Cell culture
HUVECs were obtained from Cambrex (Shanghai Biological Technology Co., Ltd., China) and were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated FBS (Hangzhou Sijiqing biological engineering materials Co., Ltd., China) at 37°C in a humidified atmosphere of 5% CO2. Cells were used at passage 4–6 for all experiments.
Propidium iodide (PI) staining
HUVECs were cultured in 6 well plates (BD Falcon, USA) at a density of 2.0 × 105 cells/well in DMEM supplemented with 10% FBS. One day after plating, the cells were washed and incubated in serum-free medium for 12 hours. The cells were then washed again and incubated with medium containing various concentrations of H2O2 (0.1, 0.5, 1.0 mM) for 12 hours. The cells were trypsinized, washed with PBS, and centrifuged at 1000 rpm/min for 5 min. The cells were then resuspended at a density of 1 × 106 cells/ml, and the suspensions were fixed with 70% precooled ethanol at 4°C for 1 h. Next, the cells were centrifuged at 1000 rpm/min for 5 min, resuspended in 1 ml diluted PI (Shanghai Biological Technology Co., Ltd., China) and incubated in the dark at 4°C for 30 min. Flow cytometry was performed using a FACSCalibur (Backmancoulter, USA). Data were analyzed using CellQuest software (Becton–Dickinson). The amount of necrosis was determined as the percentage of PI-positive cells.
Annexin-V/PI assay
Annexin-V/PI assays were performed using a commercial apoptosis assay kit (Roche, Switzerland) according to the manufacturer's instructions. Briefly, HUVECs were cultured in 6 well plates (BD Falcon, USA) at a density of 2.0 × 105 cells/well and incubated in DMEM supplemented with 10% FBS. One day later, the cells were washed and incubated in serum-free medium for 12 hours. The cells were then washed again and incubated in medium with various concentrations of H2O2 (0.1, 0.5, 1.0 mM) for 12 hours. After incubation, the cells were trypsinized and washed with PBS. After centrifugation at 1000 rpm/min for 5 min, the cells were resuspended in 500 μL binding buffer at a concentration of 1 × 106 cells/ml. The suspensions were transferred to 1.5-mL tubes, and 5 μL of Annexin V and 10 μL of PI solution were added. The cells were incubated in the dark at room temperature for 20 min, and flow cytometry was performed using a FACSCalibur (Beckmancoulter, USA). Data were analyzed using CellQuest software (Becton–Dickinson). The amount of apoptosis was determined as the percentage of annexin V-positive cells/PI-negative cells.
MTT assay
As a measure of overall levels of cell death, HUVECs were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HUVECs were plated onto 96-well plates and incubated in DMEM supplemented with 10% FBS. One day later, the cells were washed and incubated in serum-free medium for 12 hours. The cells were then were randomly divided into 6 groups: the normal control group (untreated cells), the model control group (H2O2 only), and the H2O2 plus allicin (98% purity, Shaanxi Ciyuan Biotech Co., Ltd, China) groups (1 μg/mL, 10 μg/mL, 20 μg/mL or 40 μg/mL allicin). These concentrations of allicin were selected to reflect a range of biological activities of the drug in HUVECs. Thirty minutes prior to the end of the incubation period, MTT was diluted 1:500 in 0.5% FBS DMEM culture medium and 200 μl was administered to each well. The plates were wrapped in aluminum foil to protect them from light and read using an enzyme-labeled instrument (Biotek ELX 800/FLX800).
Western blot assay
For the extraction of proteins, cells were placed in RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China) and centrifuged at 13000 rpm/min for 30 min at 4°C. Protein concentrations were assayed with a NanoDrop instrument, and 40 μg of protein from each sample were run on a 15% SDS-PAGE gels. The separated proteins were transferred onto PVDF membranes. After blocking with 5% nonfat dry milk in double-distilled water at room temperature for 1 h, membranes were washed 3 times with PBS containing 0.05% Tween (PBS-T) and incubated overnight at 4°C with primary mouse monoclonal antibody (anti-PARP, anti-pro-Caspase-3, anti-Bax or anti-β-actin) (Santa Cruz Biotechnology, USA) at a 1:500 dilution. The membranes were washed 3 times with PBS-T, followed by 1 h incubation at room temperature in a 1:5000 dilution of goat anti-mouse-IgG-HRP (Santa Cruz Biotechnology, USA). After incubation, membranes were washed 3 times in PBS-T. Antigen-antibody complexes were analyzed by ECL, and protein levels were quantified by densitometry. Data were normalized to the β-actin content of the same sample.
Measurement of oxidative activity
The concentrations of malondialdehyde (MDA), sodium oxide dismutase (SOD) and nitric oxide (NO) were assessed using dedicated kits (Nanjing Jiancheng Biological Engineering Institute, China) according to the manufacturer’s protocols.
Reverse transcription PCR (RT-PCR) assay
Total cellular RNA was extracted from HUVECs by the Trizol method (Bio Basic Inc., Canada). PCR amplification was performed in a 20 μL reaction volume. The primer sequences were as follows: eNOS forward, 5’-CCAGCTAGCCAAAGTCACCAT-3’, eNOS reverse, 5’-GTCTCGGAGCCATACAGGATT-3’; iNOS forward, 5’-AGCGGTAACAAAGGAGATAG-3’, iNOS reverse, 5’-CCCGAAACCACTCGTATT-3’; GAPDH forward, 5’-GTCATCCATGACAACTTTGG-3’, GAPDH reverse, 5’-GAGCTTGACAAAGTGGTCGT-3’. After an initial denaturation at 95°C for 5 min, the PCR conditions were as follows: 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30s. The PCR products were electrophoresed on a 1% agarose, and stained with ethidium bromide solution.
Real-time quantitative PCR assay
Levels of endothelial nitric oxide (eNOS) mRNA expression were determined by real-time quantitative PCR. Triplicate reactions were run in a volume of 20 μL, containing 2 μL cDNA, 10 μL 2 × SYBR Green mix, 6 μL ddH2O, 1 μL PCR forward primer (10 μM), and 1 μL PCR reverse primer (10 μM). After an initial denaturation at 95°C for 5 min, the PCR conditions were as follows: 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30s.
The ΔΔCt (threshold cycle) method was used to calculate eNOS mRNA expression levels for each sample, with GAPDH as the reference gene.
Statistical analysis
All data are expressed as mean ± SEM. Statistical analysis was performed using the Student’s t-test and ANOVA. Significance was accepted at p <0.05.