role of quercetin in modulating rat cardiomyocyte gene expression profile

1

Role of quercetin in modulating rat cardiomyocyte gene

expression profile

C. Angeloni 1, E.Leoncini 1, M. Malaguti 1, S. Angelini 2, P. Hrelia 2, S. Hrelia 1

1 Department of Biochemistry “G.Moruzzi” University of Bologna, Via Irnerio 48, 40126

Bologna (Italy)

2 Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna,

(Italy)

Running head: Quercetin modulates rat cardiomyocyte gene expression

Corresponding Author:

Prof. Silvana Hrelia

Dipartimento di Biochimica “G.Moruzzi”

Alma Mater-Università di Bologna

Via Irnerio, 48

40126 BOLOGNA (Italy)

Tel: +39 051 2091233

Fax: +39 051 2091235

e-mail: [email protected]

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Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (January 4, 2008). doi:10.1152/ajpheart.01091.2007

Copyright © 2008 by the American Physiological Society.

mailto:[email protected]

2

ABSTRACT

Despite extensive studies, the fundamental mechanisms responsible for the development

and progression of cardiovascular diseases have not yet been fully elucidated. Recent

experimental and clinical studies have suggested that reactive oxygen species (ROS) play a

major pathological role. Oxidative stress reduction induced by flavonoids has been

regarded by many as the most likely mechanism in the protective effects of these

compounds; however, there is an emerging view that flavonoids may also exert modulatory

actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid

present in the human diet, has been widely studied and its biological properties are

consistent with its protective role in the cardiovascular system. However, it remains

unknown whether the cardioprotective effects of quercetin may also occur through the

modulation of genes involved in cell survival. The main goal of this study was to examine

the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin

using DNA-microarrays and to relate these data to functional effects. Results showed

distinct temporal changes in gene expression induced by quercetin and a strong up-

regulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect

antioxidant mechanism.

Keywords: flavonoids; phase 2 enzymes; oxidative stress.

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INTRODUCTION

Heart diseases are the leading cause of morbidity and mortality in industrialized countries

(55). Despite extensive studies, the fundamental mechanisms responsible for their

development and progression are still under debate. Recent experimental and clinical

studies have suggested that reactive oxygen species (ROS) play a major pathological role

(4, 13). The cellular sources of ROS generation within the heart include cardiac

myocytes, endothelial cells, and neutrophils. Potential rources of ROS in cardiac

myocytes include mitochondrial electron transport, NADPH oxidase, and xantine

dehydrogenase/xantine oxidase (50). One of the strongest lines of evidence for the

involvement of ROS in cardiovascular diseases is the ability of a number of structurally

unrelated compounds with antioxidant properties to protect against cardiovascular

pathophysiology, including myocardial ischemia-reperfusion injury, cardiomyopathy, and

arterial atherogenesis (14, 23, 26, 29).

Recently, much of the attention has been focused on flavonoids, naturally occurring

polyphenolic compounds, as food factors that may be beneficial for cardiovascular

disease prevention. Various epidemiological studies have shown an inverse correlation

between the consumption of flavonoid-rich foods and cardiovascular disease risk (6, 28,

37). Oxidative stress reduction by flavonoids has been regarded as the most likely

mechanism in the protective effects of these compounds; however, there is an emerging

view that flavonoids may also exert modulatory effects acting through protein kinase and

lipid kinase signaling pathways (5, 59). Quercetin (Q), a major flavonoid present in the

human diet, has been widely studied and its biological properties are consistent with its

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protective role in the cardiovascular system (30). In a previous paper, we have

demonstrated that Q is able to protect cardiac cells from oxidative stress acting both as an

antioxidant and a modulator of the signal transduction pathway related to apoptosis (5).

However, it remains unknown whether the cardioprotective effects of Q may also occur

through the modulation of genes involved in cell survival. Nutrigenomics is the scientific

study by which some of these questions can be answered. The concept of nutrigenomics

has rosen thanks to the development of new techniques, like global gene expression by

microarray technology, successfully applied to study the transcriptional changes

occurring in heart, vessels, and blood cells in different cardiovascular disorders. DNA

microarray technology could promote an increased understanding of how Q influences

gene expressions and regulates those genes responsible for cardioprotection. To our

knowledge, the cardiomyocyte expression profile of genes associated with response to Q

has not yet been reported. The present study was therefore designed to examine the gene

expression profiling of cultured rat primary cardiomyocytes treated with Q using DNA-

microarrays and to relate these data to functional effects. The results showed distinct

temporal changes in gene expression induced by Q and a strong up-regulation of phase 2

enzymes. In addition, to demonstrate the physiological relevance of the gene expression

analysis, cell viability and ROS production were measured in cardiomyocytes treated

with Q for selected time periods in the presence of oxidative stress.

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MATERIALS AND METHODS

Chemicals

CelLytic™ M, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT),

2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), ferrozine, Q, H2O2, digitonin,

glucose-6-phosphate, glucose-6-phosphate dehydrogenase, bovine serum albumin,

NADP, FAD, DMSO, FeCl3, menadione, monochlorobimane (MCB), 1-chloro-2,4-

dinitrobenzene (CDNB), 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB), glutathione (GSH),

and all other chemicals of the highest analytical grade were purchased from Sigma

Chemical Co. (St. Louis, MO, USA), unless otherwise stated. Q was dissolved in DMSO

at a concentration of 30 mM and kept at −20 °C until use.

Cell culture and treatments

Ventricular cardiomyocytes were derived from the heart of 2–4 days old Wistar rats, as

previously reported (7). Cells were seeded at a density of 1.2x106cells/mL and were

grown in DMEM F12 culture medium treated with 10 % fetal calf serum, 10 % horse

serum (complete medium), and 1% sodium pyruvate. Cells were routinely grown in a

humidified incubator (95 % humidity) with 5 % CO2 at 37 °C until complete confluence.

In some dishes, 30 µM Q was added to the culture medium for 6, 12 and 24 h. The low

micromolar concentration of Q employed in this study is similar to that used by other

investigators (30, 32, 53).

RNA extraction

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Total RNA was extracted using Total RNA Isolation Mini Kit (Agilent Technologies,

Palo Alto, CA), following the manufacturer’s protocol. The yield and purity of the RNA

were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies,

Inc., Rockland, DE, USA). The 28s and 18s ribosomal bands were checked using Agilent

2100 bioanalyzer with an RNA 6000 Nano LabChip Kit.

mRNA amplification and labeling

For each sample, mRNA was amplified starting from 1.5 µg of totRNA by Amino Allyl

MessageAmp I aRNA Kit (Ambion, Woodward Austin, TX, USA) to obtain amino allyl

antisense RNA following the method developed by Eberwine et al. (54). Only one round

of amplification was performed, according to the manufacturer’s protocol, with minor

modification. Briefly, mRNA was reverse transcribed into cDNA single strand; after the

second strand synthesis, cDNA was in vitro transcribed in aaRNA including an amino

allyl modified nucleotide (aaUTP). Both dsDNA and aaRNA underwent a purification

step using columns provided with the kit. Labeling was performed using NHS ester Cy3

or Cy5 dies (Amersham Biosciences, Piscataway, NJ, USA) able to react with the

modified RNA. At least 5 µg of mRNA, for each sample, were labeled and purified with

columns. mRNA quality, concentration and labeling were checked by RNA 6000 Nano

LabChip assays (Agilent Technologies) and Agilent 2100 Bioanalyzer. Concentrations

were also checked by NanoDrop ND-1000 Spectrophotometer.

Microarray hybridization and scanning

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One hybridization with dye-swap duplication was performed to compare samples versus

reference. Reverse labeling was performed to reduce dye-specific biases in signal

intensity. The same quantity of differentially labeled sample and reference (0.75 µg) was

put together, fragmented and hybridized to high-density rat arrays containing 22,575 (60

mer) oligo-nucleotide probes representing over 20,000 well characterized rat genes,

ESTs, and EST cluster. All steps were performed using the In Situ Hybridization kit-plus

(Agilent Technologies) and following the 60-mer oligo microarray processing protocol

(Agilent Technologies). Then, slides were washed with the SSC wash procedure (Agilent

Technologies) and scanned with the dual-laser microarray scanner Agilent B (Agilent

Technologies) at 5 µm resolution. Feature extraction and data normalization were

performed with the Agilent Feature Extraction software.

Data analysis

Raw result files were then loaded into the Resolver SE System (Rosetta Biosoftware,

Seattle, WA, USA) for data processing and normalization using the Agilent platform-

specific error model. Replicated expression profiles were combined to form ratio

experiments where each gene is associated to an expression fold-change and a p-value

that assesses the statistical significance of its modulation in the treated sample compared

to the control reference. Sequences with absolute fold-change ≥ 2 and p-value ≤ 0.001

were considered as differentially expressed.

For all selected genes, information were obtained from the NCBI websites UniGene,

OMIM and PubMed (www.ncbi.nlm.nih.gov). All the details about the microarrays and

data on gene expressions have been deposited in NCBIs Gene Expression Omnibus, and

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http://www.ncbi.nlm.nih.gov/

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are accessible through GEO Series accession number GSE7222

(www.ncbi.nlm.nih.gov/geo/).

Reverse Transcription-Polymerase Chain reaction (RT-PCR) analysis of mRNA

expression

NAD(P)H:quinone oxidoreductase (NQO1), Heme Oxygenase 1 (HO-1), Thioredoxin

reductase 1 (TR), and glutathione-S-transferase (GST) genes were validated using the

End-Point RT-PCR Assay, designed for conventional gel-based detection (Superarray,

Frederick, MD, USA). Total RNA was extracted as previously reported. cDNA was

synthesized from 1 µg of total RNA using the ReactionReady™ First Strand cDNA

Synthesis Kit, according to manufacturer’s direction (Superarray). cDNA was reverse

transcribed at 37°C for 60 min, finally the reaction was stopped by heating at 95° for 5

min. To ensure optimal results, two different dilutions (1:5 e 1:10) of cDNA were

prepared from each samples. PCR reaction was carried out in 25 µl volume containing

ReactionReady™ HotStart Sweet PCR master mix (10 mM Tris-HCl: 50 mM KCl, 1.5

mM MgCl2, 0.2 mM dNTPs and Taq DNA polymerase), 0.4 µM of each primer

(Superarray) and 1 µl diluted cDNA. The cDNA amplification was started by

denaturating the samples for 10 min at 95°C, followed by 35 cycles of 15 s at 95°C, 30 s

at 55°C and 30 s at 72°C. Finally, samples were held at 72°C for 7 min to ensure the

complete extension of the PCR products. Fragments size were predicted on the basis of

the mRNA sequence as reported in Table 1.

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http://www.ncbi.nlm.nih.gov/geo/

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6 µL of the amplified products were separated by electrophoresis on 10% polyacrylamide

gel in TBE buffer (pre-cast gel, Bio-Rad). The bands were stained with ethidium bromide

and visualized under UV light using a Versa-Doc 4000 Imaging system (Bio-Rad).

NAD(P)H:quinone oxidoreductase enzymatic activity assay

NQO1 enzymatic activity was measured as described previously (40). Briefly,

cardiomyocytes were plated in 96-well plates (2.5x103 cells/well) and grown under

normal condition until confluence. The plates were treated with Q for 6, 12 and 24 h, then

lysated with a solution containing 0.8% digitonin. 200 µL of reaction mix (0.025 mM

Tris-HCl, 0.67 mg/mL bovine serum albumin, 0.01% Tween-20, 5µM FAD, 1 mM

glucose 6-phosphate, 30 µM NADP, 2 U/mL yeast glucose-6-phosphate dehydrogenase,

0.3 mg/mL MTT, 50 µM menadione) were added to each well and the reaction was

arrested after 5 min by the addition of a solution containing 0.3 mM dicoumarol, 0.5%

DMSO and 5 mM potassium phosphate buffer. The plates were then scanned at 610 nm

with a microplate spectrophotometer VICTOR3 V™ Multilabel Counter (Perkin Elmer).

NOQ1 activity was expressed as nmol x min-1 x mg protein-1.

Total glutathione S-Transferase enzymatic activity assay

Total GST activity was assayed using CDNB according to the procedure of Habig et al.

(15). This assay measures the total GST activity because all the different GST isoforms

catalyze the conjugation of GSH with CDNB (16). After Q treatment, cells were lysated

with CelLytic M, and centrifuged (10,000 x g for 10 min). 10 µL of supernatant were

added to 990 µL of reaction mix (100 mM phosphate buffer pH 6.5 with 1 mM EDTA, 2

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mM GSH, 2 mM CDNB) and absorbance was read at 340 nm at 30 sec intervals over 5

minutes. GST activity was expressed as nmol x min-1 x mg protein-1.

Thioredoxin Reductase enzymatic activity assay

TR activity was assayed by an in vitro reduction of DTNB to 5’-thionitrobenzoic acid

(TNB) using a procedure adapted from Holmgren and Bjornstedt (19). Briefly, after Q

treatment, cells were lysated with CelLytic M, centrifuged at 10,000 x g for 10 min and

10 µL of supernatant was added to 990 µl of reaction mix (0.25 mM DTNB, 0.24 mM

NADPH, 10 mM EDTA, 100 mM phosphate buffer pH 7.5). The conversion of DTNB to

TNB was measured spectrophotometrically at 412 nm at 10 sec intervals over 1 min.

GST activity was expressed as mU/mg protein. 1 unit of thioredoxin reductase will cause

an increase in A412 of 1.0 per minute per mL (when measured in a non-coupled assay

containing DTNB alone) at pH 7.0 at 25 °C.

Heme Oxygenase activity assay

HO-1 activity was determined as reported in (3) with slightly modification. Briefly, cells

were lysated with CelLytic M, centrifuged (10,000 x g for 10 min) and 30 µL of

supernatant were added to 70 µL of iron-detection reagent (6.5 mM ferrozine, 6.5 mM

neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid). The incubation was

carried out for 30 min at 37 °C and the absorbance was measured at 540 nm on a

microplate reader. The iron content of the sample was calculated comparing its

absorbance to that of a standard curve obtained with FeCl3, and normalized against the

protein concentration.

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Protein concentration

The protein concentration of the cell lysates was determined by the Bio-Rad Bradford

protein assay (Bio-Rad Laboratories, Hercules, CA, USA).

Reduced glutathione levels

Reduced glutathione levels were determined with a fluorometric assay. GSH is

specifically conjugated with MCB to form a fluorescent bimane–GSH adduct, in a

reaction catalyzed by glutathione S-transferase (45). The concentration of the bimane–

GSH adducts increases during the initial 10- to 12-min period of this reaction with first-

order kinetic, before leveling off (66). After Q treatment, culture medium was removed

and cells were washed twice with 0.2 mL PBS and incubated for 30 min at 37°C in 0.1

mL fresh PBS containing 50 µM MCB. After incubation, fluorescence was measured at

355 nm (excitation) and 460 nm (emission) with a microplate spectrofluorometer.

Reduced glutathione levels were expressed as percentage of control cells (control

cells=100%).

Detection of intracellular reactive oxygen species

The formation of intracellular ROS was evaluated using a fluorescent probe, DCFH-DA,

as described by Wang et al. (57). Briefly, cardiomyocytes were treated for 6, 12, and 24 h

with 30 µM Q. The cells were washed with PBS and then incubated with 5 µM DCFH-

DA in PBS for 30 min. After DCFH-DA removal and further washing, the cells were

incubated with 100 µM H2O2 for 30 min. At the end of incubation, cell fluorescence from

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each well was measured using a microplate spectrofluorometer VICTOR3 V™

Multilabel Counter (Perkin Elmer, Wellesley, MA, USA) (λexcitation=485 nm and

λemission=535 nm). Intracellular antioxidant activity was expressed as the percentage of

inhibition of intracellular ROS produced by H2O2 exposure.

Cell viability measurement

Cardiomyocyte viability in the presence of Q was measured using the MTT assay. After

the treatment for 6, 12 and 24 h with 30 µM Q, cells were washed twice with PBS and

exposed to 100 µM H2O2 for 30 min. Controls received equivalent volumes of DMSO

(vehicle). Cellular damage elicited by H2O2 treatment was evaluated by measuring MTT

reduction. MTT was added to the medium (final concentration 0.5 mg/mL) and incubated

for 1 h at 37°C. After incubation, MTT solutions were removed, DMSO was added and

the absorbance was measured at 595 nm using a microplate spectrophotometer VICTOR3

V™ Multilabel Counter (Perkin Elmer). Data were expressed as percent of viable cells

with respect to controls.

Statistics

Each experiment was performed at least four times, and all values are represented as

means ± SD. Student’s t-test was used to analyze a statistical significance of the results

(Prism 4, GraphPad Software Inc., San Diego, CA, U.S.A.). Values of p<0.05 were

considered as statistically significant.

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RESULTS

Modulation of gene expression by Q

Q treatment did not alter the expression of most of the ~22.000 studied genes. A total of

91 genes (Fig. 1) were up- or down regulated with most of the genes significantly

changed after 6 h treatment. The number of the up-regulated genes was greater than that

of the down regulated. A total of 8 genes were up-regulated at any time exposure, but

only 2 genes were up-regulated following both 6 and 12 h treatment and 1 gene following

12 and 24 h treatment (Fig. 1 A). The overlapping region of the Venn diagram of Fig. 1 B

shows that there were no down-regulated genes shared by 6, 12 and 24 h Q treatment.

Eleven genes were modulated in both the 12 and 24 h treatment. Fig. 2 shows the

hierarchical clustering display of data for treated cardiomyocytes based on the 91 filtered

genes. Each gene is represented by a single row of colored bars. The genes were grouped

into 4 main clusters. Cluster number 1 shows a gene up-regulation only after 6 h Q

treatment, while the expression level profile was not significantly different from control

cells at 12 and 24 h Q treatment. Cluster 2 evidences an induced gene expression at each

treatment time. Cluster 3 shows a gene down-regulation at 12 and 24 h while cluster 4

shows a down regulation only at the shortest treatment time.

Table 2 shows the list of the significant up- or down-regulated genes in cardiomyocytes

treated with 30 µM Q for 6, 12 and 24 h, related to their function. We focused our

attention on genes codifying for antioxidant/detoxification enzymes. Interestingly most of

these genes belong to cluster 2, underlying a common functional regulation. In particular

we have chosen 6 genes critically involved in antioxidant/detoxification mechanisms

related to cell survival: NQO1, HO-1, TR, GSTa3, Mgst1, GSTp2.

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To verify the accuracy and validity of the array data obtained for the 6 selected genes, we

performed RT-PCR in independent samples. Gene expression levels, reported in Fig. 3,

are in agreement with the changes of gene expression observed in the microarray data.

Effects of Q on NQO1, HO-1, TR, total GST activities and cellular GSH content

NQO1, HO1, TR, total GST activities in cardiomyocytes treated with Q are shown in Fig.

4. The activities of all the 4 enzymes significantly increased at any exposure time

compared to control cells. The increase in activities, although different in magnitude, is in

agreement with the up-regulation of the corresponding genes at any treatment time.

Fig. 5 represents GSH levels in cultured cardiomyocytes treated with Q. GSH levels

significantly increased after 12 and 24 h Q treatments, with the highest increase at the

longest time exposure. After 6 h treatment no differences in GSH levels were observed in

respect to control cells. This increase is in agreement with the increased expression of the

gene codifying for the glutamate-cysteine ligase catalytic subunit as obtained by

microarray data analysis.

Q reduces ROS accumulation induced by H2O2

To verify whether antioxidant genes up regulated by Q can influence cell ability to

counteract oxidative stress, we verified the effect of Q treatment on ROS production

induced by H2O2. A significantly increased formation of ROS, as detected by DCFH-DA

assay, was observed in cells exposed to H2O2 (Fig. 6). ROS levels were markedly

reduced in Q treated cells at any exposure time, with the highest inhibition after 24h

treatment.

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Q protects against H2O2-induced damage

To verify whether the up-regulation of antioxidant genes after Q treatment could lead to

cell protection against oxidative damage, we determined cell viability after exposure to

H2O2. Incubation of cardiomyocytes with H2O2 led to a significant decrease in cell

viability, as detected by MTT reduction assay (Fig. 7). Q is responsible for a marked

protection at all the treatment time, as cell viability levels of Q treated cardiomyocytes

were comparable to controls.

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DISCUSSION

Many evidences underline a critical role for oxidative stress in the development of

various forms of cardiovascular disorders, including myocardial ischemia-reperfusion

injury, congestive heart failure, coronary arterial atherosclerosis, and chemical induced

cardiotoxicity (21, 29, 47, 52, 58). In this context, administration of some exogenous

antioxidants, such as flavonoids, has been shown to exert protective effects against

oxidative stress in different “in vitro” and “in vivo” models (1, 5, 11, 23, 26, 29, 59). The

ability of flavonoids to protect cardiac cells has been classically ascribed to their

antioxidant activity as ROS scavengers. Recently it has been demonstrated that

flavonoids are also able to modulate important signaling pathways activated by ROS in

cardiac myocytes, namely the mitogen-activated protein kinase cascades and the

phosphoinositide 3-kinase/protein kinase B (Akt) pathway (5, 55, 59). One of the major

representatives of flavonoids is Q, found in many edible plants. In Western populations

the intake has been estimated as 18 mg/day (41). The potential protective effects of Q

have been documented in various studies (5, 36). However, the exact mechanisms

underlying Q protective effects in cardiac cells remain to be elucidated. To better clarify

these aspects, we have evaluated the ability of Q to modulate the cardiomyocyte global

gene expression. Microarray technology was used to determine the expression levels of

22,000 genes after Q treatment. Since dietary components can also modify the translation

of RNA to proteins and the post-translational events, which can affect protein activity, we

have also evaluated the activity of the enzymes coded by the selected genes. Moreover,

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we have evaluated the existence of a time dependent relationship between induced

enzyme activities and ability of Q to protect cells from H2O2 elicited damage.

The results of this study demonstrated for the first time the predominant induction of

antioxidant/detoxifying genes in cardiomyocytes after treatment. In particular, a group of

cellular antioxidant/phase 2 enzymes was significantly up-regulated, including GSTa3,

GSTp2, Mgst1, NQO1, TR, HO-1, glutamate-cysteine ligase. Interestingly, the

hierarchical cluster analysis obtained with the microarray data revealed that all these

genes were classified in the same cluster, evidencing a similar time dependent regulation

pattern of expression and highlighting a possible common functional regulation. NQO1,

HO, TR, and GST activities and GSH content are critically involved in the regulation of

cellular reduction/oxidation status, and have been suggested to play important roles in

protecting against oxidative cardiovascular pathophysiology (16, 23, 46, 60).

Surprisingly, Q treatment significantly down-regulated metallothionein 1a (Mt1a) and

metallothionein 2 (MT-2) gene expressions after 12 and 24 h. Other Authors

demonstrated that Mt1a and MT-2 mRNA levels were enhanced by Q treatment in

different cell systems. These discrepancies are due to the use of DMSO as vehicle for Q.

DMSO is a potent inducer of Mt1a and MT-2 mRNA transcription, as reported by

Conklin et al. (10), who also demonstrated that MT levels in DMSO plus Q treated cells

were indistinguishable from levels in their respective controls. As we performed

competitive microarrays in which control and treated cells are hybridized on the same

microarray, the observed down-regulation of Mt1a and MT-2 was only apparent. From

the data reported in this paper it is not possible to speculate about the ability of Q to

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modulate MT gene expression in the heart. Further studies, specifically targeted to

flavonoid modulation of MT expression levels are necessary to clarify this aspect.

Although it has been previously demonstrated that Q is capable of inducing several genes

encoding for endogenous antioxidants and phase 2 enzymes in transformed cells (31, 32),

the inducibility of these important antioxidant-related genes has not yet been reported in

normal cells.

Regulation of the cellular reduction/oxidation (redox) balance is critically determined by

several antioxidant systems and its impairment alters multiple cell pathways. The

ubiquitously expressed thiol-reducing systems include the thioredoxin (TRX),

glutaredoxin, and glutathione systems (35, 39). TRX is a small and ubiquitously

expressed thiol-disulfide oxidoreductase, and functions as an important redox regulator in

cells from Escherichia coli to mammals (18, 34). The reduction of the catalytic center in

TRX is catalyzed by TR, using NADPH as cofactor. TRX functions as scavenger for

ROS at the cellular level (2). In addition, TRX interacts with various proteins in a redox

dependent manner, and modulates intracellular signaling pathways and transcription

factor activity (44, 62). Turoczi et al. (51) showed that TRX-over-expressing mouse

hearts had improved post-ischemic ventricular recovery and reduced myocardial infarct

size compared to normal hearts. It has been demonstrated that TRX overexpression in

adult rat cardiomyocytes prevents α-adrenergic receptor-stimulated hypertrophy (22),

suggesting a protective role of endogenous TRX in failing heart. Our results evidenced a

marked up-regulation of TR after Q treatment contributing to explain the ability of this

flavonoids to protect cardiomyocytes from oxidative stress.

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Glutathione is the major non enzymatic regulator of intracellular redox homeostasis,

ubiquitously present in all cell types at millimolar concentration (27). It is synthesized

enzymatically by glutamate-cysteine ligase and glutathione synthetase, with the former

being the rate-limiting enzyme (24). Glutathione exists in cell both in reduced (GSH) and

oxidized (GSSG) forms. Under normal cellular redox conditions, the major portion is in

reduced form. Our data showed both a significant up-regulation of glutamate-cysteine

ligase expression level and a marked increase of GSH content after Q treatment,

indicating a clear role of Q in increasing thiol-related compound levels. Thus, our results

strongly suggest that the elevation of GSH by Q is probably due to an induction of

glutamate-cysteine ligase and not of glutathione reductase, the enzyme responsible for the

production of GSH from GSSG. In fact, we have not evidenced any significant up-

regulation of glutathione reductase after Q treatment. Accordingly, Myhrstad et al. (33)

have shown that Q is able to increase the intracellular concentration of GSH by

approximately 50% in COS-1 cells. They also demonstrated that Q increased the

expression of both the regulatory and the catalytical subunit of glutamate-cysteine ligase.

In a recent study, Q and other flavonoids were found to efficiently protect neuronal cells

from oxidative glutamate toxicity and other forms of oxidative injury (20). Interestingly,

the increased production of GSH was one of three mechanisms suggested by the Authors

for the protective effects of Q (20).

GSH is also a cofactor for GST, an abundant cellular enzyme in mammalian tissues. GST

is generally viewed as a phase 2 enzyme, primarily involved in the detoxification of

electrophilic xenobiotics through the formation of GSH–electrophile conjugates (17, 48).

Recently, several studies have also reported that GST plays an important role in

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protecting cells against ROS-mediated injury catalyzing the decomposition of lipid

hydroperoxides generated from oxidative damage of cellular lipids (61, 63).

In this study we have demonstrated the induction of GST by Q in cardiomyocytes, which

may contribute to the increased cell resistance to H2O2-elicited toxicity.

The observed strong induction of NQO1 by Q may also be involved in the cytoprotective

effects on H2O2-induced cytotoxicity. In this context, NQO1 may act as an antioxidant

enzyme via its ability to maintain the cellular levels of ubiquinol and vitamin E, two

important non-protein antioxidants (42).

HO catalyzes the rate limiting step in the heme degradation yielding to biliverdin, carbon

monoxide (CO) and free iron. Biliverdin is subsequently reduced to bilirubin by

biliverdin reductase. Three isoforms of HO have been characterized: the inducible HO-1,

the constitutive HO-2, and the recently cloned HO-3, which is only marginally active in

heme degradation (43). In this study we demonstrated the ability of Q to induce HO-1

gene expression and to increase HO activity. HO-1 is also one of the heat shock proteins

(HSP32) and is highly up-regulated by various factors causing oxidative stress such as

heat shock, cytokines, hypoxia, and ischemia/reperfusion (25, 49, 56). The induction of

HO-1 is considered beneficial, as it may protect against apoptotic cell death in fibroblasts

and endothelial cells (8, 12, 38). Moreover, HO-1 deficient mice developed larger

myocardial infarcts in response to hypoxia, whereas cardiac-specific HO-1

overexpression has been shown to protect against ischaemia-reperfusion injury (64, 65).

Clark et al. (9) demonstrated that HO-1 upregulation before ischemia ameliorates

myocardial function and reduces infarct size on reperfusion of isolated rat hearts.

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Previous studies have demonstrated that Q is able to protect H9c2 cardiomioblasts against

oxidative stress, not only by a direct antioxidant mechanism, but also through the

modulation of pro-survival protein kinases, such as Akt/PKB and ERK1/2 (5). The data

here presented complete these findings showing also the enhancement of

antioxidant/detoxifying enzyme activities by Q.

Considering the ability of Q to increase GST, NQO1, TR, HO activities and GSH

content, important cellular defenses against oxidative stress, we investigated whether

their induction after Q treatment leads to protection against ROS-mediate cytotoxicity.

The results clearly showed that treatment of cardiomyocytes with Q resulted in a

complete protection against H2O2 mediated cytotoxicity and intracellular accumulation of

ROS already at the shortest treatment time.

The protective role of Q against H2O2 toxicity at short exposure times could be ascribed

to its well known antioxidant activity. The ability to decrease ROS levels and maintain

cell viability also at longer times could be due to both modulation of intracellular

signaling pathways, as previously demonstrated (5), and regulation of some important

phase 2 enzymes, evidencing its indirect antioxidant activity. These results suggest that

cardiac cells exposed to Q through diet may be protected against oxidative stress, having

higher levels of phase 2 detoxifying enzymes. In conclusion, cardiomyocytes could be

better prepared to subsequent toxic insults after Q nutritional intake. In particular Q

intake may have a role in the counteraction and prevention of cardiac stress related to

diseases.

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GRANTS

Supported by MIUR (Italy) and Fondazione del Monte di Bologna e Ravenna (Italy).

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31

FIGURE LEGENDS

Figure 1. (A) Number of up-regulated genes (FoldChange ≥ 2) in cardiomyocytes treated

with 30 µM Q for 6, 12 and 24 h compared to the controls. (B) Number of down-

regulated genes (FoldChange ≤ -2) in cardiomyocytes treated with 30 µM Q for 6, 12 and

24 h compared to the controls. The overlapping regions denote the number of common

genes up- or down-regulated. The nonoverlapping regions denote the number of unique

genes up- or down-regulated.

Figure 2. Hierarchical clustering display of data in cardiomyocytes treated with 30 µM Q

for 6, 12 and 24 h based on the significant 91 genes. The clustering display was

performed using Rosetta Resolver SE software as reported in Methods. Each gene is

represented by a single row of colored bars. The red color indicates up-regulation of the

gene expression and the green color denotes the down-regulation of the gene expression

compared to the controls.

Figure 3. Gene expression levels of NQO1 (A), HO-1 (B), TR (C), GSTa3 (D), Mgst1

(E), and GSTp2 (F) in cardiomyocytes. Cardiomyocytes were treated with 30 µM Q for

6, 12 and 24 h and gene expression was obtained by RT-PCR, as reported in Methods,

and reported as fold change relative to the control (not-treated) cells after normalization

using the GADP gene expression level. Data are means ± S.D. of four different cell

cultures. Statistical analysis was performed by the Student’s t test comparing

cardiomyocytes treated with Q with control cardiomyocytes: * at least p < 0.05.

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32

Figure 4. Effect of Q treatment on NQO1 (A), HO (B), TR (C) and GST (D) activities in

cardiomyocytes. Cardiomyocytes were treated with 30 µM Q for 6, 12 and 24 h and

enzyme activities were evaluated as reported in Methods. Data are means ± S.D. of four

different cell cultures. Statistical analysis was performed by the Student’s t test

comparing cardiomyocytes treated with Q with control cardiomyocytes: * at least p <

0.05.

Figure 5. Effects of Q treatment on GSH levels. Cardiomyocytes were treated with 30

µM Q for 6, 12 and 24 h and GSH level was determined using the fluorescent probe

MCB as reported in Methods. Data are expressed as % GSH level in comparison to

controls. Data are means ± S.D. of four different cell cultures. Statistical analysis was

performed by the Student’s t test comparing cardiomyocytes treated with Q with control

cardiomyocytes: * at least p < 0.05.

Figure 6. Effect of Q treatment on intracellular ROS production. Cardiomyocytes were

treated with 30 µM Q for 6, 12 and 24 h before the addition of H2O2 (100 µM, 30 min)

and then the level of intracellular ROS was determined using the peroxide-sensitive

fluorescent probe DCFH-DA as reported in Methods. Data are expressed as % inhibition

of ROS produced compared to H2O2 exposure. Data are means ± S.D. of four different

cell cultures. Statistical analysis was performed by the Student’s t test: * at least p<0.05

in comparison to H2O2 exposed cells.

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33

Figure 7. Protection against peroxide-induced cell damage by Q. Cardiomyocytes were

treated with 30 µM Q for 6, 12 and 24 h before the addition of H2O2 (100 µM, 30 min)

and cellular damage was assessed by the MTT assay and reported as % cell viability in

comparison to controls. Data are means ± S.D. of four different cell cultures. Statistical

analysis was performed by the Student’s t test: * at least p<0.05 in comparison to H2O2

exposed cells.

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34

TABLE LEGENDS

Table 1. Primers used in RT-PCR.

Table 2. Up-regulated (≥2.0 fold) and down-regulated (≤-2.0 fold) genes in

cardiomyocytes treated with Q for 6, 12 and 24 h.

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Gene RefSeq Accession # 2 Amplicon (bp)

GSTa3 NM_031509 124

Mgst1 NM_134349 96

GSTp2 NM_138974 61

NQO1 NM_017000 137

TR NM_031614 87

HO-1 NM_012580 95

GADP J04038 390

1 Internal normalizer.

2 NCBI Nucleotide Sequence Database accession number.

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GenBank Fold Change Gene Name Cluster

6 h 12 h 24 h

Signal transduction

NM_017327 2.3 1.2 -1.4 Guanine nucleotide binding protein 1

BU671109 2.1 -1.1 -1 Similar to Mouse zinedin (Zin) (EST) 1

BF400606 1.2 1.5 2.3 Sequestosome 1 (Sqstm1) 4

Cell cycle

CA511295 2.3 1.4 1.1 Similar to Retinoblastoma-binding protein 2 (RBBP-2) 1

BQ207155 2.2 1.5 1 Similar to Retinoblastoma-binding protein 2 1

AW529579 1.7 2.3 1.5 similar to putative tumor suppressor 101F6 (LOC362017) 2

NM_138504 3.9 4.1 3.4 pregnancy-induced growth inhibitor (Okl38) 2

NM_133306 2.3 -2.6 -2.2 oxidised low density lipoprotein (lectin-like) receptor 1 3

NM_012987 1.5 -1.4 -2.5 Nestin (Nes) 3

NM_133306 2.3 -2.6 -2.2 oxidised low density lipoprotein (lectin-like) receptor 1 3

CB545890 1.1 1.1 2.1similar to dJ862K6.2.2 (splicing factor, arginine/serine-

rich 6 (SRP55-2)(isoform 2))4

AI175779 -1.1 -1.1 3 Polymerase (DNA directed), alpha 2 (Pola2) 4

NM_017105 -2.1 -1.2 1.1 bone morphogenetic protein 3 4

NM_012578 -2.8 1 1.2 Rattus norvegicus Histone H1-0 (H1f0) 4

Antioxidant /detoxification

NM_013215 1.6 2.3 1.3 aflatoxin B1 aldehyde reductase 2

S82820.1 2.6 4.6 4.1 GSTA5=glutathione S-transferase Yc2 2

NM_138974 3.1 5 2.5 glutathione S-transferase, pi 2 2

BQ202809 2.1 1.8 1.4 glutamate-cysteine ligase catalytic subunit 2

NM_017000 7.2 7.7 4.8 Diaphorase (NADH/NADPH) (Dia4), mRNA. 2

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AA892527 1.4 1.9 2.2 similar to Glutathione S-transferase, theta 3 2

D00688.1 2.1 1.7 1.5 Rattus sp. mRNA for monoamine oxidase, partial cds. 2

NM_012940 2.0 1.5 1.4 Cytochrome P450 1b1 (Cyp1b1) 2

AA874884 3.7 1.6 1.7 heme oxygenase 2

NM_031509 3.1 5.6 5.5 glutathione S-transferase, alpha 1 2

NM_031614 2.1 1.2 1.2 thioredoxin reductase 1 2

NM_012580 2.6 1.2 1.6 heme oxygenase 1 2

NM_138826 1.76 -2.11 -3.38 metallothionein 1a (Mt1a) 3

BF556648 1.9 -2.6 -3.7 metallothionein-2 3

NM_134349 1.1 2.3 3 microsomal glutathione S-transferase 1 (Mgst1) 4

NM_138547 -2.8 -1.4 1.3 Rattus norvegicus 3-alpha-hydroxysteroid dehydrogenase 4

Receptor

NM_017333 4.5 1.4 1.1 endothelin receptor type B (Ednrb) 1

NM_031628 2.1 -1.3 -1.2 nuclear receptor subfamily 4, group A, member 3 1

M58040.1 2 1.4 -1.3 transferrin receptor, 3 end. 1

NM_013037 1.3 -2.6 -2.5 interleukin 1 receptor-like 1 3

Miscellaneous

NM_080399 2.3 3.1 2.7 Smhs1 protein (Smhs1) 2

BM386003 3.2 1.3 1 apelin, AGTRL1 ligand (EST) 1

CB606442 2 1.4 1.1 ubiquitin-conjugating enzyme E2D 1

NM_012953 3.2 -1.1 1.1 Fos-like antigen 1 (Fosl1) 1

AA818267 2 1.5 -1.1 similar to SI:dZ142B24.4) 1

NM_053420 2.3 1.4 -1.1 BCL2/adenovirus E1B 19 kDa-interacting protein 3 1

NM_030834 2.2 1.4 -1.1 monocarboxylate transporter 1

BQ210515 2.6 -1.3 -1.4 Hypoxia induced gene 1 (Hig1) (EST) 1

AA891949 2.6 -1.2 -1.5 Adenylate kinase 3-like 1 (Ak3l1) (EST) 1

AI406759 2.2 -1.1 -1.7 Similar to Human carbonate dehydratase 1

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NM_013126 2.4 -3.2 -2.9 Diacylglycerol kinase 3 (gamma) (Dgkg) 1

AI044063 2.8 2.4 -1.6 similar to Mouse mRNA for IZP6 (EST) 1

CB544664 1.4 2.2 1.5 similar to phospholipase A2 2

NM_138863 3.2 5.1 3.1 dithiolethione-inducible gene-1 (Dig1) 2

NM_080399 2.3 3.1 2.7 Smhs1 protein (Smhs1) 2

BF420687 2.4 1.6 1.3 similari to protein ref:NP_000421.1 (H.sapiens 2

NM_013083 1.5 -1.3 -2 heat shock 70kD protein 5 (Hspa5) 3

BQ191772 1 -2.7 -3 similar to mouse annexin A8 3

BE126741 1.3 -2 -2.9 tenascin C 3

BM387134 1.3 -1.8 -2.8 tenascin C 3

AW531866 1.3 -1.9 -2.2 similar to mouse RIKEN cDNA 1810020G14 gene 3

AI044054 1.8 -3.3 -2 tissue factor pathway inhibitor 2 3

AJ132352.1 -1 -2.3 -1.1 Rattus norvegicus mRNA for 45 kDa secretory protein 3

BE116861 -1.1 1.3 2.2 Heterogeneous nuclear ribonucleoprotein A1 (Hnrpa1) 4

NM_080892 -2.1 1.4 1.4 Rattus norvegicus selenium binding protein 2 (Selenbp2) 4

NM_031545 -2.9 1 1.6 natriuretic peptide precursor type B 4

NM_019280 -2.4 -1.1 1.3 gap junction membrane channel protein alpha 5 4

BQ202499 -2.3 1.2 1.5 Aldo-keto reductase family 1, member E1 (predicted) 4

NM_053536 -2.5 1.1 1.6 Kruppel-like factor 15 (Klf15) 4

NM_012889 -2.4 -1.1 1.1 Vascular cell adhesion molecule 1 (Vcam1) 4

Unknown

BF523428 2.3 1.2 -1.4 similar to Mouse DNA sequence from clone RP23-40G2 1

AM:ARGI1-

037175-CYA2.1 1 -1 similar to Translation of nuc:U14172_1 Mus musculus 1

BE102806 2.2 1 -1.1 similar to Mouse DNA sequence from clone RP23-344D2 1

CA338760 2.5 1.6 1.1 similar to Mouse, ribosome binding protein 1 1

AA818967 2.3 -1 -1.4 unknown function 1

BU671646 2.8 2.1 1.3 similar to protein ref:NP_005830.1 (H.sapiens 2

BM385792 5 3.1 2.6 similar to Sulfiredoxin 1 homolog (S. cerevisiae) 2

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AA875256 1.6 -1.9 -2.6 transcribed sequence 3

AI043706 1.2 -1.9 -2.2 unknown function 3

CB548350 1.1 -2.3 -2.3 Transcribed sequence 3

BF522774 -1 -2.3 -1.1 Transcribed sequence 3

AA926036 -1 -2.2 -1.1 unknown function 3

CB544646 -1 -2.1 -1 similar to NP_082393.2 dermatan-4-sulfotransferase-1 3

BM392090 -1.1 -2.7 -2.9 similar to G protein-coupled receptor family C 3

CA340365 1.1 1.2 2.2 NISC_lz02g05.y1 NCI_CGAP_Pr33 Rattus norvegicus 4

AW918961 -2 1.1 1.4 Rattus norvegicus transcribed sequences 4

AB072252.1 -1.6 1.2 2.1 Rattus norvegicus cDNA, clone:bA7, 4

BE110944 -1.7 1.8 3 Similar to NP_001809.2 aldo-keto reductase family 1 4

BQ194588 -1.1 2 1.8 similar to Blvrb protein 4

BE102241 -2 1.1 1.2 similar to Zinc finger CCCH type 4

BF557949 -2.3 1.4 1.4 similar to Protein:NP_077170 4

CA506727 -2.9 1.1 1.1 Rat clone RP31-202M22 strain Brown Norway 4

BF284739 -2.3 1.2 1.2 transcribed sequences 4

AI712694 -2.1 1.6 -1 shypothetical protein FLJ10156 4

BF283760 -2.2 -1.3 -1.1 similar to NP_034828.2 LIM domain binding 2 4

AI009713 -2.1 1.1 -1.1 unknown function 4

12847730 -2.2 -1.2 -1.2 unknown function 4

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Figure 1. (A) Number of up-regulated genes (FoldChange 2) in cardiomyocytes treated with 30 µM Q for 6, 12 and 24 h compared to the controls. (B) Number of down-regulated

genes (FoldChange -2) in cardiomyocytes treated with 30 µM Q for 6, 12 and 24 h compared to the controls. The overlapping regions denote the number of common genes up- or down-regulated. The nonoverlapping regions denote the number of unique genes

up- or down-regulated. 80x53mm (600 x 600 DPI)

of 46


Figure 2. Hierarchical clustering display of data in cardiomyocytes treated with 30 µM Q for 6, 12 and 24 h based on the significant 91 genes. The clustering display was

performed using Rosetta Resolver SE software as reported in Methods. Each gene is represented by a single row of colored bars. The red color indicates up-regulation of the

gene expression and the green color denotes the down-regulation of the gene expression compared to the controls. 88x135mm (600 x 600 DPI)

of 46


Figure 3. Gene expression levels of NQO1 (A), HO-1 (B), TR (C), GSTa3 (D), Mgst1 (E), and GSTp2 (F) in cardiomyocytes. Cardiomyocytes were treated with 30 µM Q for 6, 12

and 24 h and gene expression was obtained by RT-PCR, as reported in Methods, and reported as fold change relative to the control (not-treated) cells after normalization

using the GADP gene expression level. Data are means ± S.D. of four different cell cultures. Statistical analysis was performed by the Student's t test comparing

cardiomyocytes treated with Q with control cardiomyocytes: * at least p < 0.05. 159x207mm (600 x 600 DPI)

of 46


Figure 4. Effect of Q treatment on NQO1 (A), HO (B), TR (C) and GST (D) activities in cardiomyocytes. Cardiomyocytes were treated with 30 µM Q for 6, 12 and 24 h and

enzyme activities were evaluated as reported in Methods. Data are means S.D. of four different cell cultures. Statistical analysis was performed by the Student's t test

comparing cardiomyocytes treated with Q with control cardiomyocytes: * at least p < 0.05.

120x120mm (600 x 600 DPI)

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Figure 5. Effects of Q treatment on GSH levels. Cardiomyocytes were treated with 30 µM Q for 6, 12 and 24 h and GSH level was determined using the fluorescent probe MCB as

reported in Methods. Data are expressed as % GSH level in comparison to controls. Data are means S.D. of four different cell cultures. Statistical analysis was performed by the Student's t test comparing cardiomyocytes treated with Q with control cardiomyocytes: *

at least p < 0.05. 70x66mm (600 x 600 DPI)

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Figure 6. Effect of Q treatment on intracellular ROS production. Cardiomyocytes were treated with 30 M Q for 6, 12 and 24 h before the addition of H2O2 (100 M, 30 min)

and then the level of intracellular ROS was determined using the peroxide-sensitive fluorescent probe DCFH-DA as reported in Methods. Data are expressed as % inhibition of

ROS produced compared to H2O2 exposure. Data are means S.D. of four different cell cultures. Statistical analysis was performed by the Student's t test: * at least p<0.05 in

comparison to H2O2 exposed cells. 70x59mm (600 x 600 DPI)

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Figure 7. Protection against peroxide-induced cell damage by Q. Cardiomyocytes were treated with 30 M Q for 6, 12 and 24 h before the addition of H2O2 (100 M, 30 min) and cellular damage was assessed by the MTT assay and reported as % cell viability in comparison to controls. Data are means S.D. of four different cell cultures. Statistical analysis was performed by the Student's t test: * at least p<0.05 in comparison to H2O2

exposed cells. 70x75mm (600 x 600 DPI)

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role of quercetin in modulating rat cardiomyocyte gene expression profile

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