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. Briefly, the stock solution (1mg/mL) of esculetin was prepared in methanol. Concentration of 5, 25, 50 and 100µg/mL were used along with ascorbic acid as reference standard. The test were then incubated with 50µL of 0.1mM DPPH solution and made up to the final volume to 3mL 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 517nm [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 and 50 μg/mL of esculetin at 37oC 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 4h, 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].
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 96 well plates to ~75% confluence were treated in triplicate. The cells were treated with esculetin at 0.1, 1, 5 and 10µM. After 12h, 10µM arsenic was added to the plate. The cells were incubated for an additional 30 min at 37°C and after PBS wash cells were incubated with 20µM DCFH-DA 30min 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].
RNA isolation, cDNA synthesis and qPCR to assess mRNA expression of TNF-α, IL-6, NF-κb
After arsenic and esculetin treatment, the total RNA was isolated from H9C2 cells by using Trizol reagent (Thermo Fisher Scientifi, Inc.). The isolated RNA was quantified by using a nano-drop spectrophotometer and complementary DNA (cDNA) was synthesized from 1µg 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 sec 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].
In vivo study
Animals
Male wistar rats weighed between 230-280 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±20C with 55±5% relative humidity and 12hrs 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 rats were divided into five groups of six each. Rats of group I received the normal saline and served as normal control, group II was received ISO (100mg/kg body weight) for last two consecutive days of the study and served as disease control. Groups III and IV received esculetin 10 and 20 mg/kg body weight respectively, once a day orally for 28 days of the study along with ISO for last two consecutive days of the study and serve as test groups.
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 10mM of Tris buffer, 20mM of potassium chloride, 125mM of sodium chloride, 1mM of EDTA and 3mM of ATP. To the incubation mixture, the reaction was initiated by the addition of 0.2mL 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. 1.0mL of supernatant was made up to 4.3mL with distilled water and added 1.0ml 3mM of ammonium molybdate reagent. The tubes were incubated at room temperature for 10 minutes, and later 0.4ml 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 0.1M 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 0.5µm. 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.