International Journal of Nutrition, Pharmacology, Neurological Diseases

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 11  |  Issue : 2  |  Page : 174--179

Cardioprotective Effects of Gallic Acid on an Isoprenaline-Induced Myocardial Infarction Rat Model


Abdelbaset Taher Abdelhalim1, Sayed A.M Mahmoud2, Nuruddin Mohammed Nur3, Mossad Abdelhak Shaban4, Sherif Mansour5, Suhaidah Ibrahim6,  
1 Pharmacology Department, University College WIDAD, Kuantan, Pahang, Malaysia
2 Medical biochemistry Department, Faculty of Medicine, Alazhar University, Cairo, Egypt
3 Biochemistry Department, Universiti Islam Antarabangsa Sultan Abdul Halim Mua’dzam Shah (UniSHAMS), 09300 Kuala Ketil, Kedah Darul Aman, Malaysia
4 Paediatrics Department, International Islamic University of (UIA), Kuantan, Pahang, Malaysia
5 Pharmacology Department, Faculty of Medicine, Alazhar University, Assuit, Egypt
6 Physiology Department, Universiti Islam Antarabangsa Sultan Abdul Halim Mua’dzam Shah (UniSHAMS), 09300 Kuala Ketil, Kedah Darul Aman, Malaysia

Correspondence Address:
Abdelbaset Taher Abdelhalim
Department of Pharmacology, Faculty of Medicine, University College WIDAD, Kuantan, Pahang
Malaysia

Abstract

The use of antioxidants to protect against a wide range of human disease, including ischemic heart disease, has moved to the forefront in cardiovascular research. Gallic acid has shown promising effects against oxidative stress-induced disease; however, its effect in ischemic heart disease has not been well-studied. We designed the current work to investigate the potential protective effect of gallic acid against isoprenaline (ISO)-induced myocardial infarction (MI). Rats were injected subcutaneously with ISO, 100 mg/kg for 2 days, to induce MI. Gallic acid treated rats received 15 mg/kg gallic acid orally for 10 days prior to ISO injection. The histopathological examination of the Hematoxylin and Eosin-stained heart sections from the ISO treated rats shows karyopyknosis, hypereosinophilia, loss of striation, infiltration of macrophage in the interstitium, and thrombosis of the blood vessels, all of which indicate the induction of MI. In addition, ISO treatment significantly increased the plasma level of malondialdehyde and troponin-I, as well as the activity of alanine aminotransferase, lactate dehydrogenase, and creatine kinase, compared to untreated controls. Pretreatment with gallic acid significantly attenuated the ISO-induced biochemical and histopathological changes, compared to untreated controls. Our results show that ISO induced oxidative stress-mediated MI, and that gallic acid protects the rat heart from MI, at least in part, through antioxidant mechanisms.



How to cite this article:
Abdelhalim AT, Mahmoud SA, Nur NM, Shaban MA, Mansour S, Ibrahim S. Cardioprotective Effects of Gallic Acid on an Isoprenaline-Induced Myocardial Infarction Rat Model.Int J Nutr Pharmacol Neurol Dis 2021;11:174-179


How to cite this URL:
Abdelhalim AT, Mahmoud SA, Nur NM, Shaban MA, Mansour S, Ibrahim S. Cardioprotective Effects of Gallic Acid on an Isoprenaline-Induced Myocardial Infarction Rat Model. Int J Nutr Pharmacol Neurol Dis [serial online] 2021 [cited 2022 May 19 ];11:174-179
Available from: https://www.ijnpnd.com/text.asp?2021/11/2/174/314376


Full Text



 Introduction



Myocardial infarction (MI) is a common presentation of ischemic heart disease, which accounts for up to 12.2% of deaths worldwide. Ischemic heart disease is the leading cause of death in high- and middle-income countries, followed by lower respiratory infections in lower income countries.[1] Age, diabetes, atherosclerotic vascular disease, hypertension, smoking, and drinking alcohol have consistently been associated with increased risks of MI. [2],[3]

A healthier lifestyle has been shown to prevent and lower the incidence of MI. For instance, physical activity and an antioxidant-rich diet including herbs such as ginseng, curcuma, ginkgo, rosemary, green tea, grape, ginger, and garlic have been associated with a lower risk profile.[4],[5] Previous studies proved that antioxidants have a protective effect against cellular damage from oxidative stress, thus lowering the risk of chronic diseases.[6],[7],[8],[9] Furthermore, antioxidants such as ascorbate, tocopherols, tocotrienols, flavonoids, and carotenoids show promise as cardio protective compounds.[10]

Gallic acid, 3,4,5-trihydroxybenzoic acid, is a phenolic compound found in many plants, including gallnuts, sumac, witch hazel, tea leaves, oak bark, and grape seeds. Gallic acid has shown a variety of pharmacological activities, including antifungal, antiviral, anti-inflammatory, and anticancer.[11],[12],[13] Gallic acid possesses significant antioxidant activity and may protect the liver from the harmful effects of free radicals that are formed as a result of various metabolic processes in the body[14] however, its ability to protect against ischemic heart disease has not been delineated. In this study, we investigated the potential role of gallic acid in protecting rats against isoprenaline (ISO)-induced MI. Our results provide evidence that gallic acid attenuates the oxidative damage induced by ISO and protects the heart against MI.

 Materials and Methods



Experimental design: Thirty age-matched male albino rats, with an initial body weight ranging from 150 to 200 g, were bred in the animal house of the Department of Pharmacology at Al-Azhar University College of Medicine. All animal procedures and handling were performed in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee at Al-Azhar University, which comply with the international regulations for the use and care of laboratory animals. Rats were maintained on a balanced diet and water was given ad libitum. Rats were divided into three groups, 10 animals each: (1) control untreated group; (2) MI group, where animals received subcutaneous injection of ISO (dissolved in water; Sigma/Aldrich, USA) at a dose of 100 mg/kg body weight every 24 hours for 2 days; and (3) gallic acid/MI group where rats were pretreated with gallic acid (dissolved in water; Oxford laboratory, India) at a dose of 15 mg/kg body weight every 24 hours for 10 days through oral gavage, and then subcutaneously injected with ISO at a dose of 100 mg/kg every 24 hours for 2 days.[15] Ten animals were used for blood-based biochemical analysis; six animals were used for heart tissues-based biochemical analysis; and four animals were used for histopathological study.

Sampling: 12 hours after the second ISO dose, rats were anesthetized with ether and sacrificed by cervical decapitation. Blood was collected in dry glass tubes and centrifuged at 5000 rpm for 10 minutes. Serum was separated and used for various biochemical endpoints. The hearts were excised, weighed and homogenized in TRIS HCl buffer (10 mmol/L; pH 7.4) at a ratio of 1:10 (w/v). The homogenates were centrifuged at 10,000 rpm for 20 minutes and the clear supernatant was used to determine lipid peroxidation, superoxide dismutase and reduced glutathione levels.

Determination of serum aspartate transaminase and alanine aminotransferase activities: The activity of serum aspartate transaminase (AST) and alanine aminotransferase (ALT) was determined using an enzymatic colorimetric method as described earlier.[16] Briefly, 100 μL of serum was added to 0.5 mL of a warm solution containing 100 mM L-aspartate and 4 mM α-oxoglutarate in 100 mM phosphate buffer pH7.4 and incubated at 37.0°C for 30 minutes. Then 0.5 mL of 4 mM 2.4-dinitrophenylhydrazine was added, tubes were mixed well and incubated at 20 to 25°C for another 20 minutes. 0.5 mL of 4 M NaOH was added, followed by mixing, tubes were left at the room temperature 5 minutes, and the absorbance at 546 nm was measured at 37.0°C against a serum-free reagent as a blank. The enzymatic activity (IU/L) of AST and ALT was estimated using a standard curve of commercially available standard AST and ALT.

Determination of serum lactate dehydrogenase activity: Serum lactate dehydrogenase (LDH) activity was determined using the enzymatic–colorimetric method as described earlier.[17] Briefly, 100 μL serum was added to 3 mL of 4:1 v/v of a solution containing 65 mM Imidazole and 0.6 mM pyruvate and a solution containing 0.18 mM NADH. Tubes were incubated at 25°C for 1 minute and the absorbance was measured at 340 nm every minute for 3 minutes. The average absorbance difference per minute (ΔA/min) was calculated. The LDH activity (U/L) was determined using the following equation: LDH (U/L) = ΔA/min. x 4925.

Determination of serum creatine kinase level: Serum level of creatine kinase (CK) was determined calorimetrically, using a commercially available kit (Chema Diagnostica, Italy). 40 μL of serum sample was mixed with reagents supplied with the kit according to the manufacturer’s instructions. The absorbance was measured at 340 nm after 1 minute incubation. Then samples were incubated again at 37°C for another 5 min, and the absorbance was taken three times at 1 minute intervals. CK serum activity was calculated using the following equation: CK U/L=ΔA/min x 4127.

Determination of serum troponin-I (CTnI) level: A commercially available kit (Monobind Inc., USA), utilizing an immunoenzymometric method, was used to determine the serum level of CTnl.[18] 25 μL serum samples were used to carry out the assay performed in a microtiter plate according to the manufacturer’s instructions.

Determination of reduced glutathione level: The level of reduced glutathione (GSH) in heart homogenate was determined colorimetrically following the manufacturer’s instructions provided with the assay kit (Bio-diagnostic, Egypt).[19] The absorbance was measured at 405 nm and GSH values were calculated according to the manufacturer’s instructions.

Determination of superoxide dismutase level: The activity of superoxide dismutase (SOD) in heart homogenate was determined, using nitroblue tetrazolium colorimetric assay, following the manufacturer’s instructions provided with the assay kit (Bio-diagnostic, Egypt).[20] The absorbance was measured at 560 nm and SOD values were calculated according to the manufacturer’s instructions.

Determination of malondialdehyde: The level of malondialdehyde (MDA) in heart homogenates was determined colorimetrically following the manufacturer’s instructions provided with the assay kit (Bio-diagnostic, Egypt). The absorbance was read at 534 nm and MDA values were was calculated according to the manufacturer’s instruction manual using the following equation.

[INLINE:1]

Histopathology: Rats were sacrificed; hearts were rapidly dissected out, washed immediately with saline, fixed in 10% neutral buffered formalin, and embedded in paraffin. 5-µm-thick heart sections were stained with Hematoxylin and Eosin (H&E) and examined under the microscope for histopathological changes.

Statistical analysis: One–way analysis of variance (ANOVA) followed by the Turkey-Kramer test was used to test the significance between groups using SPSS software. Data is presented as mean ± standard error of mean (SEM). P < 0.05 was used as a limit for statistical significance.

 Results



[Table 1] shows that gallic acid significantly decreased the activity (IU/l) of AST (49.32 ±2.82), ALT (40.59 ± 4.19), LDH (105.07 ± 4.02), and CK (197.06 ± 6.96) in rat serum treated with ISO, compared to ISO-alone-treated animals. However, gallic acid treatment did not decrease the enzyme activities to their normal levels shown in control untreated animals.{Table 1}

The levels of GSH and SOD activity were significantly lower in heart homogenates of ISO-treated rats, compared to ISO-gallic acid pre-treated group. The tissue homogenates content of GSH and SOD activity of gallic acid-treated rats show 3.91 mmol/g tissues and 6.03 U/g of tissue, respectively, compared to that of ISO-treated animals (8.41 mmol/g and 11.25 U/g of tissue for GSH and SOD, respectively). Gallic acid treatment did not decrease these values to their normal levels shown in control untreated animals (12.62 mmol/g and 17.47 U/g of tissue for GSH and SOD, respectively).

Histopathological examination revealed the presence of focal area of coagulative necrosis within the cardiac muscle of ISO-treated rats. Myofibers of ISO-treated rats also showed karyopyknosis, hypereosinophilia, loss of striation, infiltration of macrophage in the interstitium, and thrombosis of blood vessels [Figure 1]. Tissue sections of control untreated rats showed normal striation of myocardium [Figure 2]cc.{Figure 1}{Figure 2}

Examination of H&E-stained myocardial tissue sections of ISO-treated rats pre-treated with gallic acid showed only congestion of blood vessels with normal myofibers [Figure 2].

 Discussion



Understanding the pathogenesis of MI and developing a better therapeutic regimen require an animal model that mimics the human disease. Using chemicals, open surgery, or closed-chest catheter has been used to induce MI in animals.[21] However, these methods either invasive or result in a low success rate. ISO has shown many advantages when used to induce MI in experimental animals. ISO-induced MI is a simple noninvasive technique with a high success rate, low mortality rate, and shows ‘infarct-like’ myocardial necrosis that resembles human myocardial infarction.[22],[23]

ISO in large doses has been reported to produce free radicals through activation of catecholamine oxidative metabolism.[24] Cardiotoxicity of ISO in this study resulted from oxidative stress[25].

Free radicals are molecules containing one or more unpaired electrons in atomic or molecular orbitals.[26] Oxygen and nitrogen-derived free radicals are called reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively. Under physiological conditions, ROS are produced in low concentrations and play important roles in normal cellular function through regulation of cell signaling, apoptosis, gene expression, and ion transportation.[27] Among major endogenous ROS-producing sources are mitochondrial respiration, NADPH oxidases, xanthine oxidoreductase, and uncoupled nitric oxide synthases.[17],[28],[29] Excessive ROS generation causes harmful effects to many cellular molecules through their ability to damage the nucleic acids, protein amino acid side chains, and double bonds in unsaturated fatty acids. Phospholipids, glycolipids, cholesterol esters, cholesterol and proteins are major components of the cell membrane. Under pathological conditions of oxidative stress, ROS attack these cellular components and cause damage to tissue cellular membrane, as observed in this study. Our results suggest that ISO generated ROS that led to damage to the integrity of cellular tissue structure which caused leakage of the cellular enzymes CK, AST, ALT, and LDH into blood stream thus increasing their concentration in the serum. Such damage may have led to MI.

Serum CK, AST, ALT and LDH are well known MI markers. When myocardial cells are damaged or destroyed due to deficient oxygen supply or glucose, the cardiac membrane becomes permeable or may rupture. This membrane damage causes a leakage of these enzymes in the blood stream, leading to an increase of their plasma concentration.[30]

Antioxidants neutralize free radicals by accepting or donating electron(s) to eliminate the unpaired condition of the radical. The antioxidant may also react with the reactive radicals and either destroy them or convert them to a less active, longer-lived, and less dangerous form which may be reneutralized by other antioxidants or other mechanisms to terminate their radical status.[31] Cells are normally able to defend themselves against ROS damage using intracellular natural antioxidant enzymes SOD, CAT, and glutathione peroxidase (GPX).[7] These endogenous antioxidants provide an important defense against free radicals to keep ROS at a low level.[32],[33] Oxidative stress lowers the intracellular natural antioxidants SOD and GSH, as observed in rat sera treated with ISO. Gallic acid, as observed in this study, was able to minimize the damaging effects of ROS on tissue structure and reduce the leakage of cellular enzymes into blood stream. Our results suggest the beneficial cardioprotective effect of gallic acid through its ability attenuate the oxidative stress induced by ISO and restoring the activity of the endogenous natural antioxidative enzymes. Antioxidants have been shown to increase myocyte autophagy after MI, suggesting that oxidative stress mediates reduction in myocyte autophagy that contributes to post-MI remodeling.[34]Antioxidants are known to play a role in mitochondrial disease, glycogen storage disease, Kwashiorkor, therapeutic hypothermia, and cardiovascular damage in chronic renal failure. Antioxidants have been evaluated for both primary and secondary prevention of coronary heart disease and have been proven as effective cardioprotective agents. It has also been shown that cardiovascular protection has been associated with dietary intake of antioxidants through a daily use of fruits and vegetables.[35]

 Conclusion



The current study concludes that gallic acid attenuates ISO-induced biochemical and histopathological changes in MI rat model. Gallic acid protects the heart against ISO-induced oxidative stress-mediated MI. Our results provide evidence that gallic acid protects the heart against ISO-induced MI, at least in part, through antioxidant mechanisms.

Acknowledgment

The authors like to thank Dr. Abdelhamid Gadmour, senior lecturer of Pathology, College of Medicine, WIDAD University College, Malaysia, for his great and kind help in accomplishing the pathological part of this study.

Financial support and sponsorship

Nil.

Conflict of interest

There are no conflicts of interest.

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