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Inhibition of Endothelial Adhesion Molecule Expression by Monascus purpureus-fermented Rice Metabolites, Monacolin
K, Ankaflavin, and Monascin
Journal: Journal of the Science of Food and Agriculture Manuscript ID: JSFA-10-1026.R1
Wiley - Manuscript type: Original Article Date Submitted by the
Author: n/a
Complete List of Authors: Lin, Chih-Pei; Taipei Veterans General Hospital, Department of Pathology and Laboratory Medicine
Lin, Yun-Lian; National Research Institute of Chinese Medicine, National Research Institute of Chinese Medicine
Huang, Po-Hsun; Taipei Veterans General Hospital, Division of Cardiology
Tsai, Hui-Szu; Taipei Veterans General Hospital, Department of Pathology and Laboratory Medicine
Chen, Yung-Hsiang; China Medical University, Graduate Institute of Integrated Medicine
Key Words: Cell adhesion molecule, Monascus purpureus rice (red yeast rice), Inflammation, Oxidative stress, Nuclear factor-ĸB
For Peer Review
Journal of the Science of Food and Agriculture
(JSFA-10-1026.R1)
Inhibition of Endothelial Adhesion Molecule Expression by Monascus
purpureus-fermented Rice Metabolites, Monacolin K, Ankaflavin, and Monascin
Chih-Pei Lin,1,2,* Yun-Lian Lin,3 Po-Hsun Huang,4,5 Hui-Szu Tsai,1 Yung-Hsiang Chen6,*
1Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan; 2Department of Biotechnology and Laboratory Science in Medicine and Institute of Biotechnology in
Medicine, National Yang-Ming University, Taipei, Taiwan; 3National Research Institute of Chinese Medicine, Taipei, Taiwan 4Division of Cardiology, Taipei Veterans General Hospital, Taipei, Taiwan;
5Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; 6Graduate Institute of
Integrated Medicine, China Medical University, Taichung, Taiwan;
*Address for reprint requests and other correspondence: C.-P. Lin and Y.-H. Chen. Taipei Veterans General Hospital, Taipei 11217, Taiwan; National Yang-Ming University, Taipei 11221, Taiwan. FAX: +886-4-22037690 (e-mail: [email protected] and [email protected])
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Abstract
BACKGROUND: Inflammation is an independent risk factor of cardiovascular
diseases and associated with endothelial dysfunction. Monascus purpureus-fermented
rice, containing naturally-occurring statins and various pigments, has lipid-modulating,
anti-inflammatory, and antioxidative effects.
RESULTS: The effects of monacolin K, ankaflavin, and monascin, as the metabolites
from Monascus-fermented rice on the expression of cell adhesion molecules
(intercellular adhesion molecule-1/ICAM-1, vascular cell adhesion
molecular-1/VCAM-1, and E-selectin) by tumor necrosis factor (TNF)-α-treated
human aortic endothelial cells (HAECs) were investigated. Supplement of HAECs
with these Monascus-fermented rice metabolites significantly suppressed cellular
binding between the human monocytic cells U937 and TNF-α-stimulated HAECs.
Immunoblot analysis showed that Monascus-fermented rice metabolites significantly
attenuated TNF-α-induced of VCAM-1 and E-selectin but not ICAM-1 protein
expression. Gel shift assays showed that Monascus-fermented rice metabolites
treatment reduced TNF-α-activated transcription factor nuclear factor (NF)-κB.
Furthermore, Monascus-fermented rice metabolites also attenuated reactive oxygen
species (ROS) generation in vitro and in TNF-α-treated HAECs. Supplement with an
ROS scavenger N-acetyl-cysteine gave similar results as compared with 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Monascus-fermented rice metabolites.
CONCLUSION: Monascus-fermented rice metabolites reduced TNF-α-stimulated
endothelial adhesiveness as well as down-regulating intracellular ROS formation,
NF-κB activation, and VCAM-1/E-selectin expression in HAECs, supporting the
notion that the various metabolites from Monascus-fermented rice might have
potential implications in clinical atherosclerosis disease.
Keywords: Cell adhesion molecule; Monascus purpureus rice (red yeast rice); Inflammation; Nuclear factor-κB; Oxidative stress
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Introduction
Red yeast rice, a fermented product of rice and red yeast (Monascus purpureus),
has been used by Chinese for many centuries to make rice wine, as a food
preservative for maintaining the taste and the color in meat and fish, and for its
medicinal properties.1-3 Cholestin is a dietary supplement related to red yeast rice
that has been reported to have lipid-lowing effects and considered beneficial in
subjects with hyperlipidemia.1 The pharmacological preparation from red yeast rice
that has been publicly used in China, United States, and many other countries is
composed, in part, of 734 g kg-1 starch, 58 g kg-1 protein, less than 20 g kg-1 fat, and a
number of compounds named monacolins (~4 g kg-1), which are inhibitors of
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.4 It has also been
reported that Monascus-fermented rice contains 20 – 60 g kg-1 fatty acids including
linoleic acid, oleic acid, palmitic acid, and stearic,5 where some of them have lipid
lowing properties.6 Monascus species have been proven to produce many functional
secondary metabolites. These pigments (yellow pigment: ankaflavin and monascin;
orange pigment: monascorubrin and rubropunctanin; red pigment: monascorubramine
and rubropuctamine) were investigated and applied to the food colorant in the
previous studies.7, 8 In current study, Monascus-fermented product was gradually
regarded as the functional food because the monacolins (lipid-lowering agents),9 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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γ-aminobutyric acid (GABA) (hypotensive agent),10 dimerumic acid,11-13 and
dihydromonacolin-MV (antioxidants)14 were found.15 These secondary metabolites
have been identified with anti-inflammatory or antioxidative activities.
The salutary effect of HMG-CoA reductase inhibitors (statins) on reducing
mortality rate in patients with coronary artery disease (CAD) has been evidenced.16, 17
The pharmacological benefit of statins is explained by their lipid-modulating effects;
but recent experimental and clinical evidence demonstrates that the
anti-atherosclerosis activity of statins also includes cholesterol-independent
mechanisms.18, 19 Red yeast rice contains a family of naturally occurring statins that
has a marked modulating effect on lipids1, 20 and the extract of red yeast rice has been
shown with free radical scavenging properties,11, 13, 21 Recently, a
Monascus-fermented rice extract was found to decrease C-reactive protein and protect
endothelial function through lipid-lowing, anti-inflammatory, or antioxidative
mechanisms.22-26
Elevated endothelial expression of adhesion molecules as mediators of
subintimal leukocyte accumulation in atherosclerosis27, 28 and increased oxidative
stress may play the cardinal role in the inflammatory mechanisms for the progression
of atherosclerosis.29 More recently, it was reported that Cholestin extract reduced
homocysteine-stimulated endothelial adhesiveness as well as down-regulating 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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intracellular ROS formation, supporting the notion that the natural compound
Cholestin might have potential implications in clinical atherosclerosis disease.30
Because the concentrations of statins used in the previous study were markedly higher
than that in Cholestin-treated group, it was speculated that Cholestin was not only an
impure form of statin drug and chemical components other than monacolins might be
responsible for the observation. The antioxidative components could possibly
contribute to the anti-athergenetic effects of Cholestin. Inflammatory cytokine tumor
necrosis factor (TNF)-α has been shown to promote the adhesion of leukocytes to
endothelial cells through oxidative stress-related mechanism.31, 32 Since
Monascus-fermented rice metabolites, like statins, may also exhibit a “pleiotropic”
effect on vascular protection, in the present study, the ability of Monascus-fermented
rice metabolites, monacolin K (MK) and two yellow pigments – ankaflavin and
monascin, was tested in modulating the expression of adhesion molecules and the
activation of redox-sensitive transcription factor nuclear factor-κB (NF-κB) by
TNF-α-treated human aortic endothelial cells (HAECs). 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Materials and methods
Cell culture
Human aortic endothelial cells (HAECs, Cascade Biologics) were grown in
Medium 200 (M200) (Cascade Biologics) supplemented with low serum growth
supplement (Cascade Biologics) in an atmosphere of 950 ml l-1 air and 50 ml l-1 CO2
at 37°C in plastic flasks. The final concentrations of the components in M200
contained 20 ml l-1 FBS, 1 µg/ml hydrocortisone, 10 ng/ml human epidermal growth
factor, 3 ng/ml human fibroblast growth factor, 10 µg/ml heparin, and 10 ml l-1
antibiotic-antimycotic mixture (GibcoBRL). At confluence, the cells were subcultured
at a 1:3 ratio and used at passage numbers 3 through 8. The human monocytic cell line
U937 (American Type Culture Collection) was grown in suspension culture in
RPMI-1640 (JRH Bioscience) containing 100 ml l-1 FBS, 25 ml l-1
[N-(2-hydroxyethyl) piperazine-N’-(2-ethenesulphonic acid)] (HEPES) buffer and 10
ml l-1 antibiotic-antimycotic mixture in an atmosphere of 950 ml l-1 air and 50 ml l-1
CO2 at 37°C. The cells were routinely subcultured at a 1:4 ratio. TNF-α,
N-acetyl-cysteine (NAC), and MK were purchased from Sigma Chemical Co. (MO,
USA). Ankaflavin and monascin were purchased from reseaLIFE (Switzerland).
MTT assay for cell viability 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,
Sigma, USA) assay was used to measure cell viability 33. The principle of this assay is
that mitochondrial dehydrogenase in viable cells reduces MTT to a blue formazan.
Briefly, cells were grown in 96-well plates and incubated with various concentrations
of TNF-α, Monascus-fermented rice metabolites, or NAC. After washing HAECs by
PBS 2 times, 100 µl medium containing MTT (0.5 mg/ml) was added to each well and
incubation continued at 37°C for an additional 4 h. The medium was then carefully
removed, so as not to disturb the formazan crystals formed. 100 µl DMSO, which
solubilizes the formazan crystals, was added to each well and the absorbance of the
solubilized blue formazan read at 540 nm using a microplate reader (Multiskan Ex,
ThermoLabsystems) where DMSO as the blank. The reduction in optical density
caused by drugs was used as a measurement of cell viability, normalized to cells
incubated in control medium, which were considered 100% viable.
Monocytic cell-endothelial cell adhesion assay
The adherence of monocytic cells U937 to TNF-α-activated HAECs was
examined under static conditions. HAECs were grown to sub-confluence in 6-well
plates; cells were incubated with Monascus-fermented rice metabolites or NAC for 18
h followed by 6-h stimulation with TNF-α. HAECs in 6-well plates were incubated 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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with U937 (106 cells/ml) for 30 min. Finally, 2 ml (20 ml l-1) gluteraldehyde was
added to each well to fix the whole cells. Non-adherent cells were removed, and
plates were gently rocking washed 5 min twice with HEPES-HBSS (1:50, HEPES 20
mM; HBSS with Ca2+ and Mg2+, with out EDTA). The numbers of adherent cells were
recognized and determined under inverted microscopy (OLYMPUS) with computer
software, ImagePro Plus 4.0 (USA). Under 40X objective lens, twenty randomly
chosen fields were counted per well. Experiments were performed in duplicate or
triplicate and were repeated at least 3 times.
Western blot analysis
Protein extracts were prepared as previously described.34 Briefly, HAECs were
lysed in 100 µl lysis buffer with protein: protease inhibitor (PIERCE), after washing
by PBS, then centrifuge in 4°C, 8,000×g for 30 min to harvest the supernatant. The
cell total protein was quantified by Bio-Rad protein assay reagent. The whole-cell
lysates were subjected to SDS-polyacrylamide (100 g kg-1) gel electrophoresis,
followed by electroblotting onto PVDF membrane (Amersham Biosciences).
Membranes were probed with a goat monoclonal antibody directed to ICAM-1,
VCAM-1, or E-selectin (R&D, USA) and incubated with horseradish
peroxidase-labeled secondary antibody, and then washed with PBS containing 1 ml l-1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Tween 20. Bands were visualized by chemiluminescence detection reagents
(PerkinElmer, USA). Anti-β-actin antibodies were used as loading control.
Densitometic analysis was conducted with software, ImageQuant (Promega), to
semiquantify Western blot data.
Nuclear extract preparation and electrophoretic mobility shift assay (EMSA)
Nuclear protein extracts were prepared as previously described.35 Briefly, after
washing with PBS, the cells were scraped off the plates in 0.6 ml of ice-cold buffer A
[HEPES 10 mM, pH 7.9, KCl 10 mM, dithiothreitol (DTT) 1 mM,
phenylmethylsulphonylfluoride (PMSF) 1 mM, MgCl2 1.5 mM, and 2 µg/ml each of
aprotinin, pepstatin, and leupeptin]. After centrifugation at 300×g for 10 min at 4°C,
the cells were resuspended in buffer B (80 µl of 1 ml l-1 Triton X-100 in buffer A), left
on ice for 10 min, then centrifuged at 12,000×g for 10 min at 4°C. The nuclear pellets
were resuspended in 70 µl of ice-cold buffer C (HEPES 20 mM, pH 7.9, MgCl2 1.5
mM, NaCl 0.42 M, DTT 1 mM, EDTA 0.2 mM, PMSF 1 mM, 250 ml l-1 glycerol, and
2 mg/ml each of aprotinin, pepstatin, and leupeptin), then incubated for 30 min at 4°C,
followed by centrifugation at 15,000×g for 30 min at 4°C. The resulting supernatant
was stored at -70°C as the nuclear extract. Protein concentrations were determined by
the Bio-Rad method. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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EMSA was carried out with the DIG Gel Shift Kit (Roche Diagnostics) following
the user’s manual. In the first step, single-stranded complementary oligonucleotides
containing the binding sequences for transcription factors were annealed and
end-labeled with digoxygenin. The NF-κB probe used in the gel shift assay was a
31-mer synthetic double-stranded oligonucleotide (5’-ACA AGG GAC TTT CCG
CTG GGG ACT TTC CAG G-3’; 3’-TGT TCC CTG AAA GGC GAC CCC TGA
AAG GTC C-5’) containing a direct repeat of the κB site. The labeled probes (48
fmol of double-stranded oligonucleotides) were then incubated for 30 min at 4°C with
10 µg of nuclear extract proteins in 40 mM HEPES buffer, pH 7.9 containing 100 mM
KCl, 12.5 mM MgCl2, 1 mM EDTA, 200 ml l-1 glycerol, 1 mM DTT, 2 µg of
poly(dI–dC), 0.2 µg of poly-(L)-lysine. Then the mixtures were subjected to
electrophoresis on a 60 g kg-1 polyacrylamide gel with 0.5× TBE running buffer. The
DIG-oligonucleotide/protein complexes were transferred to a Hybond-N blotting
membrane (Amersham Life Science, Germany) and the shift bands were visualized.
Densitometic analysis was conducted with software, ImageQuant (Promega), to
semiquantify EMSA data.
Detection of intracellular ROS production
The effect of Monascus-fermented rice and NAC on ROS production in HAECs 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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was determined by a fluorometric assay using 2’,7’-dichlorofluorescein diacetate
(DCFH-DA, Molecular Probe) as the probe.35 This method is based on the oxidation
by H2O2 of nonfluorescent DCFH-DA to fluorescent DCF. Briefly, 15 µM DCFH-DA
was added to the medium in the last 20 min of incubation (37°C, 18 h), while the
incubation ended up, HAECs were washed by HBSS (with out Ca2+, Mg2+) containing
100 g kg-1 BSA. Then 250 µl cell lysis buffer (PBS containing 200 ml l-1 EtOH, 1 ml
l-1 Tween 20) was added to each well. After centrifuging, the supernatant was
transferred to measure the fluorescence intensity (relative fluorescence units) at 485
nm excitation and 530 emission using a fluorescence microplate reader (Victor II).
Ultraweak chemiluminescence (uwCL) monitoring of oxygen-derived free radicals
For superoxide anion (⋅O2-)-generating system, the following reaction mixture in
a total volume of 2.1 ml consisting of 1.0 ml of 2.0 mM lucigenin; 1.0 ml of
phosphate-buffered saline, pH 7.4; 0.05 ml of 1.0 M arginine; 0.05 ml of 1.4 µM
methylglyoxal was used. After gently mixing the above-mentioned components, the
reaction mixture was added to a quartz round-bottom cuvette in the black-box unit of
the uwCL analyzer equipped with a high-sensitivity detector [3.3 × 10-15 W/(cm2·
count)] form Jye Horn Co. (Taipei, Taiwan).36
For hydroxyl radical (·OH) generating system. The reaction mixture used 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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consisting the following: 1.0 ml of 3 µM indoxyl-β-glucuronide (IBG), 0.1 ml FeSO4,
1.6 ml of 30 ml l-1 H2O2, and 0.05 ml of 10 mM EDTA. All the above-mentioned
reagents were added to the quartz round-bottom cuvette in the black-box unit of the
uwCL analyzer in a sequential order of EDTA, IBG, H2O2, and FeSO4.37
For hydrogen peroxide (H2O2)-generating system, the following reaction mixture
were used: 1.0 ml of 2 mM luminol, containg sodium borate, pH 7.3; 1.0 ml of PBS,
pH 7.4 and 1.0 ml of 12 ml l-1 H2O2. The total volume of the reaction mixture was
3.00 ml. All the above-mentioned reagents were then added to the quartz
round-bottom cuvette and uwCL was measured using BJL uwCL analyzer.38 To
standardize the system, we use Trolox as the standard; thus, the IC50 value of a test
compound can be converted.
Statistical analyses
Results were expressed as mean ± SEM, and data were analyzed using ANOVA
followed by Dunnett’s test or Student’s t-test for significant difference. Statistical
significance was defined as p<0.05. All statistical procedures were performed with
SigmaStat version 3.1 (Jandel, USA). 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Results
MTT assay for MK, ankaflavin, and monascin on HAECs
Cell viability was assessed using the MTT assay. Treatment of HAECs with low
dose MK, ankaflavin, and monascin for 24 h did not result in cytotoxicity, whereas
high concentration Monascus-fermented rice metabolites (≥ 60 µM ankaflavin and ≥
100 µM MK and monascin) significantly inhibited cell survival (Fig. 1). In addition,
cell viability did not significantly change under the conditions of 50 µM MK,
ankaflavin, and monascin as well as 5 mM NAC treatment for 18 h followed by 10
ng/ml TNF-α treatment for 6 h (data not shown). The results indicate that the notable
cytotoxic effects on HAECs are found in high-dose various Monascus-fermented rice
metabolites. The non-cytotoxic working concentrations of MK, ankaflavin, and
monascin (≤ 50 µM) in the following tests were used to avoid possible interferences
on cell viability.
Monascus-fermented rice metabolites inhibits U937 adhesiveness to
TNF-αααα-activated endothelial cells
TNF-α increases early events of the atherosclerotic process by modulating
monocyte adhesion and transmigration.27 Fig. 2 shows that incubation of HAECs with 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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TNF-α (10 ng/ml for 6 h) significantly increased U937 adhesiveness. The ability of
various Monascus-fermented rice metabolites was then tested to modulate U937
adhesiveness to TNF-α-activated endothelial cells. As shown in Fig. 2, un-stimulated
control HAECs showed minimal binding to U937 cells, but endothelial adhesiveness
to U937 was substantially increased (10.4 fold increase, p<0.05) when the HAECs
were treated with TNF-α. Supplement of HAECs with various Monascus-fermented
rice metabolites dose-dependently inhibited U937 adhesion to HAECs treated with
TNF-α; supplement of HAECs with 5 mM NAC (an ROS scavenger antioxidative
control) for 18 h similarly inhibited U937 adhesion to TNF-α-activated HAECs.
Inhibition of TNF-αααα-induced VCAM-1 and E-selectin expressions by Monascus-fermented rice metabolites
To determine the optimal conditions for TNF-α-induced adhesion molecule
expression by HAECs, dose-response studies were performed, in which HAECs were
cultured with various concentrations of TNF-α for various time intervals in a pilot
study; in accordance with the previous studies,27 when HAECs treated with TNF-α
(10 ng/ml for 6 h), the cell adhesion molecules, ICAM-1, VCAM-1, and E-selectin
expressions on HAECs were significantly increased. Next, the effect of various
Monascus-fermented rice metabolites on TNF-α-induced cell adhesion molecule 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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expressions on HAECs was investigated. HAECs were pre-treated with 50 µM MK,
ankaflavin, or monascin for 18 h and followed by 10 µg/ml TNF-α for 6 h. The results
showed that expression of VCAM-1 and E-selectin but not ICAM-1 protein, (Fig. 3; 5
mM of NAC was used as an ROS scavenger control) in TNF-α-stimulated HAECs
were significantly suppressed by various Monascus-fermented rice metabolites. This
result suggests that endothelial VCAM-1 and E-selectin rather than ICAM-1
expression, are more critical to monocyte adhesion in this in vitro model.
Inhibition of TNF-αααα-induced activation of NF-κκκκB by Monascus-fermented rice metabolites
Transcriptional regulation involving NF-κB activation has been implicated in the
TNF-α-induced endothelial dysfunction.27, 39 To examine whether or not
Monascus-fermented rice metabolites inhibited NF-κB activation, gel-shift assays
were performed with the consensus NF-κB binding sequence. This pilot study showed
that incubation of HAECs with 10 ng/ml TNF-α caused significant activation of
NF-κB at 30 min. The activation of NF-κB induced by TNF-α could be suppressed by
ROS scavenger NAC as detected with DNA binding activity. Supplement with 50 µM
MK, ankaflavin, and monascin showed that TNF-α-caused NF-κB shifted bands were
significantly reduced (Fig. 4). The results suggest that various Monascus-fermented 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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rice metabolites, by down-regulating the NF-κB activation, may inhibit
TNF-α-induced VCAM-1 or E-selectin expression in the HAECs, with the result of
suppressing monocyte adhesiveness to endothelial cells.
Inhibition of TNF-αααα-induced intracellular ROS generation by Monascus-fermented rice metabolites
Inflammatory cytokine TNF-α could activate NF-κB in endothelial cells via
oxidative stress.27 The effect of Monascus-fermented rice metabolites on intracellular
ROS generation in HAECs was studied. Fig. 5 shows the effects of 2 – 50 µM MK,
ankaflavin, and monascin on intracellular ROS production induced by TNF-α (10
ng/ml for 6 h) in HAECs. Treatment with NAC or various Monascus-fermented rice
metabolites dose-dependently inhibited TNF-α-induced ROS production in HAECs.
Ultraweak Chemiluminescence for radical-scavenging abilities of
Monascus-fermented rice metabolites
Probe-based uwCL technique was used to measure the production of a panel of
three oxygen-derived free radicals.30 As shown in the Table, Monascus-fermented rice
metabolites exhibited the major radical-scavenging abilities on .OH, whereas less
effect was found on O2-. (with no suppressible activity for MK and ankaflavin), and 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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there were no obviously suppressible activity for H2O2 scavenging (vitamin E analog,
Trolox, was used as an experimental standard for uwCL technique). 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Discussion
The present study showed that supplement of HAECs with Monascus-fermented
rice metabolites, including MK, ankaflavin, and monascin, significantly suppressed
cellular binding between the human monocytic cells U937 and TNF-α-stimulated
HAECs. Monascus-fermented rice metabolites significantly attenuated
TNF-α-induced VCAM-1 and E-selectin protein expressions. Monascus-fermented
rice metabolites treatment also reduced TNF-α-activated redox-sensitive transcription
factor NF-κB. Furthermore, Monascus-fermented rice also attenuated intracellular
ROS generation in TNF-α-treated HAECs. Probe-based uwCL technique showed that
Monascus-fermented rice metabolites exhibited the major radical-scavenging abilities
on .OH.
The results confirmed that expression of VCAM-1 and E-selectin proteins in
HAECs was significantly elevated by TNF-α stimulation; furthermore, this elevation
could be suppressed by various Monascus-fermented rice metabolites supplement,
suggesting that endothelial VCAM-1 and E-selectin, rather than ICAM-1 expression,
was more critical to monocyte adhesion in this in vitro model. These results also
demonstrate that Monascus-fermented rice metabolites may decrease TNF-α-induced
endothelial adhesiveness to monocytes, at least in part, via VCAM-1 and E-selectin
modulation on HAECs. It’s known that VCAM-1 and E-selectin expression is focally 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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elevated in endothelial cells in vascular regions prone to atherogenesis;28, 40 the data
might reflect a link between elevated TNF-α levels and increased leukocyte
infiltration in atherosclerosis development; and supplement of Monascus-fermented
rice may have therapeutic potential attenuating inflammation-related atherogenesis.
Transcriptional regulation involving NF-κB activation has been implicated in the
TNF-α-induced endothelial dysfunction.27 Supplement with Monascus-fermented rice
metabolites showed that TNF-α-caused NF-κB shifted bands were significantly
reduced, suggesting that Monascus-fermented rice metabolites, by down-regulating
the NF-κB activation, might inhibit TNF-α-induced VCAM-1 and E-selectin
expressions in the HAECs, with the result of suppressing monocyte adhesiveness to
endothelial cells. Since Monascus-fermented rice has shown antioxidative
properties,11, 13, 21 this study demonstrates a similar pattern of Monascus-fermented
rice-sensitive inactivation of VCAM-1 and E-selectin expressions and NF-κB activity
in HAECs.
Inflammatory cytokine TNF-α could activate NF-κB in endothelial cells via
oxidative stress.27 It has been shown that statins have intrinsic antioxidant activity
with both anti-hydroxyl and peroxyl radical activity.41 The Table shows the in vitro
(cell-free model) RSA of various Cholestin derivates using the uwCL method. By
contrast, Figure 5 shows the ex vivo (cell culture model) inhibitory effects of 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Cholestin metabolites on TNF-α-induced ROS production in HAECs by a fluorescent
probe DCFH-DA assay. These data suggest that all MK, ankaflavin, and monascin
have the ability to reduce intracellular ROS production. However,
Monascus-fermented rice pigments further exhibited the major radical-scavenging
abilities on .OH, but MK failed to inhibit ROS directly in vitro. Recently, a novel
antioxidant mechanism by which statins reduce ROS in endothelial cells has been
demonstrated, and statin-mediated S-nitrosylation of thioredoxin has enhanced the
enzymatic activity of thioredoxin, resulting in a significant reduction in intracellular
ROS.42 These results suggest that the inhibitory effect of Monascus-fermented rice on
adhesion molecule expressions and NF-κB activation may be due to its direct or
indirect properties on ROS scavenging. Further study for investigating the direct or
indirect radical scavenging ability of various Monascus-fermented rice metabolites is
carried on to distinguish the action mechanism between monacolins and different
pigments.
Monascus-fermented rice contains 4 g kg-1 HMG-CoA reductase inhibitors
belonging to the statin class.1 The effective dose in the previous study, 50 µg/ml
Cholestin, contains approximately 0.2 mg l-1 compounds of the statin class and has
similar effect on homocysteine-induce endothelial dysfunction as compared to 10 µM
simvastatin or pravastatin.30 The other antioxidative components, such as sterols, 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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isoflavones,1 pigments,43 and dimerumic acid,11, 13 could possibly contribute to the
anti-athergenetic effects of Monascus-fermented rice.
In the study, although these three components are from the same Monascus
purpureus-fermented rice, the levels of TNF-α-stimulated endothelial adhesiveness,
intracellular ROS formation, NF-κB activation, and VCAM/E-selectin expression are
different among MK, ankaflavin, and monascin. These data suggest that there are still
some different mechanisms involved in these metabolites. The MK significantly
inhibited the activation of NF-κB and ROS production, but only partially reduced the
expression of adhesion molecules. By contrast, ankaflavin and monascin significantly
inhibited the activation of NF-κB and ROS production, but seemed to be more
effective in reducing TNF-α-induced monocyte adhesion and adhesion molecule
expressions. These findings are also compatible with the present understanding that
the activation of NF-κB by cytokines, such as TNF-α, could be caused through both
redox-dependent and -independent pathways.44, 45 Other intracellular signaling
pathways, such as mitogen-activated protein kinases or activator protein-1, might be
involved and warrant further investigation.
In conclusion, the present study demonstrates that TNF-α markedly increases
VCAM-1 and E-selectin expressions as well as the adhesiveness of U937 monocytic
cells to endothelial cells. Moreover, supplement of Monascus-fermented rice 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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metabolites, MK, ankaflavin, and monascin, or NAC is useful for endothelial
dysfunction induced by TNF-α. These data suggest that Monascus-fermented rice
supplement may be a potential implication to attenuate TNF-α-stimulated activation
of the endothelium and may help reduce the risk of vascular disease associated with
inflammation.
Acknowledgments
This study was supported by grant VGH 97C1-157 from Taipei Veterans General
Hospital, Taipei, Taiwan, grant NSC 97-2320-B-039-022-MY3 from the National
Science Counsel, Taiwan. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review
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Figure legends
Fig. 1. HAEC viability after culture with various Cholestin metabolites, MK,
ankaflavin, and monascin, for 24 h as determined by MTT assay. Data are expressed
as percentage (mean ± SEM) of survival cells using the untreated group as control
(viability = 100%). The results are from 3 separate experiments, *p<0.05, compared
to control.
Fig. 2. Effects of Cholestin metabolites, MK, ankaflavin, and monascin, on
TNF-α-stimulated adhesiveness of HAECs to U937 monocytic cells. Incubation of
HAECs with 10 ng/ml TNF-α increased U937 adhesiveness. HAECs were
pre-incubated for 18 h with various Cholestin metabolites or NAC followed by
stimulated with TNF-α (10 ng/ml for 6 h) and adhesion assay was performed. The
results for 3 separate experiments, each performed in triplicate, are expressed as mean
percentage of untreated control ± SEM. *p<0.05, compared to untreated control group; #
p<0.05, compared to TNF-α-treated group. Representative photomicrographs show
the effects of Cholestin metabolite treatments on the TNF-α-induced adhesion of
U937 cells to HAECs. The scale bar length = 100 µm.
Fig. 3. Western blot analysis of ICAM-1, VCAM-1, and E-selectin expressions in 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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cultured HAECs. Cells were pre-incubated for 18 h with 50 µM MK, ankaflavin,
monascin, or 5 mM NAC followed by stimulated with TNF-α (10 ng/ml for 6 h);
Western blot analysis was performed as described in “Methods”. Densitometric
analysis was conducted with software to semiquantify Western blot data. Three
independent experiments gave similar results. The summarized data (mean ± SEM)
from 3 separate experiments is shown in the bar graph. *p<0.05, compared to
untreated control group; #p<0.05, compared to TNF-α-treated group.
Fig. 4. EMSA for NF-κB activation in cultured HAECs. HAECs pre-incubated with
for 18 h with 50 µM MK, ankaflavin, monascin, or 5 mM NAC followed by
stimulated with TNF-α (10 ng/ml for 30 min) and nuclear protein extracts were
prepared and gel shift assay was performed using DIG-labeled oligonucleotides
containing consensus NF-κB. Densitometric analysis was conducted with software to
semiquantify EMSA data. Three independent experiments gave similar results. The
summarized data (mean ± SEM) from 3 separate experiments is shown in the bar
graph. *p<0.05, compared to untreated control group; #p <0.05, compared to
TNF-α-treated group.
Fig. 5. Inhibitory effects of Cholestin metabolites on TNF-α-induced ROS production 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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in HAECs. Cells were pre-incubated for 18 h with MK, ankaflavin, monascin, or
NAC followed by stimulated with TNF-α (10 ng/ml for 30 min) and intracellular
ROS generation (DCF assay) was performed. HAECs were labeled with
H2O2-sensitive fluorescent probe DCFH-DA (15 µM) for 20 min. Fluorescence
intensity of cells was measured with fluorescence microplate. Data are shown as the
mean ± SEM of 3 independent analyses. *p<0.05, compared to untreated control
group; #p<0.05, compared to TNF-α-treated group. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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Table. The radical scavenging ability (RSA) of various Cholestin derivates and Trolox (a water soluble vitamin E analog for comparison) using probe-based ultraweak chemiluminescence (uwCL) method
RSA (IC50 value, µµµµM) Superoxide (O2-⋅) Hydroxyl radical (⋅OH) Hydrogen peroxide (H2O2)
Monacolin K Nil# Nil# Nil#
Ankaflavin Nil# 27.84 Nil#
Monascin 654.10 41.32 Nil#
Trolox 9.51 2.16 395.94
#
No suppressible activity. The highest concentration used was 700 µM. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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HAEC viability after culture with various Cholestin metabolites, MK, ankaflavin, and monascin, for 24 h as determined by MTT assay 409x406mm (72 x 72 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
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Effects of Cholestin metabolites, MK, ankaflavin, and monascin, on TNF-alpha-stimulated adhesiveness of HAECs to U937 monocytic cells
416x874mm (72 x 72 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
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Western blot analysis of ICAM-1, VCAM-1, and E-selectin expressions in cultured HAECs 323x507mm (72 x 72 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
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EMSA for NF-κB activation in cultured HAECs 325x466mm (72 x 72 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
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Inhibitory effects of Cholestin metabolites on TNF-α-induced ROS production in HAECs 404x398mm (72 x 72 DPI) 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56
