Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent

Apr 30, 2018 - Key Laboratory of Functional Small Organic Molecule, Ministry of Education; Key Laboratory of Green Chemistry, Jiangxi Province and Col...
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Inhibitive effects of quercetin on myeloperoxidase-dependent hypochlorous acid formation and vascular endothelial injury Naihao Lu, Yinhua Sui, Rong Tian, and Yiyuan Peng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01537 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Journal of Agricultural and Food Chemistry

Inhibitive effects of quercetin on myeloperoxidase-dependent hypochlorous acid formation and vascular endothelial injury Naihao Lu a, *, Yinhua Sui a, Rong Tian a, *, Yi-Yuan Peng a a

Key Laboratory of Functional Small Organic Molecule, Ministry of Education; Key Laboratory

of Green Chemistry, Jiangxi Province and College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, China

*Corresponding Author Phone/fax: 86-791-88120380 (Lu N and Tian R) E-mail: [email protected]; [email protected] (Lu N) ; [email protected] (Tian R)

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Abstract 1

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human

2

inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl).

3

As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and

4

anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited

5

MPO-mediated HOCl formation (75.0±6.2% for 10 µM quercetin versus 100±5.2% for control

6

group, P 0.05, all cases). Moreover, quercetin inhibited HOCl generation by

8

stimulated neutrophils (a rich source of MPO) and protected endothelial cells from

9

neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial

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dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial

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cells (P 98%) were purchased from Shanxi Huike Botanical Development Co. Ltd.

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2.2 Effect of quercetin on MPO activity

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MPO chlorinating activity was measured by taurine chloramine formation.3, 4, 12, 17 In the

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absence or presence of quercetin, H2O2 (500 µM) was added to a solution containing NaCl (100

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mM), MPO (0.6 µM) and taurine (1 mM). Then, the taurine chloramine was measured by 3,3',5,5'-

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tetramethylbenzidine (TMB) method.12, 27 HOCl was significantly generated in short time when

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MPO-H2O2 were used at high concentrations, and high concentrations of quercetin and MPO were,

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therefore, selected in this study.3,4, 8,16

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Cys-thiol levels in proteins were determined spectrophotometrically by 5, 5'-dithiobis

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(2-nitrobenzoic) acid method, and the change of absorption at 412 nm would reflect the change of

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thiol contents.28

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Neutrophil cells were cultured with different concentrations of quercetin,16, 29 and these cells

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were stimulated with LPS (0.2 mg/ml) for 60 min. Then, the formation of HOCl was determined

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by taurine chloramine assay. 4

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2.3 Interaction between MPO and quercetin by molecular docking

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The original X-ray structure of human MPO (PDB ID: 5FIW) was selected and one monomer

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(chains B and D) of protein was kept. Quercetin or rutin was docked to MPO by AutoDock

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software.16, 29-31

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2.4 Effect of quercetin on MPO-induced human umbilical vein endothelial cells (HUVEC)

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injury

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HUVEC were cultured in DMEM containing NaCl (100 mM), glucose (5.6 mM). Different

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amounts of quercetin were first added to cells for 5 min, and then MPO (1.5 U/mL) and glucose

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oxidase (10 mU/mL) were added and maintained for 2 additional hours. In the presence of

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neutrophils, HUVEC were cultured with quercetin and stimulated with LPS (0.2 mg/ml) for 2 h.

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Then, HOCl generation and cell viability were measured by taurine chloramine and MTT method,

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respectively.

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2.5 Effect of quercetin on HOCl-mediated HUVEC injury

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Different concentrations of quercetin were first added to HUVEC cells for 5 min. Then,

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NaOCl (80 µM) was added and cells were maintained for 20 min. NO formation and cell viability

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were measured by commercial kit and MTT method, respectively.

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2.6 Vascular endothelial function in inflammation and in vitro

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To further investigate the potential relevance of quercetin to endothelial function in vivo,

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inflammation in male mice was induced by intraperitoneally injection of LPS and the dose of LPS

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used in this study (10 mg/kg) was sufficient to cause endotoxaemia.23, 26 Mice were randomly

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divided into five groups: (I) the control group treated with saline; (II) the quercetin (Qu) group;

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(III) the LPS group (animal models of inflammation) treated with LPS at 10 mg/kg (i.p.) and

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(IV-V) the LPS + Qu groups treated with Qu (20 and 50 mg/kg, i.p.) 1 h before LPS

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administration. After 6 h treatment, aortas from these animals were isolated and cut into individual

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ring segments. Acetylcholine (ACh) was added to induce endothelium-dependent relaxation

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(vascular endothelial function), as previously described.7, 8 Meanwhile, the expression and activity

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of MPO was determined as described previously.8

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Aortic rings from control mice were incubated with quercetin (20 µM) for 2 h and washed

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with vehicle, and then treated with 50 µM HOCl for 30 min. Then, the vascular function was 5

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measured described above.7, 8

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2.7 Statistical analysis

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The results were presented as the means ± SD of at least three independent experiments.

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One-way ANOVA was performed for statistical analyses, and P< 0.05 was considered significant.

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3. Results

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3.1 Quercetin inhibited MPO-catalyzed HOCl production and cytotoxicity in HUVEC

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Firstly, we used HUVEC to confirm that MPO system could cause significant cell injuries by

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generating HOCl. Glucose oxidase/glucose system was used to generate H2O2,4 neither H2O2 nor

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MPO alone barely decreased the cell viability. However, the presence of both MPO and H2O2

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could result in significant loss of cell viability, and the decrease of cell viability was effectively

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inhibited by 4-aminobenzoic acid hydrazide (ABAH, a well-known MPO inhibitor) (Fig. 2).

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Then, we investigated if quercetin could protect endothelial cells from MPO-dependent

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injury. It was found that quercetin could dose-dependently inhibit MPO-induced cytotoxicity to

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HUVEC (Fig. 2). Moreover, quercetin at the concentration used (up to 50 µM) did not show

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cytotoxicity to HUVEC (Fig. S1).

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In addition, we investigated if quercetin could inhibit MPO-mediated HOCl formation in

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vitro. As shown in Fig. 3A, quercetin could inhibit MPO-catalyzed HOCl production in a

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dose-dependent manner. Compared with MPO-catalyzed HOCl production, quercetin at 5 and 10

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µM reduced HOCl production by 16 and 25%, respectively. Then, we mixed quercetin (5 and 10

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µM) with HOCl and analyzed the remaining HOCl content. Our data showed that quercetin at 10

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µM significantly scavenged HOCl by 16% (Fig. 3B), which was less than the reduction of

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MPO-mediated HOCl production by quercetin at the same concentration (25%, Fig. 3A).

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Meanwhile, quercetin at 5 µM did not scavenge HOCl (Fig. 3B), while quercetin at this

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concentration could effectively inhibit MPO-mediated HOCl formation (Fig. 3A), demonstrating

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that inhibition of MPO activity was the prior pathway for quercetin at low concentration. These

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results suggested that quercetin at high concentration reduced HOCl generation by both

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scavenging HOCl and inhibiting MPO activity, and significant inhibition of MPO-mediated HOCl

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generation was achieved by quercetin at low concentration (5 µM) that could not be explained as 6

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scavenging HOCl. Consistent with the protective effects on MPO cytotoxicity, quercetin

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effectively inhibited MPO-catalyzed HOCl production even in the presence of HUVEC (data not

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shown).

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MPO has 12 cysteine residues per monomer and these cysteine residues are critical to MPO

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activity.1, 2 Quercetin could be readily oxidized by MPO system to form quinone.17 However, no

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information has been available concerning the possible interaction between MPO cysteine

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residues and the oxidation products of quercetin. Therefore, we measured the loss of Cys-thiol

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contents in MPO to assess whether a covalent binding of oxidized quercetin to cysteine residues

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was formed. As shown in Fig. S2, incubation of MPO with H2O2 resulted in a significant decrease

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of protein thiol group, and the addition of quercetin further decreased the level of Cys-thiol in

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MPO. Control experiments demonstrated that quercetin alone did not influence the level of

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Cys-thiol in MPO (data not shown).

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3.2 Docking of quercetin to MPO

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Fig. 4A showed that quercetin fit in the active site of MPO. This simulation showed that

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quercetin could be oriented in such a way that the A and C-ring was above the iron-heme (active

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site) of MPO (Fig. 4B) with the shortest distance of 5.0 Å. These results showed that quercetin

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directly bound into the iron-heme site of MPO.

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As revealed by the docking, the homologous and conserved amino acids Arg239 and Phe407

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could form non-covalent interactions with quercetin (Fig. 4C). The flavonoid A-ring of quercetin

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stacked onto the active heme cavity. In addition, the phenyl groups of A-ring formed hydrophobic

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interaction with Arg239. However, these amino acid residues (Arg239 and Phe407) are near the

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catalytic heme center of MPO.2, 17, 31 Therefore, quercetin interacted with the active heme site and

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might block the substrate channel, providing the possible theoretical explanation for its inhibition

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on MPO activity.

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3.3 Quercetin inhibited MPO activity in neutrophils and protected HUVEC from

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neutrophils-induced injury

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The release of MPO was induced by LPS in neutrophil cells, 3, 29 and the effects of quercetin

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on MPO-induced HOCl production were investigated. LPS-stimulated neutrophils could generate

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high level of HOCl, while little HOCl was produced by unstimulated (i.e. resting) neutrophils. 7

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Then, quercetin effectively inhibited HOCl formation in activated neutrophils (Fig. 5A). Moreover,

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quercetin also inhibited ROS formation from activated neutrophils (data not shown). These data

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suggested that quercetin inhibited MPO activity in activated neutrophils.

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Similar to MPO/H2O2/Cl--induced cytotoxicity in HUVEC, incubation of LPS-activated

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neutrophils with endothelial cells also induced significant loss of cell viability (Fig. 5B).

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Quercetin protected cytotoxicity from activated neutrophils as well. In addition, quercetin

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inhibited these HOCl formations by activated neutrophils (data not shown). These results

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confirmed that quercetin inhibited MPO-mediated HOCl formation even in the existence of

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HUVEC.

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3.4 Quercetin inhibited HOCl-induced endothelial dysfunction in HUVEC

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To further demonstrate the effects of quercetin on MPO-mediated cell injury, effects of

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quercetin on HOCl-induced endothelial dysfunction were examined (Fig. 6A). OCl- (80 µM)

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significantly caused the loss of cell viability, and the presence of quercetin before OCl- addition

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could dose-dependently inhibit OCl--mediated cytotoxicity. However, if OCl- was first incubated

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with the cells for 10 min followed by quercetin addition, quercetin could not reverse OCl--induced

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loss of cell viability (Fig. 6A).

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Reduced production or availability of NO is a common feature of endothelial dysfunction.9

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Consistent with the loss of cellular viability, HOCl could decrease NO formation in endothelial

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cells (Fig. 6B). However, the addition of quercetin could inhibit HOCl-induced decrease of NO

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formation. Therefore, these results demonstrated that quercetin could inhibit HOCl-mediated

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endothelial dysfunction in HUVECs such as the decreases in cell viability and NO formation (Fig.

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6).

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3.5 Effects of rutin on MPO-dependent HOCl production in vitro

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As the glycoside of quercetin, rutin is also commonly found in the human diet.30 Compared

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with quercetin, rutin showed less effective effects on MPO-catalyzed HOCl production in vitro

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(Fig. S3), which was consistent with the weaker free radical scavenging ability of rutin.14, 19, 30

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Moreover, docking study demonstrated that the strong binding of rutin to MPO was also observed,

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and the B-ring of rutin was near to the heme of MPO with the shortest distance of 6.3Å (Fig. S4),

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which was longer than the shortest distance of A-ring of quercetin to the active heme centre of 8

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MPO (5.6Å) (Fig. 4). Consistent with the inhibitory effect on MPO-dependent HOCl formation,

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rutin also inhibited MPO/neutrophil-mediated cytotoxicity to HUVEC (data not shown).

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3.6 Quercetin attenuated aortic endothelial dysfunction in inflammation and in vitro

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There is evidence that vascular-bound MPO is potent inducers for vascular injury.6-8 As

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shown in Fig. 7A, MPO expression and activity in normal aortas was very low. However, MPO

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expression and activity in aortas from LPS-treated mice was significantly higher, presumably

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demonstrating that MPO was secreted into the blood vessels by activated neutrophils and then

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permeated vascular tissue in inflammation. Consistent with the increased MPO expression and

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activity, the ACh-mediated vessel relaxation (determined as vascular endothelial function) was

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significantly impaired in aortas from LPS-treated mice (Fig. 7B). Therefore, MPO contributed

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importantly to endothelial dysfunction associated with inflammation produced by LPS. In contrast

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to the causal role of MPO in vascular endothelial dysfunction,6-8 a recent study reported that MPO

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did not induce endothelial dysfunction after LPS treatment.

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be related to gender and age differences.32

32

These different conclusions might

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However, compared with LPS-treated mice, the pretreatment of quercetin dose-dependently

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improved the vascular endothelial function (Fig. 7B) and attenuated the increase of MPO activity

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and expression (Fig. 7A) in inflammatory vasculature, demonstrating that quercetin attenuated the

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adhesion and infiltration of MPO to the vascular wall and subsequently inhibited MPO activity in

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vivo. Control experiments demonstrated no significant effects of quercetion on aortic endothelial

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function and MPO in normal mice. To investigate the effect of quercetin on HOCl-mediated

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endothelial dysfunction, the flavonoid was pre-treated with vessel for 2 h and then washed away

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before the addition of HOCl. It was found that HOCl alone could inhibit ACh-mediated vessel

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relaxation and pre-treatment with quercetin effectively prevented HOCl-mediated endothelial

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dysfunction in isolated aortas (Fig. 7C). These data revealed that quercetin might attenuate

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vascular endothelial dysfunction during inflammation by inhibiting MPO-dependent HOCl

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formation and endothelial dysfunction in vivo.

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4. Discussion

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As a widely present polyphenol flavonoid, quercetin exhibits numerous beneficial effects 9

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against various diseases.16,

17, 21-26

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neutrophils)-mediated HOCl formation in vitro (Fig. 3 and 5A). Quercetin protected endothelial

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cells from MPO (or neutrophil)-induced damage through inhibition of MPO activity, while it

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effectively inhibited HOCl-induced endothelial dysfunction in HUVEC (Fig. 6). The protection of

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quercetin was observed in LPS-induced vascular endothelial dysfunction in parallel with the

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decrease of MPO activity in vivo but our experiments did not confirm that there was a causal

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relationship between both effects. Therefore, quercetin exhibited the therapeutic ability in vivo

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through mediating MPO-dependent endothelial dysfunction and vascular inflammation (Fig. 7).

Our results showed that quercetin inhibited MPO (or

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The inhibitive effects of quercetin on MPO-catalyzed HOCl production in vitro and in

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activated neutrophils were firstly demonstrated. The protective effect on MPO-mediated HOCl

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formation was related to the reducing reactivity of quercetin towards MPO redox

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intermediates.16-18 As a one-electron reducing substrate of MPO, quercetin has been found to react

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with both reactive compounds, and the second order rate constants for its reaction with MPO

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compound II was 7.0×106 M-1 s-1 which is higher than other flavonoids such as (-)-epicatechin

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(3.5×106 M-1 s-1) and (+)-eriodictyol (1.3×106 M-1 s-1).31 Consistent with this result herein, several

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studies have shown that some other flavonoids can also inhibit MPO chlorination activity by

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acting as peroxidase substrates. 16-18 Therefore, quercetin was an effective substrate for MPO

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intermediates and significantly competed with Cl- for MPO.

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Moreover, docking studies showed that the A-ring bound to the active heme pocket (Fig. 4A

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and B). The hydrophobic interactions between A, C ring in quercetin and amino acid residues

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(Phe407, Arg 239) were present (Fig. 4C). As the part of the catalytic sites, both Arg 239 and

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Phe407 are located close to active heme iron.2, 17, 29 Quercetin interacted with the hydrophobic

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region at active heme pocket and served as substrates for the MPO. 16-18 Therefore, quercetin acted

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as a competitive inhibitor to block other substrate to access the active site of MPO. In addition,

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2-thioxanthine derivatives have recently been reported to irreversibly inhibit MPO activity by

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covalently modifying the heme prosthetic group of the enzyme.33 It was possible that the binding

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of quercetin to the heme pocket might exhibit the similar mechanism for inactivation by

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2-thioxanthine derivatives. However, this presume should be experimentally verified in future.

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Modifications in the quercetin structure result in different inhibitory effects. Rutin, differing 10

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from quercetin by the 3-hydroxyl group in the C ring, was less effective at inhibiting MPO activity

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than its parent molecule (Fig. S3). Moreover, it was interesting to note that the substitution of

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3-hydroxyl group by more hydrophilic group (i.e. rhamnosylglucoside) dramatically showed the

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longer distance to active center heme of MPO, as compared to quercetin which was a lipophilic

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compound (Fig. S3 and 4). It appears that hydrophobicity is an essential requirement for the

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inhibitive effect of MPO. 16-18 It was likely, that in compounds having the same basic chemical

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structure, the effective inhibition on MPO activity was related to the C3 hydroxylation and the

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glycoside in the flavonoid structure. 19 The presence of rutinose at position C-3 in rutin would

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cause the reduction in the access to the active heme site of MPO and consequently resulted in the

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less effective effects on MPO activity.

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On the other hand, quercetin could be readily oxidized by MPO system and the presence of a

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quinone as the main intermediate in the oxidation of quercetin was confirmed by demonstrating

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that glutathione (GSH) formed a hydroquinone conjugate with oxidized flavonoid.17, 18 Thereafter,

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we were concerned the interaction between MPO cysteine residues and the oxidation products of

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quercetin. Compared with the level of Cys-thiol in H2O2-incubated MPO, oxidation of quercetin

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promoted the loss of Cys-thiol content in MPO (Fig. S2) which was accompanied by the inhibition

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of MPO activity (Fig. 3A). Therefore, besides its role as a cosubstrate of MPO in reducing the

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access of other substrates to the active site of MPO, quercetin may irreversibly inactivated MPO

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via formation of covalent bond(s) between oxidized quercetin (quinone form) and cysteine

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residues. However, this presume was not verified and the identification of quercetin derivatives

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with MPO cysteine residues were not shown in this study and need further study.

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There are at least two pathways to ameliorate inflammatory injury caused by MPO, the

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scavenging of HOCl and the inhibition of the enzyme itself.2 Of course, we showed a certain

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HOCl-scavenging effect of quercetin at high concentration (Fig. 3). The second order rate constant

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for the reaction between quercetin and HOCl is 1.4×105 M-1 s-1.34, 35 Thus, the reaction between

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this MPO compound II and flavonoid (7.0×106 M-1 s-1) most likely prevails, than that between

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HOCl and quercetin. Besides HOCl scavenging ability, quercetin could inhibit MPO-induced

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injury by participating in the regulation of HOCl generation.

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There is evidence that neutrophils-derived MPO plays an important role in endothelial 11

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dysfunction and vascular injury by its ability to produce reactive HOCl.6-8 Because endothelial

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cells do not express MPO, the activated neutrophils-released MPO can bind and infiltrate into the

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vascular wall directly (Fig. 7A). In this study, we found that quercetin effectively inhibited

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MPO/neutrophils-induced cytotoxicity in endothelial cells without causing any toxicity (Figs. 2

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and 5). Moreover, quercetin prevented HOCl from causing injuries to endothelial dysfunction such

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as loss of cell viability, and decrease of nitric oxide formation (Fig. 6). Consistent with these in

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vitro data, quercetin attenuated LPS-induced endothelial dysfunction and increase of MPO activity

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in mouse aortas, while this flavonoid improved endothelial function of HOCl-treated aortic rings

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in vitro. In vivo, quercetin inhibited the adhesion of MPO (or neutrophil) to the vascular wall and

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therefore attenuated MPO activity (Fig. 7A and B). On the other hand, quercetin was incubated

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with isolated aortic rings for 2 h and the flavonoid was then washed away before the addition of

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HOCl. It was found that the “quercetin-washed” aortas also effectively prevented HOCl-induced

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endothelial dysfunction (Fig. 7C). It could be revealed that quercetin could also adhere and

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infiltrate to the vascular wall and influence MPO/HOCl-induced endothelial dysfunction.

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Therefore, it was proposed that quercetin inhibited the adhesion of MPO to the vascular wall (Fig.

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7), and subsequently attenuated endothelial injury in inflammatory vasculature via inhibition of

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vascular-bound MPO-mediated HOCl formation. Moreover, quercetin did not induce cytotoxicity

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to HUVEC (Fig. S1) and dietary administration of high doses of quercetin to mice was not toxic.36

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Compared with the toxic side effects of other effective inhibitors (hydroxamic acids, hydrazides

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and azides),2,

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bioapplication in vivo.

12, 37

the nontoxic effects and natural source for quercetin could increase their

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It should be noted that quercetin detected in plasma is mostly in conjugated forms under

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normal physiological conditions, and quercetin aglycone (i.e. free quercetin) is only found in the

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sub-micromolar concentration range.21, 24, 26, 38 In tissues, however, quercetin aglycone can be the

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predominant form. It has been reported that both monocytes and macrophages can metabolize

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quercetin glucuronides to the parent aglycone compound, quercetin.24,

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information, therefore, it was proposed that quercetin could inhibit the adhesion and infiltration of

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MPO to the vascular wall, and attenuated vascular-bound MPO-dependent endothelial injury in

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inflammatory vasculature. In this work, we did not pursue the identification of quercetin 12

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metabolites, but it was possible that quercetin in free form could contribute to the inhibition on

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MPO activity and vascular endothelial injury in inflammatory vasculature.

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In conclusion, quercetin was a potent nontoxic inhibitor of MPO activity that might be

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beneficial to human health. As quercetin significantly inhibited MPO-dependent HOCl generation

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and protected HUVEC from MPO/neutrophil-induced injury (Fig. 8), quercetin was an effective

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inhibitor for mediating MPO-dependent endothelial dysfunction in inflammatory vasculature.

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Moreover, the inhibitive effect of quercetin on MPO-mediated HOCl formation would render this

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flavonoid as potential nutriment to decrease the risk of cardiovascular diseases, which was

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different from the widespread anti-oxidant and anti-inflammatory mechanisms, i.e. radical

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scavenging, metal-chelating, oxidative stress and cell signaling pathways etc.17, 21-26, 38 Our results

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could also represent a novel mechanism to explain, at least in part, the anti-inflammatory property

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of flavonoids and nutrient phenomenon that high consumption of flavonoids-rich food can

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significantly reduce the risks of cardiovascular diseases.

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Conflict of interest

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The authors declare no competing financial interest.

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Acknowledgments

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Financial support from the National Natural Science Foundation of China (Nos. 31760255,

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31560255, 31260216, 31100608), the Natural Science Foundation of Jiangxi province (No.

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20171BCB23041, 20161BAB215215).

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Figure captions

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Fig. 1. (A) Peroxidase and chlorination cycles of MPO.1, 3, 4, 16 MPO catalyzes two competitive

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oxidative reactions: Cl- to OCl- (chlorination cycle) and flavonoid to quinone and dimer

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(peroxidase cycle). (B) Schematic structure of quercetin.

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Fig. 2. Cytotoxicity of MPO and the protective effects of quercetin. HUVEC were cultured in

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DMEM containing NaCl (100 mM), glucose (5.6 mM). Different concentrations of quercetin were

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preincubated with cells for 5 min. In the absence or presence of MPO inhibitor (ABAH, 50 µM),

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MPO and glucose oxidase (10 mU/mL) were then added and incubated for 2 h. H2O2 was

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generated by glucose oxidase/glucose system. The Blank values were set to 100%, to which other

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values were compared. Values are means ± S.D. of three independent determinations, **P