The study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Pub. No. 85–23, revised 1996). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Huazhong University of Science and Technology (Permit number: S249).
Reagents and materials
Green tea polyphenols (70% catechins, 10% flavonols, and 20% polymeric flavonoids) were kindly provided by Unilever Health Institute (Vlaardingen, The Netherland). Goat polyclonal antibody (anti-NADPH oxidase, p22phox, and p67phox) and rabbit anti-β-actin antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, USA). Fluorescein isothiocyanate (FITC)-dextran (40 kDa, anionic) was from Molecular Probes, Inc. (Eugene, USA). Dihydroethidium (DHE) and 2′, 7′-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probes were from Beyotime Institute of Biotechnology (Jiangsu, China). Other reagents used were of the highest grade commercially available.
Wistar rats were from B&K Universal Group Limited (Shanghai, China). After one week’s acclimation, thirty male Wistar rats, weighting 40-60 g, divided into 5 groups randomly. Four groups of rats fed on a modified HF diet (60% standard chow, 12% sugar, 12% lard, 8% yolk powder, 6% peanuts powder, and 1% milk powder, w/w). Since the 4th week, GTPs solutions of different concentrations (0, 0.8, 1.6, and 3.2 g/L) were served instead of deionized water. One group fed on standard chow as control.
Measurement of endothelial permeability in rat aorta
At the end of the 26th week, animals were anaesthetized with intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg). Anaesthetic monitoring such as testing of rear foot reflexes and observation of respiratory pattern throughout the procedure was performed. After anaesthesia, Evans blue dye (3%, 1 mL/kg bw) was injected into the tail vein of the rats, thirty minutes later, the animals were sacrificed. The thoracic part of the aorta was isolated, rinsed in normal saline, and weighed. Thirty milligrams of fresh aorta tissue incubated in 0.9 mL dimethylformamide at 50°C for 24 h was used for analysis. The concentration of Evans blue dye was measured spectrophotometrically at 620 nm and presented as μg Evans blue dye per mg wet tissue.
Measurement of ROS in rat aorta
The ROS levels were measured in situ by DHE fluorescent probe, which reacts with ROS and forms ethidium bromide (ETH) that binds to DNA. Fresh cross-sections (10 μm) of frozen aortic arch were incubated with 5 M DHE (37°C, 15 min) in a humidified chamber, then red fluorescence signal was detected with a fluorescence microscope. ROS level was presented as integrated optical density (IOD) per unit area.
Endothelial cell culture and treatment
Bovine aortic endothelial cells (BAECs, No. C-003-5C) were purchased from Health Science Research Resources Bank (Osaka, Japan). BAECs maintained at 37°C in 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum. Cells at passages 3–10 were used in this study. For protein expression analysis, confluent cultures were treated with 0.4 μg/mL or 4.0 μg/mL GTPs along with 33 mmol/L glucose in medium (HG group) for 24 h. Cells cultured in 5.5 mmol/L glucose medium were used as controls.
Cell permeability assay
A previously described method was used for the cell permeability assay . Briefly, BAECs were seeded in the upper chambers of 0.4 μm polycarbonate Transwell filters of a 24-well filtration microplate (Whatman Inc., Clifton, USA). Upon confluence, three groups of the cells were treated with HG (high-glucose, 33 mmol/L) and GTP (0, 0.4 and 4.0 μg/mL) for 24 h. The fourth group was treated with HG alone for 23 hours followed by one hour co-treatment with DPI (10 μmol/L), an inhibitor of NADPH oxidase. After treatment, medium was replaced with fresh phenol red-free DMEM in the presence of FITC-dextran (1.0 μmol/L). The filtration microplate was removed after 2 h incubation, and fluorescence in the medium of the 24-well feeder tray was evaluated at 494 nm excitation and 521 nm emission.
Measurement of ROS in BAECs
ROS level was determined using a method previously described . Briefly, 1 × 105 endothelial cells per well were seeded onto a 96-well plate, cultured overnight, then exposed to HG with GTPs (0, 0.4, and 4 μg/mL) for 24 h. After exposure, 10 μmol/L DCFH-DA was added to the culture followed by 30 min incubation in the dark. The fluorescence intensity was measured with excitation wavelength at 488 nm and emission wavelength at 525 nm.
Electrophoresis and immunoblotting
Whole cell extracts were prepared by lysing these cells in extraction buffer (containing 50 mmol/L Tris/HCl, pH 8.0, 150 mmol/L NaCl, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% SDS, 0.1 mmol/L DTT, 0.05 mmol/L PMSF, 0.002 mg/mL aprotinin, 0.002 mg/mL leupeptin, and 1 mmol/L NaVO3). The protein concentration was quantified with BIO-RAD Dc protein assay reagent (Bio-Rad, Hercules, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological blotting were performed according to the method provided by Amersham Biosciences. Immunoreactive bands were detected by an ECL plus Western Blotting Detection System (Amersham Biosciences, Little Chalford, UK) according to manufacturer’s instructions. Protein expression was visualized with a chemiluminescent detection system (Syngen, Cambridge, UK) and analyzed by Gel Pro3.0 software (Biometra, Goettingen, Germany).
Quantitative values were expressed as mean ± SEM and the data were analyzed using one-way ANOVA followed by the Bonferroni post hoc test at α = 0.05. For the statistical analysis, SPSS 14.0 was used.