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IMPACT OF EGCG SUPPLEMENTATION ON THE PROGRESSION OF DIABETIC NEPHROPATHY IN RATS: AN INSIGHT INTO FIBROSIS AND APOPTOSIS Kalaiselvi Periandavan, Thangarajeswari Mohan, Prema Velusamy, Lakshmi Narasimhan Chakrapani, Ashok Kumar Srinivasan, Abhilasha Singh, and Thanka Johnson J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017
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Journal of Agricultural and Food Chemistry
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IMPACT OF EGCG SUPPLEMENTATION ON THE PROGRESSION OF
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DIABETIC NEPHROPATHY IN RATS: AN INSIGHT INTO FIBROSIS AND
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APOPTOSIS
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Thangarajeswari Mohan1, Prema Velusamy1, Lakshmi Narasimhan Chakrapani1, Ashok Kumar Srinivasan1, Abhilasha Singh1, Thanka Johnson2, Kalaiselvi Periandavan1*
10 11
1
Department of Medical Biochemistry, Dr.ALMPGIBMS, University of Madras, Taramani Campus, Chennai. 2 Department of Pathology, Sri Ramachandra University, Chennai.
*-Corresponding author Abstract
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Apoptosis is an active response of cells to altered microenvironments which is
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characterized by cell shrinkage, chromatin condensation and DNA fragmentation, in variety
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of cell types such as renal epithelial cells, endothelial cells, mesangial cells and podocytes.
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Hyperglycemia is among the micro environmental factors that may facilitate apoptosis which
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plays a decisive role in the initiation of diabetic nephropathy. Transforming growth factor-β,
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emerges as a powerful fibrogenic factor in the development of renal hypertrophy. Although, a
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number of potential treatment strategies exist for diabetic nephropathy, considering the ease
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of use and bioavailability, phytochemicals stands distinct as the preeminent option. EGCG, a
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green tea catechin is one such phytochemical which possess hypoglycemic and anti-fibrotic
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activity. The present study aims to explore the potential of EGCG to prevent apoptosis in
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high-fat diet and STZ induced diabetic nephropathy rats by assessing renal function, pro-
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fibrotic marker, and the expression of apoptotic and anti-apoptotic proteins. Our results
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validate EGCG as a potential anti-apoptotic agent evidently by improving renal function via
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down regulating TGF-β, consequently ameliorating diabetic nephropathy. In accordance,
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EGCG might be regarded as a prospective therapeutic candidate in modulating diabetic
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nephropathy, thus being a promising treatment.
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Key words: Diabetic nephropathy, EGCG, High-fat diet, STZ, TGF-β, Apoptosis.
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Introduction
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Apoptosis contributes to the development of diabetic nephropathy leading to the
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functional deterioration of the kidney. Epithelial cells, endothelial cells, mesangial cells and
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podocytes play a crucial role in the maintenance of structure and function of the glomerulus.
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Injury to the glomerulus is usually characterized by effacement of foot processes leading to
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proteinuria,1 accumulation of extracellular matrix, mesangial matrix expansion, and tubular
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fibrosis.
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Transforming growth factor-β (TGF-β) is shown to induce podocyte loss via apoptosis
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in diabetic nephropathy contributing to the development of glomerulosclerosis2 and regulate
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urinary albumin excretion by increasing both glomerular permeability and decreasing
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reabsorption in the proximal tubules.3 Even though, a number of potential treatment strategies
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have shown benefit in improving surrogate markers like albuminuria, but the translation to
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preserve GFR and prevent ESRD has not always been followed.4
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Therefore, any phytochemical that addresses glycemic control, podocytes loss by
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preventing apoptosis and regulate TGF-β levels will be a promising therapeutic agent that
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targets diabetic nephropathy. EGCG (Epigallocatechin-3-gallate) is shown to possess anti-
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hyperglycemic effect5,6 and anti-fibrotic effect,7,8 also has an afforded protective effect
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against ROS production in diabetic nephropathy induced mice kidneys.9 Therefore, we
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intended to investigate the role of EGCG in ameliorating apoptosis in the kidneys of rats
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induced with diabetic nephropathy.
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Materials and Methods
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Chemicals
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Epigallocatechin-3-gallate and Bovine Serum Albumin were procured from Sigma–
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Aldrich, USA. Streptozotocin (STZ) was purchased from Cayman chemicals, Europe. All
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other chemicals used were of analytical grade and were obtained from SRL (Sisco Research
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Laboratories Pvt. Ltd) and CDH (Central Drug House Pvt. Ltd., Mumbai, India). Enhanced
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chemiluminescence (ECL) kit was purchased from BioRad, USA.
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Laboratory animals
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Male albino rats of Wistar strain (100-110g) were obtained from Central Animal
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House facility, University of Madras, Taramani campus, and experiments were conducted in
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accordance with guidelines approved by the Institutional Animal Ethical Committee (IAEC
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No.
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conditions of controlled temperature (25±2 °C) with 12/12 h light/dark cycle and were given
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food and water ad libitum. The animals were maintained on a commercial rat feed
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manufactured by Hindustan Lever Ltd., Mumbai under the trade name “GOLD MOHUR
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RAT FEED”. The feed contained 5% fat, 21% protein, 55% nitrogen free extract, 4% fibre
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(w/w) with adequate vitamins and minerals.
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Experimental design
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01/07/2016). The animals were housed two per cage in large spacious cages under
Diabetic nephropathy model was established by High-fat Diet (HFD) comprising of
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the normal rat chow
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30% coconut oil and 30% fructose water. After sixty days of dietary manipulation, HFD fed
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rats was injected intraperitoneally (i.p.) with low dose of streptozotocin (STZ, 30 mg/kg,
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Cayman chemicals, Europe) dissolved in 0.1 M citrate buffer, pH 4.3. The fasting blood
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glucose was measured 3 days after the STZ injection. Rats with the fasting blood glucose
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16.7 mmol/L and above were considered as diabetic rats. Then these rats are left for fortnight
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to establish diabetic nephropathy and allowed to continue to feed on their respective diets
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until the end of the study.
supplemented with 4% cholesterol and 1.5% cholic acid along with
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Group 1: Normal control rats were given saline orally for 28 days. Group 2:
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Diabetic nephropathy induced HFD fed rats. Group 3: Diabetic nephropathy induced HFD
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fed rats treated with EGCG (100mg/kg body weight) through oral gavage for 28 days. Group
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4: Rats were treated with EGCG (100mg/kg body weight) through oral gavage for 28 days.
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Urine was collected from the rats housed in metabolic cages for 24hr. (Based on the literature
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survey, bioavailability of EGCG is about 1.6% accounting to approximately 320µg for a rat
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weighing 200 g).
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At the end of the experimental period, rats were anesthetized with ketamine (22
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mg/kg body weight, i/p) and kidneys were excised immediately, immersed in ice-cold
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physiological saline and 10% cortex of the kidney tissue homogenate were prepared using
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Tris-HCl buffer (0.01 M, pH 7.4), followed by centrifugation at 12,000 rpm for 10 min and
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the supernatant was used for the analysis of various parameters. Rest of the tissue was stored
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at -80oC for protein expression studies. For histological examination, the kidney was
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collected and fixed in 10 % neutral buffered formalin, embedded in paraffin. Standard
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sections of 5 μm thickness were cut, which were then stained with hematoxylin and eosin
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(H&E) and Masson Trichrome Stains.
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Fasting blood glucose measurements
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Fasting blood glucose levels were measured weekly on lateral tail vein blood samples TM
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using an One touch select simple
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treatment period.
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Determination of glycosylated hemoglobin (HbA1c)
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Glycosylated
hemoglobin
meter and system (Life scan, Scotland) throughout the
was
assessed
using
the
Hemoglobin
Analyzer
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(D-10 - Biorad) based on chromatographic separation of the analytes by ion exchange high
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performance liquid chromatography (HPLC) and the changes in the absorbance measured at
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415 nm.
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Urinary parameter
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The 24 h urine sample collected from each rat was centrifuged and the supernatant
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was used for further analysis. Creatinine levels were determined using Kit method
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(Medsource Ozane Biomedicals). Glomerular Filtration Rate (GFR) was calculated on the
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basis of urinary creatinine, serum creatinine and urine volume using the following equation:
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GFR (mL/min) = [urinary creatinine (mg/dl × urine volume (mL)/serum creatinine (mg/dl)]
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×1/1440 (min)]. Albumin creatinine ratio (ACR) was calculated from the urinary creatinine
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and urinary albumin values.
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Assay of urinary albumin by ELISA
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Urinary proteins were extracted using 10% TCA and High affinity 96 well microtitre
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plates were coated with 10 µg of protein in 50µl of 0.2 M sodium carbonate/bicarbonate
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buffer (pH 9.6) per well and incubated at 4°C for overnight. After that plates were washed
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thrice with 100 µl of 0.01M phosphate buffered saline (pH 7.4) and then blocked for 2 hours
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with 200 µl of 1% BSA in PBS at 37°C. Following four washes as above in PBS, appropriate
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primary antibody diluted in PBS were added and incubated at 4°C for overnight. Further,
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plates were washed thrice with phosphate buffered saline (pH 7.4) in 0.2 % Tween 20 (PBS-
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T) and incubated with 100 µl of streptavidin-horse radish peroxidase for 1 hour.
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Subsequently after washing, the reaction was visualized by the addition of 100µl/well 2,2’
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Azinobis [3-ethylbenzothiazoline-6sulfonic acid] diammonium salts (ABTS) substrate for
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20min at room temperature. The reaction was stopped by the addition of 100µl/well 2N
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H2SO4 optical densities were determined at 415 nm using iMark™ Microplate Absorbance
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Reader (Biorad). The urinary albumin was expressed as ng/mL. 10
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Analysis of serum lipid profile
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Total Cholesterol, Triglycerides and High density lipoproteins (HDL) in serum were
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analyzed using commercial kits from Spin react in the auto analyzer (Rx Monza, Randox,
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U.K). The values of total cholesterol, triglycerides, LDL (Low Density Lipoprotein), VLDL
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(Very Low Density Lipoprotein) and HDL are expressed as mg/dl.
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Biochemical analysis of protein carbonyl and lipid peroxidation in kidney cortex tissue
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The estimation of protein carbonyl in kidney cortex tissue were determined
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using 2, 4-dinitrophenyl hydrazine (DNPH) with slight modifications of the method of
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Levine et al.,11 and expressed as nmoles of protein carbonyl/mg protein/ml.
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The level of lipid peroxides was assayed by the method of Devasagayam and
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Tarachand, 12 and expressed as nmoles of MDA released/g of tissue.
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Western blot analysis
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Cortical kidney tissue samples were homogenized in Tris-HCl buffer (0.01 M, pH
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7.4) and protein concentrations were determined.13 For immunoblotting, aliquots of lysate
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were mixed with loading buffer containing dithiothreitol and maintained at 95oC for 10 min
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before loading on 10% SDS-PAGE. Following SDS-PAGE separation, proteins were
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transferred to polyvinyl difluoride membrane (PVDF). Membranes were blocked in Tris-
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buffered saline with Tween-20 containing 5% (w/v) non-fat milk for 1 h at room temperature.
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Membrane strips were incubated with primary antibodies (TGF-β1, Bax, Bcl-2 and cleaved
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caspase-3) overnight at 4oC. Following extensive washing, membrane strips were incubated
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with horseradish peroxidase conjugated secondary antibodies for 1 h. Protein bands were
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detected by the detection reagent mixture (an equal volume of luminol with H2O2) using
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standard enhanced chemiluminescence method in Bio-rad Chemidoc XRS imaging system.
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Statistical analysis
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Data are presented as mean ± Standard Error of Mean (SEM) of the results obtained
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from the average of at least three or six independent experiments. Differences between
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groups were analyzed by one-way analysis of variance (ANOVA) using the SPSS software
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package for Windows (Version: SPSS 20.0). Post hoc testing was performed for inter-group
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comparisons using Least Significant Difference (LSD) test. Values are statistically significant
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at the level of p
