Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous

Mar 31, 2017 - Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorou...
0 downloads 7 Views 476KB Size
Subscriber access provided by University of Newcastle, Australia

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

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

1

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

2

ABSTRACT

3

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

4

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

5

an inverse correlation between plant polyphenol consumption and the incidence of

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

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

15

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

16

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

17

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

18

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

19

may participate in regulation of immune responses at inflammatory sites.

20 21 22 23

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

24

2

ACS Paragon Plus Environment

Page 2 of 23

Page 3 of 23

Journal of Agricultural and Food Chemistry

25

INTRODUCTION

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

and subsequently are unsuitable therapeutic drugs.

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

MPO/neutrophil-induced cytotoxicity to endothelial cells.

51

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

52

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

53

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 23

54

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

55

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

56

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

57

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

58

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

59

MATERIALS AND METHODS

60

Chemicals

61

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

62

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

63

purchased from Sigma-Aldrich.

64

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

65

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

9, 22

In the

66

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

67

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

68

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

69

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

70

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

22

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

described previously.22-24

4

ACS Paragon Plus Environment

Page 5 of 23

Journal of Agricultural and Food Chemistry

82

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

83

(HUVEC) injury

84

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

85

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

86

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

87

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

88

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

89

using MTT assay and taurine chloramine assay, respectively.

90

Statistical analysis

91

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

92

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

93

RESULTS

94

Effects of EGCG on MPO-catalyzed HOCl production

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

activity-mediated HOCl formation by EGCG.

104

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

105

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

106

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

107

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

108

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

109

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

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

110

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

111

compound I to form compound II.

112

Docking of EGCG in the active site of MPO

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

formed a hydrogen bond with Arg 239.

122

EGCG inhibited MPO activity in neutrophils

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

EGCG protected HUVEC from MPO-induced injury

136

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

137

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

138

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

ACS Paragon Plus Environment

Page 6 of 23

Page 7 of 23

Journal of Agricultural and Food Chemistry

139

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

140

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

141

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

142

associated with caspase-3 activation (Figure S2).

143

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

144

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

145

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