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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
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Gouty inflammation results from the stimulation of monosodium urate (MSU).
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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
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cells.
MSU-triggered
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Introduction
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Monosodium urate (MSU) shows needle-shape crystals and is derived from uric
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acid.1 Uric acid is the metabolites of purine metabolism via xanthine oxidase enzyme
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converting hypoxanthine and xanthine.1 The high level of blood uric acid contributes
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to over-saturation and crystallization of MSU crystals.1 MSU crystals that precipitate
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in the articular tissues could trigger acute gout attack, and the location of gout attack
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occurs predominately in the large joints of big toe.1,2 The main clinical manifestations
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of early gout attack are neutrophil infiltration and pro-inflammatory cytokine IL-1β
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secretion.3-5 During gout flare, colchicine and non-steroidal anti-inflammatory drugs
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(NSAIDs) are used as the clinical treatment of gout to ameliorate inflammatory
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responses.2-4 New drugs for gout treatment have been developed to block
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interleukin-1β (IL-1β).3,5 Anakinra (IL-1 receptor antagonist), rilonacept (IL-1 trap),
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canakinumab (IL-1β antibody) and VX-765 (caspase-1 inhibitor) have been employed
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as new drugs for gout therapy.5
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The regulations of caspase-1 and IL-1β depend on the nucleotide-binding
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oligomerization domain-like receptor containing pyrin domain 3 (NLRP3)
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inflammasome.
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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
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Reactive oxygen species (ROS) that is generated by NLRP3 inflammasome activators
NLRP3
inflammasome comprises the
3
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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
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(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
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with antioxidant activities, anti-inflammation and immunological regulation.12
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Previous reports have demonstrated that naturally occurring phenolic compounds with
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potent
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inflammasome.13,14 Epigallocatechine gallate (EGCG) prevents NLRP3-mediated
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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.
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summarized
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inflammasome-related diseases, including renal injury, diabetes, infection, and liver
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diseases.15 However, it remains unclear whether the phenolic compounds exert the
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inhibitory effects on MSU-induced inflammation. We examined and selected the
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potential agents in several phenolic compounds, and this study aimed to investigate
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the effects and underlying mechanisms of these candidate molecules on MSU-induced
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inflammation and inflammasome activation in vivo and in vitro.
antioxidant
that
profiles
the
natural
exhibit
inhibitory
compounds
may
effects
be
67 68 69 4
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on
potential
the
to
NLRP3
improve
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Materials and methods
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Chemicals
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Cinnamic
acid,
o-coumaric
acid,
m-coumaric
acid,
ferulic
acid,
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p-hydroxybenzoic acid, vanillic acid, syringic acid, protocatechuic acid, gentisic acid,
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gallic acid, catechin, monosodium urate (MSU), 2',7'-dichlorofluorescin diacetate
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(DCFH-DA),
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4-(2-hydroxyethyl)-1-piperazineethanesulfonic
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
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sulforhodamine B (SRB), β-mercaptoethanol and nitro blue tetrazolium (NBT) were
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purchased from Sigma-Aldrich (St. Louis, MO, USA). The purity of the phenolic
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compounds was over 95%. RPMI-1640 medium and fetal bovine serum (FBS) were
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obtained from Gibco BRL (Grand Island, NY, USA). Penicillin-streptomycin and
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sodium pyruvate solution were obtained from Hyclone (Logan, UT, USA). Antibodies
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against caspase-1, β-actin, Bcl-2, DJ-1 and TRX were obtained from Cell Signaling
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Technology (Beverly, MA, USA). NLRP3 antibody was purchased from Abgent (San
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Diego, CA, USA). Dihydroethidium (DHE), MitoSOX red mitochondrial superoxide
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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
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The free radical scavenging assay was performed according to the methods of
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Orallo et al.16 Briefly, free radical scavenging was measured as the absorbance at 570
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nm using a FLUOstar Omega spectrophotometer (BMG Labtechnologies, Germany)
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after incubation with 200 µL working solution (50 mM phosphate buffer, pH 7.4, 100 5
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µM EDTA-2Na, 100 µM xanthine and 100 µM NBT), 25 µL sample solution (5 µM,
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10 µM or 25 µM) and 25 µL xanthine oxidase (0.066 U) for 10 min. Allopurinol was
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used as a positive control.
98 99
Animal treatment
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The animal experiments were conducted in accordance with the guidelines of the
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National Institutes of Health and approved by the Institutional Animal Care and Use
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Committee (IACUC) of National Chung Hsing University (No: 101-81). Male
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C57BL/6 mice were purchased from the National Laboratory Animal Center (Taipei,
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Taiwan). The animals were provided a chow diet and water ad libitum under
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conditions of 22-24°C, 40-70% relative humidity and a 12 h light-dark cycle. Acute
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gout inflammation was induced by intraperitoneal injection with 3 mg MSU in 0.5 mL
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phosphate-buffered saline (PBS), and the mice were subcutaneously treated with or
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without phenolic compounds at 100 mg/Kg body weight (BW) (n=5). After 6 h, all
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mice were sacrificed using carbon dioxide (CO2), and the mice were intraperitoneally
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injected with 4 mL PBS.17 The peritoneal fluid was then collected, and the volume of
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peritoneal fluid was about 2.5-3.0 mL from each mouse.
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Identification of MSU crystal-elicited neutrophils
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The collected peritoneal exudate cells were washed and resuspended in PBS. The
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cells were stained with fluorescent antibodies against the specific surface markers
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Ly-6G (eBioscience, San Diego, CA, USA) and 7/4 (AbD serotec, Raleigh, NC, USA)
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for 20 min at 37°C in the dark, and the cell populations were sorted using a FACScan
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flow cytometer (Becton-Dickinson, San Jose, CA).
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Cell culture
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The THP-1 cell line was purchased from the Bioresource Collection and
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Research Center (BCRC 60430, Food Industry Research and Development Institute,
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Hsinchu, Taiwan) and cultured in RPMI-1640 media with 10% (v/v) FBS, 100 µg/mL
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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
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atmosphere of 5% CO2. THP-1 cells were treated with 100 nM PMA for 3 h to initiate
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NF-κB pathway. The cells were subsequently treated with 75 µg/mL MSU crystals in
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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
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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
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manufacturer’s instructions.
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Immunoprecipitation assay
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Immunoprecipitation was performed using the Catch and Release Reversible
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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.
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The immunoprecipitated proteins were washed twice in 1X wash buffer, and the
155
bound proteins were eluted in 70 µL elution buffer.
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Detection of superoxide anion and MtROS production
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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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Intracellular calcium levels were determined using Fluo3-AM stain. The cells
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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
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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%
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2-mercaptoethanol], and the samples were subjected to SDS-polyacrylamide gel
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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
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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.
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Results
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Gallic acid and catechin exhibited potent free radical scavenging
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Previous studies have suggested that redox molecules are major signals for
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MSU-induced inflammation.8-10 This study investigated potential inflammation
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inhibitors, including cinnamic acid, o-coumaric acid, m-coumaric acid, ferulic acid,
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p-hydroxybenzoic acid, vanillic acid, syringic acid, protocatechuic acid, gentisic acid,
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gallic acid, and catechin. The results showed that gallic acid and catechin exhibited
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higher free radical scavenging activity than other phenolic compounds (Figure. 1).
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Therefore, the effects of gallic acid and catechin on MSU-induced inflammation were
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further explored using MSU-challenged models in vivo and in vitro.
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Effects of gallic acid and catechin on murine peritoneal inflammation in
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C57BL/6 mice
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Intraperitoneal injection of MSU (3 mg) significantly (p
