Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent

Article Cite This: J. Agric. Food Chem. 2018, 66, 4933−4940

pubs.acs.org/JAFC

Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury Naihao Lu,*,† Yinhua Sui,† Rong Tian,*,† and Yi-Yuan Peng† †

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 330022, China S Supporting Information *

ABSTRACT: Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures (P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells (P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas (P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPOmediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation. KEYWORDS: myeloperoxidase, quercetin, hypochlorous acid, neutrophils, endothelial cells high density lipoprotein.11 As the major product of MPO, HOCl plays the crucial roles in endothelial dysfunction through the induction of cell death and endothelial nitric oxidase synthase uncoupling, and decrease of nitric oxide (NO) bioavailability, etc.9 The activation, adhesion, and infiltration of neutrophils into the vessel wall are an important component in the development of vascular endothelial dysfunction.6−8 MPO, MPO-derived 3-chlorotyrosine, and HOCl-modified low density lipoprotein are highly detected in human and animal atherosclerotic vessels.1,5 Thus, MPO is usually considered as a biomarker of inflammation and cardiovascular diseases.1,10 Some inhibitors (hydrazides, azides, and hydroxamic acids) have been developed to inhibit the deleterious effects of excessive MPO activity and subsequently prevent HOCldependent oxidative damage.2,12,13 Although these inhibitors have effective abilities to inhibit MPO activity in vitro,2,12 they are potentially toxic and therefore are undesirable therapeutic drugs in medical use. Epidemiological studies have suggested that the high consumption of flavonoid-rich food would result in a low incidence of cardiovascular diseases.14,15 Usually, a variety of mechanisms including radical scavenging, metal-chelating, antiinflammatory, and antioxidant properties have been demonstrated for the beneficial effects of these polyphenols in vitro

1. INTRODUCTION Myeloperoxidase (MPO) is abundantly expressed in activated neutrophils and in certain types of macrophages that play important roles in bacterial killing and host defense.1,2 Neutrophils are rapidly recruited during the inflammation process and secrete MPO. As a heme peroxidase, ferric MPO reacts with H2O2 to form compound I, which could be reduced back to the ferric state either directly during Cl− oxidation or peroxidase cycle (Figure 1A).1,3,4 MPO plays the physiological role in bacterial killing by generating hypochlorous acid (HOCl). However, evidence has emerged that excessive MPO intermediates and HOCl contribute to tissue injury and the initiation and propagation of inflammatory disease.1,5−11 MPO oxidizes Tyr 192 of apolipoprotein A-I, leading to impaired cholesterol efflux of

Figure 1. (A) Peroxidase and chlorination cycles of MPO.1,3,4,16 MPO catalyzes two competitive oxidative reactions: Cl − to OCl − (chlorination cycle) and flavonoid to quinone and dimer (peroxidase cycle). (B) Schematic structure of quercetin. © 2018 American Chemical Society

Received: Revised: Accepted: Published: 4933

March 25, 2018 April 27, 2018 April 30, 2018 April 30, 2018 DOI: 10.1021/acs.jafc.8b01537 J. Agric. Food Chem. 2018, 66, 4933−4940

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Journal of Agricultural and Food Chemistry and vivo.14−16 Flavonoids have been proposed to limit MPO activity because these polyphenols could act as reducing agents and compete with the other substrates (such as Cl−) for reactive MPO compounds (Figure 1A).16−20 It has been shown that a metabolite of quercetin, one of the most abundant flavonoids in the human diet,21 specifically accumulates in activated macrophages expressing MPO in human atherosclerotic arteries, but not in the normal aorta.17,22 Therefore, we presumed that this flavonoid could prevent cardiovascular diseases through inhibition of MPO activity, but the molecular mechanisms were still not demonstrated. Quercetin is a flavonoid that occurs naturally in plants and is widely used as a nutritional supplement due to its antioxidant and anti-inflammatory properties.17,21−26 In this study, we were interested to investigate the effects of quercetin (Figure 1B) on MPO-induced HOCl formation and cytotoxicity to vascular endothelial cells. Here, quercetin was found to be an effective inhibitor on MPO-mediated HOCl formation and counteracted the deleterious effects of MPO on endothelial injury in inflammatory vasculature.

saline; (II) the quercetin (Qu) group; (III) the LPS group (animal models of inflammation) treated with LPS at 10 mg/kg (i.p.); and (IV−V) the LPS + Qu groups treated with Qu (20 and 50 mg/kg, i.p.) 1 h before LPS administration. After 6 h treatment, aortas from these animals were isolated and cut into individual ring segments. Acetylcholine (ACh) was added to induce endothelium-dependent relaxation (vascular endothelial function), as previously described.7,8 Meanwhile, the expression and activity of MPO was determined as described previously.8 Aortic rings from control mice were incubated with quercetin (20 μM) for 2 h, washed with vehicle, and then treated with 50 μM HOCl for 30 min. Then the vascular function was measured described above.7,8 2.7. Statistical Analysis. The results were presented as the means ± SD of at least three independent experiments. One-way ANOVA was performed for statistical analyses, and P < 0.05 was considered significant.

3. RESULTS 3.1. Quercetin Inhibited MPO-Catalyzed HOCl Production and Cytotoxicity in HUVEC. First, we used HUVEC to confirm that MPO system could cause significant cell injuries by generating HOCl. Glucose oxidase/glucose system was used to generate H2O2,4 and neither H2O2 nor MPO alone barely decreased the cell viability. However, the presence of both MPO and H2O2 could result in significant loss of cell viability, and the decrease of cell viability was effectively inhibited by 4-aminobenzoic acid hydrazide (ABAH, a wellknown MPO inhibitor) (Figure 2). Then we investigated if quercetin could protect endothelial cells from MPO-dependent injury. It was found that quercetin could dose-dependently inhibit MPO-induced cytotoxicity to HUVEC (Figure 2). Moreover, quercetin at the concentration

2. MATERIALS AND METHODS 2.1. Chemicals. Myeloperoxidase (MPO), taurine, sodium hypochlorite (NaOCl), glucose oxidase, and lipopolysaccharide (LPS) were purchased from Sigma. Quercetin (Qu, purity> 98%) and rutin (Ru, purity> 98%) were purchased from Shanxi Huike Botanical Development Co. Ltd. 2.2. Effect of Quercetin on MPO Activity. MPO chlorinating activity was measured by taurine chloramine formation.3,4,12,17 In the absence or presence of quercetin, H2O2 (500 μM) was added to a solution containing NaCl (100 mM), MPO (0.6 μM) and taurine (1 mM). Then the taurine chloramine was measured by 3,3′,5,5′tetramethylbenzidine (TMB) method.12,27 HOCl was significantly generated in short time when MPO-H2O2 were used at high concentrations, and high concentrations of quercetin and MPO were, therefore, selected in this study.3,4,8,16 Cys-thiol levels in proteins were determined spectrophotometrically by 5,5′-dithiobis (2-nitrobenzoic) acid method, and the change of absorption at 412 nm would reflect the change of thiol contents.28 Neutrophil cells were cultured with different concentrations of quercetin,16,29 and these cells were stimulated with LPS (0.2 mg/mL) for 60 min. Then the formation of HOCl was determined by taurine chloramine assay. 2.3. Interaction between MPO and Quercetin by Molecular Docking. The original X-ray structure of human MPO (PDB ID: 5FIW) was selected and one monomer (chains B and D) of protein was kept. Quercetin or rutin was docked to MPO by AutoDock software.16,29−31 2.4. Effect of Quercetin on MPO-Induced Human Umbilical Vein Endothelial Cells (HUVEC) Injury. HUVEC were cultured in DMEM containing NaCl (100 mM), glucose (5.6 mM). Different amounts of quercetin were first added to cells for 5 min, and then MPO (1.5 U/mL) and glucose oxidase (10 mU/mL) were added and maintained for 2 additional hours. In the presence of neutrophils, HUVEC were cultured with quercetin and stimulated with LPS (0.2 mg/mL) for 2 h. Then HOCl generation and cell viability were measured by taurine chloramine and MTT method, respectively. 2.5. Effect of Quercetin on HOCl-Mediated HUVEC Injury. Different concentrations of quercetin were first added to HUVEC cells for 5 min. Then NaOCl (80 μM) was added and cells were maintained for 20 min. NO formation and cell viability were measured by commercial kit and MTT method, respectively. 2.6. Vascular Endothelial Function in Inflammation and in Vitro. To further investigate the potential relevance of quercetin to endothelial function in vivo, inflammation in male mice was induced by intraperitoneally injection of LPS and the dose of LPS used in this study (10 mg/kg) was sufficient to cause endotoxaemia.23,26 Mice were randomly divided into five groups: (I) the control group treated with

Figure 2. Cytotoxicity of MPO and the protective effects of quercetin. HUVEC were cultured in DMEM containing NaCl (100 mM), glucose (5.6 mM). Different concentrations of quercetin were preincubated with cells for 5 min. In the absence or presence of MPO inhibitor (ABAH, 50 μM), MPO and glucose oxidase (10 mU/ mL) were then added and incubated for 2 h. H2O2 was generated by glucose oxidase/glucose system. The blank values were set to 100%, to which other values were compared. Values are means ± SD of three independent determinations, ∗∗P < 0.01, ∗P < 0.05 compared to control group (MPO/H2O2 added). 4934

DOI: 10.1021/acs.jafc.8b01537 J. Agric. Food Chem. 2018, 66, 4933−4940

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Journal of Agricultural and Food Chemistry

Figure 3. Effects of quercetin on (A) MPO-dependent HOCl formation and (B) scavenging of HOCl. The HOCl generation by MPO/H2O2/Cl− system or remaining HOCl was determined in the presence of quercetin. Values are means ± SD of three independent determinations, ∗P < 0.05, ∗∗P < 0.01 compared to respective control (MPO/H2O2/Cl−(or HOCl)-treated) group.

Figure 4. Docking of quercetin in the active site of MPO (PDB ID: 5FIW). (A) Quercetin on the top of heme of MPO active center and (B) A and C-ring of quercetin was almost parallel to the distal heme pocket of MPO. The blue molecule was the heme of MPO and the central iron ion was shown as a purple sphere, the red molecule was quercetin. The distance of A-ring to the distal pyrrole ring and the central iron ion of the enzymatic heme was 5.0 and 5.6 Å, respectively, in this lowest energy and best bound conformation. (C) Interactions of quercetin with ligand amino acid in MPO. The amino acid residues were shown as green.

used (up to 50 μM) did not show cytotoxicity to HUVEC (Figure S1). In addition, we investigated if quercetin could inhibit MPOmediated HOCl formation in vitro. As shown in Figure 3A, quercetin could inhibit MPO-catalyzed HOCl production in a dose-dependent manner. Compared with MPO-catalyzed HOCl production, quercetin at 5 and 10 μM reduced HOCl production by 16 and 25%, respectively. Then we mixed quercetin (5 and 10 μM) with HOCl and analyzed the remaining HOCl content. Our data showed that quercetin at 10 μM significantly scavenged HOCl by 16% (Figure 3B), which was less than the reduction of MPO-mediated HOCl production by quercetin at the same concentration (25%,

Figure 3A). Meanwhile, quercetin at 5 μM did not scavenge HOCl (Figure 3B), while quercetin at this concentration could effectively inhibit MPO-mediated HOCl formation (Figure 3A), demonstrating that inhibition of MPO activity was the prior pathway for quercetin at low concentration. These results suggested that quercetin at high concentration reduced HOCl generation by both scavenging HOCl and inhibiting MPO activity, and significant inhibition of MPO-mediated HOCl generation was achieved by quercetin at low concentration (5 μM) that could not be explained as scavenging HOCl. Consistent with the protective effects on MPO cytotoxicity, quercetin effectively inhibited MPO-catalyzed HOCl production even in the presence of HUVEC (data not shown). 4935


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Journal of Agricultural and Food Chemistry MPO has 12 cysteine residues per monomer and these cysteine residues are critical to MPO activity.1,2 Quercetin could be readily oxidized by MPO system to form quinone.17 However, no information has been available concerning the possible interaction between MPO cysteine residues and the oxidation products of quercetin. Therefore, we measured the loss of Cys-thiol contents in MPO to assess whether a covalent binding of oxidized quercetin to cysteine residues was formed. As shown in Figure S2, incubation of MPO with H2O2 resulted in a significant decrease of protein thiol group, and the addition of quercetin further decreased the level of Cys-thiol in MPO. Control experiments demonstrated that quercetin alone did not influence the level of Cys-thiol in MPO (data not shown). 3.2. Docking of Quercetin to MPO. Figure 4A showed that quercetin fit in the active site of MPO. This simulation showed that quercetin could be oriented in such a way that the A- and C-ring was above the iron-heme (active site) of MPO (Figure 4B) with the shortest distance of 5.0 Å. These results showed that quercetin directly bound into the iron-heme site of MPO. As revealed by the docking, the homologous and conserved amino acids Arg239 and Phe407 could form noncovalent interactions with quercetin (Figure 4C). The flavonoid A-ring of quercetin stacked onto the active heme cavity. In addition, the phenyl groups of A-ring formed hydrophobic interaction with Arg239. However, these amino acid residues (Arg239 and Phe407) are near the catalytic heme center of MPO.2,17,31 Therefore, quercetin interacted with the active heme site and might block the substrate channel, providing the possible theoretical explanation for its inhibition on MPO activity. 3.3. Quercetin Inhibited MPO Activity in Neutrophils and Protected HUVEC from Neutrophils-Induced Injury. The release of MPO was induced by LPS in neutrophil cells,3,29 and the effects of quercetin on MPO-induced HOCl production were investigated. LPS-stimulated neutrophils could generate high level of HOCl, while little HOCl was produced by unstimulated (i.e., resting) neutrophils. Then quercetin effectively inhibited HOCl formation in activated neutrophils (Figure 5A). Moreover, quercetin also inhibited ROS formation from activated neutrophils (data not shown). These data suggested that quercetin inhibited MPO activity in activated neutrophils. Similar to MPO/H2O2/Cl−-induced cytotoxicity in HUVEC, incubation of LPS-activated neutrophils with endothelial cells also induced significant loss of cell viability (Figure 5B). Quercetin protected cytotoxicity from activated neutrophils as well. In addition, quercetin inhibited these HOCl formations by activated neutrophils (data not shown). These results confirmed that quercetin inhibited MPO-mediated HOCl formation even in the existence of HUVEC. 3.4. Quercetin Inhibited HOCl-Induced Endothelial Dysfunction in HUVEC. To further demonstrate the effects of quercetin on MPO-mediated cell injury, effects of quercetin on HOCl-induced endothelial dysfunction were examined (Figure 6A). OCl− (80 μM) significantly caused the loss of cell viability, and the presence of quercetin before OCl− addition could dosedependently inhibit OCl−-mediated cytotoxicity. However, if OCl− was first incubated with the cells for 10 min followed by quercetin addition, quercetin could not reverse OCl−-induced loss of cell viability (Figure 6A). Reduced production or availability of NO is a common feature of endothelial dysfunction.9 Consistent with the loss of cellular viability, HOCl could decrease NO formation in

Figure 5. Effects of quercetin on (A) neutrophil-dependent HOCl formation and (B) cytotoxicity to HUVEC. (A) In the absence or presence of LPS stimulation, the generation of HOCl was assessed in quercetin-preincubated neutrophils. (B) Neutrophils were added into HUVEC in the presence of quercetin. The cells were stimulated with LPS for 2 h. Values are means ± SD of three independent determinations, (A) ∗P < 0.05 compared to activated neutrophilstreated group; (B) ∗∗P < 0.01 compared to Blank group (no neutrophils/quercetin added), P < 0.05 compared to neutrophilstreated group.

endothelial cells (Figure 6B). However, the addition of quercetin could inhibit HOCl-induced decrease of NO formation. Therefore, these results demonstrated that quercetin could inhibit HOCl-mediated endothelial dysfunction in HUVECs such as the decreases in cell viability and NO formation (Figure 6). 3.5. Effects of Rutin on MPO-Dependent HOCl Production in Vitro. As the glycoside of quercetin, rutin is also commonly found in the human diet.30 Compared with quercetin, rutin showed less effective effects on MPO-catalyzed HOCl production in vitro (Figure S3), which was consistent with the weaker free radical scavenging ability of rutin.14,19,30 Moreover, docking study demonstrated that the strong binding of rutin to MPO was also observed, and the B-ring of rutin was near to the heme of MPO with the shortest distance of 6.3 Å (Figure S4), which was longer than the shortest distance of Aring of quercetin to the active heme center of MPO (5.6 Å) (Figure 4). Consistent with the inhibitory effect on MPOdependent HOCl formation, rutin also inhibited MPO/ neutrophil-mediated cytotoxicity to HUVEC (data not shown). 3.6. Quercetin Attenuated Aortic Endothelial Dysfunction in Inflammation and in Vitro. There is evidence 4936


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Figure 6. Effects of quercetin on NaOCl-mediated endothelial dysfunction, that is, (A) loss of cellular viability and (B) decrease of NO formation. Different concentrations of quercetin were added to HUVEC for 5 min before NaOCl addition. Control group represented that cells were incubated with NaOCl alone. Values are means ± SD of three independent determinations, (A) ∗P < 0.05, ∗∗P < 0.01 compared to control group (NaOCl added); (B) P < 0.05, P < 0.01 compared to respective control group.

that vascular-bound MPO is a potent inducer for vascular injury.6−8 As shown in Figure 7A, MPO expression and activity in normal aortas was very low. However, MPO expression and activity in aortas from LPS-treated mice was significantly higher, presumably demonstrating that MPO was secreted into the blood vessels by activated neutrophils and then permeated vascular tissue in inflammation. Consistent with the increased MPO expression and activity, the ACh-mediated vessel relaxation (determined as vascular endothelial function) was significantly impaired in aortas from LPS-treated mice (Figure 7B). Therefore, MPO contributed importantly to endothelial dysfunction associated with inflammation produced by LPS. In contrast to the causal role of MPO in vascular endothelial dysfunction,6−8 a recent study reported that MPO did not induce endothelial dysfunction after LPS treatment.32 These different conclusions might be related to gender and age differences.32 However, compared with LPS-treated mice, the pretreatment of quercetin dose-dependently improved the vascular endothelial function (Figure 7B) and attenuated the increase of MPO activity and expression (Figure 7A) in inflammatory vasculature, demonstrating that quercetin attenuated the adhesion and infiltration of MPO to the vascular wall and subsequently inhibited MPO activity in vivo. Control experiments demonstrated no significant effects of quercetion on aortic endothelial function and MPO in normal mice. To investigate the effect of quercetin on HOCl-mediated endothelial dysfunction, the

Figure 7. Effects of quercetin on (A) MPO activity and expression in the aorta and (B) ACh-mediated relaxations (determined as vascular endothelial function) of aortic rings from mice treated with LPS for 6 h. Quercetin (20 and 50 mg/kg, i.p.) was injected 1 h before LPS injection (10 mg/kg, i.p.). Insert A: MPO expression in the presence of 50 mg/kg Qu. Values are means ± SD of three independent determinations, ∗∗P < 0.01 compared to control group; ##P < 0.01, #P < 0.05 compared to LPS group. (C) Effects of quercetin on NaOCl-mediated vascular function in isolated mouse aortas. Quercetin was incubated with isolated aortas for 2 h before NaOCl addition. ∗∗P < 0.01 compared to control group (no Qu/NaOCl added); ##P < 0.01 compared to NaOCl-treated group.

flavonoid was pretreated with vessel for 2 h and then washed away before the addition of HOCl. It was found that HOCl alone could inhibit ACh-mediated vessel relaxation and pretreatment with quercetin effectively prevented HOClmediated endothelial dysfunction in isolated aortas (Figure 7C). These data revealed that quercetin might attenuate vascular endothelial dysfunction during inflammation by inhibiting MPO-dependent HOCl formation and endothelial dysfunction in vivo. 4937


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4. DISCUSSION As a widely present polyphenol flavonoid, quercetin exhibits numerous beneficial effects against various diseases.16,17,21−26 Our results showed that quercetin inhibited MPO (or neutrophils)-mediated HOCl formation in vitro (Figures 3 and 5A). Quercetin protected endothelial cells from MPO (or neutrophil)-induced damage through inhibition of MPO activity, while it effectively inhibited HOCl-induced endothelial dysfunction in HUVEC (Figure 6). The protection of quercetin was observed in LPS-induced vascular endothelial dysfunction in parallel with the decrease of MPO activity in vivo, but our experiments did not confirm that there was a causal relationship between both effects. Therefore, quercetin exhibited the therapeutic ability in vivo through mediating MPO-dependent endothelial dysfunction and vascular inflammation (Figure 7). The inhibitive effects of quercetin on MPO-catalyzed HOCl production in vitro and in activated neutrophils were first demonstrated. The protective effect on MPO-mediated HOCl formation was related to the reducing reactivity of quercetin toward MPO redox intermediates.16−18 As a one-electron reducing substrate of MPO, quercetin has been found to react with both reactive compounds, and the second order rate constants for its reaction with MPO compound II was 7.0 × 106 M−1 s−1, which was higher than other flavonoids such as (−)-epicatechin (3.5 × 106 M−1 s−1) and (±)-eriodictyol (1.3 × 106 M−1 s−1).31 Consistent with this result herein, several studies have shown that some other flavonoids can also inhibit MPO chlorination activity by acting as peroxidase substrates.16−18 Therefore, quercetin was an effective substrate for MPO intermediates and significantly competed with Cl− for MPO. Moreover, docking studies showed that the A-ring bound to the active heme pocket (Figure 4A,B). The hydrophobic interactions between A-, C-ring in quercetin and amino acid residues (Phe407, Arg 239) were present (Figure 4C). As the part of the catalytic sites, both Arg 239 and Phe407 are located close to active heme iron.2,17,29 Quercetin interacted with the hydrophobic region at active heme pocket and served as substrates for the MPO.16−18 Therefore, quercetin acted as a competitive inhibitor to block other substrate to access the active site of MPO. In addition, 2-thioxanthine derivatives have recently been reported to irreversibly inhibit MPO activity by covalently modifying the heme prosthetic group of the enzyme.33 It was possible that the binding of quercetin to the heme pocket might exhibit the similar mechanism for inactivation by 2-thioxanthine derivatives. However, this presume should be experimentally verified in future. Modifications in the quercetin structure result in different inhibitory effects. Rutin, differing from quercetin by the 3hydroxyl group in the C ring, was less effective at inhibiting MPO activity than its parent molecule (Figure S3). Moreover, it was interesting to note that the substitution of 3-hydroxyl group by more hydrophilic group (i.e., rhamnosylglucoside) dramatically showed the longer distance to active center heme of MPO, as compared to quercetin which was a lipophilic compound (Figures S3 and S4). It appears that hydrophobicity is an essential requirement for the inhibitive effect of MPO.16−18 It was likely that in compounds having the same basic chemical structure, the effective inhibition on MPO activity was related to the C3 hydroxylation and the glycoside in the flavonoid structure.19 The presence of rutinose at position C-3 in rutin would cause the reduction in the access to

the active heme site of MPO and consequently resulted in the less effective effects on MPO activity. On the other hand, quercetin could be readily oxidized by MPO system and the presence of a quinone as the main intermediate in the oxidation of quercetin was confirmed by demonstrating that glutathione (GSH) formed a hydroquinone conjugate with oxidized flavonoid.17,18 Thereafter, we were concerned the interaction between MPO cysteine residues and the oxidation products of quercetin. Compared with the level of Cys-thiol in H2O2-incubated MPO, oxidation of quercetin promoted the loss of Cys-thiol content in MPO (Figure S2), which was accompanied by the inhibition of MPO activity (Figure 3A). Therefore, besides its role as a cosubstrate of MPO in reducing the access of other substrates to the active site of MPO, quercetin may irreversibly inactivated MPO via formation of covalent bond(s) between oxidized quercetin (quinone form) and cysteine residues. However, this presume was not verified and the identification of quercetin derivatives with MPO cysteine residues were not shown in this study and need further study. There are at least two pathways to ameliorate inflammatory injury caused by MPO, the scavenging of HOCl and the inhibition of the enzyme itself.2 Of course, we showed a certain HOCl-scavenging effect of quercetin at high concentration (Figure 3). The second order rate constant for the reaction between quercetin and HOCl is 1.4 × 105 M−1 s−1.34,35 Thus, the reaction between this MPO compound II and flavonoid (7.0 × 106 M−1 s−1) most likely prevails over that between HOCl and quercetin. Besides HOCl scavenging ability, quercetin could inhibit MPO-induced injury by participating in the regulation of HOCl generation. There is evidence that neutrophils-derived MPO plays an important role in endothelial dysfunction and vascular injury by its ability to produce reactive HOCl.6−8 Because endothelial cells do not express MPO, the activated neutrophils-released MPO can bind and infiltrate into the vascular wall directly (Figure 7A). In this study, we found that quercetin effectively inhibited MPO/neutrophils-induced cytotoxicity in endothelial cells without causing any toxicity (Figures 2 and 5). Moreover, quercetin prevented HOCl from causing injuries to endothelial dysfunction such as loss of cell viability and decrease of nitric oxide formation (Figure 6). Consistent with these in vitro data, quercetin attenuated LPS-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid improved endothelial function of HOCl-treated aortic rings in vitro. In vivo, quercetin inhibited the adhesion of MPO (or neutrophil) to the vascular wall and therefore attenuated MPO activity (Figure 7A and B). On the other hand, quercetin was incubated with isolated aortic rings for 2 h and the flavonoid was then washed away before the addition of HOCl. It was found that the “quercetin-washed” aortas also effectively prevented HOCl-induced endothelial dysfunction (Figure 7C). It could be revealed that quercetin could also adhere and infiltrate to the vascular wall and influence MPO/HOClinduced endothelial dysfunction. Therefore, it was proposed that quercetin inhibited the adhesion of MPO to the vascular wall (Figure 7), and subsequently attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation. Moreover, quercetin did not induce cytotoxicity to HUVEC (Figure S1) and dietary administration of high doses of quercetin to mice was not toxic.36 Compared with the toxic side effects of other effective inhibitors (hydroxamic acids, hydrazides and azides),2,12,37 the 4938


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represent a novel mechanism to explain, at least in part, the anti-inflammatory property of flavonoids and nutrient phenomenon that high consumption of flavonoids-rich food can significantly reduce the risks of cardiovascular diseases.

nontoxic effects and natural source for quercetin could increase their bioapplication in vivo. It should be noted that quercetin detected in plasma is mostly in conjugated forms under normal physiological conditions, and quercetin aglycone (i.e., free quercetin) is only found in the submicromolar concentration range.21,24,26,38 In tissues, however, quercetin aglycone can be the predominant form. It has been reported that both monocytes and macrophages can metabolize quercetin glucuronides to the parent aglycone compound, quercetin.24,26 In line with this information, therefore, it was proposed that quercetin could inhibit the adhesion and infiltration of MPO to the vascular wall, and attenuated vascular-bound MPO-dependent endothelial injury in inflammatory vasculature. In this work, we did not pursue the identification of quercetin metabolites, but it was possible that quercetin in free form could contribute to the inhibition on MPO activity and vascular endothelial injury in inflammatory vasculature. In conclusion, quercetin was a potent nontoxic inhibitor of MPO activity that might be beneficial to human health. As quercetin significantly inhibited MPO-dependent HOCl generation and protected HUVEC from MPO/neutrophilinduced injury (Figure 8), quercetin was an effective inhibitor

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01537. Effect of quercetin on cellular viability, thiol content; effects of rutin and quercetin on MPO-dependent HOCl formation; docking of rutin in the active site of MPO (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected]. Phone: 86-791-88120380. Fax: 86-791-88120380. *E-mail: [email protected]. Phone: 86-791-88120380. Fax: 86-791-88120380. ORCID

Naihao Lu: 0000-0003-3336-2502 Yi-Yuan Peng: 0000-0003-3471-8566 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (Nos. 31760255, 31560255, 31260216, 31100608) and the Natural Science Foundation of Jiangxi province (Nos. 20171BCB23041, 20161BAB215215).

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Figure 8. Novel role for quercetin as an essential mediator of MPOdependent HOCl formation and vascular endothelial injury. MPO, secreted mainly from activated neutrophils, plays central roles in cell damage and vascular endothelial dysfunction through the production of HOCl by this enzyme. The presence of quercetin could inhibit MPO (or neutrophil)-mediated HOCl formation in vitro and accordingly protected endothelial cells from MPO (or neutrophil)induced injury. In inflammation, the endothelial dysfunction in aortas from LPS-treated mice was attenuated by quercetin in parallel with the inhibitive effects of quercetin on MPO expression and activity in inflammatory aortas. Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation.

for mediating MPO-dependent endothelial dysfunction in inflammatory vasculature. Moreover, the inhibitive effect of quercetin on MPO-mediated HOCl formation would render this flavonoid as potential nutriment to decrease the risk of cardiovascular diseases, which was different from the widespread antioxidant and anti-inflammatory mechanisms, that is, radical scavenging, metal-chelating, oxidative stress and cell signaling pathways etc.17,21−26,38 Our results could also 4939


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Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent

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