Hesperidin Methylchalcone Suppresses Experimental Gout Arthritis in

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Bioactive Constituents, Metabolites, and Functions

Hesperidin methylchalcone suppresses experimental gout arthritis in mice by inhibiting NF-kB activation Kenji W Ruiz-Miyazawa, Felipe Almeida Pinho-Ribeiro, Sergio M Borghi, Larissa StaurengoFerrari, Victor Fattori, Flávio Amaral, Mauro Martins Teixeira, José Carlos Faria AlvesFilho, Thiago Mattar Cunha, Fernando Q Cunha, Rubia Casagrande, and Waldiceu A Verri J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00959 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Journal of Agricultural and Food Chemistry

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Hesperidin methylchalcone suppresses experimental gout arthritis in mice by inhibiting NF-

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κB activation

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Kenji W. Ruiz-Miyazawa†, Felipe A. Pinho-Ribeiro†, Sergio M. Borghi†, Larissa Staurengo-

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Ferrari†, Victor Fattori†, Flavio A. Amaral‡, Mauro M. Teixeira‡, Jose C. Alves-Filho§, Thiago M.

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Cunha§, Fernando Q. Cunha§, Rubia Casagranded, and Waldiceu A. Verri, Jr †,*

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†

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Garcia Cid, Km 380, PR445, 86057-970, Cx. Postal 10.011, Londrina, Paraná, Brazil.

Departamento de Ciências Patológicas, Universidade Estadual de Londrina-UEL, Rod. Celso

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‡

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Imunofarmacologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

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§

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Bandeirantes s/n, 14050-490, Ribeirão Preto, São Paulo, Brazil.

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¥

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Robert Koch, 60, Hospital Universitário, 86038-350, Londrina, Paraná, Brazil.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Laboratório de

Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Avenida

Departamento de Ciências Farmacêuticas, Universidade Estadual de Londrina-UEL, Avenida

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*Corresponding author: Waldiceu A. Verri Jr - Department of Pathology, Biological Sciences

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Center, Londrina State University. Rod. Celso Garcia Cid, Pr 445, Km380, Londrina, Paraná,

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Brazil, Cx. Postal 10.011, CEP 86057-970, Tel: + 55 43 33714979, FAX: +55 43 33715828, e-mail

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addresses: [email protected]; and [email protected].

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1 ACS Paragon Plus Environment


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ABSTRACT

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Gout arthritis is a painful inflammatory disease induced by monosodium urate (MSU) crystals. In

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this work, we evaluate the therapeutic potential of the flavonoid hesperidin methylchalcone (HMC)

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in a mouse model of gout arthritis induced by intra-articular injection of MSU (100 µg/10 µL).

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Orally given HMC (3-30 mg/kg, 100 µL) reduced in a dose-dependent manner the MSU-induced

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hyperalgesia (44%, p < 0.05), edema (54%, p < 0.05) and leukocyte infiltration (70%, p < 0.05).

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HMC (30 mg/kg) inhibited MSU-induced infiltration of LysM-eGFP+ cells (81%, p < 0.05),

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synovitis (76%, p < 0.05), and oxidative stress (increased GSH, FRAP and ABTS by 62%, 78% and

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73%, respectively; and reduced O2- and NO by 89% and 48%, p < 0.05), and modulated cytokine

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production (reduced IL-1β, TNF-α, IL-6 and IL-10 by 35%, 72%, 37% and 46%, respectively, and

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increased TGF-β by 90%, p < 0.05). HMC also inhibited MSU-induced NF-κB activation (41%,

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p < 0.05), gp91phox (66%, p < 0.05) and NLRP3 inflammasome components mRNA expression in

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vivo (72%, 77%, 71% and 73% for NLRP3, ASC, pro-caspase-1 and pro-IL-1 β, respectively,

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p < 0.05), and induced Nrf2/HO-1 mRNA expression (3.9 and 5.1 fold increase, respectively,

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p < 0.05). HMC (30, 100 and 300 µM) did not inhibit IL-1β secretion by macrophages primed by

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LPS and challenged with MSU (450 µg/mL), demonstrating that the anti-inflammatory effect of

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HMC in gout arthritis depends on inhibiting NF-κB, but not on direct inhibition of inflammasome.

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The pharmacological effects of HMC indicate its therapeutic potential for the treatment of gout.

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KEYWORDS: Gout arthritis, cytokines, NLRP3 inflammasome, NF-κB, oxidative stress.

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1. INTRODUCTION

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Gout is an arthritic disease characterized by articular and peri-articular deposition of

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monosodium urate (MSU) crystals. Deposition of MSU crystals affects mainly distal joints because

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low temperatures facilitate crystal precipitation, causing intense pain and articular inflammation.1,2

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The incidence of gout is associated with increasing of age, affecting mainly middle-aged or older

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patients, reaching a plateau in men after 70 years old.3 In women, gout is more commonly observed

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after menopause due to the reduction in estrogen levels since this hormone regulates the elimination

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of uric acid by the kidneys.4 Current treatments for gout arthritis include colchicine, corticosteroids

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NSAIDs (and non-steroidal anti-inflammatory drugs).5,6 Side effects of these drugs include gastritis,

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hormonal imbalance as well as bleeding and renal dysfunction, which support the need of

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developing novel pharmacological approaches with reduced side effects.7

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In gout arthritis, there is an intense inflammatory response and immune cell activation. This

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is because MSU crystals induce cytokine production by tissue resident cells, including macrophage-

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like synoviocytes, and by recruited cells, including monocytes and neutrophils.1,6,7 Evidence

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indicates that MSU crystals activate macrophages via CD14 (cluster of differentiation 14) and

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TLR4 (toll-like receptor 4), which triggers the production of the pro-inflammatory mediators tumor

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necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-8 and monocyte chemotactic factors.8,9

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These cytokines increase the production of reactive species such as superoxide anion and nitric

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oxide (NO) by NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and iNOS

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(inducible nitric oxide synthase), respectively.10 These reactive oxygen species (ROS) contribute to

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tissue lesion observed in gout arthritis.11,12

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Importantly, both ROS and MSU are well-known activators of NLRP3 (NACHT, LRR and

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PYD domains-containing protein 3) inflammasome.1,13,14 Macrophages phagocytize MSU crystals,

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which causes the rupture of the phagolysosome membrane and the release of cathepsin G into the



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cytoplasm. In turn, cathepsin G activates the NLR receptor NLRP3 that recruits the adaptive

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molecule ASC (apoptosis-associated speck-like protein containing CARD), which interacts with

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pro-caspase-1 resulting in its activation to cleave pro-IL-1β to generate its mature form IL-1β.1,13–15

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Then, released IL-1β induces inflammation and further activates the transcription factor NF-

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κB.1,13,15 This activity is reciprocal since NF-κB signaling stimulates the expression of more pro-IL-

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1β, as well as inflammasome components NLRP3, ASC, pro-caspase-1.15,16 As a consequence,

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enhanced NF-κB activity together with MSU-induced activation of NLRP3 induces pronounced IL-

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1β release that orchestrates gout arthritis. Proving the importance of this concept, the treatment with

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IL-1 receptor antagonist (IL-1ra) known as Anakinra reduces acute gout arthritis flares in clinical

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practice.7 Therefore, NLRP3 inflammasome activation and NF-κB activation are central

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mechanisms in the development of gout arthritis.13,17 This NF-κB and NLRP3 inflammasome

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system can be investigated in vitro in cultured macrophages as demonstrated in the seminal

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manuscript that unveiled this essential pathologic mechanism in gout arthritis shaping our current

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understanding of this disease.13 In this in vitro macrophage system, lipopolysaccharide (LPS) is

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signal 1 to trigger NF-κB activation and induction of the expression of NLRP3 inflammasome

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components, and MSU is signal 2 that activates NLRP3 inflammasome.13,18

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Hesperidin, or 3,5,7-trihydroxy flavanone 7-rhamnoglucoside, is a flavonoid belonging to

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the class of flavanones, which is found in vegetables and fruits such as oranges and grape fruit,

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especially in citrus fruits.19 In general, flavonoids present several biological properties and have

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antioxidant, anti-allergic, anti-tumoral, antimicrobial, anti-inflammatory and analgesic effects.20

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Several studies demonstrated that hesperidin reduces oxidative stress and inflammation.21,22

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However, the bioactivity of hesperidin is profoundly affected due to its poor water solubility and

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low intestinal absorption when compared with other flavonoids. Methylation under alkaline

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conditions produces hesperidin methylchalcone (HMC), or (E)-1-[4-[[6-O-(6-Deoxy-α-L-

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mannopyranosyl)-ß-D-glucopyranosyl]oxy]-2-hydroxy-6-methoxyphenyl]-3-(3-hydroxy-44 ACS Paragon Plus Environment

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methoxyphenyl)-2-propen-1-one, that presents greater bioavailability, metabolic stability and tissue

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distribution than hesperidin.23

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HMC induces vasodilation, vascular permeability and stabilizes prostaglandin E.24 In fact,

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HMC is clinically used as a vasoprotective agent similarly to other drugs used to treat vascular

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disorders such as Cyclo 3 Fort and Cirkan.25,26 Recently, our group showed that HMC presents

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analgesic, antioxidant, and anti-inflammatory activity in mouse models of inflammatory pain. These

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effects have been associated with inhibition of cytokine production and NF-κB activation.22 In the

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current study, we investigate the therapeutic potential of HMC in a murine model of gout arthritis

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and its effects on pro-inflammatory and pro-oxidative pathways known to contribute to gout

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disease.

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2. MATERIALS AND METHODS

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2.1. Chemicals. NaCl 0.9% was supplied by Fresenius Kabi Brasil Ltda. (Aquiraz, CE,

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Brazil). NaOH was obtained from Labsynth Ltda. (Diadema, SP, Brazil). Hesperidin

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methylchalcone (HMC, PubChem CID: 6436550, MW: 624.592 g/mol, ≥95% purity) from Santa

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Cruz Biotechnology (Santa Cruz, CA, USA). ELISA Ready-SET-Go! Kits from eBioscience (San

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Diego, CA, USA). PathScan® kits from Cell Signaling Technology (Beverly, MA, USA). ABTS

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[2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate)], NBT (nitroblue tetrazolium), 2,4,6-tripiridil-s-

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triazine (TPTZ), ferric chloride hexahydrate, 6-hydroxy- 2,5,7,8-tetramethylchroman-2-11

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carboxylic (trolox) and monosodium urate crystals (MSU) from Millipore Sigma (St. Louis, MO,

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USA), and quercetin (PubChem CID: 5280343, MW: 302.238 g/mol, 95% purity) was from Acros

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(Pittsburg, PA, USA).

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2.2. Animals. Swiss mice (male, 25-30 g) were obtained from the animal facility of

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Universidade Estadual de Londrina (Londrina, PR, Brazil), and LysM-eGFP+ mice (male, 20-25 g)

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from animal facility of Ribeirão Preto Medical School, University of São Paulo (Ribeirão Preto, SP,

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Brazil). The heterogeneous Swiss mice strain is currently used by our laboratory to study the

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molecular and cellular mechanism related to inflammatory diseases and inflammatory pain12,22 and

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was used for all experimental analysis in the present study, with exception of the experiment

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evaluating the participation of LysM+ leukocytes in the model, in which LysM-eGFP+ mice were

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used. LysM-eGFP+ strain was generated through its background C57BL/6 mouse and present eGFP

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(enhanced green fluorescent protein) expression controlled by the lysozyme M promoter (LysM),

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enzyme that is found predominantly in neutrophils granules and secreted upon cell activation.27

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LysM-eGFP+ mice were used to corroborate the data on leukocyte recruitment using total and

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differential cell counts. The difference in the background of mice (Swiss and C56BL/6) is a likely


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explanation for the difference in the intensity of cellular recruitment observed in the results section.

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Animals were housed in standard clear plastic cages with water and food ad libitum, in a 12/12h

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light/dark cycle and controlled temperature (21ºC). Behavioral measurements were acquired

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between 9 a.m. and 5 p.m. in a temperature controlled room (21ºC). Euthanasia was performed with

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deep isoflurane anesthesia (5% in oxygen, Abbott Park, IL, USA) followed by cervical dislocation

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and decapitation. All procedures were previously approved by institutional Ethics Committee on

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Animal Use (CEUA) of Universidade Estadual de Londrina (process n. 14600.2013.73). Every

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effort was made to avoid animal stress and discomfort and reduce the number of mice used. We did

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not observe unexpected death of mice during the study.

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2.3. Preparation of MSU crystal. We prepared the MSU crystals following the method

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described previously.12 Monosodium urate (800 mg) was dissolved in boiling milli-Q water (155

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ml) and NaOH (5 ml). The solution was then adjusted to the pH 7.2 and gradually cooled at room

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temperature. After centrifugation (3,000 g, 2 min, 4◦C), the aqueous portion was evaporated and

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crystals sterilized at 180◦C for 2 hours. Crystals were collected and kept sterile until use. Presence

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of endotoxin in the MSU crystals was assessed using a ToxinSensor™ Single Test Kit (GenSricpt)

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following manufacturer’s instructions. This is a qualitative in vitro end-point endotoxin test used for

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pre-clinical studies and supplies a series of kits with different sensitivities of which we used the

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highest sensitivity testing tube (0.015 EU/mL). Each kit contains a lysate of Limulus Polyphemus

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(LAL) that detect the labeled concentration (EU/mL) of the FDA Reference Standard Endotoxin.

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Briefly, 200 µL of sample was added to tubes containing LAL reagent and incubated in a water bath

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(60 min, 37°C). The presence of endotoxin results in the formation of a viscous gel. Entodotoxin

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free water was used as negative control. We tested titrated concentrations of MSU diluted in

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endotoxin free water (10, 5, 2, 1 and 0.5 mg/mL). The test was positive for 200 µL of a 10 mg/mL

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solution (2000 µg of MSU crystals), and negative for 200 µL of a 5 mg/ml solution (1000 µg of



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MSU crystals) and solutions with lower concentrations of MSU crystals. These results indicate that

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the endotoxin concentration in 200 µL of a 10 mg/mL solution of MSU is between 0.015 and 0.03

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EU/mL. The in vivo dose of MSU used in this study was 100 µg/ 10 µL and in vitro concentration

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of MSU was 450 µg/mL. Therefore, the selected dose and concentration of MSU did not present

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detectable endotoxin levels.

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2.4. Induction of articular joint inflammation. MSU (100 µg/10µL, i.a.) injection of the

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right knee was performed to induce joint inflammation in mice. Sterile saline was administered in

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the control group.12 To reduce suffering and stress, all i.a. injections were performed under

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anesthesia (isoflurane 5% in oxygen, Abbott Laboratories, IL, USA).

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2.5. Electronic pressure-meter test. Flexion-induced pain was performed using the

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electronic pressure-meter test as previously described.12 Briefly, tibiotarsal joint flexion was

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induced by application of pressure in the plantar surface using a non-nociceptive tip probe (area of

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4.15 mm2 ). The pressure necessary to evoke flexion-elicited paw withdrawal response was

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automatically recorded in grams (n = 6 mice/group/experiment, experiments were performed in

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twice).

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2.6. Edema. A Mitutoyo caliper (Suzano, SP, Brazil) was used to measure thickness of knee

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joint before (time-point zero) and after the i.a. injection of MSU or saline at different time-points (n

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= 6/group/experiment, experiments were performed twice). Increases in articular thickness were

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calculated as the individual differences between the measurements after injection and the starting

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value (time-point zero), and edema presented as ∆ reaction in mm.22

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2.7. Total and differential cell counts. Knee joint cavity was washed with 3.3 µL of PBS +

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1 mM EDTA, three times (one knee joint wash per sample, n = 6/group/experiment, experiments

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were performed twice), Samples were diluted in Turk`s solution and used for total leukocyte

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quantification in a Neubauer chamber. Cytocentrifuge stained slides (Rosenfeld) and Cytospin 4

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(Shandon, Pittsburg, PA, USA) were used for differential cell counts. Widefield light microscope

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was used for quantification and results presented as mean ± SEM of cells (x103) per cavity.12

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2.8. Immunofluorescence assay. Articular fluids of LysM-eGFP+ mice were collected in

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sterile slides 15 hours after MSU i.a. injection into the knee joints using the same procedures

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presented in sub-section 2.7., and processed for immunofluorescence assay (one knee joint wash per

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sample, n = 6/group/experiment, experiments were performed twice). DAPI fluorescent stain was

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added to slides for localization of nucleus in each sample. The representative images and

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quantitative analysis were performed using a confocal microscope (SP8, Leica Microsystems,

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Mannheim, Germany). The intensity of fluorescence was quantified in randomly selected fields of

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different groups by a blind evaluator. Result is presented as the percentage of GFP fluorescent

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intensity.

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2.9. Histopathological analysis. Knee joints were dissected 15 hours after MSU or saline

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injection (one whole knee joint per sample, n = 6/group/experiment, experiments were performed

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twice) and fixed in 10% buffered formalin in PBS. After fixation, decalcification of samples was

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performed by incubation in EDTA for 10 days. Decalcified samples were embedded in paraffin,

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sectioned, and stained with hematoxylin and eosin (H&E) for histopathological analysis.

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Conventional morphological analysis was performed in 10 aleatory fields by an investigator blinded

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to the experimental groups.

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2.10. Measurement of reduced glutathione (GSH) levels. Knee joints were dissected and

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samples collected 15 h after MSU injection (one whole knee joint per sample, n =

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6/group/experiment, and experiments were performed twice) and stored at -80 ºC for 48 h or more.

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Samples were homogenized in 0.02 M EDTA (200 µL), and trichloroacetic acid (25 µL, 50% v/v)

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added the homogenate. Samples were vortexed three times over 15 min, centrifuged (15 min, 1500

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g, 4 ºC), and supernatant collected. Supernatants were mixed with TRIS buffer (200 µL, 0.2 M, pH

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8.2) and DTNB (10 µL, 0.01 M) and incubated for 5 min at room temperature before absorbance

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measurement at 412 nm (Multiskan GO, Thermo Scientific). Samples were normalized to

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absorbance of wells with reagent only. GSH values were calculated using a standard GSH curve and

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expressed as GSH/mg of protein.22

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2.11. ABTS and FRAP assays. Samples from knee joints were dissected 15 h after injection

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of MSU or saline and used to evaluate the antioxidant capacity by ABTS and FRAP microplate-

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adapted assays as described before.22 After dissection, samples were homogenized in 500 µL of ice-

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cold KCl buffer (1.15% w/v), centrifuged (200 g, 10 min, 4 °C), and supernatants collected (one

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whole knee joint per sample, n = 6/group/experiment, and experiments were performed twice). For

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ABTS assay, 10 µL of supernatant were mixed with 200 µL of ABTS solution, incubated for 6 min

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at 25 °C, and absorbance determined (730 nm). For FRAP assay, 10 µL of supernatant were mixed

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with 150 µL of fresh FRAP reagent, incubated for 30 min at 37 °C, and absorbance determined at

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595 nm (Multiskan GO Thermo Scientific). Antioxidant capacity of the samples was compared with

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a standard curve of antioxidant Trolox (0.02 – 20 nmol).

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2.12. Production of superoxide anion. Knee joint homogenates (10 mg/mL) were prepared

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in 1.15% KCl, and superoxide anion (O2-) production measured using the microplate-adapted

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nitroblue tetrazolium (NBT) reduction assay (one whole knee joint per sample, n =


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6/group/experiment, experiments were performed twice). Samples were collected 15 h after MSU

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injection.22 Absorbance was measured at 600 nm, and results normalized by the tissue weight.

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2.13. Production of Nitrite. Knee joint was dissected 15 h after MSU or saline injection

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(one whole knee joint per sample, n = 6/group/experiment, experiments were performed twice),

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homogenized in 500 µL of saline, and the concentration of nitrite (NO2-) determined using the

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Griess reaction.28 Homogenates (100 µL) were incubated with Griess reagent (100 µL) for 5 min

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(25 °C), and the absorbance at 550 nm compared to a standard curve of NaNO2 solution. Tissue

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weight was used for normalization, and the results expressed as µmol of NO2- /mg.

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2.14. RT-qPCR. Samples of articular tissue were homogenized in Trizol reagent and used

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for RT-qPCR (real time and quantitative polymerase chain reaction) as previously described.12. SV

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Total RNA Isolation System (Promega Corporation, Madison, WI, USA) was used for RNA

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extraction (one whole knee joint per sample, n = 6/group/experiment, experiments were performed

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twice). All reactions were performed in triplicate. Cycling conditions: 2 min at 50° C, 2 min at

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95°C, and 40 cycles of 15 s at 95° C and 30s at 60° C (LightCycler Nano Instrument, Roche,

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Mississauga, ON, USA) using the Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, USA).

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The primers used were GAPDH forward: CAT ACC AGG AAA TGA GCT TG, reverse: ATG

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ACA TCA AGA AGG TGG TG; Nrf2, forward: TCA CAC GAG ATG AGC TTA GGG CAA,

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reverse: TAC AGT TCT GGG CGG CGA CTT TAT; gp91phox, forward: AGC TAT GAG GTG

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GTG ATG TTA GTG G, reverse: CAC AAT ATT TGT ACC AGA CAG ACT TGA G; Nlrp3,

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forward: AGC TAT GAG GTG GTG ATG TTA GTG G, reverse: CAC AAT ATT TGT ACC

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AGA CAG ACT TGA G; HO-1, forward: CCC AAA ACT GGC CTG TAA AA, reverse: CGT

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GGT CAG TCA ACA TGG AT; pro-caspase-1: forward: TGG TCT TGT GAC TTG GAG GA,

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reverse: TGG CTT CTT ATT GGC ACG AT; pro-IL-1β, forward: GAA ATG CCA CCT TTT



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GAC AGT G, reverse: TGG ATG CTC TCA TCA GGA CAG; ASC, forward: ATG GGG CGG

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GCA CGA GAT G, reverse: GCT CTG CTC CAG GTC CAT CAC. mRNA data was normalized

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by GAPDH expression in each sample.

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2.15. Cytokine measurement. Knee joint was dissected 15 h after saline or MSU injection

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and homogenized in 500µL of buffer containing protease inhibitor (1 mM phenylmethanesulfonyl

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fluoride, Sigma Aldrich) (one whole knee joint per sample, n = 6/group/experiment, experiments

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were performed twice. Levels of TNF-α, TGF-β, IL-1β, IL-6, and IL-10 were measured by ELISA

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using eBioscience kits and manufacture instructions, and the results expressed in picograms

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(pg)/100 mg of tissue22

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2.16. Activation of NF-κB. Knee joints were dissected 15 h after saline or MSU injection

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(one whole knee joint per sample, n = 6/group/experiment, experiments were performed twice) and

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homogenized at 4°C in lysis buffer (Cell Signaling). Samples were centrifuged (14000 rpm, 10 min,

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4 °C) and supernatants collected. Total and phosphorylated NF-κB (p65 subunit) levels were

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determined by ELISA PathScan® kits (Cell Signaling). Absorbance was measured at 450 nm and

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results presented as total p65/phospho-p65 ratio as described previously.12

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2.17. Isolation and culture of bone marrow-derived macrophages for inflammasome

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activation assay. Mouse bones (femora and tibiae) were flushed with RPMI 1640 media and bone

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marrow cells suspension cultured in supplemented RPMI 1640 medium (10% FBS + 15% L929 cell

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conditioned medium). At day 7, bone marrow-derived macrophages (BMDMs) were harvested and

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seeded in 96-well plates (1.5x105 cells per well). BMDMs were pre-treated with lipopolysaccharide

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(LPS, 500 ng/mL) from Escherichia coli (Santa Cruz Biotechnology) and stimulated with 450

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µg/mL of MSU 3 h later to induce activation of NLRP3 inflammasome as previously described.13


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BMDMs were treated with HMC (30, 100 or 300 µM), or Quercetin (30 µM), 30 min before MSU

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stimulation. After 5 h of MSU application, cell culture supernatants were collected and IL-1β

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concentration quantitated by ELISA. Lactate dehydrogenase (LDH) release in the supernatant was

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used as a marker of cellular viability (n = 6 wells/group/experiment, experiments were performed

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twice).

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2.18. Experimental procedures. HMC or saline (vehicle) was administered orally (3- 30

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mg/kg) 1 h before stimulus with MSU (100 µg/10 µL, i.a.). Edema and mechanical hyperalgesia

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were measured at different time points after MSU or saline injection. Total and differential

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quantification of articular leukocytes was evaluated 15 h after MSU injection. The dose of 30 mg/kg

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of HMC was used in subsequent experiments. Mice received HMC (30 mg/kg) or saline, and

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stimulated with MSU (100 µg/10 µL, i.a. injection) for the determination of LysM-eGFP+

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neutrophil infiltration in the knee joint lavage, histopathological analysis, cytokine production

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(TNF-α, TGF-β, IL-1β, IL-6, and IL-10), oxidative stress (GSH, FRAP, ABTS, superoxide anion,

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and NO assays), NF-κB activation (total NFκB p65/phosphorylated NFκB p65 ratio), and gp91phox,

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HO-1 (heme-oxigenase-1), Nrf2 (nuclear factor (erythroid-derived 2)-like 2)), NLRP3 (NACHT,

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LRR and PYD domains-containing protein 3), ASC (apoptosis-associated speck-like protein

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containing a CARD), pro-caspase-1 and pro-IL-1β mRNA expression in the knee joint samples

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collected 15 h after i.a. stimulus with MSU crystals. In vitro analyses using LPS-primed BMDMs

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treated with HMC (30, 100 or 300 µM) were performed 5 hours after subsequent stimulation with

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MSU crystals (450 µg/mL) for the determination of mature IL-1β secretion. Figure 1 summarizes

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these in vivo (A) and in vitro (B) experimental procedures. Unlike humans, mice express the

322

peroxisomal enzyme uricase that degrades urate. In fact, evidence demonstrates that humans have

323

higher levels of urate than mice (≈240-360 µM vs ≈30-50 µM, respectively).17,29 Uricase efficiently

324

oxidizes urate to allantoin, reducing the levels of uric acid in the mouse. Consequently, mice do not



325

develop gout arthritis under natural conditions, and therefore, the inflammatory response has a

326

shorter duration in mice than in humans.17,29 This is an explanation for the time point of evaluation

327

at 15h, which was standardized in previous studies of our group.30,31 Furthermore, this is also a

328

limitation of the mouse model of gout arthritis, which without the expression of uricase could

329

present a prolonged inflammatory response.17,29

330 331

2.19. Data analyses. Results are presented as means ± standard error mean (SEM) of

332

measurements (n = 6/group/experiment, two independent experiments). Two-way ANOVA was

333

used for statistical comparison of two or more groups at multiple time points (Fig. 2). One-way

334

ANOVA was used for statistical comparison of three or more groups at a single time point (Fig. 3-

335

10). Tukey’s post hoc test was used. Analyzed factors include time, treatments (groups), and time

336

versus treatment interaction. Statistical differences were considered when p < 0.05. GraphPad Prism

337

v5.0 was used for statistical analysis.

338 339 340 341 342 343 344 345 346 347 348 349


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350


3. RESULTS

351 352

3.1. HMC reduces mechanical hyperalgesia and articular edema induced by MSU.

353

Intra-articular injection of MSU crystals caused mechanical hyperalgesia as expected. At the dose

354

of 3 mg/kg, HMC showed no protective effects on hyperalgesia (Figure 2A). At the medium dose

355

(10 mg/kg), HMC was able to reduce significantly the hyperalgesia at 7h and 15 h after MSU

356

injection when compared to groups that received vehicle or the low dose of HMC (Figure 2A). At

357

the higher dose (30 mg/kg), HMC inhibited hyperalgesia between 5-15 h (up to 44%) after MSU

358

injection. The higher dose of HMC was more efficient than the medium dose at 15 h time point

359

(Figure 2A). In the same treatment schedule, the low dose of HMC (3 mg/kg) had no effect on

360

articular edema, while the medium dose (10 mg/kg) of HMC significantly inhibited MSU-induced

361

edema between 7-15 h (Figure 2B). In parallel with its effect on hyperalgesia, the higher dose of

362

HMC (30 mg/kg) was also more efficient than the other two doses in reducing articular edema (5-15

363

h, 54%) (Figure 2B). Thus, HMC dose-dependently inhibits MSU-induced hyperalgesia and edema.

364 365

3.2. HMC inhibits intra-articular recruitment of leukocytes induced by MSU. After

366

intra-articular injection of MSU, a significant increase of total leukocytes (Figure 3A), mononuclear

367

(Figure 3B) and neutrophil (Figure 3C) counts was observed in the knee joint exudate. HMC

368

presented the same profile of inhibition of MSU-induced intra-articular leukocytes (total leukocytes,

369

mononuclear cells, and neutrophils) (Figure 3A-C) in which the lower dose (3 mg/kg) presented no

370

effect, medium dose (10 mg/kg) induced a tendency (not significant) of reduction, and the higher

371

dose (30 mg/kg) induced a reduction of 60% in total leukocytes (Figure 3A), 70% in mononuclear

372

cells (Figure 3B), and 56% in neutrophils (Figure 3C). Thus, we selected the higher dose of HMC

373

(30 mg/kg) for the next tests. In a separate protocol, we also performed experiments using samples

374

of articular washes of LysM-eGFP+ mice. LysM is a reliable marker of neutrophil population.32 In



375

fact, this gene-targeted mouse expressing eGFP is considered an efficient tool to determine the

376

neutrophils/monocytes counts in models of diseases.27,32 In this sense, our primary aim in using this

377

specific tool was to determine the dynamics of neutrophils in experimental gout and whether

378

treatment with HMC would be able to reduce the neutrophilic infiltration, and not compare the

379

response obtained in the two different strains used in the present study (Swiss vs LysM-eGFP+).

380

MSU induced a robust infiltration of LysM+ neutrophils when compared to control animals, which

381

was inhibited by HMC treatment (Figure 3D-M).

382 383

3.3. HMC reduces MSU-induced knee joint synovitis. Histopathological analysis show

384

that saline group presented regular histological appearance (Figure 4A). In turn, MSU induced

385

significant joint synovitis with marked increase of inflammatory cells (Figure 4B), which was

386

decrease by the treatment with HMC by 76% (Figure 4C) as observed by inflammatory cell counts

387

per field in experimental groups (Figure 4D).

388 389

3.4. HMC inhibits MSU-induced depletion of endogenous antioxidant apparatus and

390

oxidative stress. MSU induced oxidative stress in the knee joint as observed by a significant

391

decrease in antioxidant defense in the knee joint, which was prevented by HMC treatment as

392

observed by GSH (62%, Figure 5A), FRAP (78%, Figure 5B) and ABTS (73%, Figure 5C) assays.

393

Corroborating the increase of antioxidant defenses, HMC was able to inhibit MSU-induced

394

superoxide anion (89%, Figure 5D) and NO (48%, Figure 5E) production. Thus, HMC inhibited

395

MSU-induced oxidative stress.

396 397

3.5. HMC inhibits the expression of gp91phox and increases expression of Nfr2 and HO-

398

1 in the knee joint after MSU injection. MSU enhanced the expression of gp91phox mRNA, and

399

this increase was significantly inhibited by HMC in 66% (Figure 6A). While no differences in Nrf2


Page 16 of 48

Page 17 of 48


400

and HO-1 mRNA expression were observed after MSU injection, HMC enhanced their expression

401

by approximately 4 and 5 fold, respectively (Figure 6B and 6C, respectively). Thus, HMC inhibited

402

the MSU-induced expression of a gene involved in promoting the production of superoxide anion,

403

and induced the expression of genes involved in promoting antioxidant effects.

404

3.6. HMC modulates MSU-induced cytokine production in the knee joint. Following

405 406

the intra-articular injection of MSU, increased levels of the pro-inflammatory cytokines TNFα, IL-

407

1β and IL-6 were observed, and this was inhibited by HMC treatment (35%, 72% and 37%,

408

respectively) (Figure 7A-C). HMC also efficiently reduced the levels of IL-10 after MSU injection

409

(46%, Figure 7D). Nevertheless, HMC induced approximately 2 fold increase of MSU-induced

410

TGF-β production, which is also an anti-inflammatory cytokine in gout arthritis (Figure 7E). Thus,

411

HMC inhibited pro-inflammatory cytokines production and also modulated in a different manner

412

the levels the anti-inflammatory cytokines, reducing IL-10 and enhancing TGF-β.

413

3.7. HMC inhibits the activity of NF-κB in the knee joint after MSU injection. Using

414 415

ELISA kits, we observed that MSU decreased the total NF-κB p65 subunit/phosphorylated NF-κB

416

p65 subunit ratio indicating NF-κB activation, which was inhibited by HMC treatment (41%)

417

(Figure 8). Thus, HMC inhibited the activation of NF-κB induced by MSU injection in the knee

418

joint.

419 420

3.8. HMC inhibits MSU-induced increase of inflammasome components expression in

421

the knee joint. MSU stimulus increased mRNA expression of inflammasome components

422

(NLRP3, ASC, pro-caspase-1, and pro-IL-1β) compared to saline control group (Figure 9A-D,

423

respectively). In turn, HMC treatment inhibited the expression of all components of inflammasome

424

(NLRP3 by 72%, ASC by 77%, pro-caspase-1 by 71%, and pro-IL-1β by 73%) (Figure 9A-D,



425

respectively). Thus, HMC inhibited the MSU-induced increase of inflammasome platform

426

components mRNA expression.

427 428

3.9. HMC does not inhibit the release of mature IL-1β by BMDMs induced by MSU.

429

Stimulation of BMDMs with LPS (priming, signal 1) followed by MSU (signal 2) resulted in

430

increased levels of IL-1β in the supernatant, and this effect was not inhibited by HMC treatment

431

(30-300 µM; Figure 10). On the other hand, treatment of these cells with the positive control

432

quercetin (30 µM) inhibited IL-1β secretion induced by MSU stimulation (Figure 10). The

433

quercetin result is a control demonstrating that this system works properly.18 None of the treatments

434

affected the LDH levels in the culture supernatant, which indicates that cell viability was not

435

affected (data not shown). Isolated stimulation of BMDMs with LPS or MSU was not able to

436

promote increases in supernatant levels of mature IL-1β (data not shown). Thus, HMC could not

437

inhibit the activity of inflammasome in BMDMs cells stimulated with MSU in vitro, indicating that

438

directly targeting inflammasome activation is not a mechanism of action of HMC in gout arthritis.

439 440 441 442 443 444 445 446 447 448 449


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450


4. DISCUSSION

451 452

HMC is a safe drug with therapeutic applications for vascular diseases. HMC improves

453

vascular tonus and reduces symptoms of acute hemorrhoid and other venous diseases.33,34 Further,

454

evidence shows that HMC has anti-inflammatory and analgesic properties.10,22,35 In this sense,

455

repurpusing of HMC for treatment of pain and inflammation seems reasonable. The present data

456

support the potential of HMC to inhibit inflammation and pain in an animal model of gout arthritis

457

by blocking NF-kB activation without directly affecting inflammasome activation. The lack of

458

effect of HMC over inflammasome activation indicates that HMC would present an additive effect

459

with the current IL-1ra therapy since these molecules act by distinct though complementary

460

mechanisms.

461

Although HMC inhibitory effects upon inflammatory pain have already been demonstrated

462

by our group using classical models of pain such as formalin and carrageenan22, this MSU intra-

463

articular injection model differs from previously used models since represents specifically the

464

physiopathological mechanisms and therapeutic targets of gout arthritis. Oral treatment with HMC

465

dose-dependently inhibited mechanical hyperalgesia, joint edema, and recruitment of leukocytes

466

induced by intra-articular MSU. Thus, HMC consistently inhibited MSU clinical inflammatory

467

signs. HMC also inhibited MSU-induced neutrophil recruitment and synovitis that are important

468

parameters indicating reduction of tissue lesion. Activation of tissue resident macrophages and the

469

recruitment of other leukocytes (e.g. neutrophils) represent an important mechanism of

470

pathogenesis in acute gout. Leukocytes actively contribute to the gout disease36 by releasing varied

471

mediators including ROS, proteolytic enzymes and pro-inflammatory cytokines (e.g. TNFα, IL-1β),

472

which contribute to cartilage degradation and joint damage.11,12,37 Here, the treatment with HMC

473

reduced the accumulation of neutrohils into the joint, as identified by morphology and

474

immunofluorescence using LysM-eGFP+ mice. Macrophages also express LysM, but to a lesser



475

extent than neutrophils.27 Therefore, the HMC inhibition of leukocyte infiltration, joint edema, pain

476

and synovitis demonstrates the therapeutic potential of this flavonoid.

477

Active NADPH oxidase in phagocytes produces large quantities of superoxide anion

478

generating various ROS that target proteins, lipids and nucleic acids.14 Nitric oxide (NO) is also

479

produced and contributes to arthritis by reacting with superoxide anion generating peroxynitrite that

480

causes tissue lesion.38 Synovial tissue from gout arthritis patients exhibits an increase of NO

481

production, and MSU crystals increases NO production by monocytes/macrophages of gout

482

patients.39 The excessive production of ROS causes depletes GSH and promotes oxidation of

483

cysteine in proteins, resulting in oxidative stress-induced lesioning of tissues. ROS also activate

484

NF-κB and NLRP3 inflammasome signaling pathways, which are crucial in gout arthritis

485

pathogenesis.14 Cytokines and oxidative stress contribute to edema40, leukocyte recruitment41, and

486

further pro-inflammatory cytokines production and pain.42 HMC inhibited MSU-induced oxidative

487

stress and re-established endogenous antioxidant levels and activity. The antioxidant effects of

488

HMC were not solely explained by its structural antioxidant chemical groups.20 HMC also induced

489

Nrf2 mRNA expression, an important transcription factor that induces HO-1 and GSH43 lining up

490

with the results of enhanced HO-1 mRNA expression and GSH levels by HMC treatment. Hence,

491

HMC inhibited gp91phox mRNA expression (NADPH oxidase) and the formation of its product

492

superoxide anion. Therefore, HMC has antioxidant chemical structures20 and up-regulates

493

endogenous antioxidant and anti-inflammatory mechanisms.

494

Pro-inflammatory cytokines present a pivotal role in gout.37 HMC inhibited MSU-induced

495

pro-inflammatory cytokines production (TNFα, IL-1β and IL-6). These cytokines induce the

496

activation of NADPH oxidase and the production of superoxide anion, NO and other ROS.10

497

Furthermore, these cytokines induce pain, edema and leukocyte recruitment.44,45 Therefore, the

498

HMC inhibition of MSU-induced cytokine production accounted to reducing oxidative stress, pain,

499

edema and leukocyte recruitment. The production of the anti-inflammatory cytokine IL-10 is


Page 20 of 48

Page 21 of 48


500

concomitant with the production of pro-inflammatory cytokines, which helps to limit their

501

production and activity.46 Flavonoids have been shown to induce IL-10 production, which explains

502

in part the anti-inflammatory actions of these molecules.47 However, HMC did not induce an

503

increase of IL-10, but rather reduced its production. It is likely that as HMC inhibited the pro-

504

inflammatory cytokines production, IL-10 release was also reduced since its limiting actions over

505

inflammatory cytokines were not necessary.35 Furthermore, these results show that not all

506

flavonoids act by the same mechanisms. MSU-induced knee joint inflammation was also

507

accompanied by a slight non-significant increase of TGF-β production. HMC treatment enhanced

508

MSU-induced TGF-β production. TGF-β increases the phagocytosis of apoptotic neutrophils by

509

monocytes/macrophages. This clearance of cell debris is very important for resolution of

510

inflammation.48 Therefore, the increase of TGF-β production in response to HMC administration

511

may be a contributing mechanism to its anti-inflammatory and analgesic effect.

512

NF-κB and NLRP3 inflammasome signaling pathways have pivotal roles in gout.13,49

513

Activation of the transcription factor NF-κB increases the expression of cytokines and enzymes that

514

produce ROS.12,22,50 Cytokines and ROS, in turn, further activate NF-κB, and this reciprocal circle

515

amplifies the inflammatory response.51 HMC inhibited the pro-inflammatory cytokines production

516

and the oxidative stress induced by MSU. Accordingly, HMC inhibited MSU-induced NF-κB

517

activation in the knee joint, thus, HMC targets an essential transcription factor in the inflammatory

518

response explaining the prominent effect of this flavonoid in reducing MSU-induced knee joint

519

inflammation and pain. Activation of NF-κB also induces the expression of the components of

520

NLRP3 inflammasome (NLRP3, ASC, pro-caspase-1, and pro-IL-1β).16,52 In agreement with the

521

HMC inhibition of NF-κB activation, this flavonoid reduced NLRP3, ASC, pro-caspase-1, and pro-

522

IL-1β mRNA expression induced by MSU in vivo. This result is important since it demonstrates

523

that HMC reduces the up-regulation of an inflammatory platform that is essential in gout

524

pathogenesis. 21 ACS Paragon Plus Environment


Flavonoids such as quercetin inhibit ASC oligomerization and the activation of

525 526

inflammasome, which contributes to reduce inflammasome-dependent inflammation in a model of

527

vasculitis18 and gout arthritis.12 Therefore, we reasoned that it was rational to evaluate whether

528

HMC would also inhibit inflammasome activation. The flavonoid quercetin decreased secretion of

529

IL-1β by MSU-stimulated, LPS-primed BMDMs, corroborating previous data18 and that this model

530

was working properly in our experimental conditions. However, HMC did not affect MSU-induced

531

inflammasome-dependent IL-1β secretion. Therefore, inhibiting MSU-induced NLRP3 activation is

532

not a mechanism of action of HMC. Other studies with various inflammasome activators are

533

necessary to investigate the extent of inflammasome inhibition by flavonoids, but our results do

534

suggest that not all flavonoids inhibit inflammasome activation, and therefore, not all flavonoids

535

have the same mechanism of action. Further studies addressing structure-relationship of flavonoids

536

and inflammasome, and also applying other inflammasome activators will be necessary to

537

understand these differences in greater detail.

538

In conclusion, our study demonstrates that HMC inhibits MSU-induced acute knee joint

539

inflammation and pain. The mechanism of action of HMC is not dependent on inhibiting MSU

540

crystals-induced inflammasome activation, but rather it depends on inhibiting NF-κB activation and

541

induces Nrf2/HO-1 pathway. As a consequence of these effects, HMC inhibits the recruitment of

542

cells and production of ROS and cytokines, effects that explain its anti-inflammatory and analgesic

543

effects.

544 545 546 547 548 549


Page 22 of 48

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550

AUTHOR INFORMATION

551

Corresponding author

552

*Phone: + 55 43 33714979. E-mail: [email protected]; [email protected].

553

Authors contributions

554

R.C. and W.A.V.J. designed the study. K.W.R.M., F.A.P.R., S.M.B., L.S.F., V.F., F.A.A., M.M.T.,

555

J.C.A.F., T.M.C., F.Q.C., R.C. and W.A.V.J. planned experiments and analyze the data. K.W.R.M.,

556

F.A.P.R., S.M.B., L.S.F., and V.F. performed the experiments. F.A.A., M.M.T., J.C.A.F., T.M.C.

557

and F.Q.C. provided essential materials. K.W.R.M., F.A.P.R., S.M.B., R.C. and W.A.V.J. wrote the

558

manuscript. W.A.V.J. supervised the study. All authors read and approved the manuscript.

559 560

Acknowledgment

561

This work was supported by grants from Coordenadoria de Aperfeiçoamento de Pessoal de Nível

562

Superior (CAPES), Financiadora de Estudo e Projetos–Apoio à Infraestrutura (CT-INFRA 01/2011;

563

process 01.13.0049.00), Central Multiusuária de Laboratórios de Pesquisa da UEL (CMLP-UEL),

564

São Paulo Research Foundation (FAPESP), Center for Research on Inflammatory Diseases (CRID),

565

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Pesquisa para o Sistema

566

Único de Saúde (PPSUS) grant supported by Ministério da Ciência, Tecnologia e Inovação (MCTI),

567

Secretaria da Ciência, Tecnologia e Ensino Superior (SETI), Decit/SCTIE/MS through CNPq with

568

the support of Fundação Araucária and Secretaria da Saúde do Estado do Paraná (SESA-PR), and

569

Parana State Government (Brazil).

570 571

Notes

572

There are no competing financial interests to be declared.

573 574



575

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Figures caption

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Figure 1. Schematic representation of in vivo (a) and in vitro (b) protocols used in the study.

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Figure 2. HMC inhibits MSU-induced articular mechanical hyperalgesia and edema. Mice were

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treated with HMC (3, 10 or 30 mg/kg/saline, p.o.) or vehicle (saline) 1 h before MSU (100 µg/10

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µL) i.a injection. Mechanical hyperalgesia (A) and edema (B) were assessed 1-15 h after MSU

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administration using an electronic pressure test and a caliper, respectively. Results are presented as

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means ± SEM of 6 mice per group per experiment and are representative of 2 independent

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experiments. *p < 0.05 compared to saline group; #p < 0.05 compared to MSU + vehicle group; ƒp

Hesperidin Methylchalcone Suppresses Experimental Gout Arthritis in

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