Protective Effects of Catechin against Monosodium Urate-Induced

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Journal of Agricultural and Food Chemistry

Protective effects of catechin against monosodium urate-induced inflammation through the modulation of NLRP3 inflammasome activation

Jhih-Jia Jhang1, Chi-Cheng Lu1, Cheng-Ying Ho1, Yu-Ting Cheng1 and Gow-Chin Yen1, 2*

1

Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan

2

Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan

*Author to whom correspondence should be addressed. Tel: 886-4-2287-9755, Fax: 886-4-2285-4378, E-Mail: [email protected]

Running title: Catechin attenuates MSU-induced inflammation

Keywords: monosodium urate; IL-1β; NLRP3; catechin; inflammation

Abbreviations: IL-1β, interleukin-1β; MSU, monosodium urate; MtROS, mitochondrial reactive oxygen species; NLRP3, nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3; TRX, thioredoxin; TXNIP, thioredoxin interaction protein 1

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Abstract

2

Gouty inflammation results from the stimulation of monosodium urate (MSU).

3

Interleukin-1β (IL-1β) secretion is the primary clinical manifestation of MSU attack,

4

and MSU activates IL-1β through nucleotide-binding oligomerization domain-like

5

receptor containing pyrin domain 3 (NLRP3) inflammasome. This study investigated

6

the protective effect and underlying mechanism of natural occurring phenolic

7

compounds on MSU-induced inflammation in vivo and in vitro. A screening of

8

phenolic compounds revealed that gallic acid and catechin exhibited the most potent

9

free radical scavenging activities. Subcutaneous injection of gallic acid or catechin

10

significantly reduced MSU-induced IL-1β and IL-6 secretion in C57BL/6 mice.

11

However, only catechin inhibited MSU-induced IL-1β secretion and NLRP3

12

inflammasome

13

mitochondrial reactive oxygen species (MtROS) production and intracellular calcium

14

levels were significantly decreased by treatment with catechin in THP-1 cells.

15

Catechin treatment also up-regulated Bcl-2 levels and restored MSU-induced

16

mitochondrial transmembrane potential impairment. These results indicate that the

17

protective effects of catechin on MSU-induced IL-1β secretion are associated with

18

modulation of mitochondrial damage. It also suggests that catechin has potential to

19

protect gout attack by modulation of NLRP3 inflammasome activation.

activation

in

MSU-challenged

THP-1

2


cells.

MSU-triggered

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Introduction

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Monosodium urate (MSU) shows needle-shape crystals and is derived from uric

22

acid.1 Uric acid is the metabolites of purine metabolism via xanthine oxidase enzyme

23

converting hypoxanthine and xanthine.1 The high level of blood uric acid contributes

24

to over-saturation and crystallization of MSU crystals.1 MSU crystals that precipitate

25

in the articular tissues could trigger acute gout attack, and the location of gout attack

26

occurs predominately in the large joints of big toe.1,2 The main clinical manifestations

27

of early gout attack are neutrophil infiltration and pro-inflammatory cytokine IL-1β

28

secretion.3-5 During gout flare, colchicine and non-steroidal anti-inflammatory drugs

29

(NSAIDs) are used as the clinical treatment of gout to ameliorate inflammatory

30

responses.2-4 New drugs for gout treatment have been developed to block

31

interleukin-1β (IL-1β).3,5 Anakinra (IL-1 receptor antagonist), rilonacept (IL-1 trap),

32

canakinumab (IL-1β antibody) and VX-765 (caspase-1 inhibitor) have been employed

33

as new drugs for gout therapy.5

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The regulations of caspase-1 and IL-1β depend on the nucleotide-binding

35

oligomerization domain-like receptor containing pyrin domain 3 (NLRP3)

36

inflammasome.

37

apoptosis-associated speck like protein containing a caspase recruitment domain

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(ASC) and a caspase-1 domain.6,7 Upon stimulation, the inflammasome-forming NLR

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proceeds conformational change, allowing for NLR binding to ASC via the pyrin

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domains. ASC acts as an adaptor protein and binds to pro-caspase-1 through caspase

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recruitment domains.7, 8 Finally, the large NLRP3 complex provides the platform for

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the activation of caspase-1 by proteolytic cleavage, and active caspase-1 then cleaves

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pro-forms of IL-1β and IL-18, allowing for their secretion and biological activity.7,8

44

Reactive oxygen species (ROS) that is generated by NLRP3 inflammasome activators

NLRP3

inflammasome comprises the

3


NLRP3

domain, an


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has been shown to modulate NLRP3 inflammasome activation.8,9 Robust ROS

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dissociates the conjugation of thioredoxin (TRX) and thioredoxin interaction protein

47

(TXNIP),

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inflammasome, leading to the release of IL-1β.10 IL-1β secretion is increased by the

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knock-down of TRX expression or decreased by the depletion of TXNIP.9,11 In

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addition, mitochondrial ROS (MtROS) is also associated with NLRP3 inflammasome

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activation.10 The specific inhibitors for complex I and III of the mitochondrial

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respiratory activate MtROS generation and NLRP3 inflammasome in THP-1 cells.10

9

and the released TXNIP further recruits and binds to the NLRP3

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The phenolic compounds have multiple health-promoting properties associated

54

with antioxidant activities, anti-inflammation and immunological regulation.12

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Previous reports have demonstrated that naturally occurring phenolic compounds with

56

potent

57

inflammasome.13,14 Epigallocatechine gallate (EGCG) prevents NLRP3-mediated

58

lupus nephritis,13 and quercetin ameliorates streptozotocin-induced kidney injury

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through the down-regulation of the NLRP3 inflammasome.14 Chuang et al.

60

summarized

61

inflammasome-related diseases, including renal injury, diabetes, infection, and liver

62

diseases.15 However, it remains unclear whether the phenolic compounds exert the

63

inhibitory effects on MSU-induced inflammation. We examined and selected the

64

potential agents in several phenolic compounds, and this study aimed to investigate

65

the effects and underlying mechanisms of these candidate molecules on MSU-induced

66

inflammation and inflammasome activation in vivo and in vitro.

antioxidant

that

profiles

the

natural

exhibit

inhibitory

compounds

may

effects

be

67 68 69 4


on

potential

the

to

NLRP3

improve

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70

Materials and methods

71

Chemicals

72

Cinnamic

acid,

o-coumaric

acid,

m-coumaric

acid,

ferulic

acid,

73

p-hydroxybenzoic acid, vanillic acid, syringic acid, protocatechuic acid, gentisic acid,

74

gallic acid, catechin, monosodium urate (MSU), 2',7'-dichlorofluorescin diacetate

75

(DCFH-DA),

76

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

77

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

78

sulforhodamine B (SRB), β-mercaptoethanol and nitro blue tetrazolium (NBT) were

79

purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of the phenolic

80

compounds was over 95%. RPMI-1640 medium and fetal bovine serum (FBS) were

81

obtained from Gibco BRL (Grand Island, NY, USA). Penicillin-streptomycin and

82

sodium pyruvate solution were obtained from Hyclone (Logan, UT, USA). Antibodies

83

against caspase-1, β-actin, Bcl-2, DJ-1 and TRX were obtained from Cell Signaling

84

Technology (Beverly, MA, USA). NLRP3 antibody was purchased from Abgent (San

85

Diego, CA, USA). Dihydroethidium (DHE), MitoSOX red mitochondrial superoxide

86

indicator, the TXNIP antibody and Fluo3-AM were purchased from Life Technologies

87

(Carlsbad, CA, USA). The JC-1 Mitochondrial Transmembrane Potential Assay Kit

88

was obtained from Cayman Chemical Co. (Ann Arbor, MI, USA).

xanthine

oxidase,

phorbol

12-myristate acid

13-acetate

(PMA),

(HEPES),

colchicine,

bromide

(MTT),

89 90

Scavenging free radical assay

91

The free radical scavenging assay was performed according to the methods of

92

Orallo et al.16 Briefly, free radical scavenging was measured as the absorbance at 570

93

nm using a FLUOstar Omega spectrophotometer (BMG Labtechnologies, Germany)

94

after incubation with 200 µL working solution (50 mM phosphate buffer, pH 7.4, 100 5



95

µM EDTA-2Na, 100 µM xanthine and 100 µM NBT), 25 µL sample solution (5 µM,

96

10 µM or 25 µM) and 25 µL xanthine oxidase (0.066 U) for 10 min. Allopurinol was

97

used as a positive control.

98 99

Animal treatment

100

The animal experiments were conducted in accordance with the guidelines of the

101

National Institutes of Health and approved by the Institutional Animal Care and Use

102

Committee (IACUC) of National Chung Hsing University (No: 101-81). Male

103

C57BL/6 mice were purchased from the National Laboratory Animal Center (Taipei,

104

Taiwan). The animals were provided a chow diet and water ad libitum under

105

conditions of 22-24°C, 40-70% relative humidity and a 12 h light-dark cycle. Acute

106

gout inflammation was induced by intraperitoneal injection with 3 mg MSU in 0.5 mL

107

phosphate-buffered saline (PBS), and the mice were subcutaneously treated with or

108

without phenolic compounds at 100 mg/Kg body weight (BW) (n=5). After 6 h, all

109

mice were sacrificed using carbon dioxide (CO2), and the mice were intraperitoneally

110

injected with 4 mL PBS.17 The peritoneal fluid was then collected, and the volume of

111

peritoneal fluid was about 2.5-3.0 mL from each mouse.

112 113

Identification of MSU crystal-elicited neutrophils

114

The collected peritoneal exudate cells were washed and resuspended in PBS. The

115

cells were stained with fluorescent antibodies against the specific surface markers

116

Ly-6G (eBioscience, San Diego, CA, USA) and 7/4 (AbD serotec, Raleigh, NC, USA)

117

for 20 min at 37°C in the dark, and the cell populations were sorted using a FACScan

118

flow cytometer (Becton-Dickinson, San Jose, CA).

119 6


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Cell culture

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The THP-1 cell line was purchased from the Bioresource Collection and

122

Research Center (BCRC 60430, Food Industry Research and Development Institute,

123

Hsinchu, Taiwan) and cultured in RPMI-1640 media with 10% (v/v) FBS, 100 µg/mL

124

streptomycin, 100 U/mL penicillin, 10 mM HEPES, 1 mM sodium pyruvate, 1.5 g/L

125

sodium bicarbonate, and 50 µM β-mercaptoethanol at 37°C in a humidified

126

atmosphere of 5% CO2. THP-1 cells were treated with 100 nM PMA for 3 h to initiate

127

NF-κB pathway. The cells were subsequently treated with 75 µg/mL MSU crystals in

128

the presence or absence of 20 µM phenolic compounds for 6 h as our previous

129

methods.18

130 131

Cell viability assay

132

Cell viability was examined using MTT and sulforhodamine B (SRB) assays. For

133

the MTT assay, the cells were cultured in 0.5 mg/mL MTT for 2 h at 37°C, and

134

formazan blue formation in cells was dissolved using dimethyl sulfoxide (DMSO).

135

The optical density (OD) was detected at 570 nm using a FLUOstar Omega

136

spectrophotometer. For the SRB assay, the cells were fixed in cold 10%

137

trichloroacetic acid at 4°C. A solution of SRB in 1% acetic acid was added to each

138

well and incubated for 30 min. Cell-bound SRB was dissolved in 10 mM Tris base

139

solution, and the absorbance was measured at 510 nm using a FLUOstar Omega

140

spectrophotometer.

141 142

Cytokine ELISA assay

143

Human IL-1β, mouse IL-1β and mouse IL-6 cytokine secretions were measured

144

using the ELISA kits (eBioscience, San Diego, CA, USA) according to the 7



145

manufacturer’s instructions.

146 147

Immunoprecipitation assay

148

Immunoprecipitation was performed using the Catch and Release Reversible

149

Immunoprecipitation System (Millipore, Billerica, MA, USA) according to the

150

manufacturer’s instructions. Treated THP-1 cells were washed with PBS and

151

resuspended in lysis buffer containing 1X protease inhibitor cocktail (Sigma-Aldrich,

152

St. Louis, MO, USA). Each cell lysate (500 µg) was incubated with 4 µg anti-TXNIP

153

antibody and 10 µL antibody capture affinity ligand for 30 min at room temperature.

154

The immunoprecipitated proteins were washed twice in 1X wash buffer, and the

155

bound proteins were eluted in 70 µL elution buffer.

156 157

Detection of superoxide anion and MtROS production

158

The levels of superoxide anion and MtROS production were determined after

159

treatment with 8 µM DHE and 2.5 µM MitoSOX red mitochondrial superoxide

160

indicator, respectively, for 20 min at 37°C using the FL-2 channel (excitation 488 nm

161

and emission 585 nm) of a FACScan flow cytometer.

162 163

Detection of mitochondrial transmembrane potential

164

The mitochondrial transmembrane potential was determined using the Cayman

165

JC-1 Assay Kit according to the manufacturer’s protocol. The mitochondrial

166

transmembrane potential was detected using the FL-2 channel of a FACScan flow

167

cytometer.

168 169

Detection of intracellular calcium levels 8


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170

Intracellular calcium levels were determined using Fluo3-AM stain. The cells

171

were treated with 5 µM Fluo3-AM for 40 min, and the fluorescent intensity was

172

analyzed at 485/530 nm using a FLUOstar Galaxy fluorescence plate reader (BMG

173

Labtechnologies, Offenburg, Germany).

174 175

Western blot

176

The protein lysates were boiled in 4X protein loading dye [8% SDS, 40%

177

glycerol, 200 mM Tris-HCl (pH 6.8), 0.04% Coomassie blue R-250 and 10%

178

2-mercaptoethanol], and the samples were subjected to SDS-polyacrylamide gel

179

electrophoresis. The proteins were transferred to nitrocellulose membrane (Sartorius

180

Stedim Biotech, Aubagne Cedex, France) and incubated with specific primary

181

antibodies at 4°C. The membranes were washed three times with Tris-buffered saline

182

containing

183

peroxide-conjugated secondary antibody before analysis using a chemiluminescence

184

ECL detection system (Millipore, Billerica, MA, USA). The levels of protein were

185

normalized to the β-actin or TXNIP signals and quantitated using Vision Works LS

186

6.3.3 (UVP, Cambridge, UK).

0.05%

Tween-20

(TBST)

and

incubated

with

horseradish

187 188

Statistical analysis

189

All results are expressed as the means ± SD from at least three independent

190

experiments. ANOVA was used to evaluate the differences between multiple groups.

191

Significant differences (p < 0.05) between the means were determined using Duncan’s

192

multiple range test or Student’s t-test. All statistical analyses were performed with

193

SPSS 12.0 software.

194 9



195

Results

196

Gallic acid and catechin exhibited potent free radical scavenging

197

Previous studies have suggested that redox molecules are major signals for

198

MSU-induced inflammation.8-10 This study investigated potential inflammation

199

inhibitors, including cinnamic acid, o-coumaric acid, m-coumaric acid, ferulic acid,

200

p-hydroxybenzoic acid, vanillic acid, syringic acid, protocatechuic acid, gentisic acid,

201

gallic acid, and catechin. The results showed that gallic acid and catechin exhibited

202

higher free radical scavenging activity than other phenolic compounds (Figure. 1).

203

Therefore, the effects of gallic acid and catechin on MSU-induced inflammation were

204

further explored using MSU-challenged models in vivo and in vitro.

205 206

Effects of gallic acid and catechin on murine peritoneal inflammation in

207

C57BL/6 mice

208

Intraperitoneal injection of MSU (3 mg) significantly (p

Protective Effects of Catechin against Monosodium Urate-Induced

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