic, hypolipidemic, and anti-inflammatory properties.[13] We therefore, have selected this polyphenol to address its effect on experimentally induced cardiac hypertrophy as its study on CVDs is scarce. Atorvastatin, a mainstay in cholesterol lowering therapy, is chosen in this study as standard drug as well as for combination therapy, because of its known properties of plaque stabilization preservation in endothelial function, scavenging of free radicals, anti-proliferative, anti-inflammatory, and anti-apoptotic properties, responsible for its cardioprotective effects.[14151617]

Materials and Methods

Drugs

Resveratrol was purchased from Zenith Nutrition's Pvt. Ltd., Bangalore, India. Atorvastatin was received as a gift sample from Ranbaxy Laboratories Limited, New Delhi, India. Isoproterenol (ISO) was obtained from Sigma-Aldrich, Germany.

Experimental procedure

This study was approved by Jamia Hamdard, Animal Ethics Committee. Albino rats (Wistar strain) of either sex, weighing 200–300 g were procured from the Central Animal House Facility, Jamia Hamdard, New Delhi. The animals were kept in polypropylene cages (five rats in each cage) under standard laboratory conditions and had a free access to commercial pellet diet and tap water. Rats were randomized into the following eight groups.

On 26th day, animals were sacrificed and various biochemical parameters in serum and tissues were estimated. Hearts were also examined for histological changes and infarct size in all the groups.

Serum parameters

Creatine phosphokinase-MB (CK-MB isoenzyme), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) were estimated using commercially available kits (Reckon Diagnostics, Baroda, India).

Tissue parameters

The hearts were removed and washed with ice-cold saline. Weighed amount of heart tissue was mixed and 10% homogenate was prepared in ice-cold 0.15 M KCl for thiobarbituric acid reactive substances (TBARS) estimation;[18] in 0.02 M EDTA for tissue reduced glutathione (GSH) estimation;[19] and in phosphate buffer (pH 7.4) for superoxide dismutase (SOD);[20] and catalase (CAT)[21] estimation by using Teflon homogenizer.

Histopathology

Hearts were excised out and placed in 0.9% normal saline solution until histopathology is done. Myocardial tissue was fixed in 10% formalin, processed and impregnated with paraffin. Paraffin sections were cut, stained with hematoxylin and eosin and examined under a light microscope.[22]

Infarct size

Hearts were kept in the freezer (−20°C) at least for 1–2 h. Once the tissue solidified, it was sliced into approximately 2–3 mm thickness and incubated in the tetrazolium stain at 37°C in a water bath for 20 min. The heart slices were agitated in between at least once a minute. Infarct scarred area and the total area of left ventricular (LV) myocardium were traced manually in the digital images and measured automatically by the J image software in the computer. Infarct size, expressed as a percentage, was calculated by dividing the sum of infarcted areas from all sections by the sum of LV areas from all sections (including those without infarct scars) and multiplying by 100.[2324]

Statistical analysis

Statistical analysis was carried out using Graph pad prism 3.0 (graph pad software San Diego, CA, USA). All results were expressed as mean ± Standard error of the mean. Groups of data were compared with the analysis of variance (ANOVA), followed by Dunnett's t-test.

Results

All the serum parameters are shown in Table 1. Administration of ISO significantly increased the level of LDH, AST, and CK-MB in serum. Treatment with resveratrol decreased the level of these enzymes indicating improvement, but its combination with atorvastatin, however, showed better efficacy in lowering down the level than the drugs alone. Treatment with atorvastatin also inhibited the level of this enzyme but to a lesser extent as compared to other treated groups [Table 1].

Table 1

Biochemical observations of the serum parameters in different groups

Antioxidant enzymes (superoxide dismutase, catalase), glutathione and Lipid peroxidation (MDA)

There was a significant decrease in GSH, SOD, and CAT and increase in lipid peroxidation (MDA) showing a decrease in antioxidant level when the rats were challenged with ISO. These levels however, were reversed to almost normal when they were treated with resveratrol, atorvastatin, and their combination [Table 2].

Table 2

Biochemical observations in the heart tissues of different groups

Histopathology of heart

ISO administration resulted into cytoplasmic vacuolation, infiltration of chronic inflammatory cells, disarrangement in myocardial cells, and angiogenesis in blood vessels whereas normal groups showed regular and well oriented myocardial fibers. Resveratrol and its combination with atorvastatin showed marked reduction in vacuolation and infiltration of inflammatory cells as compared to ISO treated group. Atorvastatin treated group showed mild hypertrophy with an increase in the size of nuclei, mild inflammatory cells, and vacuolation [Photomicrographs 1-8].

Photomicrograph 1

High power photomicrograph of control group showing normal architecture of myocardial fibers with no infiltration of inflammatory cells (hematoxylin and eosin ×400)

Photomicrograph 8

High power photomicrograph of resveratrol + atorvastatin per se group showing regular and well oriented myocardial fibers (hematoxylin and eosin ×400)

High power photomicrograph of toxic group showing cytoplasmic vacuolation, infiltration of chronic inflammatory cells and disarrangement in myocardial cells (hematoxylin and eosin ×400)

High power photomicrograph of resveratrol-treated group showing marked reduction in disarray of cells and infiltration of inflammatory cells (hematoxylin and eosin ×400)

High power photomicrograph of atorvastatin treated group showing mild hypertrophy with increase in the size of nuclei, mild inflammatory cells, and vacuolation (hematoxylin and eosin ×400)

High power photomicrograph of resveratrol per se group showing normal morphology of myocardial fibers (hematoxylin and eosin ×400)

High power photomicrograph of atorvastatin per se group showing normal oriented myocardial fibers (hematoxylin and eosin ×400)

High power photomicrograph of resveratrol + atorvastatin treated group showing mild hypertrophy with increase in the size of nuclei (hematoxylin and eosin ×400)

Infarct size study showed the same trend of improvement as indicated by various biochemical parameters. Our result clearly indicated that the large infracted area seen in the toxic group was considerably reduced when treated with the test drugs alone or in combination [Figure 1].

Figure 1

Infarct size in various groups. Each column is mean ± standard error of the mean for five rats showing percentage of the infracted area in rat myocardium (toxic vs. treated group, **P < 0.01 and *P < 0.05)

Discussion

The present study showed that the administration of ISO (5 mg/kg/day, s.c) to the Wistar Albino rats produced significant cardiotoxicity, which is evident by the increased levels of serum marker enzymes (LDH, AST, and CK-MB) and cardiac tissue TBARS and decreased level of myocardial endogenous antioxidant markers (GSH, SOD, and CAT). Histopathological observations showed cytoplasmic vacuolation, infiltration of chronic inflammatory cells, disarrangement in myocardium cells thereby confirmed the damage by ISO in rats.

Pretreatment with resveratrol (20 mg/kg/day, p.o) for 25 days showed cardioprotection as it reversed the rise in biochemical parameters (LDH, AST, CK-MB, and TBARS) showing membrane integrity and restored the myocardial endogenous antioxidants level (GSH, SOD, and CAT) by improving the antioxidant status of the cells. It also minimized vacuolation and maintained the integrity of myofibrils as observed in the light microscope is also indicative of the same.

Pretreatment with (resveratrol + atorvastatin) combination (10 + 10 mg/kg/day, p.o) for 25 days showed cardioprotection as substantiated by the significant decline in the elevated levels of serum marker enzymes LDH, AST, and CK-MB; abating lipid peroxide levels and also by maintaining the level of other endogenous antioxidants and also by minimizing vacuolation.

Pretreatment with atorvastatin (20 mg/kg/day/p.o) for 25 days also showed cardioprotective effect as evident by the restoration of all the biochemical parameters toward normal and histopathology showing mild hypertrophy with increase in the size of nuclei, mild inflammatory cells and vacuolation.

Infarct size study also showed the reduction in the infarct size when treated with resveratrol or atorvastatin or their combination indicating the preventive role of these drugs in cardiotoxicity.

Conclusion

Comparing all the data obtained in relation with atorvastatin, resveratrol, and their combination, we conclude that all of them showed cardioprotection, but resveratrol and (resveratrol + atorvastatin) combination showed better protection than atorvastatin. However, among resveratrol and (resveratrol + atorvastatin) treated groups, there were no significant changes in the extent of protection. Hence, resveratrol appears to be a better drug in protecting ISO-induced cardiac injury.

Clinical significance

Resveratrol, a herbal nutritional supplement having negligible side-effects, easy availability, moderate treatment dose and lastly a cheap drug having proved for lipid-lowering activity, hypertension, and other indications appears to be a better option considering a huge population suffering from cardiac and other lifestyle related disorders.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.