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Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (–)-Epigallocatechin gallate Rong Tian, Yun Ding, Yiyuan Peng, and Naihao Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00631 • Publication Date (Web): 31 Mar 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Journal of Agricultural and Food Chemistry

Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (–)-Epigallocatechin gallate Rong Tian †, Yun Ding †, Yi-Yuan Peng †, Naihao Lu †, * †

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

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

*Corresponding Author Tel/Fax: 86-791-88120380 E-mail: [email protected]; [email protected]

Running title: Inhibition of myeloperoxidase-mediated injury by EGCG

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ACS Paragon Plus Environment


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ABSTRACT

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Myeloperoxidase (MPO) plays important roles in various diseases by its unique chlorinating

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activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate

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an inverse correlation between plant polyphenol consumption and the incidence of

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cardiovascular diseases. Here we showed that (–)-epigallocatechin gallate (EGCG), the main

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flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in

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vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 µM EGCG versus 100 ± 5.6% for control,

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P < 0.01). UV-Vis spectral and docking studies indicated that EGCG bound to the active site

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(heme) of MPO and resulted in the accumulation of compound II, which was unable to produce

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HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl

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formation: 65.0 ± 5.6% for 20 µM EGCG versus 100 ± 6.2% for control, P < 0.01) without

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influencing MPO and Nox2 release, and superoxide formation, suggesting that EGCG specifically

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inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG

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inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial

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cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced

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injury (P < 0.05, all cases), while it did not induce cytotoxicity to HUVEC (P > 0.05, all cases).

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Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity, and

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may participate in regulation of immune responses at inflammatory sites.

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Key words: EGCG; myeloperoxidase; hypochlorous acid; neutrophils; endothelial cells

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INTRODUCTION

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As a heme peroxidase, myeloperoxidase (MPO) is abundantly expressed in activated

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neutrophils that play important roles in host defense.1-3 During the inflammation process,

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neutrophils are rapidly recruited at sites of infections and secrete MPO. The reaction of ferric

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MPO with hydrogen peroxide (H2O2) could generate compound I which is able to oxidize chloride

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to produce the strong oxidant, hypochlorous acid (HOCl) (Figure 1A).1-4

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MPO plays the physiological role in killing fungal and bacterial pathogens by producing

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HOCl. However, growing evidence indicates that the excessive reactive intermediates and HOCl

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play important roles in oxidative stress and the pathogenesis of disease.1, 5-7 Epidemiological

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studies have shown that MPO could be considered as a risk factor in some cardiovascular

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diseases.1, 2 Due to the causal role of MPO in various diseases, some inhibitors have been used to

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prevent the deleterious effects of MPO.1, 5-8 Although a lot of drugs (i.e., azides, hydrazides, and

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hydroxamic acids) are effective for inhibiting MPO activity in vitro,1, 8-10 they are inherently toxic

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and subsequently are unsuitable therapeutic drugs.

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Recently, flavonoids have been widely used to ameliorate or prevent cardiovascular

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diseases.5, 7, 11-14 The free radical scavenging and metal-chelating properties of flavonoids have

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been usually proposed for the antioxidant mechanisms in vitro and vivo. As natural phenolic

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compounds in plants and vegetables, flavonoids (such as quercetin, catechin and myricitrin) have

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been also used to inhibit MPO activity in vitro because they could act as powerful reducing agents

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and as competitive substrates for MPO intermediate compounds (Figure 1A).5, 7, 15, 16 Moreover,

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plant polyphenols (such as resveratrol, curcuminoids, catechins) have been shown to inhibit

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MPO/activated neutrophil-dependent HOCl formation or MPO/HOCl-induced biomolecule

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damage.17-21 However, the precise molecular mechanisms are still unclear. Herein, we were

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interested to investigate the effects of (–)-epigallocatechin gallate (EGCG, the main flavonoid

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present in green tea, Figure 1B) not only on MPO activity in vitro and neutrophils, but also on

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MPO/neutrophil-induced cytotoxicity to endothelial cells.

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In this study, we showed that EGCG effectively inhibited MPO/neutrophil-mediated HOCl

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formation in vitro as well as MPO-induced cytotoxicity to human umbilical vein endothelial cells.

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The inhibitory mechanism was further investigated and indicated that EGCG bound to the active 3



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site (heme) of MPO and resulted in the accumulation of compound II, which was unable to

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produce HOCl. Therefore, EGCG is a potent inhibitor of MPO activity and may counteract the

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deleterious effects of circulating and/or tissue-accumulated MPO. The inhibition of MPO activity

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by EGCG would partially explain the epidemiological phenomenon that a low incidence of

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cardiovascular disease is associated with the high consumption of green tea.

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MATERIALS AND METHODS

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Chemicals

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Myeloperoxidase (MPO) from human leukocytes, (–)-epigallocatechin gallate (EGCG),

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taurine, glucose oxidase (GO), 4-aminobenzoic acid hydrazide (ABAH) and zymosan were

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purchased from Sigma-Aldrich.

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Effect of EGCG on chlorinating activity of MPO in vitro and in neutrophils

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The chlorinating activity of MPO was determined as previously described.6,

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In the

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presence or absence of EGCG, H2O2 (500 µM) was incubated with a mixture of taurine (1 mM),

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NaCl (100 mM) and MPO (0.6 µM) in 20 mM phosphate-buffered saline (PBS, Na2HPO4, pH 7.0)

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for 10 min. Moreover, high concentrations of MPO-H2O2 and EGCG were used to conveniently

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investigate the effects of EGCG on MPO-mediated HOCl formation.4, 6, 7

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Human neutrophils were isolated and cultured according to these previous studies.9,

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Neutrophils were mixed with different amounts of EGCG in DMEM containing NaCl (100 mM).

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The cells were stimulated with serum-opsonized zymosan (SOZ) and incubated for 30 min. The

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formations of HOCl and O2•- were measured by taurine chloramine assay and the superoxide

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dismutase-inhibitable cytochrome c reduction assay, respectively.9, 22 The expressions of MPO and

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Nox2 were analyzed by Western blot with respective antibodies (Supplementary Methods).

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Interaction between MPO and EGCG by UV-Vis spectra and molecular docking

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H2O2 (15 µM) was added into MPO solution in PBS. After incubation for certain time, EGCG

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was added and then NaCl (100 mM) was added. UV-Vis spectra of MPO were determined by

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Hitachi U-3310 spectrophotometer at 25°C. All assays were performed in three independent

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replications. EGCG was docked to human MPO (PDB ID: 5FIW) using the AutoDock software, as

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described previously.22-24

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Effect of EGCG on MPO (or neutrophil)-mediated human umbilical vein endothelial cells

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(HUVEC) injury

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HUVEC (purchased from CCTCC, Wuhan, China) were cultured in DMEM containing

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glucose (5.6 mM) and NaCl (100 mM). EGCG with different concentrations were preincubated

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with cells for 5 min, MPO and GO (10 mU/mL) were then added and incubated for 2 h.

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Neutrophils were added into HUVEC in the presence or absence of EGCG. The cells were

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stimulated with SOZ for 2 h. After that, cellular viability and HOCl formation were measured by

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using MTT assay and taurine chloramine assay, respectively.

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Statistical analysis

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All data were the means ± SD of three independent experiments. One-way ANOVA was used

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for statistical analysis, and P< 0.05 was considered statistically significant.

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RESULTS

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Effects of EGCG on MPO-catalyzed HOCl production

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To determine whether EGCG affected MPO activity, HOCl production by purified MPO

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was assessed. Figure 2A showed that EGCG dose-dependently inhibited HOCl production at these

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concentrations (5-20 µM). EGCG at the concentration of 10 µM and 20 µM inhibited HOCl

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production by 25% and 50%, respectively. 4-aminobenzoic acid hydrazide (ABAH), a suicide

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substrate for MPO, inhibited production of HOCl by nearly 80% at a concentration of 20 µM.

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These results suggested that EGCG reduced HOCl formation by inhibiting MPO activity.

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Meanwhile, the addition of EGCG decreased MPO-dependent H2O2 consumption (Figure S1), and

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this inhibition of H2O2 consumption was once again consistent with reduction of MPO

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activity-mediated HOCl formation by EGCG.

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The UV-Vis spectra of active MPO were analyzed to investigate the inhibitive mechanism of

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EGCG on MPO activity. First, the addition of H2O2 to MPO for 2 min could result in the shift of

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the Soret band from 430 nm to 456 nm which was the characteristic for MPO compound II (Figure

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2B). However, after 4 min incubation, the addition of EGCG caused a decrease at 417 nm and an

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increase of absorbance at 455 nm, suggesting the MPO compound II formation.4, 6 Furthermore,

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the Soret band of active MPO did not change after the subsequent addition of NaCl, indicating the

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absence of MPO compound I. Therefore, these data demonstrated that EGCG could reduce MPO

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compound I to form compound II.

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Docking of EGCG in the active site of MPO

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The lowest binding energy calculated by AutoDock was −9.0 kcal/mol and revealed strong

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binding of EGCG to MPO (Figure 3). Consistent with MPO spectra analyses (Figure 2B), these

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results further showed that EGCG directly bound into the active site (iron-heme) of MPO. In

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addition, the amino acids His95, Glu102, Arg239, Phe366 and Phe407 in MPO were found to

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form hydrophobic interactions and hydrogen bond with EGCG (Figure 3C). Meanwhile, the π-π

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stacking hydrophobic interactions between polyphenol (i.e. A, C ring) in EGCG and aromatic

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residues (i.e. Phe366, Phe407) in MPO were observed. In addition, the C3′ and C4′ hydroxyl

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groups in B ring interacted with His95 and form two hydrogen bonds, while C5′ hydroxyl group

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formed a hydrogen bond with Arg 239.

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EGCG inhibited MPO activity in neutrophils

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SOZ was used to stimulate the release of MPO from neutrophils, and the effects of EGCG on

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MPO-mediated HOCl formation were determined. Without SOZ stimulation, neutrophils produced

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little MPO and HOCl. However, after SOZ stimulation, neutrophils induced significant release of

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MPO and accordingly generated high level of HOCl (Figure 4 A and C). Figure 4A showed that

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adding EGCG to the SOZ-stimulated neutrophils effectively inhibited neutrophil-mediated HOCl

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formation. To test the specific inhibition on MPO activity, EGCG was also assessed for its effect

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on Nox2 expression and O2•- production. EGCG did not have significant effects on the expressions

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of MPO and Nox2, and O2•- production in SOZ-stimulated or unstimulated (i.e. resting)

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neutrophils (Figure 4 B and C), demonstrating that EGCG did not inhibit NADPH

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oxidase-mediated O2•- generation in neutrophil cells. Therefore, these results suggested that EGCG

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could effectively inhibit MPO activity rather than NADPH oxidase activity, and resulted in the

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decrease of HOCl formation by stimulated neutrophils (Figure 4D).

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EGCG protected HUVEC from MPO-induced injury

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Further, we used HUVEC to confirm MPO-induced endothelial cell injuries. To mimic H2O2

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formation in vivo, we used glucose oxidase/glucose to generate H2O2.6 Under this condition,

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neither MPO or H2O2 alone barely decreased the cell viability. However, the coexistence of MPO 6


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and H2O2 significantly resulted in the loss of cell viability to ≈ 65% (Figure 5A). This decrease of

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cell viability was also inhibited by ABAH (a classic MPO inhibitor). Consistent with previous

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studies,1, 6, 9, 25, 26 MPO-induced HOCl could result in extensive cell death by apoptosis, which was

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associated with caspase-3 activation (Figure S2).

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Next, we determined if EGCG could effectively protect cells from MPO-induced injury. As

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shown in Figure 5B, EGCG could dose-dependently protect HUVEC from MPO/H2O2/Cl--induced

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cell injury. Meanwhile, control experiments demonstrated that EGCG at these concentration (

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