Cardiovascular diseases (CVDs) comprise disorders of the heart and blood vessels and still represent a major cause of death globally. CVDs have shown to be responsible for approximately 17.9 million deaths each year, which accounts for 31% of all deaths worldwide. Among the various CVDs, myocardial infarction (MI) is a major cause of mortality and morbidity across the world. Any blockade in the coronary artery leads to insufficient blood supply to heart, causing the heart muscle that is being supplied by the artery to get infarcted ultimately resulting in ischemic tissue necrosis in addition to other pathological and structural changes. The pathogenesis of MI includes hyperlipidemia, oxidative stress, peroxidation of membrane lipids, and loss of plasma membrane integrity [1].
Isoproterenol (ISO), a synthetic catecholamine and β-adrenergic agonist is well-known to cause severe stress in the myocardium by generating free radicals which in turn stimulates lipid peroxidation and perhaps the major contributing factor for the irreversible damage to the myocardial membrane [2]. ISO upon administration causes increase in heart rate leading to increased oxygen demand, high calcium burden and accumulation besides causing alterations in the morphology and membrane integrity of the myocardium with elevated cAMP levels in the myocardial cells [3].
Induction of myocardial infarction was previously performed by surgical procedures, but it has incidence of morbidity, mortality and animals were prone to pneumothorax infections [4]. Isoproterenol-induced myocardial infarction is simple and non-invasive model, considered as one of the most widely used experimental model to study the beneficial effects of many drugs and cardiac function [2] and it is similar to those pathophysiological changes observed in human myocardial infarction [5].
Natural products have high universal demands due to their claimed advantage in terms of both safety and efficacy against various diseases like MI. Plant based coumarins are low- molecular weight phenolic compounds that has been used for the prevention and treatment of various thromboembolism MI and stroke [6]. Esculetin (6, 7- di hydroxyl coumarin) is a natural coumarin derivative isolated from many plants such as Artemisia capillaries, Citrus limonia, Solanum surrettence and Euphorbia lathyris [7] with multiple pharmacological & biochemical properties [8]. There is numerous treatment approaches that have been developed to attenuate the risk of myocardial infarction, but most of them fail when translated from the bench to the bedside. Therefore, there is a need to find new drugs which hostile MI as well as be used as suitable therapeutic candidate and also can be translated to clinical use in the future [9].
The present study was intended to investigate whether esculetin can provide protection against free radical generation by ISO and its associated alterations in the membrane bound enzymes and myocardial lysosomal enzyme activities in experimental rats. Further, we also tried to explore the probable mechanism of action of the esculetin for its cardio protection in H9C2 cell lines.
Experimental procedures
Chemicals
Isoproterenol (CAS Number 5984-95-2), and Esculetin (CAS Number 305–01-1) was purchased from Sigma Aldrich. Co, St. Louis, USA. All the chemicals used in the present study were of analytical grade and indigenous.
In vitro studies
2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
The antioxidant activity of esculetin was measured using DPPH radical scavenger assay in triplicate. Concentration of 5, 25, 50 and 100 μM/mL were used along with 10 μM of ascorbic acid as reference standard. The test were then incubated with 50 μL of 0.1 mM DPPH solution and made up to the final volume to 3 mL with methanol. A blank was prepared using DPPH solution and methanol. The reaction mixture was incubated for 30 min at room temperature in the dark followed by measuring absorbance at 517 nm [10].
H9C2 cell culture
H9C2 myoblast cells from rat’s myocardium were acquired from National Centre for Cell Sciences, Pune, India. The myoblast cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) medium supplemented with 10% FBS and 10 ml/l100 × antibiotic–antimycotic solution containing 10,000 units of penicillin and 10 mg/ml streptomycin in 0.9% normal saline in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Measurement of cell viability by MTT assay
The cells were seeded in 96-well culture plates at a density of 7 × 104 cells/well. When the cells reached 80% confluence, they were treated with 1‰ dimethyl sulfoxide (DMSO) solution, and 5, 25 50 and 100 μg/mL of esculetin at 37 °C for 24 h. The cells were then incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/mL) solution for 4 h, and the resulting formazan was solubilized with 150 μL of DMSO for 30 min. The absorbance of each well was measured at 570 nm, and the absorbance of control cells was considered to indicate 100% cell viability [10, 11]. Scheme represented in Fig. 1.
Intracellular reactive oxygen species (ROS) measurement
The generation of intracellular reactive oxygen species (ROS) was measured by using the ROS-sensitive fluorescence indicator called Dichlorofluorescin diacetate (DCFH-DA) as per our previous protocol. H9C2 cells grown in 12 well plates 0.1× 106 to ~ 75% confluence were treated in triplicate. The cells were treated with esculetin at 25 and 50 μM/mL concentrations for 12 h. After incubation, 10 μM arsenic were added to esculetin treated wells and incubated for 2 h at 37 °C. All the wells including control were washed with PBS and incubated with 20 μM DCFH-DA for 30 min at 37 °C in the dark. After, Cells were washed, and analyzed by flow cytometer. The florescence intensity was calculated using the FAC Suite software [10]. Scheme represented in Fig. 1.
RNA isolation, cDNA synthesis and qPCR to assess mRNA expression of TNF-α, IL-6, NF-κb
H9C2 cells grown in 12 well plates 0.1× 106 to ~ 75% confluence were treated in triplicate. After arsenic and esculetin treatment, the total RNA was isolated from H9C2 cells by using Trizol reagent (Thermo Fisher Scientific, Inc.). The isolated RNA was quantified by using a nano-drop spectrophotometer and complementary DNA (cDNA) was synthesized from 1 μg of RNA was used for reverse transcription reaction using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed by using the SYBR green reagent according to the manufacturer’s protocol (MilliporeSigma).
The primer sequences used for qPCR were as follows: TNF-α forward, 5′ GAACTGGCAGAAGAGGCACT-3′ and reverse, 5′-GGTCTGGGCCATAGAACTGA-3′; IL-6 forward, 5′-CCGGAGAGGAGACTTCACAG-3′ and reverse, 5′-CAG AATTGCCATTGCACA-3′; NF-κB forward 5′-CCCACACTATGGATTTCCTACTTATGG’-3 and reverse 5′ CCAGCAGCATCTTCACGTCTC-3′. RT-qPCR reactions were performed under the conditions like, 50 °C for 35 min, 85 °C for 12 min, followed by 60 cycles of 95 °C for 23 s and 60 °C for 1.5 min. The selected gene expression level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′ CTTTGGTATCGTGGAAGG ACTC-3′ and reverse, 5′ GTAGAGGCAGGGATGATGTTCT-3′ as internal loading control [12, 13]. Scheme represented in Fig. 1.
In vivo study
Animals
Male wistar rats weighed between 230 and 280 g were used in this study. Rats were housed under standard conditions and fed with standard pellet with drinking water ad libitum. The animals were kept in polypropylene cages and maintained at a room temperature of 25 ± 2 °C with 55 ± 5% relative humidity and 12 h light/dark cycle. The study was carried out in compliance with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines (“Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Academic Press 1996; NIH publication number #85–23, revised 1996). All experimental procedures and methods were approved by the Institutional Animal Ethical Committee (IAEC), Sri Padmavathi School of Pharmacy, constitute as per the directions of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
Induction of myocardial infarction
Myocardial infarction was induced by dissolving isoproterenol (100 mg/kg) in normal saline and injected subcutaneously to rats for last two consecutive days of the experimental schedule [14].
Experimental schedule
The treatment schedule was fixed for 28 days and the 24 rats were divided into four groups of six each. Same timing preferable morning 10 am to 10:30 a m was maintained while dosing every day.
Group 1 | Normal saline serve as control |
Group 2 | Isoproterenol 100 mg/kg body weight dissolved in 1 mL of normal saline and administered by subcutaneously for last two consecutive days of the study and serve as disease control |
Group 3 | Esculetin 10 mg/kg body weight dissolved in 1 mL normal saline once daily oral and serve as test group at low dose |
Group 4 | Esculetin 20 mg/kg body weight dissolved in 1 mL normal saline once daily oral and serve as test group at high dose |
Blood sample collection and analysis
At the end of treatment blood was collected from retro orbital plexus by anesthetizing the rats with thiopental sodium (35 mg/kg body weight, intra peritoneal) [15] and serum was separated by centrifugation at 2000 rpm. Serum was used to analyze various biochemical parameters such as determinations of cardiac biomarkers lactate dehydrogenize (LDH), and creatinine kinase MB (CK-MB) by using commercial diagnostic kits (Agappe Pvt. Ltd., Kerala, India).
Na+ /K+ ATPase activity of myocardial membrane
The myocardial membrane Na+ /K+ ATPases activity was determined according to procedure done by Periyathambi and Ponnian 2007. The incubation mixture contained 10 mM of Tris buffer, 20 mM of potassium chloride, 125 mM of sodium chloride, 1 mM of EDTA and 3 mM of ATP. To the incubation mixture, the reaction was initiated by the addition of 0.2 mL of tissue homogenate and the contents were incubated at 37 °C for 15 min. To stop the reaction of 10% trichloro acetic acid (TCA) was added. The tubes were centrifuged and supernatant was used for the estimation of liberated Pi. 1.0 mL of supernatant was made up to 4.3 mL with distilled water and added 1.0 ml 3 mM of ammonium molybdate reagent. The tubes were incubated at room temperature for 10 min, and later 0.4 ml of amino naptholsulphonic acid reagent was added to develop the color and the Pi released recorded using a standard Pigraph [16].
Preparation of lysosomal sub cellular fractions
Lysosomal subcellular fractions were isolated according to the method of Venkatachalem et al.,2003. The heart tissue sample was cut open and placed in isotonic saline to remove the blood. Then the heart tissue was rinsed in ice cold 0.25 M sucrose, blotted, weighed and minced. The enzyme extracts were prepared by homogenizing the tissue samples in 0.25 M sucrose at 4 °C. The portion of the homogenate was subjected to differential centrifugation, and the different fractions were separated as follows: structural proteins, nucleus, and cell debris at 600×g for 10 min; mitochondria at 5000×g for 10 min; lysosomes at 15,000×g for 10 min. Myocardial sub-fractions were treated with Triton X-100 (final concentration 0.2% v/v) in ice for 15 min prior to the determination of enzymatic activity [17] .
The activities of the lysosomal enzymes including β-glucuronidase [18], β-glucosidase and β-galactosidase [19], and acid phosphatase [20] were determined.
Determination of tissue antioxidants
At the end of the experimentation hearts were excised from rats and homogenate in 0.1 M Tris buffer (pH 7.4) and the separated homogenates were used for estimation of heart antioxidants like super oxide dismutase (SOD) [21], Reduced glutathione (GSH) [22], Catalase [23] and lipid peroxidation (LPO) [24].
Histopathological studies of heart
After removal of myocardial tissue immediately washed with ice cold saline to remove all the blood and fixed in 10% buffered neutral formalin solution. After fixation was complete, tissues were embedded in paraffin and serial sections were cut in to 0.5 μm. Each section was stained with hematoxylin and eosin. The sections were examined under light microscope and histograms were taken.
Statistical analysis
Results were expressed as mean ± standard error mean multiple comparisons of the significant analysis of variance (ANOVA) followed by the Dennett’s test as post parametric test using computer based fitting program (Prism graph pad 5.0). A p value of < 0.05 was considered as statistically significant.
