The assay was performed using DuoSet VCAM-1/CD106 enzyme linked-immunosorbent assay (ELISA) and ancillary reagent kit (R&D System, Minnesota, USA). HAEC were seeded onto 24-well plates at a density of 2x105 cells/well overnight. The cells were then treated with A. sessilis ethanolic extract at 25, 50, 100 and 200 µg/ml, 2 µM of simvastatin or 10 mM of NAC for 24 h. NAC was prepared by dissolving NAC powder in water to a stock concentration of 612.8 mM. NAC stock solution was kept at 4°C, while working solution at a concentration of 10 mM was freshly prepared prior to each experiment. Next, the cells were induced with 10 ng/ml TNF-α for 6 h. The cell culture supernatant was collected and centrifuged at 1500 rpm
36
for 10 min at 4 ºC. Then, the supernatant was collected into new tubes and stored at – 80 ºC if not assayed on the same day. Repetitive freeze-thaw cycle was avoided.
A 96-well ELISA plate was coated with 2 µg/ml mouse anti-human VCAM-1 capture antibody diluted in PBS without carrier protein. After overnight incubation, the wells were washed with wash buffer containing 0.05% Tween 20 in PBS for three times. The plate was then incubated with reagent diluent made of 1% bovine serum albumin (BSA) in PBS for at least 1 h and the wells were washed again thrice. Then, recombinant human VCAM-1 standards and samples diluted with reagent diluent at a dilution factor of 2 were added into each well and incubated for 2 h. The wells were washed thrice with wash buffer and 100 µl of 200 ng/ml of biotinylated sheep anti-human VCAM-1 detection antibody was added into each well. Then, the plate was incubated for another 2 h. The washing step was repeated and 100 µl of streptavidin conjugated with horseradish-peroxidase (Steptavidin-HRP D) was then added to each well and incubated for 20 min. After washing the wells, substrate solution which consisted of 1:1 mixture of H2O2
and tetramethylbenzidine was then added into each well for 20 min. The reaction was stopped by adding a stop solution containing 2 N H2SO4 into each well. The absorbance was immediately read at 450 nm, and corrected at 540 nm using a microplate reader (Infinite M200, TECAN, Switzerland).
Standard curves were generated using Microsoft Excel by plotting the absorbance of each standard on Y-axis against the concentration of 15.6 – 1000 pg/ml of recombinant human VCAM-1 on X-axis. The best fit curve was obtained with R2 > 0.95. The concentration of VCAM-1 in the samples was calculated by finding the x-value based on the absorbance reading of each sample in the standard curve.
37 3.9 Antioxidant Enzymes Production Assays 3.9.1 Measurement of SOD Activity
This assay was performed using the SOD Assay Kit (Cayman, USA) based on the instruction provided with the kit. HAECs were seeded on 12-well plates at a density of 2 x 105 cells/well.
For this assay, H2O2 was used to induce decreased SOD activity in HAEC. To optimize the H2O2 concentration and induction time, the cells were induced with H2O2 at concentrations of 50, 100, 200 and 400 µM for 30 min, 1 h and 2 h.
Cell lysates of the samples were collected using rubber cell scrapers. Media were removed from each well, and iced-cold 20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer containing 1 mM ethylene glycol tetraacetic acid (EGTA), 210 mM mannitol and 70 mM sucrose (pH7.2) was then added. Cell lysates were then collected using a rubber cell scraper and transferred into centrifuge tubes. The cell lysate was centrifuged at 1500 x g for 5 min at 4ºC. The supernatant was kept at -80ºC if not assayed on same day.
To perform the SOD assay, 200 µl of radical detector containing tetrazolium salt solution was first added into a 96-well plate. Then, 10 µl of samples and bovine erythrocyte SOD (Cu/Zn) standards were added into the plate. To initiate the reaction, 20 µl xanthine oxidase was added into each well, and the plate was incubated for 30 min at room temperature. The absorbance was read using a microplate reader (Infinite M200, TECAN, Switzerland) at 440 nm.
To study the effects of A. sessilis on SOD activity, the cells were treated with 25, 50, 100 and 200 µg/ml of A. sessilis ethanolic extract or 2 µM of simvastatin or 10 mM of NAC or 10 µM of dexamethasone for 24 h, followed by induction with 100 µM of H2O2 for 2 h. Collection of cell lysates and the measurement of SOD activity were performed as described above.
38 3.9.2 Measurement of CAT Activity
This assay was performed using the CAT Assay Kit (Cayman, USA) based on the instruction provided with the kit. HAEC were seeded on 6-well plates at a density of 3 x 105 cells/well.
H2O2 was used to induce reduced CAT activity in HAEC. To optimize the H2O2 concentration and induction time, the cells were induced with H2O2 at concentrations of 50, 100, 200 and 500 µM for 30 min, 1 h and 2 h, 4 h and 6 h.
The CAT activity was detected using cell lysate samples. Media were first removed from each well and iced-cold lysis buffer consisting of 50 mM potassium phosphate and 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 7.0 was added. The cell lysates were then harvested using a rubber cell scraper and transferred into centrifuge tubes. The cell lysate was centrifuged at 10,000 x g for 15 min at 4ºC. The supernatant was then kept at -80ºC if not assayed on the same day.
To perform the CAT assay, samples with buffer and ethanol were added into each well of a 96- well plate. To initiate the reaction, 20 µl of hydrogen peroxide was added to the well and incubated for 20 min. The reaction was terminated by adding 30 µl potassium hydroxide into the well. 30 µl of chromogen and 10 µl of potassium periodate were then added. The absorbance was read using a microplate reader (Infinite M200, TECAN, Switzerland) at 540 nm.
In the subsequent experiment, effects of A. sessilis on CAT activity were evaluated by first treating the cells with 25, 50, 100 and 200 µg/ml of A. sessilis ethanolic extract or 2 µM of simvastatin for 24 h, and this was followed by induction with 100 µM of H2O2 for 2 h.
Collection of cell lysates and the CAT assay were performed as described above.
39 3.10 Statistical Analysis
All experiments were performed in triplicates for at least three times. The results were expressed as mean of readings and standard error of means (S.E.M). Statistical analysis was performed using one-way Analysis of Variance (ANOVA) and Dunnett’s test with GraphPad Prism 7 (CA, USA).
40 CHAPTER 4
RESULTS