Received: 4 April 2018 Revised: 5 April 2019 Accepted: 11 May 2019

DOI: 10.1002/cbf.3413

RESEARCH ARTICLE

Effect of quercetin on E‐NTPDase/E‐ADA activities and

cytokine secretion of complete Freund adjuvant–induced
arthritic rats

Renata da Silva Pereira Saccol1 | Karine Lanes da Silveira1 | Stephen Adeniyi Adefegha2 |

Alessandra Guedes Manzoni3 | Leonardo Lanes da Silveira4 | Ana Paula Visintainer Coelho4 |

Livia Gelain Castilhos1 | Fátima Husein Abdalla3 | Lara Vargas Becker3 |

Nara Maria Beck Martins4 | Juliana Sorraila Oliveira3 | Emerson André Casali5 |

Daniela Bitencourt Rosa Leal1,3,4

1
Programa de Pós‐Graduação em Ciências
Farmacêuticas, Centro de Ciências da Saúde, The effect of quercetin was assessed in rats induced with complete Freund adjuvant
Universidade Federal de Santa Maria, Santa
(CFA). Arthritis scores, paw oedema, latency, activities of myeloperoxidase (MPO),
Maria, RS, Brazil
2
Functional Foods and Nutraceuticals Unit, ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase), and ectoadenosine
Department of Biochemistry, School of deaminase (E‐ADA) in lymphocytes were determined. Furthermore, nucleotide and
Sciences, Federal University of Technology,
Akure, Ondo State, Nigeria nucleoside levels as well as the secretion of pro‐ and anti‐inflammatory cytokines
3
Programa de Pós‐Graduação em Bioquímica were evaluated. Animals were treated with saline and quercetin in doses of 5, 25,
Toxicológica, Centro de Ciências Naturais e
and 50 mg/kg for 45 days. The result revealed that quercetin (50 mg/kg) reduced
Exatas, Universidade Federal de Santa Maria,
Santa Maria, RS, Brazil arthritis score and paw oedema, and increased the latency in the thermal hyperalgesia
4
Centro de Ciências da Saúde, Departamento test. Histopathological analysis showed that all the doses of quercetin reduced infil-
de Patologia, Universidade Federal de Santa
Maria, Santa Maria, RS, Brazil
tration of inflammatory cells. MPO activity was increased in the arthritis group; how-
5
Departamento de Ciências Morfológicas, ever, quercetin reduced this activity. E‐NTPDase activity was increased in
Instituto de Ciências Básicas da Saúde, lymphocytes of arthritis rats, and treatment with quercetin reversed this increase.
Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil However, E‐ADA activity was reduced in the arthritis group, and treatment with
quercetin modulated the activity of this enzyme in arthritis rat groups. Serum adeno-
Correspondence
Daniela Bitencourt Rosa Leal, Departamento sine levels were increased in arthritis, and the levels were lowered with quercetin
de Microbiologia e Parasitologia, Centro de treatment. Quercetin treatment in arthritis groups decreased the elevated levels of
Ciências da Saúde, Universidade Federal de
Santa Maria, Av. Roraima, 1000, prédio 20, cytokines in the arthritis control group. Thus, quercetin demonstrated an anti‐
97105‐900 Santa Maria, RS, Brazil. inflammatory effect, and this flavonoid may be a promising natural compound for
Email: [email protected]
the treatment of arthritis.
Significance of the study: Quercetin may represent a potential therapeutic com-
pound in the treatment of rheumatoid arthritis. Findings from this study indicate that
quercetin suppresses swelling and attenuates the underlying inflammatory responses.
This is the first report where quercetin was shown to modulate the immune response
to arthritis via attenuation of the purinergic system (E‐NTPDase and E‐ADA activities)
and the levels of IFN‐gamma and IL‐4. Thus, this work is relevant to basic research
and may be translated into clinical practice.

Cell Biochem Funct. 2019;1–12. wileyonlinelibrary.com/journal/cbf © 2019 John Wiley & Sons, Ltd. 1
2 SACCOL ET AL.

K E Y W OR D S

arthritis, complete Freund adjuvant, cytokines, ectoenzymes, quercetin

1 | I N T RO D U CT I O N on E‐NTPDase and E‐ADA activities in rat lymphocytes, determine

serum purine levels, and evaluate the secretion of pro‐ and anti‐
Rheumatoid arthritis (RA) is a chronic autoinflammatory disorder of inflammatory cytokines of complete Freund adjuvant (CFA)–induced
1 arthritic rats.
the joints. It is characterized by irreversible joint damage and destruc-
tion of cartilage and bone.1,2 It affects approximately 1% of the
world's population.3 Patients with RA often experience progressive
2 | MATERIALS AND METHODS
disabilities with increased risk of death.4 In this pathology, an intense
inflammatory process activates the immune response leading to an
imbalance of pro‐ and anti‐inflammatory cytokines, thus favouring 2.1 | Chemicals
the induction of autoimmunity.5
CFA 0.6% suspension of heat‐killed Mycobacterium tuberculosis in liq-
CD4+ T helper (Th) cells play an important role in the development
uid paraffin, ATP, ADP, adenosine, as well as bovine serum albumin,
of RA. During the activation of T cells in a particular cytokine milieu,
Trizma base, and Coomassie Brilliant Blue G were obtained from
these naive CD4+ T cells are polarized into lineages of Th cells, includ-
Sigma Chemical (St Louis, Missouri). All the other chemicals used in
ing Th1, Th2, Th17, and regulatory T cells (Treg), which are defined by
this experiment were of the highest purity.
their individual cytokine production patterns and function.6 Th1 cells
are pro‐inflammatory and secrete interferon (IFN)‐γ, tumor necrosis
factor (TNF)‐α, and interleukin (IL)‐1β, which promote the inflamma- 2.2 | Animals
tory process and joint damage,7,8 while Th2 cell‐derived agents like
IL‐4 and IL‐5 induce humoral immunity.6 Adult female heterogenic and conventional Wistar rats (n = 40; 70‐

Purinergic signalling plays an important role in modulating inflam- 90 d, 200‐300 g) obtained from the Central Animal House of the Fed-

matory and immune responses via extracellular biomolecules such as eral University of Santa Maria (UFSM), Santa Maria, Brazil, were used

adenine nucleotides, adenosine triphosphate (ATP), adenosine diphos- for this experiment. The animals were kept on a 12‐hour light/12‐hour

phate (ADP), adenosine monophosphate (AMP), and adenosine. 9,10 dark cycle, at a temperature of 22 ± 2°C, with free access to food and

The levels of extracellular ATP, ADP, AMP, and adenosine are dynam- water. The animals were handled according to the guidelines of the

ically controlled during inflammation by the actions of enzymes Committee on Brazilian Society of Animal Science Laboratory, in

expressed in immune cells.11 accordance with international guidelines. This project was approved

Ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase) is a by the Committee on the Use and Care of Laboratory Animals, Federal

membrane‐bound enzyme involved in the breakdown of ATP and ADP University of Santa Maria (UFSM), and assigned the project number

to AMP, which is sequentially hydrolyzed by E‐5′‐nucleotidase to (99700812140).

adenosine.12 Ectoadenosine deaminase (E‐ADA) is responsible for

catalysis of the irreversible deamination of adenosine and 2′‐ 2.3 | CFA‐induced arthritis
deoxyinosine, which contributes to the removal of adenosine from
the extracellular compartment.13 To investigate the effects of quercetin in chronic inflammatory pro-
Several epidemiological and experimental studies have shown that cess, the adjuvant‐induced arthritis model was used. The animals were
quercetin has antioxidant, anti‐inflammatory, antiproliferative, and mildly anaesthetized with inhaled isoflurane, and intraplantar injection
pro‐apoptotic effects. 14
In addition, quercetin can inhibit secretion of of 50 μL of CFA (0.6% suspension of heat‐killed M tuberculosis,
inflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐2,15 and can 1.0 mg/μL, in liquid paraffin) or saline (used as a control negative) into
modulate the production of Th1 and Th2 cell–derived cytokines.6,16 the right paw was carried out according to the method described by
Quercetin (3, 3′, 4′, 5, 7‐pentahydroxy flavone) is a natural flavonoid Choi et al18 to induce arthritis.
that is widely distributed in hundreds of herbs (eg, dill), vegetables
(eg, onions, broccoli, and peppers), fruits (eg, apples, various berries, 2.4 | Treatment with quercetin and saline
and grapes), and some types of tea and wine.17
Given that adjuvant arthritis is an experimental model of arthritis The treatment of animals with quercetin was performed in doses of 5,
widely used for preclinical testing of numerous antiarthritic agents, it 25, and 50 mg/kg during of 45 days. Quercetin was obtained from
is of clinical interest to investigate the therapeutic action of quercetin Sigma Chemical Co (St Louis, Missouri) and freshly prepared in 25%
as an anti‐inflammatory agent. However, there is a paucity of informa- ethanol and was administered orally, by gavage. According to the stud-
tion on the effect of quercetin on the purinergic system in arthritis. ies published in our research group, the dilution in 25% ethanol does
The purpose of this study was to investigate the effect of quercetin not interfere with the activity of purinergic system ectoenzymes.
SACCOL ET AL. 3

The choice of 5, 25, and 50 mg/kg of doses of quercetin was used, decreases of paw withdrawal latency were interpreted as indicative of
based on previous works, where the therapeutic effects of this com- heat hyperalgesia.
pound in rats were reported by Abdalla et al.19
The rats were divided into eight groups, and the groupings are as
follows: 2.5.3 | Paw oedema

To observe the development of oedema, animals were held while right

Group I Normal rats administered saline (CS)
hind paw thickness was measured using a digital caliper.23 Fifteen
Group II Normal rats administered quercetin 5 mg/kg (CQ5)
days after the induction of inflammation and 1 day after end treat-
Group III Normal rats administered quercetin 25 mg/kg (CQ25)
ment of quercetin and saline, new measurements were taken and
Group IV Normal rats administered quercetin 50 mg/kg (CQ50); compared with basal values. An increase on these measurement
Group V Rats induced with CFA and administered saline (AS) values compared with baseline was considered as representing
Group VI Rats induced with CFA and administered quercetin 5 mg/kg oedema values.
(AQ5)
Group Rats induced with CFA and administered quercetin 25 mg/kg
VII (AQ25)
2.6 | Histopathological analysis
Group Rats induced with CFA and administered quercetin 50 mg/kg
VIII (AQ50)
Sample joint tissues of the right hind paw were collected and fixed in
Rats were administered quercetin and saline for a period of 45 days 10% formalin solution and then dehydrated and embedded in paraffin,
prior to euthanasia. After the treatment period, animals were followed by sectioning and histological staining with haematoxylin and
anaesthetised with isoflurane, submitted to euthanasia and blood col- eosin. The slides were observed in optical microscope 100× to evalu-
lected by cardiac puncture. ate a possible damage. The score of joint tissues was analysed accord-
ing to the lymphocyte counts by high power field (HPF) as follows: (0):
0 to 50 lymphocytes/HPF (1): 50 to 100 lymphocytes/ HPF, (2): 100
2.5 | Evidences of arthritis induction or chronic
to 150 lymphocytes/HPF, (3): 150 to 200 lymphocytes/HPF, and (4):
inflammation evaluation
more than 200 lymphocytes/HPF.

To confirm the development of chronic inflammation and possible

anti‐inflammatory effect of quercetin, the arthritis score, thermal
hyperalgesia, and paw oedema were assessed. These tests were
2.7 | Separation of blood plasm
performed a day prior to induction of arthritis, on the 15th day after
Rats were anaesthetised with isoflurane, and blood was collected by
the induction of arthritis, and the day before the euthanasia of
cardiac puncture. The blood samples were collected in tubes contain-
the animals.
ing dipotassium ethylenediamine tetra acetic acid (EDTA) as anticoag-
ulant and centrifuged at 1,400×g for 15 minutes at room temperature
2.5.1 | Arthritis score to obtain the plasma for determination of myeloperoxidase (MPO)
activity, and lymphocytes were isolated for the subsequent enzymatic
To evaluate the inflammatory response to CFA‐induced arthritis, the assay. Additionally, blood was collected without anticoagulant to
following signs of inflammation were observed and classified accord- serum separation.
ing to the scale: oedema formation (0—normal; 1—slight swelling at
the injection site; 2—swelling at the injection site and toes or ankle;
3—swelling at the injection site, toes and ankle), redness (0—normal;
2.8 | MPO activity in plasma of arthritis‐induced rats
1—slightly red/purple; 2—red/purple), and claw position (0—normal;
1—slightly curved; 2—almost closed). Individual scores were added to The MPO activity was analysed spectrophotometrically by the
give the total arthritis score.20,21 peroxidase‐coupled assay system involving phenol, 4‐aminoantipyrine
(AAP), and H2O2.24 Briefly, 390 μL of 2.5mM AAP and 20mM phenol
2.5.2 | Thermal hyperalgesia were placed in each tube, followed by 450 μL of 1.7mM H2O2. In the
presence of H2O2 as oxidizing agent, MPO catalysed the oxidative
To evaluate the hypersensitivity to heat stimulation, the paw immer- coupling of phenol and AAP yielding a coloured product,
sion test was used of according with Dalmolin et al.22 Briefly, animals quinoneimine, with a maximum absorbance at 500nM. The millimolar
were held, and the right hind paw was immersed in a water bath at absorbance coefficient for the quinoneimine was determined to be
48°C. The time elapsed between onset of the stimulus and manifesta- 14 ± 0.1/mM/cm, close to the previously reported values.25 Results
tion of the paw withdrawal response was measured automatically and were expressed in micromolar of quinoneimine produced at
was taken as an index of the thermal nociceptive threshold. Significant 30 minutes.
4 SACCOL ET AL.

2.9 | Isolation of lymphocytes from blood performed in triplicate, and the values were expressed in nmol
NH3/min/mg protein.
Blood was collected with 7.2 mg EDTA as anticoagulant, and
lymphocyte‐rich mononuclear cell were isolated from blood collected
with EDTA and separated on Ficoll‐Histopaque density by Boyum.26
2.13 | Separation of blood serum
The percentage of lymphocytes was superior to 93% as previously
outlined by Jaques et al.27 The integrity of lymphocytes preparation The blood samples were collected in tubes without anticoagulant, and
was confirmed by determining the lactate dehydrogenase (LDH) activ- after the clot formation, samples were centrifuged at 1,400×g for
ity in intact and disrupted lymphocytes using the kinetic method of 15 minutes at room temperature. The resultant serum samples were
the LabQuest apparatus (Diagnostics Gold Analyser). The procedure aliquoted in microtubes and kept in the refrigerator until the purines
was repeated before and after the incubation period. The protocol and cytokines were quantified.
was carried out according to the manufacturer's instructions. Triton
X‐100 (1%, final concentration) was used to disrupt the lymphocytes
preparation. The enzymatic activity was expressed as units per litre,
and one unit (1 U) corresponds to 1 μmol of NADH formed per minute
2.14 | Serum purine levels
per litre. The resultant lymphocytes samples were used immediately
Purine compounds were analysed by high‐pressure liquid chromatog-
for enzymatic assays.
raphy (HPLC) according to Voelter.32 The proteins were denaturated
by 0.6 mol/L perchloric acid. All samples were then centrifuged
2.10 | Protein determination (16,000×g for 10 min at 4°C), supernatants were neutralized with
4.0N KOH and clarified with a second centrifugation (16,000×g for
Protein content was measured by the method previously described by 30 min at 4°C). After the second centrifugation, the supernatants were
Bradford,28 using bovine serum albumin as standard. collected and centrifuged again (16,000×g for 30 min at 4°C). Aliquots
of 20 μL were applied to a reversed‐phase HPLC (LC‐20AT

2.11 | E‐NTPDase activity model, Shimadzu, Kyoto, Japan) using a C18 column (Ultra C18,
25 cm × 4.6 mm × 5 μm, Restek, USA). The elution was carried out
E‐NTPDase activity in lymphocytes was determined as previously applying a linear gradient from 100% solvent A (60mM KH2PO4 and
described by Leal et al, 29
in which the reaction medium contained 5mM of tetrabutylammonium chloride, pH 6.0) to 100% of solvent B
5mM CaCl2, 1200mM NaCl, 50mM KCl, 600mM glucose, and (solvent A plus 30% methanol) over a 40‐minute period (flow rate at
500mM Tris‐HCl buffer at pH 8.0, with a final volume of 200 μL. 1.0 mL/min). Mobile phases were filtered through a 0.45 μm Millipore
Twenty microlitres of the intact mononuclear cells suspended in saline filter prior to analysis, and all reagents utilized were of HPLC grade.
solution was added to the reaction medium (0.1‐0.2 mg/mL of protein) The amounts of purines and metabolic residues were measured by
and preincubated for 10 minutes at 37°C; incubation proceeded for absorption at 254 nm. The retention time of standards was used as
70 minutes. The reaction was initiated by the addition of substrate parameter for identification and quantification by comparison of the
(ATP or ADP) at a final concentration of 2.0mM and stopped with peak area. Purine levels were expressed as nmol of different com-
200 μL of 10% trichloracetic acid (TCA). The released inorganic phos- pounds per mL of serum.
phate (Pi) was assayed by a method previously described by Chan
et al,30 using malachite green as colorimetric reagent and KH2PO4 as
standard. Controls were carried out by adding the enzyme preparation 2.15 | Serum cytokine levels
after TCA addition to correct for nonenzymatic nucleotide hydrolysis.
All samples were run in triplicate, and the specific activity is reported Serum cytokines were simultaneously measured by cytometric bead
as nmol of Pi released/min/mg of protein. array (CBA). The Rat IL‐4 and IFN‐γ Flex Set kit (BD Biosciences,
San Jose, California) was applied following manufacturer's instructions.
Quantitative results were generated using an Accuri flow cytometer
2.12 | E‐ADA activity
and FCAP Array software. Results were expressed as picogram per

E‐ADA activity in lymphocytes was measured by the method by milliliter of serum.

Giusti and Galanti,31 which is based on the direct measurement of

ammonia produced when ADA acts in excess of adenosine. Briefly,
25 μL of lymphocytes reacted with 21mM of the substrate (adeno- 2.16 | Statistical analysis
sine), pH 6.5, and incubation was carried out for 1 hour at 37°C.
The reaction was stopped by adding 106.2mM phenol and 167.8nM Data were analysed by two‐way analysis of variance (ANOVA). Post
sodium nitroprussiate and hypochlorite solution. Ammonium sulfate hoc analyses were carried out by the Tukey mean ± standard error
75μM was used as ammonium standard. All the experiments were of the mean (SEM).
SACCOL ET AL. 5

3 | RESULTS shown). The AS group (Figure 2B) showed that the joint space is
distended by oedema, filled, and expanded by an intense inflammatory

3.1 | Arthritis induction and effect of quercetin lymphocytic infiltrate, separating muscle fibres, adipose cells, and
attachments such as nerves and vessels. The AQ5 group (Figure 2C)
To investigate the anti‐inflammatory effects of quercetin treatment, showed that the inflammatory infiltrate contains fewer lymphocytes
the chronic inflammation was induced by intraplantar injection of than the AS group. Moreover, the AQ25 group (Figure 2D) showed
CFA. Fifteen days after the injection of CFA, an increase in the arthri- less marked inflammatory changes than the AQ5 group. The AQ50
tis score (Figure 1A) and paw thickness (Figure 1B) and a concomitant group (Figure 2E) demonstrated lower inflammatory infiltrate than
decrease in paw thermal latency (Figure 1C) were observed in all rat group AQ25. The histological score is shown in Figure 3. In the CS
groups administered CFA. However, treatment with 50 mg/kg of group, we observed an absence of significant inflammatory infiltrate;
quercetin caused a significant decrease in the arthritis score and paw however, in the AS group, more than 200 lymphocytes/HPF were
thickness as well as an increase in paw thermal latency in rats. observed; moreover, in the groups treated with quercetin, we verified
a reduction in the inflammatory infiltration.

3.2 | Histological analysis

3.3 | MPO activity in plasma of arthritis‐induced rats
After 45 days, joint tissue from the paw of rats from the control saline
(CS) group showed organized collagen, normal connective tissue, and Analysis of MPO activity is presented in Figure 4. MPO activity was
no inflammatory infiltrate (Figure 2A). The other control groups significantly increased in the AS group (6.5 μmol quinoneimine,
(CQ5, CQ25, and CQ50) also showed normal structures (data not SEM = 0.4, n = 5, P < 0.001) compared with the CS group (1.9 μmol

FIGURE 1 Effect of quercetin (5, 25, and

50 mg/kg) and saline on some inflammatory
changes induced by intraplantar complete
Freund adjuvant (CFA) on the arthritis score
(A), oedema formation (B), and thermal
hyperalgesia (C). ***P < 0.001—basal values
when compared with induction arthritis.
#
P < 0.05, ##P < 0.01, and ###P < 0.001—
arthritis saline treated when compared with
other groups arthritis treated. Two‐way
analysis (analysis of variance [ANOVA])
followed by Tukey post test (n = 5)
6 SACCOL ET AL.

quinoneimine, SEM = 0.2, n = 5, P < 0.001). However, treatment with

5, 25, and 50 mg/kg of quercetin significantly reduced MPO activity
(2.5 μmol quinoneimine, SEM = 0.7, n = 5, P < 0.001; 2.4 μmol
quinoneimine, SEM = 0.4, n = 5, P < 0.001; 1.7 μmol quinoneimine,
SEM = 0.3, n = 5, P < 0.001, respectively) compared with the AS group
(6.5 μmol quinoneimine, SEM = 0.4, n = 5, P < 0.001).

3.4 | Cellular integrity

Analysis of LDH activity revealed that approximately 5% of lympho-

cytes of all the rat groups was disrupted, indicating that the prepara-
tion was predominantly intact after the isolation procedure (data
not shown).

3.5 | E‐NTPDase activity

Analysis of lymphocyte E‐NTPDase activity with ATP as a substrate

is shown in Figure 5A. The hydrolysis of ATP was significantly
increased in the AS group (43.5 nmol of Pi/min/mg protein,
SEM = 4.5, n = 5, P < 0.01) compared with the CS group (7.6 nmol
of Pi/min/mg protein, SEM = 0.8, n = 5, P < 0.01). These results
demonstrated that ATP hydrolysis in the AS group was increased
approximately 5.7‐fold compared with the CS group. However,
groups that received quercetin (AQ25: 19.2 nmol of Pi/min/mg pro-
tein, SEM = 2.3, n = 5, P < 0.05; AQ50: 13.1 nmol of Pi/min/mg
protein, SEM = 3.5, n = 5, P < 0.01) showed a decrease compared
FIGURE 2 Histological image of joint tissues from treated or non‐
treated rats. Groups: CS (control/ saline, A), AS(arthritis/saline, B), with the AS group, demonstrating that ATP hydrolysis was
AQ5 (arthritis/quercetin 5mg/kg, C), AQ25(arthritis/quercetin decreased approximately 2.2‐ and 3.3‐fold, respectively. Analysis of
25mg/kg, D) and AQ50 (arthritis/quercetin 50mg/kg, E). Arrow lymphocyte E‐NTPDase activity with ADP as a substrate is shown in
demonstrates the lymphocytes and asterisks edema Figure 5B, where ADP hydrolysis was also higher in the AS group
(77.5 nmol of Pi/min/mg protein, SEM = 4.5, n = 5, P < 0.001) com-
pared with the CS group (21.2 nmol of Pi/min/mg protein, SEM = 1.2,
n = 5, P < 0.001), representing an increase of 3.6‐fold. On the other
hand, treatment with 25 and 50 mg/kg of quercetin resulted in a
decrease compared with the AS group, (AQ25: 31.7 nmol of
Pi/min/mg protein, SEM = 9.9, n = 5, P < 0.01; AQ50: 18.6 nmol
of Pi/min/mg protein, SEM = 3.8, n = 5, P < 0.001), indicating a
decrease of 2.4 and 4.2 fold, respectively.

3.6 | E‐ADA activity

Analysis of E‐ADA activity is shown in Figure 6. The deamination of

FIGURE 3 Histological scoring of joint tissues from treated or non‐
adenosine was lower in the AS group (5.1 nmol of Pi/min/mg protein,
treated rats. Lymphocytes count by high power field (HPF) in joint
tissues of control and arthritis rats and treated for 45 days with SEM = 1.1, n = 5, P < 0.001) compared with the CS group (42.6 nmol
quercetin (5, 25 and 50 mg/kg) and treated with saline. Groups: saline of Pi/min/mg protein, SEM = 5.9, n = 5, P < 0.001), indicating a
control (CS), arthritis + saline (AS), arthritis + quercetin 5 mg/kg (AQ5), decrease of 8.3‐fold. Treatment with quercetin modulated the activity
arthritis+quercetin 25 mg/kg (AQ25) and arthritis+quercetin 50 mg/kg of E‐ADA as revealed by the AQ5 (37.6 nmol of Pi/min/mg protein,
(AQ50). Score 0 (0‐50 lymphocytes/HPF), 1(50‐100 lymphocytes/
SEM = 4.8, n = 5, P < 0.001), AQ25 (46.8 nmol of Pi/min/mg protein,
HPF), 2 (100‐150 lymphocytes/HPF), 3 (150‐200 lymphocytes/HPF)
SEM = 4.9, n = 5, P < 0.001), and AQ50 (54.4 nmol of Pi/min/mg pro-
and 4 (more than 200 lymphocytes/HPF). Ash indicates significant
differences from the arthritis saline (### P<0.001). Bars represent tein, SEM = 2.8, n = 5, P < 0.001) groups, demonstrating increases of
mean + SEM (n=5). Two‐way ANOVA‐Tukey test 7.4, 9.2, and 10.7 fold, respectively.
SACCOL ET AL. 7

FIGURE 4 Myeloperoxidase (MPO) activity in plasma of rats submitted to an experimental model of arthritis treated with saline and quercetin at
doses 5, 25, and 50 mg/kg. Asterisk indicates significant differences from the control saline (***P < 0.001). Hash indicates significant differences
from the arthritis saline (###P < 0.001). Bars represent mean ± SEM (n = 5). Two‐way analysis of variance (ANOVA)–Tukey test

3.7 | Serum purine levels 3.8 | Serum cytokine levels

Purine levels in the serum were measured by HPLC (Table 1). There Serum IFN‐γ and IL‐4 levels are shown in Figure 7. Results obtained
were no significant alterations in the levels of ATP, ADP, and AMP for the pro‐inflammatory cytokine IFN‐γ are shown in Figure 7A.
between the arthritis/saline and control/saline groups as well as the Serum levels for IFN‐γ were significantly increased in the AS group
other groups. However, a higher level of adenosine was found in the (79.0 pg/mL, SEM = 4.3; n = 5; P < 0.001) compared with the CS group
AS group (8.12 nmol/mL of serum, SEM = 0.31, n = 5, P < 0.05) com- (17.7 pg/mL, SEM = 8.4; n = 5; P < 0.001). However, there was a sig-
pared with the control/saline group (4.32 nmol/mL of serum, nificant decrease in IFN‐γ levels in the arthritis group that received
SEM = 0.97, n = 5, P < 0.05). 5 mg/kg (33.7 pg/mL, SEM = 6.9; n = 5; P < 0.01), 25 mg/kg
(25.3 pg/mL, SEM = 2.9; n = 5; P < 0.001), and 50 mg/kg (17.9 pg/mL,

FIGURE 5 E‐NTPDase activity—(A) ATP and

(B) ADP hydrolysis—in lymphocytes of rats
submitted to an experimental model of
arthritis treated with saline and quercetin at
doses 5, 25, and 50 mg/kg. Asterisk indicates
significant differences from the control saline
(P < 0.001). Hash indicates significant
differences from the arthritis saline
(***P < 0.001, ##P < 0.01, and ###P < 0.001).
Bars represent mean ± SEM (n = 5). Two‐way
analysis of variance (ANOVA)–Tukey test
8 SACCOL ET AL.

FIGURE 6 E‐ADA activity (adenosine deamination) in lymphocytes of rats submitted to an experimental model of arthritis treated with saline,
quercetin at doses 5, 25, and 50 mg/kg. Asterisk indicates significant differences from the control saline (***P < 0.001). Hash indicates significant
differences from the arthritis saline (###P < 0.001). Bars represent mean ± SEM (n = 5). Two‐way analysis of variance (ANOVA)–Tukey test

TABLE 1 Purine levels in rats with arthritis induced by CFA serum and treated with quercetin and saline

ATP (nmol/mL of Serum) ADP (nmol/mL of Serum) AMP (nmol/mL of Serum) Adenosine (nmol/mL of Serum)
a a a
CS 2.11 ± 0.50 5.10 ± 0.62 24.19 ± 3.35 4.32 ± 0.97a
CQ5 1.62 ± 0.10a 6.61 ± 1.49a 31.09 ± 4.20a 5.87 ± 0.34a
a a a
CQ25 2.22 ± 0.25 5.82 ± 0.95 23.61 ± 2.04 4.31 ± 0.60a
CQ50 2.25 ± 0.62a 5.22 ± 2.29a 29.84 ± 1.79a 4.00 ± 1.14a
AS 2.58 ± 0.10a 8.38 ± 0.53a 27.58 ± 2.31a 8.12 ± 0.31b
a a a
AQ5 2.06 ± 0.41 7.83 ± 2.12 32.64 ± 1.79 6.89 ± 0.64a
AQ25 2.37 ± 0.68a 7.66 ± 1.02a 24.60 ± 3.03a 5.97 ± 0.65a
AQ50 1.84 ± 0.30a 8.40 ± 0.46a 27.22 ± 1.22a 6.15 ± 0.63a

Note. Adenine nucleotides and adenosine levels measurement in serum of control and arthritis rats and treated for 45 days with quercetin (5, 25, and
50 mg/kg) and treated with saline. Purine level measurements are reported as of nmol/mL of serum. Groups: saline control (CS), saline + quercetin
5 mg/kg (CQ5), saline + quercetin 25 mg/kg (CQ25), saline + quercetin 50 mg/kg (CQ50), arthritis + saline (AS), arthritis + quercetin 5 mg/kg (AQ5), arthri-
tis + quercetin 25 mg/kg (AQ25), and arthritis + quercetin 50 mg/kg (AQ50). Bars represent mean ± SEM. Groups with different letters are statistically dif-
ferent (P < 0.05; n = 5) (two‐way ANOVA–Tukey multiple comparison test).
Abbreviations: ANOVA, analysis of variance; CFA, complete Freund adjuvant.

SEM = 2.7; n = 5; P < 0.001) of quercetin compared with the AS group studies have shown that flavonoids have been used for the treatment
(79.0 pg/mL ± 4.3; n = 5; P < 0.001). As shown in Figure 7B, the aver- of RA. Natural flavonoids such quercetin have also been reported
age serum levels for the anti‐inflammatory cytokine IL‐4 were signifi- to exhibit anti‐inflammatory and immunosuppressive properties.
cantly increased in the AS group (29.6 pg/mL, SEM = 6.6; n = 5; However, the mechanisms of action of these actions have not been
P < 0.001) compared with the CS group (7.7 pg/mL, SEM = 3.8; fully understood.
n = 5; P < 0.001). Our results also revealed a significant decrease in CFA‐induced arthritis is a veritable model for assessing the series
IL‐4 levels in the arthritis group treated with 5 mg/kg (8.2 pg/mL, of inflammatory processes that occur in RA.33,34 Reports have shown
SEM = 4.2; n = 5; P < 0.01), 25 mg/kg (1.1 pg/mL, SEM = 0.3; n = 5; that approximately 90% of changes observed in this model are similar
P < 0.001), and 50 mg/kg (2.0 pg/mL, SEM = 0.7; n = 5; P < 0.001) to those observed in human arthritis.35 In this way, novel chemother-
of quercetin compared with the AS group (29.6 pg/mL, SEM = 6.6; apeutic agents can be easily evaluated for the treatment of chronic
n = 5; P < 0.001). inflammatory conditions.36,37 Thus, this study was performed to
assess the effect of arthritis on purinergic enzyme activity, nucleotide
and nucleoside levels, and secretion of pro‐ and anti‐inflammatory
4 | DISCUSSION cytokines, as well as the effect of three doses of quercetin.
To confirm the arthritis in rats, arthritis score, oedema formation,
This is the first in vivo study to assess the effect of quercetin on thermal hyperalgesia, histological analysis, and MPO activity were
purinergic system of CFA‐induced arthritis in Wistar rats. Several evaluated. The results of this study showed that 50 mg/kg of
SACCOL ET AL. 9

FIGURE 7 Serum levels of INF‐γ (A) and IL‐

4 (B) cytokines. Cytokines levels are reported
as of pg/mL. Groups: Saline control (CS),
arthritis + saline (AS), arthritis + quercetin
5 mg/kg (AQ5), arthritis + quercetin 25 mg/kg
(AQ25), and arthritis + quercetin 50 mg/kg
(AQ50). Bars represent mean ± SEM. Asterisk
indicates significant differences from the
control saline (***P < 0.001). Hash indicates
significant differences from the arthritis saline
(##P < 0.01, ###P < 0.001). Bars represent
mean ± SEM (n = 5). Two‐way analysis of
variance (ANOVA)–Tukey test

quercetin improved the physical features and ameliorated the progres- Alterations in the activity of ectoenzymes, such E‐NTPDase and
sion of arthritis induced by CFA. This agrees with the report of E‐ADA, have been reported in several diseases, including ischaemic
Mamani‐Matsuda et al,36 where quercetin was shown to be effective heart disease,42 HIV,43 Chagas disease,44 and multiple sclerosis,45 indi-
in reducing signs of arthritis. cating that these enzymes could be involved in the pathogenesis of
The histological analysis corroborates the effectiveness of all of many diseases, including RA.34,46-48 The increase in E‐NTPDase
the doses of quercetin tested in this study to reduce oedema activity in arthritic rats indicates that there is increased ATP and
formation and minimize inflammatory process. This is consistent with ADP hydrolysis in the extracellular milieu, which would reduce their
previous studies where quercetin was shown to inhibit inflammatory levels during the development of RA. This change is likely a result of
aspects of synovial cell function and neutrophil activation.38,39 It is a dynamic response of lymphocytes in an attempt to maintain appro-
believed that anti‐inflammatory activity includes inhibition of inflam- priate nucleotide levels.47
matory agents and mediators, such as enzymatic reactive oxygen At high concentrations, extracellular ATP activates pro‐
species and nitric oxide, regulation of the activity of cyclooxygenase inflammatory purinergic P2X7 receptors, stimulates the Th1 immune
enzymes and inducible nitric oxide synthase, reduction of the levels response, and contributes to tissue damage as well as inflammatory
and expression of cytokines, and modulation of transcription factors conditions.11,49 The increased E‐NTPDase activity in arthritis/saline
40
such as factor nuclear kappa B (NF‐κB). rats compared with the control/saline rats is consistent with the
MPO activity is an important inflammatory marker and is present results of Castilhos et al34 and da Silveira et al.48 However, in groups
in the granules of leukocytes and primarily in neutrophils and macro- treated with different doses of quercetin, we observed that this flavo-
41
phages. It is responsible for secreting hypochlorite. In this study, noid was able to reduce the activity of E‐NTPDase. At low concentra-
the AS group exhibited elevated MPO activity, which indicates a pro- tions, extracellular ATP possesses affinity for the P2Y receptor
gressive inflammatory process. This is consistent with the results of subtype on the surface of lymphocytes. These purinergic receptors,
Stamp et al,41 where MPO activity was reported to be increased in when stimulated, downregulate pro‐inflammatory cytokines and
arthritis patients, indicating its role in the pathogenesis and severity stimulate the Th2 immune response, leading to the production of
of arthritis. Conversely, animals treated with quercetin showed a anti‐inflammatory cytokines.11,44
lower MPO activity, indicating a decrease in neutrophil infiltration In addition to ATP and ADP, adenosine exhibits potent anti‐
and subsequent reduction in the inflammatory process, thereby con- inflammatory and immunosuppressive actions through the inhibition
tributing to the decreased tissue damage observed in the histopathol- of T cell proliferation, cytokine secretion, and leukocyte migration
ogy of the paw.19 through the endothelial barrier.50 Our results demonstrated that E‐
10 SACCOL ET AL.

ADA activity was decreased in lymphocytes of arthritis rats compared IFN‐γ levels, demonstrating that quercetin exerts anti‐inflammatory
with the control group. Consistent with our results, other studies have activities by regulating inflammatory cytokine production mediated
also shown a decrease in E‐ADA activity with arthritis.47,48 However, by macrophages and T lymphocytes.
treatment with quercetin (all doses) was able to reverse this effect in Another cytokine evaluated in this study was IL‐4. Our results
this study. demonstrated that IL‐4 levels were increased in the arthritis rats com-
In the study carried out by Abdalla et al,51 the effect of quercetin pared with the control group. Naciute et al58 also found increased
(1, 5, 10, 25, and 50 μM) in vitro resulted in no significant differences levels of IL‐4 in the plasma of RA patients. However, when arthritis
in lymphocyte E‐NTPDase activity when ATP or ADP was used as a rats were treated with 5, 25, and 50 mg/kg of quercetin, the levels
substrate. On the other hand, E‐ADA activity in vitro showed a signif- of IL‐4 were lower in relation to the arthritis group treated with saline.
icant decrease proportional to the increase in quercetin concentration Our results are consistent with the study of Braun et al,59 which
compared with 0 μM quercetin (control group). showed that pretreatment with quercetin reduced the levels of IL‐4
The products of nucleotide degradation were analysed in the con- in rats with hyperlipidaemia, decreased eosinophil recruitment, and
trol and arthritis group treated with saline and quercetin by HPLC. The inhibited NF‐κB activation in the inflammatory process.60
results revealed that there were no changes in the ATP, ADP, and Overall, our results demonstrate alterations in enzymes of the
AMP levels. However, adenosine levels were increased in the AS rat purinergic system and in MPO activity of lymphocytes, as well in cyto-
group. Extracellular adenosine levels mediate an autoregulatory immu- kine secretion in arthritic rats, indicating that these alterations may be
52
nosuppressive loop to protect healthy tissues. due to the inflammatory process of arthritis. Interestingly, treatment
The observed increase in adenosine levels may be attributed to the with quercetin was able to reverse the alterations, demonstrating an
decreased E‐ADA activity in the AS group, as well as the attendant anti‐inflammatory effect. The possible mechanism action of quercetin
systemic changes caused by inflammatory conditions. Adenosine plays may be partly through the inhibition of inflammatory activity/agents,
a central and direct role in the regulation of inflammatory responses as well as the cytokines secretion, which may occur via the regulation
by inhibiting lymphocyte activation and decreasing both Th1 and Th2 of NF‐κB.61 However, further studies are needed to elucidate other
cytokine secretion through A2A receptor activation.53 However, the possible mechanisms of quercetin as an antiarthritic agent.
observed decrease in adenosine levels in rats treated with quercetin
could suggest an anti‐inflammatory mechanism of quercetin. Previous ACKNOWLEDGEMENTS
studies have demonstrated that one of the mechanisms by which
This study was supported by the Conselho Nacional de
quercetin employs its beneficial effects in the central nervous system
Desenvolvimento Científico e Tecnológico (CNPq), Fundação
is through modulation of E‐ADA activity because this flavonoid
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
inhibits activity of this enzyme, thus contributing to the increased
(CAPES), and Fundo de Incentivo a Pesquisa (FIPE/UFSM), Brazil.
levels of adenosine.19
Recent studies have shown that an imbalance in T cell subsets
CONFLIC T OF INT E RE ST
plays an important role in RA. CD4+ T cells can be divided into Th1,
Th2, Th17, and Treg subsets according to differentiation and func- The authors declare that they have no conflict of interest.
54
tion. Th1 cells secrete a large amount of IFN‐γ, which is responsible
for promoting cell‐mediated immunity.54 IFN‐γ is synthesized in the
COMP LIANCE WI TH E T HICAL S TANDAR DS
cellular immune response process and is a strong immune regulator,
promoting inflammation by inducing the expression of a variety of All procedures performed in studies involving animals were in
cytokines, including TNF‐α, IL‐2, and IL‐10.55 Nair et al16 showed that accordance with the ethical standards of the institution or practice
IL‐4 and IFN‐γ play a significant role in the regulation of immune at which the studies were conducted.
responses by their mutually antagonistic mechanisms. Other parame-
ters evaluated in this study were the secretion of pro‐ and anti‐
DATA AVAILABLE STATEMENT
inflammatory cytokines in rats with arthritis.
Our results demonstrate higher levels of IFN‐γ in arthritis, indicat- Data available on request from the authors.
ing an inflammatory state. Consistent with the results of our study,
Niu et al8 also found significantly higher levels of serum IFN‐γ in an ORCID
animal model of collagen‐induced arthritis. Pavlovic et al56 also found Daniela Bitencourt Rosa Leal https://orcid.org/0000-0003-2618-
significantly elevated IFN‐γ levels in the serum of patients with early 9801
RA, indicating that this pro‐inflammatory cytokine might play a key
role in maintaining immune homeostasis in patients with RA. Our data RE FE RE NC ES
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