Trans-Chalcone attenuates pain and inflammation in

experimental acute gout arthritis in mice

Larissa Staurengo-Ferrari1, Kenji W. Ruiz-Miyazawa1, Felipe A. Pinho-Ribeiro1, Victor
Fattori1, Tiago H. Zaninelli1, Stephanie Badaro-Garcia1, Sergio M. Borghi1, Thacyana T.
Carvalho1, Jose C. Alves-Filho2, Thiago M. Cunha2, Fernando Q. Cunha2, Rubia
Casagrande1, Waldiceu A. Verri1*

1
Department of Pathology, Universidade Estadual de Londrina, Brazil, 2Faculdade de
Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil
Submitted to Journal:
Frontiers in Pharmacology

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Specialty Section:

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Inflammation Pharmacology

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ISSN:

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1663-9812

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Article type:

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Original Research Article

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Received on:
13 Jun 2018

Accepted on:
13 Sep 2018

Provisional PDF published on:

13 Sep 2018

Frontiers website link:

www.frontiersin.org

Citation:
Staurengo-ferrari L, Ruiz-miyazawa KW, Pinho-ribeiro FA, Fattori V, Zaninelli TH, Badaro-garcia S,
Borghi SM, Carvalho TT, Alves-filho JC, Cunha TM, Cunha FQ, Casagrande R and Verri WA(2018) Trans-
Chalcone attenuates pain and inflammation in experimental acute gout arthritis in mice. Front.
Pharmacol. 9:1123. doi:10.3389/fphar.2018.01123

Copyright statement:
© 2018 Staurengo-ferrari, Ruiz-miyazawa, Pinho-ribeiro, Fattori, Zaninelli, Badaro-garcia, Borghi,
Carvalho, Alves-filho, Cunha, Cunha, Casagrande and Verri. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and
reproduction in other forums is permitted, provided the original author(s) or licensor are credited
and that the original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply with these terms.
This Provisional PDF corresponds to the article as it appeared upon acceptance, after peer-review. Fully formatted PDF
and full text (HTML) versions will be made available soon.

Frontiers in Pharmacology | www.frontiersin.org

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1 Trans-Chalcone attenuates pain and inflammation in

2 experimental acute gout arthritis in mice
3 Larissa Staurengo-Ferrari1,f, Kenji W. Ruiz-Miyazawa1,f, Felipe A. Pinho-Ribeiro1,
4 Victor Fattori1, Tiago H. Zaninelli1, Stephanie Badaro-Garcia1, Sergio M. Borghi1,
5 Thacyana T. Carvalho1, Jose C. Alves-Filho2, Thiago M. Cunha2, Fernando Q. Cunha2,
6 Rubia Casagrande3, and Waldiceu A. Verri, Jr.1,*
1Departamento
7 de Ciências Patológicas, Universidade Estadual de Londrina-UEL,
8 Londrina, Paraná, PR, Brazil.
2Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo,
9
10 Ribeirão Preto, São Paulo, SP, Brazil
3Departamento de Ciências Farmacêuticas, Universidade Estadual de Londrina-UEL,
11
12 Londrina, Paraná, PR, Brazil.
ƒThese
13 authors contributed equally.

14 *Correspondence:
15 Waldiceu A. Verri Jr
16 waldiceujr@yahoo.com.br; waverri@uel.br

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17 Running title: Trans-Chalcone attenuates gout arthritis

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18 Manuscript length: 5.413 words, 7 figures

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19 ABSTRACT

20 Gouty arthritis is characterized by an intense inflammatory response to monosodium urate

21 crystals (MSU), which induces severe pain and reduction in the life quality of patients.
22 Trans-Chalcone (1,3-diphenyl-2-propen-1-one) is a flavonoid precursor presenting
23 biological activities such as anti-inflammatory and antioxidant proprieties. Thus, the aim
24 of this work was to evaluate the protective effects of trans-Chalcone in experimental gout
25 arthritis in mice. Mice were treated with trans-Chalcone (3, 10, or 30mg/kg, per oral) or
26 vehicle (Tween 80 20% plus saline) 30 minutes before intra-articular injection of MSU
27 (100 µg/knee joint, intra-articular). We observed that trans-Chalcone inhibited MSU-
28 induced mechanical hyperalgesia, edema, and leukocyte recruitment (total leukocytes,
29 neutrophils, and mononuclear cells) in a dose-dependent manner. Trans-Chalcone also
30 decreased inflammatory cell recruitment as observed in Hematoxylin and Eosin (HE)
31 staining and the intensity of fluorescence of LysM-eGFP+ cells in the confocal
32 microscopy. Trans-Chalcone reduced MSU-induced oxidative stress as observed by an
33 increase in the antioxidant defense [Glutathione (GSH), Ferric Reducing (FRAP), and
34 2,2’-Azinobis-3-ethylbenzothiazoline 6-sulfonic acid (ABTS assays) and reduction in
35 reactive oxygen and nitrogen species production [superoxide anion (NBT assay) and
36 nitrite (NO assay)]. Furthermore, it reduced in vivo MSU-induced interleukin- 1β (IL-1β),
37 Tumor necrosis factor- α (TNF-α), and IL-6 production, and increased Transforming
38 growth factor-β (TGF-β) production. Importantly, trans-Chalcone reduced nuclear factor
39 kappa-light-chain-enhancer of activated B cells (NF-κB) activation and thereby the

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40 mRNA expression of the inflammasome components Nlrp3 (cryopyrin), Asc (apoptosis-
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42
43

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associated speck-like protein containing a CARD), Pro-caspase-1 and Pro-IL-1 . In
vitro, trans-Chalcone reduced the MSU-induced release of IL-1β in lipopolysaccharide

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(LPS)-primed macrophages. Therefore, the pharmacological effects of trans-Chalcone

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44 indicate its therapeutic potential as an analgesic and anti-inflammatory flavonoid for the

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45 treatment of gout.

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Keywords: Trans-Chalcone; joint pain; gouty arthritis; gout flare; inflammation,
flavonoids, natural products; rheumatic disease
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49 Introduction
50 Gout is a painful inflammatory disease caused by the over-production of uric acid,
51 which forms deposits and crystallizes as monosodium urate (MSU) in articular and
52 periarticular tissues (Rees et al., 2014; Fattori et al., 2016). MSU crystals trigger an
53 intense inflammatory response and increase the recruitment of neutrophils to the joint
54 (Rees et al., 2014; Fattori et al., 2016). Management of gout arthritis lies on the use of
55 urate-lowering therapies and the control of acute flares. The main reason for patient
56 seeking of medical care is the intense pain in acute flares (Rees et al., 2014). Gout flares
57 in humans are self-limited and last for nearly 10 days with intense and debilitating pain
58 despite anti-inflammatory treatment (Martin and Harper, 2010). Nevertheless, if left
59 untreated, continuing MSU crystal deposition causes repetitive flares (Rees et al., 2014;
60 Dalbeth et al., 2016). More importantly, irreversible joint damage with chronic symptoms
61 and disability can be observed. In fact, advanced cases of gout are characterized by
62 chronic joint pain, movement limitation, and recurrent flares (Dalbeth et al., 2016).
63 Currently, the management of gout flares lies on the use of non-steroidal anti-
64 inflammatory drugs (NSAIDs), colchicine, corticoids, or biological agents (Rees et al.,
65 2014; Dalbeth et al., 2016). However, the use of these drugs lack safety in patients with
66 comorbidities (NSAIDs), often cause severe side effects (NSAIDs, colchicine, and
67 corticoids), present high cost (biological agents), or possess non-satisfactory analgesic
68 effects in some patients (Rees et al., 2014; Dalbeth et al., 2016).
69 MSU crystals trigger an inflammatory response that is dependent on Nacht, LRR

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70 and Pyd domains-containing protein 3 (NLRP3) inflammasome assembly and production
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of mature interleukin-1β (IL-1β) (Martinon et al., 2006; Amaral et al., 2012). In addition

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to its hyperalgesic effect, IL-1β also induces the recruitment of neutrophils that further
produce hyperalgesic mediators, such as endothelin-1 and prostaglandin E2 (PGE2) (Popa-

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74 Nita et al., 2007; Cunha et al., 2008; Amaral et al., 2012; Fattori et al., 2016). In

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75 neutrophils, IL-1 induces PGE2 production, which is a molecule responsible for

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76 sensitization of nociceptor sensory neurons (Cunha et al., 2008). Thus, the advances in
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the understanding of the pathophysiology of pain and inflammation in gout raised the

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possibility of more effective analgesic therapies.
Flavonoids are natural compounds and represent a major subset of polyphenols
widely distributed in vegetables, fruits, and herbs (Verri et al., 2012). In general, there is
evidence that flavonoids present broad biological activity such as antioxidant,
antitumoral, antimicrobial, anti-inflammatory, and analgesic (Verri et al., 2012). Trans-
Chalcone (1,3-diphenyl-2-propen-1-one) is an open-chain flavonoid and a major
84 precursor of other flavonoids in plants, which also possesses anti-inflammatory and
85 antioxidant properties (Lamoke et al., 2011; Singh et al., 2016; Martinez et al., 2017a;
86 Martinez et al., 2017b). Chalcones have a good pharmacological profile and low toxicity
87 (Karimi-Sales et al., 2018) supporting these molecules as good therapeutic candidates for
88 chronic inflammation such as the observed in rheumatic diseases.
89 Evidence shows that trans-Chalcone reduces UV-induced skin inflammation
90 (Martinez et al., 2016; Martinez et al., 2017b), ameliorates carbon tetrachloride (CCl4)-
91 induced liver damage (Singh et al., 2016), and prevents pathological neovascularization
92 in the ischemic retina (Lamoke et al., 2011). However, to the date, there is no study
93 addressing the analgesic effect of trans-Chalcone or even its effectiveness in gout
94 arthritis. Given the evidence on the biological activities of trans-Chalcone and lack of
95 data evaluating its analgesic effect, we aimed at evaluating the efficacy of this flavonoid
96 in a model of MSU-induced gout arthritis in mice.
97
98 Material and methods
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100 Animals and Ethics Statement
101 Male Swiss (25-30 g, 8 weeks) and C57BL/6 (20-25 g, 8 weeks) mice from the
102 Universidade Estadual de Londrina (Londrina, Paraná, Brazil), LysM-GFP mice (20-25
103 g, 8 weeks) and C57/BL6 from the Ribeirão Preto Medical School – University of São
104 Paulo (Ribeirão Preto, São Paulo, Brazil) were used in this study. Mice were housed in
105 standard clear plastic cages with free access to food and water with a light/dark cycle of
106 12/12h temperature of 21º +/- 1º C. All behavioral testing was performed between 9 a.m.
107 and 5 p.m. in a temperature-controlled (21º +/- 1º C) room. Animal care and handling
108 procedures were approved by the Ethics Committee of the Universidade Estadual de
109 Londrina (process number 14600.2013.73). All efforts were made to minimize animal
110 suffering and to reduce the number of animals used.
111
112
113 Experimental procedures
114 Mice were treated with trans-Chalcone (TC; 3, 10, or 30 mg/kg, p.o) or vehicle
115 (Tween 80 20% plus saline) 30 minutes before intra-articular stimulus with MSU (100
116 µg/10µL, i.a.). trans-Chalcone (at 95% purity was purchased from Santa Cruz
117 Biotechnology, #CAS 614-47-1, Dallas, TX, USA) dose range was selected upon the
118 doses previously used in the literature (Lamoke et al., 2011; Jalalvand et al., 2015; Singh
119 et al., 2016; Karimi-Sales et al., 2018). The negative control of stimulus received only
120 intraarticular injection of 10 l of saline. In experiments that the effect of trans-Chalcone
121
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investigated.

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was higher than the control responses, the effect of trans-Chalcone per se was also

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Mechanical hyperalgesia and edema were evaluated 1, 3, 5, 7, and 15h after MSU

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124 or saline injection. Fifteen hours after MSU injection, knee joint wash was collected to

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125 determine leukocyte recruitment. Based on these previous experiments, the dose of 30

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126 mg/kg was chosen for the following experiments: histopathological analysis (HE

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127 staining), neutrophil migration to the knee joint wash using LysM-eGFP mice (confocal
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microscopy), oxidative stress, determination of the expression of genes sensitives to

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oxidative stress by RT-qPCR (gp91phox, Nrf2, Ho-1), cytokine measurement by ELISA,
NF-κB activation (total p65/phosphorylated p65 ratio) by ELISA, and inflammasome
components expression Nlrp3, Asc, Pro-caspase-1, and Pro-Il-1β mRNA expression by
RT-qPCR. In all in vivo analysis, samples were collected 15h after MSU injection. Serum
samples were also collected 15.5h after trans-Chalcone or vehicle treatment to evaluate
renal and hepatic toxicity considering the same time of protocol of the all experiments
135 with trans-Chalcone . The negative control received only intraarticular injection of 10 l
136 of saline. As a positive control for liver injury (hepatic toxicity) and for kidney injury
137 (renal toxicity), acetaminophen (650 mg/kg,) and diclofenac (200 mg/kg) were used,
138 respectively. One protocol evaluated the effect of trans-Chalcone (30 mg/kg, p.o) or
139 vehicle (Tween 80 20% plus saline) treatment 30 min after MSU injection on mechanical
140 hyperalgesia and knee edema.
141 For in vitro analysis, to assess the effect of trans-Chalcone on NF-κB activation,
142 bone marrow derived macrophages (BMDMs) were pre-treated with 0.1-3 μM before 500
143 ng/mL of LPS (i.e. priming) and supernatants were collected 5h after MSU (activation)
144 stimulation and IL-1β levels were quantitated by ELISA. A selected concentration of
145 trans-Chalcone using the same protocol was also used to test the flavonoid effect over
146 Nrf2 and Ho-1 mRNA expression. Next, it was also evaluated the effect of trans-Chalcone
147 at 0.1-3 μM on BMDMs 30 min before MSU stimulation (i.e. after priming with LPS/
148 before activation with MSU) to address if TC could interfere directly with the activation
149 stimulus to prevent IL-1 secretion and inflammasome activation. The vehicle (Tween
 5

150 80 20% plus saline) treatment did not alter the responses. All experiments were performed
151 in Swiss mice except for the experiments presenting LysM-eGFP analysis and BMDM
152 cell culture.
153
154 Preparation of MSU crystal
155 The MSU crystals were prepared as described previously (Ruiz-Miyazawa et al.,
156 2017). Briefly, 800 mg of monosodium urate were dissolved in 155 ml of boiling milli-Q
157 water containing 5 ml of NaOH, and after the pH was adjusted to 7.2, the solution was
158 cooled gradually by stirring at room temperature. The crystals were collected, and then
159 centrifuged at 3,000 g for 2 min at 4◦C. The crystals were evaporated and sterilized by
160 heating at 180◦C for 2 hours and stored in a sterile environment until use. The load of
161 endotoxin present in the MSU crystals was determined using a ToxinSensor™ Single
162 Test Kit (GenSricpt). The selected dose and concentration of MSU did not present
163 detectable endotoxin levels as previously described (Ruiz-Miyazawa et al., 2018).
164
165 Induction of MSU-induced knee joint inflammation
166 Joint inflammation was induced by the intra-articular (i.a.) administration of MSU
167 (100 μg/10 µl) into the right knee joint of mice under isoflurane anesthesia. Control
168 animals received an i.a. injection of sterile saline (10 μL) (Ruiz-Miyazawa et al., 2017).
169
170 Evaluation of knee joint hyperalgesia
171 The mechanical hyperalgesia of the femur-tibial joint was evaluated by an

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172 electronic von Frey apparatus. Mice were placed in acrylic cages with a wire grid floor,
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175
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and the stimulations were performed only when the animals were quiet (and with the four

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paws on the grid floor). This method consists of an electronic pressure-meter, with a force

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transducer fitted with polypropylene tip (Insight instruments, Ribeirao Preto, SP, Brazil).

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176 To evaluate the articular pain, we used a large tip (4.15 mm2), to exclude the subcutaneous

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177 effect (Guerrero et al., 2006). An increase perpendicular force was applied to the central

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178 area of the plantar surface of the hind paw to induce flexion of the femur-tibial joint
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followed by the hind paw withdrawal. A digital analgesimeter recorded the intensity of
the force applied (in grams) when the paw was withdrawal. The test was performed at the
times indicated on figures. The investigators were blinded to the treatment groups.

Knee joint edema evaluation

Knee joint edema was determined with a caliper (Mitutoyo) before (zero time),
and after the intra-articular injection with MSU at the times indicated. Knee joint edema
186 was determined for each mouse by the difference between the time points indicated on
187 figures and the zero time (Ruiz-Miyazawa et al., 2017). The edema value is represented
188 as Δmm/joint.
189
190 In vivo leukocyte migration
191 Knee joint wash was collected 15h after MSU injection for determination of
192 leukocyte recruitment (Verri et al., 2010). Briefly, articular cavities were washed 3 times
193 with 3.3 µL of saline with 1 mM EDTA, then, diluted to a final volume of 50 L with
194 PBS/EDTA to evaluate leukocyte migration. The total number of leukocytes was
195 determined in a Neubauer chamber diluted in Turk’s solution 1:2 (used to lyse the
196 erythrocytes). Differential cell counts were determined in Rosenfeld stained slices using
197 a light microscope and results are expressed as the number of neutrophils per cavity.
198
199 Histopathological analysis
 6

200 Knee joint was collected 15h after MSU injection, fixed with 10%
201 paraformaldehyde in PBS, and then decalcified for 10 days with EDTA and embedded in
202 paraffin for histological analysis. The paraffin sections were stained with hematoxylin
203 and eosin for conventional morphological evaluation. Results are expressed as leukocytes
204 infiltrate (cell/field) counted at the inflammatory foci as indicative of synovitis. A
205 dimension used for the analysis of the slices was 569 x 633 pixels and magnification ×400
206 (Ruiz-Miyazawa et al., 2017).
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208 Fluorescence assay
209 Knee joint wash of LysM-GFP mice was collected in sterile slides 15h after MSU
210 injection into the knee joints. DAPI fluorescent stain was added to slides for localization
211 of nucleus in each sample. The representative images and quantitative analysis were
212 performed using a confocal microscope (SP8, Leica Microsystems, Mannheim,
213 Germany). The intensity of fluorescence was quantified in randomly selected fields of
214 different groups by an investigator blinded to the treatments. Results are presented as the
215 percentage of GFP fluorescent intensity.
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217 GSH levels measurement
218 Samples of articular joint were collected and maintained at -80 ºC for at least 48
219 h. The sample was homogenized with 200 μl of 0.02 M EDTA. The homogenate was
220 mixed with 25 μl of trichloroacetic acid 50% and was homogenized three times over 15
221 min. The mixture was centrifuged (15 min x 1500 g x 4 ºC). The supernatant was added

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222 to 200 μl of 0.2 M TRIS buffer, pH 8.2, and 10 μl of 0.01M DTNB. After 5 min, the
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225
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absorbance was measured at 412 nm (Multiskan GO, Thermo Scientific) against a blank

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reagent with no supernatant. A standard GSH curve was formed. The results are expressed

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as GSH per mg of protein (Ruiz-Miyazawa et al., 2017).

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226

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227 ABTS and FRAP assays

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228 The ability of samples to resist oxidative damage was determined by their free
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radical scavenging (ABTS [2,2’-Azinobis-3-ethylbenzothiazoline 6-sulfonic acid] assay)

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and ferric reducing (FRAP assay) properties. The ABTS is effective in detecting anti-
radical activity and FRAP detects reducing potential in tissue samples (Menghini et al.,
2018). The tests were adapted to a 96-well microplate format as previously described
(Ruiz-Miyazawa et al., 2017). Articular tissue samples were collected 15h after MSU i.a
injection (100 μg/10µL) and homogenized immediately in ice-cold KCl buffer (500 μl,
1.15% w/v). The homogenates were centrifuged (200 g × 10 min × 4 °C), and the
236 supernatants were used in both assays. Diluted ABTS solution (200 µl) was mixed with
237 10 μL of sample in each well. After 6 min of incubation at 25 °C, the absorbance was
238 measured at 730 nm. For FRAP assay, the supernatants (10 μl) were mixed with the
239 freshly prepared FRAP reagent (150 μl). The reaction mixture was incubated at 37 °C for
240 30 min, and the absorbance was measured at 595 nm (Multiskan GO Thermo Scientific).
241 The results of ABTS and FRAP assays were equated against a standard Trolox curve
242 (0.02 – 20 nmol).
243
244 Superoxide anion production
245 The measurement of superoxide anion production in tissue homogenates (10
246 mg/ml in 1.15% KCl) was performed using the nitroblue tetrazolium (NBT) assay
247 adapted to a microplate as described previously (Ruiz-Miyazawa et al., 2017). The NBT
248 reduction was measured at 600 nm (Multiskan GO, Thermo Scientific). The tissue weight
249 was used for data normalization.
250
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251 Nitrite production

252 Samples from knee joint were collected 15 h after MSU injection, homogenized
253 in 500 μl of saline, and nitrite (NO2-) concentration was determined by the Griess reaction
254 as an indicator of nitric oxide (NO) production (Lima-Junior et al., 2013). Briefly, 100 μl
255 of the homogenate was incubated with 100 μl of Griess reagent for 5 min at 25 °C, and
256 NO2- concentration was determined by measuring the optical density at 550 nM
257 (Multiskan GO, Thermo Scientific) in reference to a standard curve of NaNO2 solution.
258 Results are expressed as μmol of NO2- per mg of tissue.
259
260 Preparation of bone marrow-derived macrophages (BMDMs)
261 Bone marrow cells from femora and tibiae of C57BL/6 mice (8 weeks old) were
262 aspirated and flushed with a syringe filled with RPMI 1640 to extrude the bone marrow
263 into a 15 ml sterile polypropylene tube. A 5ml plastic pipette was used to homogenize the
264 bone marrow. The fresh bone marrow cells obtained were cultured with RPMI 1640
265 media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml
266 streptomycin and 15% of L929 cell conditioned medium, as source of
267 granulocyte/macrophage colony stimulating factor and seeded in non-tissue culture
268 treated Optilux Petri dishes (BD Biosciences) and incubated at 37°C in a 5% CO 2
269 atmosphere for 7 seven days (Marim et al., 2010).
270 To obtain the BMDM, the supernatants were discarded and the attached cells were
271 washed with 10 ml of sterile PBS 1x. Ten ml of ice-cold PBS were added to each plate
272 and incubated at 4°C for 10 minutes to detach the cells. The macrophages were then
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RT-qPCR and ELISA assay.

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aspirated, counted, seed and cultivated in tissue culture plates for 12 hours before further

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276 Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)

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277 Total RNA was extract from 3x106 bone marrow macrophages cells (BMDMs)

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278 5 hours after MSU activating stimulus and from knee joints at 15h after MSU injection

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279 by using TRIzol® reagent. The total RNA was isolated according to manufacturer’s
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281
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directions. The purity of total RNA was measured with a spectrophotometer and the

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wavelength absorption ratio (260/280 nm) was between 1.8 and 2.0 for all preparations.
Reverse transcription of total RNA to cDNA and qPCR were carried out using GoTaq®
2-Step RT-qPCR System (Promega) and specific primers (Applied Biosystems®). The
mRNA level of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as
reference gene. The primers used were Gapdh forward: CAT ACC AGG AAA TGA GCT
TG, reverse: ATG ACA TCA AGA AGG TGG TG; gp91phox, forward: AGC TAT GAG
287 GTG GTG ATG TTA GTG G, reverse: CAC AAT ATT TGT ACC AGA CAG ACT
288 TGA G; Nrf2, forward: TCA CAC GAG ATG AGC TTA GGG CAA, reverse: TAC
289 AGT TCT GGG CGG CGA CTT TAT; Ho-1, forward: CCC AAA ACT GGC CTG TAA
290 AA, reverse: CGT GGT CAG TCA ACA TGG AT; Nlrp3, forward: AGC TAT GAG
291 GTG GTG ATG TTA GTG G, reverse: CAC AAT ATT TGT ACC AGA CAG ACT
292 TGA G; Asc, forward: ATG GGG CGG GCA CGA GAT G, reverse: GCT CTG CTC
293 CAG GTC CAT CAC; Pro-caspase-1: forward: TGG TCT TGT GAC TTG GAG GA,
294 reverse: TGG CTT CTT ATT GGC ACG AT; Pro-Il-1β, forward: GAA ATG CCA CCT
295 TTT GAC AGT G, reverse: TGG ATG CTC TCA TCA GGA CAG. Raw data were
296 normalized to Gadph expression and were analyzed by the 2 −(ΔΔCt) method by calculating
297 the relative expression to the saline control.
298
299 Inflammasome activation/IL-1 release assay
300
 8

301 In some cultures, BMDMs were seeded at the density of 1 x 106 cells in
302 96-well plate. After 24 hours, the cells were stimulated with 500 ng/mL of
303 lipopolysaccharide (LPS) from Escherichia coli (Santa Cruz Biotechnology) and 3h later
304 treated with 450 µg/ml of MSU to stimulate NLRP3 inflammasome activation as
305 described previously (Martinon et al., 2006). To evaluate effect of trans-Chalcone on
306 NF-κB activation in vitro, BMDMs were pre-treated with TC at 0.1-3 μM 30 min before
307 LPS stimulation (priming). Supernatants were collected 5h after MSU stimulation and
308 IL-1β concentration was quantitated by ELISA. In another experimental set, BMDMs
309 were treated with TC at 0.1-3 μM 30 min after LPS priming and before MSU stimulation,
310 in order to address the direct effect on inflammasome activation. Supernatants were also
311 collected 5h after MSU stimulation to assess IL-1β concentration. The data are
312 representative of the means of three independent experiments.
313 Lactate dehydrogenase (LDH) release in the supernatant was used to determine
314 cytotoxicity using LDH Cytotoxicity Assay Kit (Cayman Chemical, Michigan, USA)
315 according to the manufacturer’s directions. Trypan blue exclusion test of cell viability
316 was also performed.
317
318 Cytokine measurement
319 Mice were anesthetized and killed, and knee joint was collected and frozen with
320 liquid nitrogen. The samples were then homogenized in 500 l of buffer containing
321 protease inhibitors, centrifuged (3600 rpm x 4C x 15 min) and the supernatants were
322 used to determine the levels of TNF-α (#88-7324-76), IL-1β (#88-8014-22), IL-6 (#88-
323
324
325
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7064-76), TGF-β (# 88-8350-76), and IL-10 (#88-7105-76) by an enzyme-linked

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immunosorbent assay (ELISA) using eBioscience/ThermoFisher kits. The levels of IL-

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1β were also assessed in BMDM culture. The results are expressed as picograms (pg) of

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326 cytokine/100 mg of tissue or pg/ml.

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327

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328 NF-κB activation
329
330
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332
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335
r o Mice were anesthetized and killed, and knee joint was collected and frozen with
liquid nitrogen. The samples were then homogenized with a tissue-tearor in 500 l of

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ice-cold lysis buffer (Cell Signaling). The homogenates were centrifuged (14000 rpm ×
10 min × 4 °C), with the supernatants used to assess the levels of total (# 7836) and
phosphorylated (#7834) NF-κB p65 subunit by ELISA using PathScan® kits (Cell
Signaling, Danvers, MA, USA) according to the manufacturer’s directions. Data were
expressed as the total NF-B p65/phospho-NF-B ratio p65 measured at 450 nm
336 (Multiskan GO Thermo Scientific).
337
338 Enzymatic Markers of Liver Injury
339 Blood samples were collected by cardiac puncture 15.5h after treatment with
340 trans-Chalcone at 30 mg/kg or 10h after stimulus with acetaminophen at 650 mg/kg and
341 added into microtubes containing anticoagulant (EDTA, 5,000 IU/mL, Sigma Chemical
342 Co., St. Louis, MO, USA). Acetaminophen was used as positive control of liver injury
343 as described previously (Fattori et al., 2017a). The plasma was separated by
344 centrifugation (200×g, 10 min, 4°C). Plasma samples were processed according to the
345 manufacturer’s instructions (Labtest Diagnóstico S.A., Lagoa Santa, MG, Brazil) to
346 evaluate ALT and AST levels as indicators of hepatoxicity. Results are presented as U/L
347 of plasma ALT or AST.
348
349 Renal function tests
350 Blood samples were collected by cardiac puncture 15.5h after treatment with
351 trans-Chalcone at 30 mg/kg or 24h after stimulus with diclofenac at 200 mg/kg and added
 9

352 into microtubes containing anticoagulant (EDTA, 5,000 IU/mL, Sigma Chemical Co., St.
353 Louis, MO, USA). Diclofenac was used as positive control of kidney injury as described
354 previously (Fattori et al., 2017a). The plasma was separated by centrifugation (200×g, 10
355 min, 4°C). Plasma samples were processed according to the manufacturer’s instructions
356 (Labtest Diagnóstico S.A., Lagoa Santa, MG, Brazil) to evaluate urea and creatinine
357 levels as indicators of nephrotoxicity. Results are presented as mg/dL of plasma urea or
358 creatinine.
359
360 Data analysis
361 Data were analyzed using GraphPad Prism statistical software (GraphPad
362 Software, Inc., USA-500.288, version 5.0). Results are presented as means +/- SEM of
363 measurements made on 12 mice per group that were obtained in two separate experiments
364 with 6 mice per group. For in vitro experiments, 6 wells were performed per group per
365 experiment, and every experiment was repeated 3 times, and results are the mean of the
366 3 experiments. Two-way ANOVA was used to compare the groups and doses at all times
367 when the parameters were measured at different times after the stimulus injection. The
368 analyzed factors were treatments, time, and time versus treatment interaction. One-way
369 ANOVA followed by Tukey’s test was performed for each time-point. P < 0.05 was
370 considered significant.
371
372 Results
373

l
374 Trans-Chalcone inhibits MSU-induced mechanical hyperalgesia and edema in a
375
376
377
a
dose-dependent manner. Severe pain and swelling around the joint are the most clinical

n
findings after acute gout in humans causing disability to the patients (Amaral et al., 2012;

sio
Ruiz-Miyazawa et al., 2017). Considering these features, in the first set of experiments

i
378 we addressed whether trans-Chalcone would inhibit MSU-induced knee joint mechanical

v
379 hyperalgesia and edema within 15 hours after MSU injection. MSU injection induced

o
380 mechanical hyperalgesia, which was not reduced by trans-Chalcone at the dose of 3
381
382
383
384
385
386
387
r
mg/kg in any evaluated time-points (Fig. 1A). trans-Chalcone at the dose of 10 mg/kg

P
reduced MSU-induced mechanical hyperalgesia between 5-15h. The dose of 30 mg/kg of
trans-Chalcone inhibited MSU-induced mechanical hyperalgesia between 3-15h, with
significant statistical difference compared to the doses of trans-Chalcone of 10 mg/kg in
the 15th h post-MSU (Fig. 1A). Only trans-Chalcone at 30 mg/kg inhibited MSU-induced
knee joint edema in more than one-time point (5-15h post-MSU) (Fig. 1B). Furthermore,
in a post-treatment protocol with trans-Chalcone (Fig. S1), the dose of 30 mg/kg reduced
388 the MSU-induced knee joint mechanical hyperalgesia at 1, 3 and 15h post-stimulus (Fig.
389 S1A). The difference in edema were observed at 7 and 15 post MSU stimulus (Fig. S1B).
390
391 Trans-Chalcone inhibits MSU-induced leukocyte recruitment to the knee joint and
392 synovitis. Leukocyte recruitment the knee joint, especially of neutrophils, is a hallmark
393 of gout arthritis pathology (Amaral et al., 2012; Fattori et al., 2016; Ruiz-Miyazawa et
394 al., 2017). Thus, the effect of trans-Chalcone on MSU-induced leukocyte recruitment was
395 evaluated. Only trans-Chalcone at the dose of 30 mg/kg reduced MSU-induced total
396 leukocyte (Fig. 2A), neutrophil (Fig. 2A), and mononuclear cell recruitment (Fig. 2A).
397 However, the dose of 10 mg/kg of trans-Chalcone inhibited MSU-induced mononuclear
398 cell recruitment (Fig. 2A). Considering the results of Fig. 1 and 2A, the dose of 30 mg/kg
399 of trans-Chalcone was selected for the next experiments. In agreement with Figs. 1 and
400 2A, the histopathological finding shows that trans-Chalcone reduced inflammatory cell
401 recruitment to the knee joint (Fig. 2B and 2C), suggesting a reduction in the synovitis, an
402 inflammation of synovial membrane. Furthermore, by using LysM-eGFP mice as another
 10

403 approach to investigate MSU-induced neutrophil recruitment, we show that trans-

404 Chalcone reduced the infiltration of LysM-eGFP+ cells that includes neutrophils and
405 monocytes/macrophages (Fig. 2D-E), as observed by reduced intensity of fluorescence
406 (Fig. 2D-E). Importantly, trans-Chalcone at 30 mg/kg did not induce liver injury (Fig
407 S2A-B) or kidney injury (Fig S2C-D). These side effects are commonly associated with
408 the use of analgesics such as acetaminophen (APAP, paracetamol) (Hohmann et al., 2015)
409 or anti-inflammatory drugs such as diclofenac (Fattori et al., 2017a).
410
411 Trans-Chalcone inhibits MSU-induced oxidative stress. Given the role of reactive
412 oxygen and nitrogen species in pain processing and inflammation (Gloire and Piette,
413 2009; Martinon, 2010; Fattori et al., 2016; Grace et al., 2016) and that flavonoids are
414 recognized by their antioxidant activity (Verri et al., 2012), the effect of trans-Chalcone
415 in MSU-induced oxidative stress was investigated. Trans-Chalcone inhibited MSU-
416 induced production of superoxide anion (NBT assay; Fig. 3A) and nitric oxide (NO2-
417 assay; Fig. 3B) in knee joint samples. In line with these results, trans-Chalcone also
418 restored the endogenous antioxidant capacity of samples by preventing depletion in the
419 GSH levels (Fig 3C) and increasing the FRAP (FRAP assay, Fig. 3D) and ABTS free
420 radical scavenging ability (ABTS assay, Fig. 3E) that were reduced by MSU injection.
421 ABTS in an effective assay to screen the presence of antioxidant compounds and FRAP
422 assay identifies the reducing ability exerted by antioxidants (Menghini et al., 2018). Thus,
423 both assays were essential tools to demonstrate the in vivo antioxidant effect of trans-
424 chalcone in gouty arthritis. Corroborating the results of oxidative stress status, trans-

l
425 Chalcone reduced MSU-induced gp91phox mRNA expression (Fig. 4A), a NADPH
426
427
428
a
oxidase subunit. Trans-Chalcone also increased the mRNA expression of the antioxidant

n
transcription factor Nrf2 (Fig. 4B) and its downstream target HO-1 (Fig. 4C). Importantly,

sio
the treatment with trans-chalcone in saline group (without MSU stimulus) did not affect

i
429 the mRNA expression of both genes, pointing that trans-chalcone does not have an effect

v
430 per se over Nrf2 and HO-1 mRNA expression (Fig. 4B and C). Furthermore, in an in vitro

o
431 system approach using BMDM culture, we also observed that the pre-treatment (30 min)
432
433
434
435
436
437
438
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with trans-chalcone in a concentration of 3 M enhanced Nrf2 as well as Ho-1 mRNA
expression under LPS plus MSU stimuli. The treatment of naive BMDM with trans-
chalcone did not alter Nrf2 (Fig. 4D) and Ho-1 (Fig. 4E) mRNA expression, which
corroborates the in vivo data. Therefore, trans-Chalcone inhibited MSU-induced pro-
oxidant enzymes and increases antioxidant sensitive pathways in the knee joints,
confirming an important effect of this flavonoids in the redox biology of gouty arthritis.

439 Trans-Chalcone modulates in vivo and in vitro MSU-induced cytokine production.

440 Next, the effect of trans-Chalcone on MSU-induced pro-inflammatory cytokine
441 production was investigated considering the importance of cytokines to gout arthritis
442 pathology (Dalbeth et al., 2016; Fattori et al., 2016). Treatment with trans-Chalcone
443 reduced in vivo MSU-induced IL-1β (Fig. 5A), TNFα (Fig. 5B), and IL-6 (Fig. 5C)
444 production. Moreover, trans-Chalcone also increased the levels of the anti-inflammatory
445 cytokine TGF-β (Fig. 5D) without changing the levels of IL-10 in knee joints (Fig. 5E).
446 Regarding TGF- levels, trans-Chalcone did not alter the levels of this anti-inflammatory
447 cytokine per se (Fig. 5D). As IL-1β production in its mature form depends on priming
448 and activation(Martinon et al., 2006) and some flavonoids can potentially inhibit NF-κB
449 activation (Vicentini et al.,2001) and IL-1 maturation (Domiciano et al., 2017), we
450 investigated whether or not trans-Chalcone reduced IL-1β production by targeting LPS-
451 induced NF-κB activation (LPS/priming) and/or interfering with inflammasome
452 activation (MSU/activation). BMDM pre-treatment (before priming with LPS) with
453 trans-Chalcone at the concentrations of 0.3, 1, and 3 μM reduced MSU-induced release
 11

454 of IL-1β in the supernatant of cell culture (Fig. 6A). In a post-treatment protocol, after
455 priming the BMDM with LPS and before secondary stimulation with MSU, the treatment
456 with trans-Chalcone at 3 μM also reduced MSU-induced IL-1β release by LPS-primed
457 BMDM (Fig. 6B). Thus, these data indicate that trans-Chalcone can either inhibit NF-κB
458 activation (before priming with LPS; Fig. 6A) and directly interfere with inflammasome
459 activation in a concentration-dependent manner (activation, treatment after LPS, but
460 before MSU; Fig. 6B). However, the effect of trans-Chalcone was more prominent in
461 inhibiting the priming with LPS (lower concentrations of trans-Chalcone) than the
462 activation by MSU (only the highest concentration of trans-Chalcone), which indicates
463 trans-Chalcone presents a more prominent effect in inhibiting NF-B activation than
464 inflammasome activation as a main mechanism. Importantly, none of the concentrations
465 used herein (up to 3 μM) of trans-Chalcone reduced cell viability as per LDH analysis
466 (Fig. S3A) and Trypan Blue (Fig. S3B)
467
468 Trans-Chalcone inhibits MSU-induced Nlrp3, Asc, Pro-caspase-1, and Pro-Il-1β
469 mRNA expression, and NF-κB activation. The assembly of NLRP3 inflammasome is
470 a crucial mechanism of gout arthritis that is dependent on NF-κB activation, which is a
471 transcription factor essential to inducing the expression of inflammasome components
472 and pro-IL-1β (Martinon et al., 2006; Amaral et al., 2012; Dalbeth et al., 2016). Given
473 trans-Chalcone reduced in vivo and in vitro IL-1β production, the effect of trans-
474 Chalcone on the mRNA expression of inflammasome components Nlrp3, Asc, and Pro-
475 caspase-1, and Pro-Il-1β was investigated. Treatment with trans-Chalcone reduced the
476
477
478
l
mRNA expression of all inflammasome components Nlrp3 (Fig. 7A), Asc (Fig. 7B), Pro-

a
caspase-1 (Fig. 7C), and Pro-Il-1β (Fig. 7D). Further, MSU induced the activation of NF-

n
B as observed by a decrease of total-p65/phosphorylated-p65 OD ratio (Fig. 7E). The

o
si
479 decrease in the ratio demonstrates the increase in the p65 subunit phosphorylation,

i
480 therefore, indicating activation of NF-κB. In turn, trans-Chalcone treatment inhibited

v
481 MSU-induced NF-B activation (Fig. 7E). Thus, these data confirmed that the effect of
482
483
484
485
486
487
488
r o
trans-Chalcone in gout arthritis is dependent on inhibiting NF-B activation, which will

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then result in reducing inflammasome platform expression.

4. Discussion
In this study, we show that the precursor of flavonoids in plants, trans-Chalcone,
ameliorates experimental gout arthritis in mice. This effect is related to the reduction of
489 MSU-induced oxidative stress, NF-κB activation and thereby inhibition of inflammasome
490 expression and production of IL-1β in its mature form. Trans-Chalcone also increased the
491 production of the anti-inflammatory cytokine TGF-β. It is also important to highlight that,
492 to our knowledge, this is the first demonstration that trans-Chalcone has analgesic
493 properties and also inhibits MSU-induced joint inflammation.
494 The management of gout acute flares lies on the use of corticosteroids, AINEs,
495 colchicine, and biological agents (Rees et al., 2014). These single target therapies possess
496 limit analgesic efficacy and when effective often produce several undesirable side effects
497 in gout arthritis, a disease with multiple physiopathological mechanisms (Rees et al.,
498 2014). Flavonoids are widely known for their high spectrum of biological action and low
499 toxicity, which are explained by the fact that flavonoids are multi-target drugs, inhibiting
500 but not abolishing endogenous pathways (Verri et al., 2012). Herein, we show that trans-
501 Chalcone did not induce toxicity in vivo and in vitro, indicating a safe preclinical profile.
502 Although the literature did not define the trans-Chalcone pharmacokinetic profile after
503 oral administration, we could observe that it inhibited gout-induced pain and
504 inflammation as a pre-treatment and post-treatment, which reinforces its therapeutic
 12

505 potential. The antioxidant activity is a hallmark of the activity of flavonoids (Verri et al.,
506 2012) and in fact, the reduction of oxidative and nitrosative stress are important
507 mechanisms by which flavonoids (Anjaneyulu and Chopra, 2003; Al-Rejaie et al., 2015;
508 Ahmed et al., 2016; Pinho-Ribeiro et al., 2016) and other molecules (Fattori et al., 2015;
509 Singh and Vinayak, 2015; Fattori et al., 2017a) act. Moreover, injection of a superoxide
510 anion donor, peroxynitrite or intrathecal delivery of ROS elicits pain behavior in mice
511 (Wang et al., 2004; Fattori et al., 2015; Fattori et al., 2017b) indicating that oxidative
512 stress plays important role in pain processing (Grace et al., 2016). Neutrophils produce
513 ROS upon recognition of MSU crystals (Desaulniers et al., 2001) and, in turn, ROS
514 contribute to neutrophil recruitment (Hattori et al., 2010; Sakai et al., 2012). Moreover,
515 nitric oxide quickly react with ROS to produce peroxynitrite and there is a correlation
516 between increased NO generation and gouty arthritis progression (Chen et al., 2004).
517 The antioxidant capacity of phenolic compounds is related to the ability to
518 generate a stable molecule after donating electrons. An important structural determinant
519 of the antioxidant capacity of flavonoids is attributed to hydroxyl C4 and C3 (Verri et al.,
520 2012). The structure-activity relationship of trans-Chalcone indicates that it does not
521 possess antioxidant properties (Martinez et al., 2017a). In fact, using a cell free in vitro
522 system, trans-Chalcone did not show antioxidant activity in the ABTS (electron transfer),
523 DPPH (hydrogen transfer), iron chelation and superoxide anion (free radical) assays
524 (Martinez et al., 2017a). Thus, electronic transfer of electrons, chelating transition metals
525 or direct scavenging activity are not mechanisms by which trans-Chalcone reduces
526 oxidative stress. However, in vivo data show that trans-Chalcone displays antioxidant

l
527 effect observed by an increase of antioxidant defense and reduction of ROS (Karkhaneh
528
529
530
a
et al., 2016; Singh et al., 2016; Martinez et al., 2017a; Martinez et al., 2017b). We also

n
observed that trans-Chalcone maintained the tissue antioxidant activities such as FRAP

sio
reducing and ABTS radical scavenging ability in knee joint samples, and promoted

i
531 glutathione system improvement, besides inhibiting the production of ROS and reducing

v
532 nitrite levels. Therefore, the antioxidant effect of trans-Chalcone is not related to a direct

o
533 chemical antioxidant structure, but rather is dependent on its anti-inflammatory effects
534
535
536
537
538
539
540
r
and the activation of Nrf2 signaling pathway (Wu et al., 2014; Martinez et al., 2017b). In

P
fact, trans-Chalcone also inhibited gp91phox mRNA expression and induced the mRNA
expression of the antioxidant transcription factor Nrf2 and its downstream target HO-1.
Our results corroborate other reports showing that trans-Chalcone acts on the antioxidant
transcription factor Nrf2 (Wu et al., 2014; Martinez et al., 2017b) and reducing Kelch-
like ECH-associated protein 1 (Keap1) activity (Kobayashi et al., 2006). However, it is
unlikely that in the present experimental condition trans-Chalcone is acting by increasing
541 Nrf2 and HO-1 per se, but rather, this increase seems to occur only during inflammation.
542 In addition to antioxidant effects, Nrf2/HO-1 signaling is an important analgesic
543 pathway (Steiner et al., 2001; Carcole et al., 2014). In fact, co-treatment with an HO-1
544 inducer increases the analgesic effects of opioids and cannabinoids through the activation
545 of cGMP/PKG/ATP-sensitive potassium channel pathway in the CFA-induced
546 inflammatory pain model (Carcole et al., 2014). Thus, the increase of Nrf2/Ho-1 mRNA
547 expression contributed to the reduction of MSU-induced oxidative stress and pain in this
548 study.
549 The main innate immune event in MSU crystals-induced inflammation is the
550 NLRP3-dependent maturation of IL-1β (Martinon et al., 2006; Amaral et al., 2012). This
551 mechanism accounts for neutrophil recruitment and pain (Amaral et al., 2012). In
552 response to MSU crystals, other macrophages-derived mediators such as TNF-α also
553 drive neutrophil migration toward the tissue (Amaral et al., 2016). Moreover, targeting
554 TNF-α, IL-1β, or IL-33 in rheumatic diseases reduce neutrophil recruitment and pain
555 (Verri et al., 2008; Verri et al., 2010; Amaral et al., 2012; Amaral et al., 2016). Trans-
 13

556 Chalcone reduced in vivo MSU-induced TNF-α, IL-1β, and IL-6; and in vitro maturation
557 of IL-1β. Therefore, the reduction of these cytokines certainly contributed to the
558 inhibition of neutrophils recruitment. This is an important finding because neutrophil
559 recruitment is a hallmark of the acute phases of all rheumatic diseases (Fattori et al., 2016)
560 and during gout flares, they are the predominant cell population in the synovial fluid
561 (Mitroulis et al., 2013). After recognition of MSU crystals by neutrophils, they
562 degranulate (Popa-Nita et al., 2007) and undergo NETosis (Mitroulis et al., 2011).
563 Moreover, activated neutrophils produce IL-1β, TNF-α, and PGE2 that contribute to pain
564 (Cunha et al., 2008; Verri et al., 2010; Amaral et al., 2012; Amaral et al., 2016). IL-1β
565 and TNF-α also activate nociceptor neuron and thereby producing pain (Jin and Gereau,
566 2006; Binshtok et al., 2008). Regarding IL-1β, two steps are required for the production
567 and release of its mature form (Martinon et al., 2006). The first step is related to
568 downstream signaling pathways that depend on NF-κB activation or a priming step,
569 whereas the second step is related to inflammasome assembly per se that depends on the
570 phagocytosis of MSU crystals and lysis of phagolysossome releasing cathepsin that
571 activates NLRP3 (Martinon et al., 2006; Amaral et al., 2012; Latz et al., 2013). Thus,
572 naturally occuring molecules (such as flavonoids) that target priming and/or activation
573 signal to inflammasome assembly without the side effect of the current therapies are likely
574 to be highly attractive as an analgesic approach in gout arthritis. In fact, the flavonoid
575 quercetin that inhibits NLRP3 inflammasome and ASC speck formation in mouse
576 vasculitis (Domiciano et al., 2017) and reduces pain in experimental gout arthritis (Ruiz-
577 Miyazawa et al., 2017). In the present study, pre-treated BMDMs (treatment before LPS

l
578 stimulus, i.e. priming) with trans-Chalcone reduced MSU-induced IL-1β production
579
580
581

sio a
suggesting that this flavonoid reduced NF-κB activation (priming). In fact, this effect was

n
further confirmed by in vivo data as demonstrated by an increase of NF-κB OD ratio by
ELISA. In agreement with the the present findings that trans-chacone inhibition of NF-

i
582 B activation in gout arthritis, evidence demonstrated that trans-Chalcone reduces NF-

v
583 κB activation in murine retina (Lamoke et al., 2011). Thus, the inhibition of NF-κB

o
584 activation explains the reduced levels of the pro-inflammatory cytokines observed.
585
586
587
588
589
590
591
r
Moreover, trans-Chalcone reduced MSU-induced IL-1β production in the supernatant of

P
LPS-primed BMDMs (that already received priming) indicating that it may also interfere
with inflammasome assembly. In fact, other naturally occuring molecules, such as the
flavonoids quercetin (Domiciano et al., 2017), apigenin (Zhang et al., 2014), and oroxylin
A (Zhou et al., 2017); and the sesquisterpene lactone parthenolide (Juliana et al., 2010)
have the ability to intefere with inflammasome assembly. This mechanim is related to the
inhibition of ASC speck formation (Zhang et al., 2014; Domiciano et al., 2017; Zhou et
592 al., 2017) or direct targeting of active site of caspase-1 (Juliana et al., 2010). In addition
593 to inhibiting pro-inflammatory cytokines production, trans-Chalcone also increased the
594 level of the anti-inflammatory cytokine TGF-β. This anti-inflammatory cytokine plays an
595 important role in the resolution phase of MSU-induced inflammation (Scanu et al., 2012)
596 by promoting efferocytosis of apoptotic neutrophils (Vieira et al., 2017). Of note, the fact
597 that trans-Chalcone sustained the production of IL-10 and inhibited pro-inflammatory
598 cytokine production is a relevant finding given the anti-hyperalgesic and anti-
599 inflammatory properties of IL-10 and that, in general, anti-inflammatory cytokines are
600 co-released during inflammation to limit the inflammatory response (Verri et al., 2006).
601 Therefore, the reduction of neutrophil recruitment and pro-inflammatory cytokine
602 production, the increase of anti-inflammatory cytokine TGF-β, and the maintenance of
603 anti-hyperalgesic cytokine IL-10 contribute to the analgesic effect of trans-Chalcone.
604 Concluding, we demonstrated that trans-Chalcone ameliorates MSU-induced pain
605 and inflammation. Trans-Chalcone reduced MSU-induced oxidative stress and thereby
606 demonstrating that this molecule possesses antioxidant effect by modulating Nrf2/HO-1
 14

607 signaling pathway. We also demonstrated that trans-Chalcone reduced MSU-induced

608 inflammasome assembly and NF-κB activation. As a consequence, it was also observed
609 a reduction of IL-1β, TNF-α, and IL-6 production. Interestingly, trans-Chalcone
610 increased the level of the anti-inflammatory cytokine TGF-β and sustained the levels of
611 the anti-hyperalgesic cytokine IL-10. To our knowledge, this is the first report on the
612 analgesic effect of trans-Chalcone. Therefore, trans-Chalcone displays a safe preclinical
613 profile with analgesic, antioxidants, and anti-inflammatory properties.
614
615 Author Contributions
616 Conceived and designed the experiments: LSF, KWRM, RC, WAVJ. Performed
617 the experiments: LSF, KWRM, FAPR, VF, THZ, SBG, SMB, TTC. Collection of data
618 and analysis: LSF, KWRM, FAPR, VF, THZ, SBG, SMB, TTC, RC, WAVJ. Contributed
619 reagents/materials/analysis tools: JCAF, TMC, FQC, RC, WAVJ. Writing-original draft:
620 LSF, KWRM, VF, WAVJ. Writing-review and editing: LSF, KWRM, VF, RC, WAVJ.
621 All authors contributed to manuscript revision, read and approved the final version of the
622 manuscript: LSF, KWRM, FAPR, VF, THZ, SBG, SMB, TTC, JCAF, TMC, FQC, RC,
623 WAVJ.
624
625 Funding
626 This work was supported by Programa para o Sistema Único de Saúde (PPSUS)
627 grant supported by Departamento de Ciência e Tecnologia da Secretaria de Ciência,
628 Tecnologia e Insumos Estratégicos, Ministério da Saúde (Decit/SCTIE/MS, Brazil)

l
629 intermediated by Conselho Nacional de Desenvolvimento Científico e Tecnológico
630
631
632
a
(CNPq, Brazil) with support of Fundação Araucária and Secretaria Estadual de Saúde,

n
Paraná (SESA-PR, Brazil); São Paulo Research Foundation under grant agreements

sio
2011/19670-0 (Thematic Project) and 2013/08216-2 (Center for Research in

i
633 Inflammatory Disease); Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior

v
634 (CAPES, Brazil); Financiadora de Estudos e Projetos and Secretaria de Estado da Ciência,

o
635 Tecnologia e Ensino Superior do Paraná under grant agreements 01.12.0294.00 (0476/11)
636
637
638
639
640
641
642
P r
(FINEP/SETI-PR, Brazil); and Universidade Estadual de Londrina, PROPPG, Escritório
de Apoio ao Pesquisador (Edital Internacionalize 2018). SMB and LSF received a CNPq
Post-Doc fellowship. We also thank the support of Central Multiusuário de Laboratórios
de Pesquisa da Universidade Estadual de Londrina (CMLP-UEL).

Conflict of Interest Statement

Authors declare no conflict of interest.
643
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843

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10.18632/oncotarget.19440.

Figure 1. Trans-Chalcone inhibits MSU-induced mechanical hyperalgesia and

edema in a dose-dependent manner. Mice were treated Trans-Chalcone (TC, 3, 10 or
848 30 mg/kg, p.o., 100 l) or vehicle (Tween 80 20% plus saline) 30 minutes before MSU
849 (100 g/10 l/knee) stimulus in the femur-tibial joint of swiss mice. (A) Mechanical
850 hyperalgesia and (B) edema were evaluated 1, 3, 5, 7, and 15h after MSU injection.
851 Results are expressed as mean ± SEM, data represent a total of 12 mice per group that
852 were obtained in two independent experiment with 6 mice per experiment. (*p < 0.05 vs.
853 control group; #p < 0.05 vs. vehicle group, two-way ANOVA followed by Tukey's post-
854 test).
855
856 Figure 2. Trans-Chalcone inhibits MSU-induced leukocyte recruitment in a dose-
857 dependent manner. Mice were treated Trans-Chalcone (TC, 3, 10 or 30 mg/kg, p.o., 100
858 l) or vehicle (Tween 80 20% plus saline) 30 minutes before MSU (100 g/10 l/knee)
859 stimulus in the femur-tibial joint of swiss mice (A) Fifteen hours after MSU, knee joint
860 wash was collected for counting total leukocytes, neutrophils, and mononuclear cells. (B,
861 C) Fifteen hours after MSU, knee joint was collected for histopathological analysis by
862 HE staining. Original magnification image of 400x of groups: saline, vehicle+MSU and
863 TC30+MSU. For panel B: arrows indicate leukocyte infiltration, (a) synovial tissue, (b)
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864 cartilage tissue, and * joint space. Panel C shows the total score of inflammatory infiltrate
865 (synovits). (D, E) Fifteen hours after MSU, knee joint washes of MSU-stimulated LysM-
866 GFP+ mice were collected for the determination of LysM-GFP+ neutrophil recruitment
867 by confocal microscopy. Original magnification 200x. Panel J shows the percentagem of
868 LysM-eGFP+ fluorescence. Results are expressed as mean ± SEM, data represent a total
869 of 12 mice per group that were obtained in two independent experiment with 6 mice per
870 experiment. (*p < 0.05 vs. control group; #p < 0.05 vs. vehicle, one-way ANOVA
871 followed by Tukey's post-test).
872
873 Figure 3. Trans-Chalcone inhibits MSU-induced oxidative stress. Mice were treated
874 Trans-Chalcone (TC, 30 mg/kg, p.o., 100 l) or vehicle (Tween 80 20% plus saline) 30
875 minutes before MSU (100 g/10 l/knee) stimulus in the femur-tibial joint of swiss mice.
876 Fifteen hours after MSU, knee joint samples were collected and the oxidative stress was
877 assessed by measuring: (A) superoxide anion (NBT assay), (B) NO2- production Griess
878 assay), (C) GSH levels, (D) Ferring reducing power (FRAP assay), and (E) ABTS
879 scavenging ability (ABTS assay). Results are expressed as mean ± SEM, data represent
880 a total of 12 mice per group that were obtained in two independent experiment with 6
881 mice per experiment. (*p < 0.05 vs. control group; #p < 0.05 vs. vehicle, one-way
882 ANOVA followed by Tukey's post-test).test.
883
884 Figure 4. Trans-Chalcone inhibits MSU-induced gp91phox mRNA expression and
885 increases Nrf2 and Ho-1 mRNA expression. Mice were treated Trans-Chalcone (TC,
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888

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30 mg/kg, p.o., 100 l) or vehicle (Tween 80 20% plus saline) 30 minutes before MSU
(100 g/10 l/knee) or sterile saline (10 l/knee) stimulus in the femur-tibial joint of
swiss mice. Fifteen hours after MSU, knee joint samples were collected for the

si
889 determination of the mRNA expression of: (A) gp91phox, (B) Nrf2 (B), and (C) HO-1 by

i
890 RT-qPCR. 3x106 BMDMs were seeded in 6 well plate and pre-treated with 3 μM of
891
892
893
894
895
896
897
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trans-chalcone 30 minutes before 500 ng/mL of LPS for priming over 3 hours, followed

o
by MSU (450 µg/mL) activation. After 5 hours, the cells were collected for determination

P
of mRNA expression of: (D) Nrf2 and (E) HO-1 by RT-qPCR. Results are expressed as
mean ± SEM, data represent a total of 12 mice per group that were obtained in two
independent experiment with 6 mice per experiment or the mean of 6 wells per group per
experiment with 3 independent experiments for in vitro assays. (*p < 0.05 vs. control
group; #p < 0.05 vs. vehicle, one-way ANOVA followed by Tukey's post-test).
898
899 Figure 5. Trans-Chalcone modulates MSU-induced cytokine production in vivo.
900 Mice were treated Trans-Chalcone (TC, 30 mg/kg, p.o., 100 l) or vehicle (Tween 80
901 20% plus saline) 30 minutes before MSU (100 g/10 l/knee) stimulus in the femur-tibial
902 joint of swiss mice. Fifteen hours after MSU, knee joint samples were collected for the
903 determination of (A) IL-1β, (B) TNF-α, (C) IL-6, (D) TGF-β and, (E) IL-10 production
904 by ELISA. Results are expressed as mean ± SEM, data represent a total of 12 mice per
905 group that were obtained in two independent experiment with 6 mice per experiment. (*p
906 < 0.05 vs. control group; #p < 0.05 vs. vehicle, one-way ANOVA followed by Tukey's
907 post-test).
908
909 Figure 6. Trans-Chalcone reduces MSU-induced IL-1β maturation in vitro. (A) To
910 assess the effect of trans-Chalcone on NF-κB activation, BMDMs were pre-treated with
911 0.1-3 μM before 500 ng/mL of LPS (i.e. before priming, panel A) and after 3h were
912 secondarily stimulated with MSU (450 g/ml, i.e. activation). (B) To assess the direct
 20

913 effect of trans-Chalcone on inflammasome activation, LPS-primed BMDMs were treated

914 with 0.1-3 μM 30 min before MSU stimulation (i.e. after priming and before activation).
915 Results are expressed as mean ± SEM, data represent the mean of 6 wells per group per
916 experiment with 3 independent experiments. (*p < 0.05 vs. control group; #p < 0.05 vs.
917 vehicle, one-way ANOVA followed by Tukey's post-test).
918
919 Figure 7. Trans-Chalcone decreases MSU-induced Nlrp3, Asc, Pro-caspase-1, and
920 Pro-Il-1β mRNA expression, and NF-κB activation. Mice were treated Trans-Chalcone
921 (TC, 30 mg/kg, p.o., 100 l) or vehicle (Tween 80 20% plus saline) 30 minutes before
922 MSU (100 g/10 l/knee) stimulus in the femur-tibial joint of swiss mice. Fifteen hours
923 after MSU, knee joint was collected for determination of mRNA expression of (A) Nlrp3,
924 (B), Asc, (C) Pro-caspase-1, (D) Pro-Il- by RT-qPCR and (E) NF-B activation by
925 ELISA. Results are expressed as mean ± SEM, data represent a total of 12 mice per group
926 that were obtained in two independent experiment with 6 mice per experiment. (*p < 0.05
927 vs. control group; #p < 0.05 vs. vehicle, one-way ANOVA followed by Tukey's post-
928 test).

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