Medicinus - Dexa Medica

extend, could also inhibit the platelet activation and
aggregation. 6 We have shown previously the activity
of DLBS1033 as antithrombotic and thrombolysis
agent. 7 DLBS1033, a bioactive fraction containing
8 different proteins have four different modes
of actions in treating the cardiovascular disease.
However, the effects of DLBS1033 on the structure
of the vasculature have not been explored, particularly
those pertaining to its effects on inflammation,
smooth muscle cells as well as plaque structure. This
present research was aimed at studying the effects
of DLBS1033 at the molecular levels.
MATERIALS AND METHODS
Cell Culture
RAW 264.7 cells obtained from American Type Culture
Collection (ATCC) were grown in a 75 ml flask
containing DMEM medium, 10% Fetal Calf Serum
(FCS), and 1% Penicillin/Streptomycin at 37 o C, 5%
CO 2 . Human aortic smooth muscle cells (HVSMC;
Clonetics, San Diego, CA) were cultured in SMC basal
medium according to the supplier's instructions.
Preparation of DLBS1033
DLBS1033 powder was diluted in phosphate buffer
pH 8 and the suspension was centrifuged at 5000 x g
for 10 minutes. The supernatant was then collected
and filtered before use as described previously by
Jessica et al. 7
Treatment of DLBS1033
Before treatment, the cells were poured into six-well
plate containing DMEM medium, 10% FCS, and 1%
Penicillin/Streptomycin and incubated at 37oC for
18 hours. On the next day, the medium was replaced
with DMEM medium containing only 1% Penicillin/
Streptomycin and 0.5 μg/ml DLBS1033. The cells
were then incubated again until the next day for
RNA isolation.
Cell Viability Assay
The number of viable cells was determined using
Cell Titer 96 Aqueous Assay (Promega, Madison, WI).
This assay was performed according to the manufacturer’s
protocol. The absorbance at 490 nm was
measured with a microplate reader type 680 (Biorad,
Hercules, CA). Each experiment was performed in
quadruplicate. The statistical analysis methods were
carried out in ProStat.
RNA Isolation and RT-PCR
RNA isolation was done using TRIzol reagent (Invitrogen)
which was then processed in RT-PCR. RT-PCR
was used to determine mRNA expression of genes
that involve in inflammation and platelet stabilization.
0.1 μg total RNAs were added to 25 μl reaction
mixture containing 5 μl AMV Reverse Transcriptase
5x reaction buffer (250 mM Tris-HCl, 250 mM KCl,
50 mM MgCl 2 , 2.5 mM spremidine and 50 mM DTT),
30 units AMV Reverse Transcriptase, 40 units Rnasin
Ribonuclease Inhibitor, 0.5 mM dNTPs, and 0.5 μg
oligo(dT) 15 primer. The cDNA was then processed
for PCR screening.
PCR for Screening and Analysis
PCR for screening the target gene was done using
GoTaq MasterMix (Promega). PCR was performed
with 2.5 μl cDNA in 25 μl reaction mixture containing
12.5 μl MasterMix, 1 μl 10 μM sense and antisense
primer each. PCR conditions were initial denaturation
95 o C for 2 min followed by 30 cycles of 95 o C 1
min, annealing for 1 min, 72 o C for 1 min and extension
72 o C for 10 min. PCR products were resolved in
2% agarose gel containing ethidium bromide. Images
were taken by a Gel Doc apparatus (BioRad) and
analyze with 2D software (BioRad).
Binding Assay with Monocytic Cells
RAW 264.7 cells were preincubated in DMEM containing
2% FBS and 5 µg/ml fluorescent BCECF-AM
at 37°C for 30 min in foil-covered tubes. Fluorescently
labeled cells were washed twice to remove
unincorporated dye and were then resuspended
in DMEM containing 0.2% BSA. Loaded monocytic
RAW cells (5x104) were added to each well of VSMC
and incubated at 37°C. After 30 min, unbound
monocytes were withdrawn and VSMC layers with
attached monocytes were gently washed twice with
DMEM and lysed with 0.2 ml of 0.1% Triton X-100 in
0.1 M Tris per well. Fluorescence (excitation 485 nm,
emission 535 nm) was measured using a microplate
reader (BioTek, Winooski, VT, United States).
Cell Migration Assay
original article
research
Chemotaxis cell migration assay was performed using
modified Boyden chambers with polyvinylpyrrolidone-free
polycarbonate filter membranes, 8-μm
pore size. 25,000–30,000 cells in serum-free SMC
basal medium were added to the upper well of the
Boyden chamber. Angiotensin II was added to the
bottom of Boyden chamber as a chemoattractant,
MEDICINUS 24(1), January 2011 19