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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)
1
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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
10
dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial
11
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
67
absence or presence of quercetin, H2O2 (500 µM) was added to a solution containing NaCl (100
68
mM), MPO (0.6 µM) and taurine (1 mM). Then, the taurine chloramine was measured by 3,3',5,5'-
69
tetramethylbenzidine (TMB) method.12, 27 HOCl was significantly generated in short time when
70
MPO-H2O2 were used at high concentrations, and high concentrations of quercetin and MPO were,
71
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
73
(2-nitrobenzoic) acid method, and the change of absorption at 412 nm would reflect the change of
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thiol contents.28
75
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
80
(chains B and D) of protein was kept. Quercetin or rutin was docked to MPO by AutoDock
81
software.16, 29-31
82
2.4 Effect of quercetin on MPO-induced human umbilical vein endothelial cells (HUVEC)
83
injury
84
HUVEC were cultured in DMEM containing NaCl (100 mM), glucose (5.6 mM). Different
85
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
87
neutrophils, HUVEC were cultured with quercetin and stimulated with LPS (0.2 mg/ml) for 2 h.
88
Then, HOCl generation and cell viability were measured by taurine chloramine and MTT method,
89
respectively.
90
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,
92
NaOCl (80 µM) was added and cells were maintained for 20 min. NO formation and cell viability
93
were measured by commercial kit and MTT method, respectively.
94
2.6 Vascular endothelial function in inflammation and in vitro
95
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
97
used in this study (10 mg/kg) was sufficient to cause endotoxaemia.23, 26 Mice were randomly
98
divided into five groups: (I) the control group treated with saline; (II) the quercetin (Qu) group;
99
(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
101
administration. After 6 h treatment, aortas from these animals were isolated and cut into individual
102
ring segments. Acetylcholine (ACh) was added to induce endothelium-dependent relaxation
103
(vascular endothelial function), as previously described.7, 8 Meanwhile, the expression and activity
104
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
115
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
117
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
119
injury. It was found that quercetin could dose-dependently inhibit MPO-induced cytotoxicity to
120
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
123
vitro. As shown in Fig. 3A, quercetin could inhibit MPO-catalyzed HOCl production in a
124
dose-dependent manner. Compared with MPO-catalyzed HOCl production, quercetin at 5 and 10
125
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
137
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
143
was formed. As shown in Fig. S2, incubation of MPO with H2O2 resulted in a significant decrease
144
of protein thiol group, and the addition of quercetin further decreased the level of Cys-thiol in
145
MPO. Control experiments demonstrated that quercetin alone did not influence the level of
146
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
154
stacked onto the active heme cavity. In addition, the phenyl groups of A-ring formed hydrophobic
155
interaction with Arg239. However, these amino acid residues (Arg239 and Phe407) are near the
156
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
168
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
170
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
182
cells (Fig. 6B). However, the addition of quercetin could inhibit HOCl-induced decrease of NO
183
formation. Therefore, these results demonstrated that quercetin could inhibit HOCl-mediated
184
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
189
(Fig. S3), which was consistent with the weaker free radical scavenging ability of rutin.14, 19, 30
190
Moreover, docking study demonstrated that the strong binding of rutin to MPO was also observed,
191
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
199
demonstrating that MPO was secreted into the blood vessels by activated neutrophils and then
200
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
204
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
209
and expression (Fig. 7A) in inflammatory vasculature, demonstrating that quercetin attenuated the
210
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
217
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
235
(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
238
intermediates and significantly competed with Cl- for MPO.
239
Moreover, docking studies showed that the A-ring bound to the active heme pocket (Fig. 4A
240
and B). The hydrophobic interactions between A, C ring in quercetin and amino acid residues
241
(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
244
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
246
covalently modifying the heme prosthetic group of the enzyme.33 It was possible that the binding
247
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
253
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
262
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
264
quercetin. Compared with the level of Cys-thiol in H2O2-incubated MPO, oxidation of quercetin
265
promoted the loss of Cys-thiol content in MPO (Fig. S2) which was accompanied by the inhibition
266
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
268
via formation of covalent bond(s) between oxidized quercetin (quinone form) and cysteine
269
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
272
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
274
for the reaction between quercetin and HOCl is 1.4×105 M-1 s-1.34, 35 Thus, the reaction between
275
this MPO compound II and flavonoid (7.0×106 M-1 s-1) most likely prevails, than that between
276
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
282
MPO/neutrophils-induced cytotoxicity in endothelial cells without causing any toxicity (Figs. 2
283
and 5). Moreover, quercetin prevented HOCl from causing injuries to endothelial dysfunction such
284
as loss of cell viability, and decrease of nitric oxide formation (Fig. 6). Consistent with these in
285
vitro data, quercetin attenuated LPS-induced endothelial dysfunction and increase of MPO activity
286
in mouse aortas, while this flavonoid improved endothelial function of HOCl-treated aortic rings
287
in vitro. In vivo, quercetin inhibited the adhesion of MPO (or neutrophil) to the vascular wall and
288
therefore attenuated MPO activity (Fig. 7A and B). On the other hand, quercetin was incubated
289
with isolated aortic rings for 2 h and the flavonoid was then washed away before the addition of
290
HOCl. It was found that the “quercetin-washed” aortas also effectively prevented HOCl-induced
291
endothelial dysfunction (Fig. 7C). It could be revealed that quercetin could also adhere and
292
infiltrate to the vascular wall and influence MPO/HOCl-induced endothelial dysfunction.
293
Therefore, it was proposed that quercetin inhibited the adhesion of MPO to the vascular wall (Fig.
294
7), and subsequently attenuated endothelial injury in inflammatory vasculature via inhibition of
295
vascular-bound MPO-mediated HOCl formation. Moreover, quercetin did not induce cytotoxicity
296
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
298
and azides),2,
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bioapplication in vivo.
12, 37
the nontoxic effects and natural source for quercetin could increase their
300
It should be noted that quercetin detected in plasma is mostly in conjugated forms under
301
normal physiological conditions, and quercetin aglycone (i.e. free quercetin) is only found in the
302
sub-micromolar concentration range.21, 24, 26, 38 In tissues, however, quercetin aglycone can be the
303
predominant form. It has been reported that both monocytes and macrophages can metabolize
304
quercetin glucuronides to the parent aglycone compound, quercetin.24,
305
information, therefore, it was proposed that quercetin could inhibit the adhesion and infiltration of
306
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
312
and protected HUVEC from MPO/neutrophil-induced injury (Fig. 8), quercetin was an effective
313
inhibitor for mediating MPO-dependent endothelial dysfunction in inflammatory vasculature.
314
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
316
different from the widespread anti-oxidant and anti-inflammatory mechanisms, i.e. radical
317
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
322
The authors declare no competing financial interest.
323
Acknowledgments
324
Financial support from the National Natural Science Foundation of China (Nos. 31760255,
325
31560255, 31260216, 31100608), the Natural Science Foundation of Jiangxi province (No.
326
20171BCB23041, 20161BAB215215).
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References
329
(1) van der Veen, B.S.; de Winther, M.P.; Heeringa, P. Myeloperoxidase: molecular mechanisms of
330
action and their relevance to human health and disease. Antioxid. Redox. Signal. 2009, 11,
331
2899-2937.
332
(2) Malle, E.; Furtmüller, P.G.; Sattler, W.; Obinger, C. Myeloperoxidase: a target for new drug
333
development? Br. J. Pharmacol. 2007, 152, 838-854.
334
(3) Vlasova, I.; Sokolov, A.V.; Arnhold, J. The free amino acid tyrosine enhances the chlorinating
335
activity of human myeloperoxidase. J. Inorg. Biochem. 2012, 106, 76-83.
336
(4) Lu. N.; Ding, Y.; Tian, R.; Peng, Y.Y. Inhibition of myeloperoxidase-mediated oxidative
337
damage by nitrite in SH-SY5Y cells: Relevance to neuroprotection in neurodegenerative diseases.
338
Eur. J. Pharmacol. 2016, 780, 142-147.
339
(5) Hazen, S.L.; Heinecke, J.W. 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed
340
oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic
341
intima. J. Clin. Invest. 1997, 99, 2075-2081.
342
(6) Zhang, C.; Yang, J.; Jacobs, J.D.; Jennings, L.K. Interaction of myeloperoxidase with vascular
343
NAD(P)H oxidase-derived reactive oxygen species in vasculature: implications for vascular
344
diseases. Am. J. Physiol. Heart Circ. Physiol. 2003, 285, H2563-2572.
345
(7) Eiserich, J.P.; Baldus, S.; Brennan, M.L.; Ma, W.; Zhang, C.; Tousson, A.; Castro, L.; Lusis,
346
A.J.; Nauseef, W.M.; White, C.R.; Freeman, B.A. Myeloperoxidase, a leukocyte-derived vascular
347
NO oxidase. Science. 2002, 296, 2391-2394.
348
(8) Tian, R.; Ding, Y.; Peng, Y.Y.; Lu, N. Myeloperoxidase amplified high glucose-induced
349
endothelial dysfunction in vasculature: Role of NADPH oxidase and hypochlorous acid. Biochem.
350
Biophys. Res. Commun. 2017, 484, 572-578.
351
(9) Xu, J.; Xie, Z.; Reece, R.; Pimental, D.; Zou, M.H. Uncoupling of endothelial nitric oxidase
352
synthase by hypochlorous acid: role of NAD(P)H oxidase-derived superoxide and peroxynitrite.
353
Arterioscler. Thromb. Vasc. Biol. 2006, 26, 2688-2695.
354
(10) Wang, Q.; Xie, Z.; Zhang, W.; Zhou, J.; Wu, Y.; Zhang, M.; Zhu, H.; Zou, M.H.
355
Myeloperoxidase deletion prevents high-fat diet-induced obesity and insulin resistance. Diabetes.
356
2014, 63, 4172-4185. 14
ACS Paragon Plus Environment
Page 14 of 29
Page 15 of 29
Journal of Agricultural and Food Chemistry
357
(11) Lu, N.; Xie, S.; Li, J.; Tian, R.; Peng, Y.Y. Myeloperoxidase-mediated oxidation targets serum
358
apolipoprotein A-I in diabetic patients and represents a potential mechanism leading to impaired
359
anti-apoptotic activity of high density lipoprotein. Clin. Chim. Acta. 2015, 441, 163-170.
360
(12) Zhang, H.; Jing, X.; Shi, Y.; Xu, H.; Du, J.; Guan, T.; Weihrauch, D.; Jones, D.W.; Wang,
361
W.; Gourlay, D.; Oldham, K.T.; Hillery, C.A.; Pritchard, K.A Jr. N-acetyl lysyltyrosylcysteine
362
amide inhibits myeloperoxidase, a novel tripeptide inhibitor. J. Lipid Res. 2013, 54, 3016-3029.
363
(13) Forbes, L.V.; Sjögren, T.; Auchère, F.; Jenkins, D.W.; Thong, B.; Laughton, D.; Hemsley,
364
P.; Pairaudeau, G.; Turner, R.; Eriksson, H.; Unitt, J.F.; Kettle, A.J. Potent reversible inhibition of
365
myeloperoxidase by aromatic hydroxamates. J. Biol. Chem. 2013, 288, 36636-36647.
366
(14) Havsteen, B.H. The biochemistry and medical significance of the flavonoids. Pharmacol.
367
Ther. 2002, 96, 67-202.
368
(15) Heim. K.E.; Tagliaferro, A.R.; Bobilya, D.J. Flavonoid antioxidants: chemistry, metabolism
369
and structure–activity relationships. J. Nutr. Biochem. 2002, 13, 572-584.
370
(16) Tian, R.; Ding, Y.; Peng, Y.Y.; Lu, N. Inhibition of Myeloperoxidase- and
371
Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by
372
(-)-Epigallocatechin gallate. J. Agric. Food Chem. 2017, 65, 3198-3203.
373
(17)
374
M.; Kawabata, K.; Ishisaka, A.; Terao, J.; Kawai, Y. Flavonoids as substrates and inhibitors of
375
myeloperoxidase: molecular actions of aglycone and metabolites. Chem. Res.Toxicol. 2008, 21,
376
1600-1609.
377
(18) Meotti, F.C.; Senthilmohan, R.; Harwood, D.T.; Missau, F.C.; Pizzolatti, M.G.; Kettle, A.J.
378
Myricitrin as a substrate and inhibitor of myeloperoxidase: implications for the pharmacological
379
effects of flavonoids. Free Radic. Biol. Med. 2008, 44, 109-120.
380
(19) Pincemail, J.; Deby, C.; Thirion, A.; de Bruyn-Dister, M.; Goutier, R. Human
381
myeloperoxidase activity is inhibited in vitro by quercetin. Comparison with three related
382
compounds. Experientia. 1988, 44, 450-453.
383
(20) Zholobenko, A.; Mouithys-Mickalad, A.; Modriansky, M.; Serteyn, D.; Franck, T.
384
Polyphenols from Silybum marianum inhibit in vitro the oxidant response of equine neutrophils
385
and myeloperoxidase activity. J. Vet. Pharmacol. Ther. 2016, 39, 592-601.
Shiba,
Y.; Kinoshita,
T.; Chuman,
H.; Taketani,
Y.; Takeda,
15
ACS Paragon Plus Environment
E.; Kato,
Y.; Naito,
Journal of Agricultural and Food Chemistry
386
(21) Boots, A.W.; Haenen, G.R.; Bast, A. Health effects of quercetin: from antioxidant to
387
nutraceutical. Eur. J. Pharmacol. 2008, 585, 325-337.
388
(22) Kawai, Y.; Nishikawa, T.; Shiba, Y.; Saito, S.; Murota, K.; Shibata, N.; Kobayashi, M.;
389
Kanayama, M.; Uchida, K.; Terao, J. Macrophage as a target of quercetin glucuronides in human
390
atherosclerotic arteries: implication in the anti-atherosclerotic mechanism of dietary flavonoids. J.
391
Biol. Chem. 2008, 283, 9424-9434.
392
(23) Kukongviriyapan, U.; Sompamit, K.; Pannangpetch, P.; Kukongviriyapan, V.; Donpunha, W.
393
Preventive and therapeutic effects of quercetin on lipopolysaccharide-induced oxidative stress and
394
vascular dysfunction in mice. Can. J. Physiol. Pharmacol. 2012, 90, 1345-1353.
395
(24) Shen, Y.; Ward, N.C.; Hodgson, J.M.; Puddey, I.B.; Wang, Y.; Zhang, D.; Maghzal, G.J.;
396
Stocker, R.; Croft, K.D. Dietary quercetin attenuates oxidant-induced endothelial dysfunction and
397
atherosclerosis in apolipoprotein E knockout mice fed a high-fat diet: a critical role for heme
398
oxygenase-1. Free Radic. Biol Med. 2013, 65, 908-915.
399
(25) Shen, Y.; Croft, K.D.; Hodgson, J.M.; Kyle, R.; Lee, I.L.; Wang, Y.; Stocker, R.; Ward, N.C.
400
Quercetin and its metabolites improve vessel function by inducing eNOS activity via
401
phosphorylation of AMPK. Biochem. Pharmacol. 2012, 84, 1036-1044.
402
(26) Lotito, S.B.; Zhang, W.J.; Yang, C.S.; Crozier, A.; Frei, B. Metabolic conversion of dietary
403
flavonoids alters their anti-inflammatory and antioxidant properties. Free Radic. Biol. Med. 2011,
404
51, 454-463.
405
(27) Dypbukt, J.M.; Bishop, C.; Brooks, W.M.; Thong, B.; Eriksso, H.; Kettle, A.J. A sensitive
406
and selective assay for chloramine production by myeloperoxidase. Free Radic. Biol. Med. 2005,
407
39, 1468-1477.
408
(28) Sedlak, J.; Lidsay, R.H. Estimation of total protein bound and nonprotein sulfhydryl group in
409
tissue with Ellman’s reagent. Anal. Biochem. 1968, 25, 192-205.
410
(29) Lu, N.; Li, J.; Tian, R.; Peng, Y.Y. Binding of human serum albumin to single-walled carbon
411
nanotubes activated neutrophils to increase production of hypochlorous acid, the oxidant capable
412
of degrading nanotubes. Chem. Res. Toxicol. 2014, 27, 1070-1077.
413
(30) Lu, N.; Ding, Y.; Yang, Z.; Gao, P. Effects of rutin on the redox reactions of hemoglobin. Int.
414
J. Biol. Macromol. 2016, 89, 175-180. 16
ACS Paragon Plus Environment
Page 16 of 29
Page 17 of 29
Journal of Agricultural and Food Chemistry
415
(31) Gau, J.; Furtmüller, P.G.; Obinger, C.; Prévost, M.; Van Antwerpen, P.; Arnhold, J.; Flemmig,
416
J. Flavonoids as promoters of the (pseudo-)halogenating activity of lactoperoxidase and
417
myeloperoxidase. Free Radic. Biol. Med. 2016, 97, 307-319.
418
(32) Golubinskaya, V.; Brandt-Eliasson, U.; Gan, L.M.; Kjerrulf, M.; Nilsson, H. Endothelial
419
function in a mouse model of myeloperoxidase deficiency. Biomed Res. Int. 2014, 2014, 128046.
420
(33) Ward, J.; Spath, S.N.; Pabst, B.; Carpino, P.A.; Ruggeri, R.B.; Xing, G.; Speers, A.E.; Cravatt,
421
B.F.; Ahn, K. Mechanistic characterization of a 2-thioxanthine myeloperoxidase inhibitor and
422
selectivity assessment utilizing click chemistry--activity-based protein profiling. Biochemistry.
423
2013, 52, 9187-9201.
424
(34) Gebicka, L.; Banasiak, E. Hypochlorous acid-induced heme damage of hemoglobin and its
425
inhibition by flavonoids. Toxicol In Vitro. 2012, 26, 924-929.
426
(35) Krych-Madej, J.; Stawowska, K.; Gebicka, L. Oxidation of flavonoids by hypochlorous acid:
427
reaction kinetics and antioxidant activity studies. Free Radic. Res. 2016, 50, 898-908.
428
(36) Ruiz, M.J.; Fernández, M.; Picó, Y.; Mañes, J.; Asensi, M.; Carda, C.; Asensio, G.; Estrela,
429
J.M. Dietary administration of high doses of pterostilbene and quercetin to mice is not toxic. J.
430
Agric. Food Chem. 2009, 57, 3180-3186.
431
(37) Békési, G.; Heinle, H.; Kakucs, R.; Pázmány, T.; Szombath, D.; Dinya, M.; Tulassay, Z.;
432
Fehér, J.; Rácz, K.; Székács, B.; Riss, E.; Farkas, A.; Gódor, F.; Illyés, G. Effect of inhibitors of
433
myeloperoxidase on the development of aortic atherosclerosis in an animal model. Exp. Gerontol.
434
2005, 40, 199-208.
435
(38) Li, C.; Zhang, W.J.; Frei, B. Quercetin inhibits LPS-induced adhesion molecule expression
436
and oxidant production in human aortic endothelial cells by p38-mediated Nrf2 activation and
437
antioxidant enzyme induction. Redox Biol. 2016, 9, 104-113.
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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