Animals and Diets
Male Sprague-Dawley rats aged 6 weeks weighing 200-220 g were purchased from Nomura Siam International Co., Ltd., Bangkok, Thailand. All rats were housed in standard cages under temperature-controlled room (23 ± 2 °C) with a relative humidity of 30 – 60 % and light/dark cycle of 12 hours. All procedures were performed in accordance with the rules of ethical guideline for the Care and Use of Laboratory Animals, which was approved by animal ethics committee of Khon Kaen University (IACUC-KKU- 74/62), based on the ethic animal experimentation of national research council of Thailand. The animals had free access to diet and water. Standard chow diet and high-fat diet were used for feeding control rats and MS rats, respectively. The standard chow diet composed of 57.81% carbohydrates, 22.9% protein and 5.72% fat while a high-fat diet composed of 46.3% carbohydrates, 13.25% protein and 24.29% fat. The composition of the standard chow and a high-fat diet were analyzed by Central Lab Thai (Central Laboratory (Thailand) Company Limited, Khon Kaen, Thailand). The control rats were given tap water while the MS rats were supplement with 15% fructose in drinking water during night-time to facilitate sings of MS.
Research Designs
After acclimatization, control rats were fed with standard chow diet and tap water for sixteen weeks (n=8). MS rats were fed with high-fat diet and 15% fructose drinking water for sixteen weeks. At the end of twelve weeks of experiment, MS rats were subdivided into 4 groups (n = 8/group), MS rats received vehicle, MS rats treated with galangin (25 mg/kg), MS rats treated with galangin (50 mg/kg), and MS rats treated with metformin (100 mg/kg). Galangin (purity ≥ 98%) was purchased from Aktin Chemicals, Inc. (Mianyang City, Sichuan, China). Metformin was obtained from Siam pharmaceutical Company Ltd. (Bangkok, Thailand). All treatments were administered orally using intragastric tube daily for the final four weeks an experiment period. Blood samples were collected via lateral rat tail vein at 12th weeks for fasting blood glucose and lipid profile measurement to confirm the characteristic of MS.
Indirect blood pressure measurements
Conscious rats were evaluated SBP changes in monthly during three month of the experimental period and weekly during the final four weeks of treatment. SBP were measured using the tail-cuff plethysmograph method (IITC/Life Science Instrument model 229 and model 179 amplifier; Woodland Hills, CA, USA). The average SBP value of three-time measurement were present.
Measurements of fasting blood glucose, serum insulin level and oral glucose tolerance test (OGTT)
Rats were fasted for 12 hours with free access to drinking water. Blood samples were collected from lateral tail veins to measure a basal glycaemic (at time 0 min (T0)) and serum insulin levels. Then, rats were fed with glucose solutions using gavage tube at a dose of 2g/kg BW. Blood glucose concentrations at 30, 60, 120 and 180 min after gavage was assessed using a glucometer (Roche Diagnostics GmbH, Mannheim, Germany). Insulin levels in serum was assessed after 12-h fasted overnight. Serum sample were obtained upon spontaneous coagulation and centrifugation (3000 g, 4°C, 30 min). Serum insulin levels were assessed by enzyme-linked immunosorbent assay (ELISA) kits (Millipore Corporation, Billerica, MA, USA). Insulin resistance was determined from the relative-value of homeostasis model (HOMA-IR) (43). HOMA-IR score was calculated using the formula as following:
HOMA-IR = (fasting blood glucose (mmol/L)) × (fasting insulin (µIU/mL))
22.5
Echocardiography
On the final day of experiment, all rats were anesthetized by 3% isoflurane. Echocardiography was performed to measure cardiac function using a commercially available echocardiography system (Model LOGIQ S7), equipped with a 10-MHz linear transducer (GE Healthcare, WI, USA). Each rat was shaved around their chest and applied a warmed resonance gel to the hairless chest. The ultrasound transducer was placed slightly left of chest then optimized for the left ventricle and aorta. Two-dimensional-guided M-mode images were recorded in accordance with the American Society of Echocardiography guideline. Three consecutive beats were measured at the five min after anesthesia, and the average of these measurements was taken for analysis. M-mode tracings to record interventricular septal end diastole and end systole (IVSd and IVSs), left ventricular internal diameter end diastole and end systole (LVIDd and LVIDs), left ventricular posterior wall end diastole and end systole (LVPWd and LVPWs) end-diastolic and systole volumes (EDV and ESV), stroke volume (SV) and ejection fraction (%EF) from three consecutive cardiac cycle were performed. LV shortening fraction (%SF) was calculated using equation: %SF= [(LVIDd-LVIDs)/LVIDd] x 100.
Direct blood pressure measurements
After cardiac function measurement, the left femoral artery was cannulated. SBP, diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) were monitored and recorded by pressure transducer using the Acknowledge Data Acquisition and Analysis Software (BIOPAC Systems Inc., California, USA).
Assessment of biochemical profiles
Following indirect blood pressure measurements, rats were euthanized by overdose of anesthesia and then blood samples were collected from the abdominal aorta and plasma was separated immediately using centrifugation at a speed of 3,000 g at 4°C for 30 min. Total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-c) levels in plasma were determined spectrophotometrically using specific commercial kits (Human Gesellschaft fuer Biochemica and Diagnostica mbH, Wiesbaden, Germany). Additionally, liver tissue was homogenized in lysis buffer for measure TC and TG using specific commercial kits as plasma. Levels of aspartate transaminase (AST) and alanine transaminase (ALT) were measured by Clinical Chemistry Laboratory Unit of Faculty of Associated Medical Sciences, Khon Kaen University, Thailand.
Tissue harvesting
After collecting blood samples, heart, liver, and visceral fat (including epididymal and retroperitoneal fats) were immediately dissected. All tissues were weighed to compare regional tissue weight (mg)/ body weight (g). A portion of the liver, heart and visceral fat was frozen at −20°C for biochemical analysis and fixed in 4% formaldehyde for histomorphology analysis.
Hematoxylin and eosin staining of cardiac and fat tissue
Myocardial tissue and epididymal fat pads were fixed in 4% paraformaldehyde for 24 hours, routinely processed, and embedded in paraffin. Briefly, all tissue paraffin blocks were cut at 5 mm thickness using a microtome. The paraffin sections (5 µm) were dewaxed and rehydrated through gradient alcohol into water. The sections were then washed with tap water, distilled water, and then stained with hematoxylin and eosin (H&E) (Bio-Optica Milano SpA., Milano, Italy). For microscopic assessment, the images of heart sections were captured by stereoscope (Nikon SMZ745T with NIS-elements D 3.2) at 1x objective lens to evaluate the LV wall thickness; cross-sectional area (CSA); the LV luminal area and wall to lumen ratio. These parameters were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
Measurement of myocardium cell size, area of cardiomyocyte was performed for 300 myocytes per group at 40x objective lens, via Digital sight DS-2MV light microscope (Nikon, Tokyo, Japan). Mean values were obtained from 300 cells/group.
Epididymal fat sections were observed using a Digital sight DS-2MV light microscope (Nikon, Tokyo, Japan) at 40x objective lens. Adipocyte was quantitated as cell sizes area (300 cells/group) using a NIS-Elements software.
Immunohistochemical stainingof myocardial sections
An immunohistochemical technique was used to evaluate TNF-α and IL-6 expression in left ventricle. The myocardial sections were deparaffinized in xylene and rehydrated through an ethanol series. Antigen retrieval was performed by tris-ethylenediaminetetraacetic acid (EDTA) buffer and used high temperature heating method to recover the antigenicity of tissue sections. The myocardial sections were incubated with hydrogen peroxide (H2O2) for blocking endogenous enzymes and then incubated with 5% bovine serum albumin in PBS for blocking nonspecific protein. Thereafter, the sections were incubated with primary antibody, mouse anti-TNF-α IgG (dilution 1:500) or mouse anti-IL-6 IgG (dilution 1:500), in moistening chamber for 4 hours at room temperature (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Goat anti-mouse IgG (HRP) dilution 1:1000 (Abcam Plc, Cam-bridge, UK) was used as secondary antibody. The brown color of 3,3'-Diaminobenzidine (DAB) was visualized as a positive control and the tissues were counterstained with hematoxylin. The myocardial sections were observed using a Digital sight DS-2MV light microscope (Nikon, Tokyo, Japan) at 40x objective lens. TNF-α and IL-6 expression were quantified using Image-Pro plus 6 software (Media Cybernetics, Inc., Rockville, MD, USA).
Assays of cytokines levels
Plasma adiponectin level was assessed using the adiponectin enzyme-linked immunosorbent assay (ELISA) kits (Millipore Corporation, Billerica, MA, USA). The serum levels of TNF-α and IL-6 were measured with ELISA kits according to the manufacturer's instructions (Sigma-Aldrich, Saint Louis, MO, USA).
Oxidative stress markers assessment
Aorta was rapidly excised for the analysis of superoxide production which determined by lucigenin enhanced chemiluminescence as described previously (44). The aorta was quickly dissected. The adherent fat and connective tissue were cleaned on ice. The vessel segments (3–5mm) were placed in Krebs-KCl buffer and allowed to equilibrate at 37°C for 30 min. Lucigenin was added to the sample tube and placed in a luminometer (Turner Biosystems, Sunnyvale, CA, USA). The photon counts were integrated every 30 sec for 5 min. The vessels were dried at the room temperature for 24 hours to determine a dry weight. Superoxide production in aorta was expressed as relative light unit counts per minute per milligram of dry tissue weight. Malondialdehyde (MDA) is an end-product of lipid peroxidation and can be as a biomarker of oxidative damage. MDA was estimated in plasma and heart tissue by using a colorimetric assay or thiobarbituric acid reactive substances (TBARS) assay as described in a previous report (45). MDA level was assessed by quantifying thiobarbituric acid (TBA) reactivity as MDA in a spectrophotometer. The resulting chromogen absorbance was determined at the wavelength of 532 nm against blank reference. The concentration of MDA was read from standard calibration curve plotted using 1, 1, 3, 3’ tetra-ethoxy propane (TEP) as a μM/L unit.
Antioxidant endogenous enzyme activity assessment
The CAT activity in plasma and heart tissue were determined using a colorimetric method. CAT is a ubiquitous enzyme that destroys hydrogen peroxides (H2O2) formed during oxidative stress. The level of CAT activity depends on monitoring the change of 405 nm absorbance at high levels of hydrogen peroxide solution. In Brief, samples were incubated with substrate (65 µmol/mL of H2O2 in 60 mmol/L sodium potassium phosphate buffer pH 7.4) in 96-well plate at 37°C for 1 min. Next step, add 32.4 mmol/L ammonium molybdate for stop reaction. The yellowish molybdate and H2O2 complex absorbance was determined at the wavelength of 405 nm and leading to calculate the CAT activity level.
SOD activities in heart tissue were measured via colorimetric analysis using a spectrophotometer with the corresponding detection kits (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocols.
Western-blotting analysis
LV tissue were homogenized in ice-cold lysis buffer. Processed samples, containing 50 μg protein, were heat-denatured in laemmli buffer and separated on 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Separated proteins were electro-transferred onto polyvinylidene difluoride (PVDF) membrane (MilliporeSigma, Merck KGaA, Darmstadt, Germany) at 90 V for 90 min. After completion of the transfer, the PVDF membranes were blocked with 5% BSA in tris-buffered saline with 0.1% Tween-20 (TBS-T) for 2 hours at room temperature. After that membranes were incubated overnight at 4°C with specific primary antibodies against AdipoR1(dilution 1:1000), COX-2 (dilution 1:500) (Abcam Plc, Cam-bridge, UK), p-NF-κB (dilution 1:1000) (Cell Signaling Technology, Inc., Danvers, USA). This was followed by incubation with appropriate secondary antibody for 2 hours at room temperature. b-actin was used was used as loading control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Bands were detected using ECLTM Prime western blotting reagents (Amersham Biosciences Corp., Piscataway, NJ, USA). The intensities of the bands were quantified using an ImageQuant™ 600 imager (GE Healthcare Life Science, Piscataway, NJ, USA) and were normalized to that of b-actin.
Statistical Analysis
All data were expressed as mean ± standard error of the mean (S.E.M.). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons analysis. All statistical analyses were performed using PRISM software version 8.3 (GraphPad Software Inc., San Diego, CA, USA). Differences were considered significant at p values < 0.05.