Ouratea spectabilis and its Biflavanone Ouratein D Exert Potent Anti-inflammatory Activity in MSU Crystal-induced Gout in Mice

• Abstract
• Introduction
• Results
• Discussion
• Material and Methods
• Extract preparation and isolation of biflavanones
• Chromatographic profile of OSpC
• Experimental model of gouty arthritis in mice and treatments
• Total and differential cell count and cell migration
• Determination of IL-1β and CXCL1 by ELISA
• Cell culture and in vitro assay
• Western blot analysis
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Gouty arthritis (GA) is an inflammatory arthritis triggered by the deposition of monosodium urate monohydrate (MSU) crystals, causing pain, inflammation, and joint damage. Several drugs are currently employed to manage acute flares of GA, but they either have limited effectiveness or induce severe adverse reactions. Ouratea spectabilis is traditionally used in Brazil to treat gastric ulcers and rheumatism. The ethanolic extract of O. spectabilis stems (OSpC) and four biflavanones (ouratein A – D) isolated thereof were evaluated in a murine model of GA induced by the injection of MSU crystals. The underlying mechanism of action of ouratein D was investigated in vitro in cell cultures by measurement of IL-1β levels by ELISA and Western blot analysis. The administration of OSpC (10, 30 or 100 mg/Kg, p. o.) reduced the migration of total inflammatory cells, monocytes, and neutrophils and diminished the levels of IL-1β and CXCL1 in the synovial tissue. Among the tested compounds, only ouratein D (1 mg/Kg) reduced the migration of the inflammatory cells and it was shown to be active up to 0.01 mg/Kg (equivalent to 0.34 nM/Kg, p. o.). Treatment of pre-stimulated THP-1 cells (differentiated into macrophages) or BMDMs with ouratein D reduced the release of IL-1β in both macrophage lines. This biflavanone reduced the activation of caspase-1 (showed by the increase in the cleaved form) in supernatants of cultured BMDMs, evidencing its action in modulating the inflammasome pathway. The obtained results demonstrate the anti-gout properties of O. spectabilis and point out ouratein D as the bioactive component of the assayed extract.

Introduction

Gouty arthritis (GA) is considered the most widespread inflammatory arthritis with prevalence rates ranging from 1% to 6.8% and incidence of 0.58 – 2.89 per 1000 people in the global population [1]. The disease is more prevalent in men than in women (3 : 1 to 10 : 1). Gout incidence and prevalence increase with each decade of life, with prevalence increasing to 11 – 13% and incidence increasing to 0.4% in individuals older than 80 years [2]. The incidence rate of gout was also found to be higher in some racial/ethnic minorities, such as in African-Americans versus whites, in Hmong Chinese men versus non-Hmong men, and in the New Zealand Maori versus those of European descent [2].

Gout is characterized by swollen, painful, red, and poor-functional joints; besides, in some patients, the disease may evolve into more severe conditions with cardiovascular and renal impairment, causing a reduction in quality of life, in addition to economic burden both for patients and health systems [3].

The inflammatory process of gout is initiated by the deposition of monosodium urate crystals (MSU) in the joints, synovial space, and surrounding tissues, causing acute episodes of pain [4]. MSU crystals are the solid form of uric acid, the final degradation product of purines. They exert pro-inflammatory action and can initiate, amplify, and sustain an intense inflammatory response through binding and activation of pattern recognition receptors (PRR) in macrophages and neutrophils [5]. Acute flares of gout occur when precipitated MSU crystals are phagocyted by resident cells, mainly macrophages, causing the assembling of the cytosolic multiprotein complex NLRP3 inflammasome and further activation of the inflammatory caspase-1, culminating with the cleavage, maturation, and secretion of IL-1β to extracellular space. This cascade of events causes a massive recruitment and accumulation of neutrophils in tissue by the production of chemoattractant factors such as CXCR2-binding chemokines, thus amplifying joint inflammation [6].

Currently, acute gout flares can be controlled by the use of non-steroidal anti-inflammatory drugs (NSAIDs) like indomethacin and naproxen to relieve pain and reduce inflammation and joint incapacitation; however, they may cause adverse reactions like stomach pain, bleeding, and ulcers [7]. Selective cyclooxygenase-2 (COX-2) inhibitors can also be used to manage acute GA, along with corticosteroids and drugs that increase uric acid excretion, such as sulfinpyrazone and probenecid [8]. In long-term therapy, the goal is to reduce hyperuricemia to prevent emergence of new crises. Of note, colchicine is one of the most-used drugs to control gouty inflammation. This alkaloid isolated from Colchicum autumnale can inhibit NLRP3 activation and reduce neutrophil chemotaxis. However, its use is related to adverse gastrointestinal effects (nausea, vomiting, diarrhea, and abdominal pain), in addition to myelosuppression, leukopenia, granulocytopenia, neutropenia, aplastic anemia, and rhabdomyolysis, because of reduced leukocyte motility to the site [9]. It is worth mentioning the need for dose adjustment in the case of chronic renal patients [10]. Therefore, alternative drugs are demanded to manage gouty arthritis [9], and various plant species have been investigated for this purpose [8]. Several plant species are traditionally used worldwide to treat inflammatory diseases, including gout, and their biological effects have been demonstrated by in vitro and in vivo assays [11], in addition to clinical trials [12].

Ouratea spectabilis (Mart.) Engl. (Ochnaceae) is an arborous species endemic in Brazil, widely distributed in the country, being found in all phytogeographic domains [13]. The species is popularly known as “folha-de-serra” [14] and is traditionally used as an anti-inflammatory agent to treat rheumatism and gastric ulcers [15]. The pioneer phytochemical investigation of O. spectabilis leaves resulted in the isolation of the biflavonoids 6,6′-bigenkanine and 7,7′-dimethoxy-agatisflavone [16]. A recent investigation of O. spectabilis barks carried out by our group afforded four new biflavanones named ouratein A – D ([Fig. 1]) [17]. We demonstrated that the ethanolic extract of O. spectabilis barks significantly reduces the release of TNF, IL-1β, and CCL2 by LPS-stimulated THP-1 cells, while ouratein D selectively inhibited CCL2 release (IC₅₀ 3.11 (2.57 to 3.76 µM) [17]. These previous findings motivated us to further explore the anti-inflammatory and antiarthritic potential of O. spectabilis extract and isolated ourateins. Therefore, we herein describe the antiarthritic activity of an ethanolic extract of O. spectabilis barks and isolated biflavanones in a murine model of gouty arthritis. In addition, we investigated in vitro the molecular mechanism of the anti-gout activity of ouratein D, the major biflavanone constituent of O. spectabilis extract.

Results

The chromatographic profile registered by HPLC-DAD for the ethanol extract of O. spectabilis bark (OSpC) revealed four major peaks ([Fig. 1]). The phytochemical investigation of OSpC afforded four new 3,3″-linked biflavanones named ouratein A, B, C, and D ([Fig. 1]) after extract fractionation over a Sephadex LH20 column and compounds isolation by preparative HPLC [7]. Ouratein D was found to be the major peak of the OSpC chromatogram ([Fig. 1]). The potential anti-inflammatory effect of OSpC was initially assessed in an experimental model of gouty arthritis in mice. The treatment with OSpC at 10, 30, or 100 mg/kg reduced the number of total leukocytes ([Fig. 2 a]) and mononuclear cells ([Fig. 2 c]), whereas the reduction in polymorphonuclear cells was observed at the doses of 10 and 100 mg/kg ([Fig. 2 b]) in comparison to the untreated arthritic group. The administration of OSpC at 30 or 100 mg/Kg also reduced the release of IL-1β ([Fig. 2 d]) and CXCL1 ([Fig. 2 e]) in the periarticular tissue of arthritic mice. Dexamethasone was used as the standard treatment, which reduced all these inflammatory parameters after MSU crystal injection.

We subsequently evaluated the anti-inflammatory activity of the ouratein A – D in the MSU crystal-induced arthritis in mice. Using the dose of 1 mg/Kg for each compound, only ouratein D reduced the accumulation of total leukocytes ([Fig. 3 a]), polymorphonuclear cells ([Fig. 3 b]), and mononuclear cells ([Fig. 3 c]) in the synovial cavity after MSU crystal challenge. Besides, ouratein D reduced significantly the levels of CCL2, IL-1β, and CXCL1 ([Fig. 3 d, e], and [f], respectively). Of note, ouratein B was able to reduce only the accumulation of mononuclear cells ([Fig. 3 b]), along with IL-1β and CXCL1 concentrations ([Fig. 3 e] and [f]).

Since ouratein D was the most active compound among the assayed biflavanones, we next tested if its lower concentrations still maintain the anti-inflammatory effect in the murine model of gouty arthritis. Interestingly, the doses of 0.01 to 1 mg/kg presented similar effects in reducing the accumulation of total leucocytes ([Fig. 4 a]), polymorphonuclear ([Fig. 4 b]), and mononuclear ([Fig. 4 c]) cells, with no significant difference among them. At 0.01 mg/kg (corresponding to 0.34 nM/kg), it reduced by 71.5 ± 18.6%, 65.3 ± 8.9%, and 86.8 ± 8.0 the migration of total leukocytes, monocytes, and polymorphonuclear cells in the knee joint, respectively, evidencing the potent effect of this flavanone.

Based on these results, ouratein D was selected for further studies aiming to access its molecular mechanism of action. The investigation was carried out in vitro using both BMDMs and THP-1 pre-stimulated cells. Ouratein D reduced the concentration of IL-1β in the supernatant of both cell lines at 3 and 10 µM ([Fig. 5 a]), while the concentration of 1 µM was effective to decrease IL-1β levels in BMDMs (39.8 ± 14.1%). Interestingly, the reduction in IL-1β release promoted by ouratein D in THP-1 cells (32.3 ± 6.2%) was lower than in BMDM cells (54.8 ± 9.3%) at 3 µM, whereas at 10 µM similar inhibition rates were obtained for both cell lines (respectively, 71.6 ± 1.0% and 82.2 ± 8.2%). To test if ouratein D can modulate the assembly of NLRP3 inflammasome, we measured the cleaved form of caspase-1, the active form of this enzyme that cleaves and matures IL-1β [18]. The concentration of 10 µM was chosen because it caused similar reduction in IL-1β in the supernatant of both cell lines ([Fig. 5 a] and [b]). As shown in [Fig. 5 c], ouratein D reduced the cleaved form of caspase-1 in BMDMs by 59.2 ± 5.3%, as evidenced by the decrease in the cleaved caspase-1 band around 20 kDa and graphically demonstrated by densitometry analysis ([Fig. 5 c]).

Discussion

We herein demonstrate for the first time the anti-gout activity of the ethanolic extract of O. spectabilis barks (OSpC) in an experimental model of MSU crystal-induced arthritis in mice. The oral administration of OSpc diminished knee inflammation in arthritic mice, as demonstrated by the reduction in total and differential leukocyte accumulation in the knee cavity and diminished levels of IL-1β and CXCL1 in the periarticular tissue. The OSpC-derived biflavanone ouratein D presented potent anti-inflammatory effect, reducing the accumulation of leukocytes even at very low doses, in addition to decreasing the concentrations of IL-1β, CCL2, and CXCL1. Furthermore, ouratein D reduced the activation of caspase-1 and the release of IL-1β in BMDMs. These results clearly show the anti-gout properties of O. spectabilis and point out that ouratein D is the compound responsible for the anti-inflammatory activity of the assayed extract.

The anti-inflammatory activity of other Ouratea species has been reported, including O. parviflora in the paw edema in mice induced by carrageenan [19] and O. semiserrata in LPS-induced murine arthritis [20], thus highlighting the potential of the genus as a source of anti-inflammatory compounds. The relevance of the anti-gout activity induced by OSpC in experimental gouty mice can be evidenced by comparison with other plant extracts. For example, a flavonoid-rich extract of Selaginella moellendorffi (100, 200, and 400 mg/Kg; p.o), which contains the biflavone amentoflavone, reduced the paw edema induced by MSU crystals in mice and the IL-1β levels in paw tissue [21]. The administration of a 70% hydroethanolic extract of Jatropha isabellei at 300 mg/kg prevented thermal hyperalgesia, mechanical allodynia, edema, and neutrophil infiltration induced by intra-articular injection of MSU crystals in rats [22]. In its turn, a 70% hydroethanolic extract of Mollugo pentaphylla (300 mg/kg) suppressed the inflammatory paw edema and pain in the same model, as well as diminished the production of TNF and IL-1β and the activation of the inflammasome NLRP3 and of the transcription factor NF-κB [23]. Here, OSpC and its biflavanone ouratein D, even at very low doses given orally, presented a remarkable anti-gout effect, highlighting the relevance of our findings.

In the inflammatory process of gout, MSU crystals accumulate in the joint and synovium, causing excessive migration of monocytes/macrophages, monocytes, and neutrophils. Monocytes play an important role both at the beginning and end of a gout crisis, as MSU crystals induce transformation of monocytes into macrophages [24]. In addition, monocytes release pro-inflammatory cytokines like IL-1β- and CXCR2-binding chemokines locally and into the peripheral circulation, guiding neutrophils to the tissue [25]. The flare of acute gout is associated with a neutrophil swarm in the joint, amplifying inflammation and pain. A lower number of neutrophils in the joints indicates a reduction in the inflammatory process and, for this reason, the count of these cells at the inflamed site is a prognostic marker of the disease [26]. Therefore, compounds capable of reducing the migration and survival of neutrophils during acute crisis contribute to reducing the inflammatory process in gout [27], [28]. Here, both OSpC and ouratein D efficaciously reduced the accumulation of neutrophils in an MSU crystal-injected joint, evidencing an important anti-inflammatory effect in gouty inflammation.

Among the biflavanones isolated from OSpC, only ouratein D reduced joint inflammation significantly after MSU crystal injection. Ourateins A, B, C, and D are structurally alike compounds, which differ only by their number of methyl groups–respectively, zero, one, two, and three ([Fig. 1]). The reduction in cell migration to the joint after MSU crystal injection by these compounds seems to be associated with the methylation grade of ouratein D. The methylation grade affects a compoundʼs lipophilicity, and an adequate value is required for a drug to be administered orally and present satisfactory bioavailability [29]. In fact, lipophilicity affects several biological and physicochemical properties of drugs, namely solubility, absorption, binding to plasma proteins, metabolite clearance, distribution volume, target interaction, renal and biliary clearance, central nervous system penetration, tissue deposit, bioavailability, and toxicity [30].

Since the animals were treated orally with the compounds, it is likely that the activity of ouratein D and the absence of significant effects induced by the other ourateins result from distinct lipophilicities. In fact, the log P values calculated for these compounds using the Virtual Computational Chemistry Laboratory (VCCL) software demonstrate the influence of methylation grade on lipophilicity: log P values of 3.20, 3.72, 4.24, and 4.76 were calculated, respectively, for ourateins A, B, C, and D. According to Lipinskiʼs rule of five, a good drug candidate should have a log P value close to 5 for favorable oral absorption [31]. Ouratein D fulfills this requirement and probably presents more favorable pharmacokinetics, which is reflected in its high anti-inflammatory potency.

The anti-inflammatory activity of flavonoids has been widely demonstrated in different experimental models [32], but only a few studies have employed gout models [33]. For instance, the administration of naringenin at 150 mg/kg to MSU-arthritic mice reduced joint pain and swelling, leukocyte recruitment, oxidative stress, and IL-1β concentration [34]. In another study, quercetin (75 mg/Kg) reduced MSU-induced knee joint edema in mice, hyperalgesia, leukocyte infiltration, IL-1β, prostaglandin E2, NO production, and COX-2 expression [35]. Regarding biflavonoids, their anti-inflammatory activity is also known, but to the best of our knowledge only robustaflavone has been reported to possess an anti-gout effect. This biflavanone (200 µM/Kg; p. o.) reduced the paw edema, cell infiltration, and IL-1β levels in the paw of MSU-arthritic mice, in addition to regulating NO and TNF levels [21]. It is worth mentioning that in this study, ouratein D, administered orally at 0.01 mg/Kg (0.34 nM/Kg), promoted a significant reduction in the total number of leukocyte, mononuclear, and polymorphonuclear cells after MSU crystal injection, whereas the positive control dexamethasone elicited a similar anti-inflammatory response at 5.0 mg/kg. These favorable preclinical data clearly demonstrate the potential of ouratein D to be investigated in further studies and be developed as an anti-inflammatory drug. One should be aware that the use of dexamethasone is associated with adverse effects like anxiety, depression, hypertension, ventricular dysfunction, gastric perforation, osteoporosis, diabetes, and renal fibrosis, among others [36], and therefore the development of alternative anti-inflammatory agents are demanded.

Aiming to undertake a preliminary investigation of the anti-inflammatory mechanism of action of ouratein D, we performed an in vitro study using two different macrophage lines. Pre-treatment of these cells with ouratein D before MSU crystal challenge reduced the release of IL-1β in the supernatant. Both THP-1 (human) and BMDMs (murine) cells are macrophages, while the monocytic line THP-1 was previously differentiated into macrophages for the assays. Resident macrophages are the main cells to first interact with MSU crystals, phagocyting the crystals and initiating the cascade of events through the NLRP3/caspase-1/IL-1β mechanism [6]. Here we found that MSU-stimulated BMDMs pre-treated with Ouratein D show reduction in caspase-1 activation, as evidenced by the decreased levels of cleaved caspase-1 seen in Western blot analysis. Activation of the NLRP3 inflammasome is considered the main event of gout and involves two steps. The first step, also known as the priming signal, activates the NF-κB pathway, thus inducing the expression of pro-IL-1β and NLRP3 mRNA. The second step, the activation signal, is transduced by PAMPs and DAMPs, resulting in the formation of the inflammasome complex with subsequent activation of caspase-1 [35]. In this work, pretreatment with ouratein D significantly inhibited IL-1β secretion and caspase-1 cleavage. These results suggest that ouratein D inhibited NLRP3 inflammasome activation.

The mechanism of action of a few biflavonoids has been described to proceed through caspase-1 inhibition, as herein reported for ouratein D. Hence, amentoflavone exerts anti-gout activity by down-regulating IL-1β, TNF, caspase-1, and NLRP3 mRNA. In MSU-stimulated THP-1 cells, robustaflavone diminished the levels of NO and LDH, suppressed the activation of NLRP3 inflammasome, decreased caspase-1 activation, repressed mature IL-1β expression, and inhibited ASC staining and NLRP3 protein expression [21]. The data here described for ouratein D highlight the anti-inflammatory potential of biflavanones, especially for developing new drugs to manage gouty arthritis. Ouratein D may have preventive or therapeutic potential against acute gout via regulation of the NLRP3/caspase-1 signaling pathway.

Material and Methods

Extract preparation and isolation of biflavanones

O. spectabilis was collected and identified by Prof. Júlio Antônio Lombardi, and a voucher specimen (48.940) was deposited at the herbarium BHCB from the Institute of Biological Sciences, UFMG, Belo Horizonte, Brazil. The dried and ground bark was extracted by percolation with ethanol 96°GL, and ourateins A, B, C, and D were isolated from this extract by chromatography over a Sephadex LH20 column, followed by purification on preparative RP-HPLC, as described in our previous publication [17].

Chromatographic profile of OSpC

A portion of OSpC (5 mg) was dissolved in 1 mL of HPLC grade MeOH and left under sonication for 10 min following centrifugation (10 min; 8400 × g). An aliquot (10 µL) of the supernatant was subject to analysis on an HPLC system (Agilent Technologies 1200 series) with a diode array detector (DAD). A Hypersil-ODS column (250 × 4.0 mm i. d.; 5 µm; Thermo Scientific) was employed in the analysis. The flow rate was set at 1 mL/min and the mobile phase consisted of deionized water (A) and acetonitrile (B), both containing 0.1% v/v formic acid. Elution was performed with a linear gradient from 5% to 95% B in 60 min, followed by isocratic elution with 95% B for10 min, and DAD detection was employed at the wavelength range of 200 – 400 nm.

Experimental model of gouty arthritis in mice and treatments

The assay was performed on C57BL/6j (wild type) female mice six to nine weeks old obtained from the Central Animal Facility of the Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil, and kept in the animal facilities of the Immunopharmacology Laboratory, Department of Biochemistry and Immunology, UFMG. The mice were maintained in a controlled environment (stable temperature and humidity) with free access to filtered water and food. All animal care and handling procedures were in accordance with the guidelines of the International Association for the Study of Pain [37], and all experiments received prior approval from the UFMG ethics committee (CEUA, certificate number 83/2015, issued on May 26th, 2015).

MSU crystals were prepared in sterile conditions by adding 1.68 g of uric acid (Sigma-Aldrich) in 500 mL of 0.01 M sodium hydroxide solution. After pH adjustment to 7.1, the solution was filtered through a 0.22 µm membrane and stirred for 24 hours. At the end of this time, crystals were obtained in the tube wall and the supernatant was discarded [27]. The mice (n = 6 per group) were fasted for 4 hours before the pre-treatment with the ethanolic extract of O. spectabilis (OSpC) (10, 30, and 100 mg/kg, p. o.), ourateins A – C (1 mg/kg, p. o.), ouratein D (1, 0.1 and 0.01 mg/kg, p. o.), and dexamethasone (5 mg/kg, p. o.; Sigma-Aldrich). All samples were dissolved in propylene glycol, employed as a negative control. In the sequence, the animals were anesthetized by a mixture of ketamine (80 mg/kg; Syntec) and xylazine (15 mg/kg; Syntec), and 90 min after treatments they were challenged by the injection into the tibiofemoral joint of 10 µL of an MSU crystal solution (100 µg/cavity) in phosphate-buffered saline (PBS). The contralateral joint was used as a control group (PBS injection), as previously described [27]. The inflammatory response was evaluated 15 hours after MSU crystal injection [28].

Total and differential cell count and cell migration

For the evaluation of cell migration, the animals were euthanized by general anesthetic overdose (mixture of ketamine (240 mg/kg; Syntec) and xylazine (45 mg/kg; Syntec) i. p.) after 15 hours, and then the articular cavity was exposed and washed with 40 µL of PBS. In the sequence, an aliquot (30 µL) of the obtained synovial fluid was diluted in 60 µL of Turk dye, and the total leukocyte count was performed in a Neubauer chamber with the aid of an optical microscope (100× magnification) and manual counter. Differential counting was performed on slides prepared by cytocentrifugation of a 70 µL aliquot of the articular lavage. The slides were stained according to the May–Grunwald–Giemsa staining technique. Cells were examined under light microscopy through the oil immersion objective (1000× magnification) using standard morphological criteria to differentiate cell types. The total number of leukocytes was used to calculate the percentage of monocytes and neutrophils. Results were expressed as the number of neutrophils × 10⁴ per articular cavity [28].

Determination of IL-1β and CXCL1 by ELISA

The contents of IL-1β and CXCL1 were quantified by using commercially available enzyme-linked immunosorbent assays (ELISA), following the manufacturerʼs instructions (Duo-Set kits; R&D Systems), 15 hours after the inflammatory stimulus. After mice euthanasia, the periarticular tissue of the lower limb knee was removed. The collected tissue was weighed and homogenized in a cytokine extraction solution (PBS containing antiproteases (0.1 mM PMSF, 0.1 nM hydrochloric benzetonium, 10 mM EDTA and 20 KI aprotinin A) and 0.05% Tween 20) using 1 mL of solution per 100 mg of tissue. The samples were centrifuged for 10 minutes at 8400 × g and the supernatant used for the quantification of IL-1β and CXCL1 in a 1 : 3 dilution in a 0.1% PBS/BSA solution.

In a 96-well plate, 100 µL/well of a capture antibody solution (anti-IL-1β and anti-CXCL1 mouse) was added, in concentrations of 1.0 and 5.5 µg/mL in PBS, respectively. The plate was incubated overnight at 4 °C. Subsequently, the plates were washed (three times) with washing buffer (PBS/0.1% Tween) in an automatic plate washer. Afterward, 200 µL/well of the blocking solution containing 1% of bovine serum albumin (BSA) in PBS was added and the plate was kept at room temperature for 2 hours. Next, the cytokine standards IL-1β (1000 to 31.25 pg/mL), or CXCL1 (1000 to 15.625 pg/mL) and samples (100 µL of the diluted supernatant per well) were added to the plate. After 18 h of incubation at 4 °C, the plates were washed and 100 µL of biotinylated detection antibody solution anti-mouse IL-1β (200 ng/mL) and anti-mouse CXCL1 (200 ng/mL) were added to them and incubated for 1 hour. In the sequence the plate was washed and 100 µL of a streptavidin–peroxidase solution (R&D system) was added to each well. After 20 to 30 minutes, the plate was washed again and 100 µL of citrate buffer containing o-phenylenediamine (OPD, Sigma; 0.4 mg/mL) and hydrogen peroxide (Merck; 0.2 µL/mL) was added. The reaction was stopped 30 minutes later by adding 50 µL of hydrochloric acid solution (1 mol/L). The OPD oxidation product was measured in a microplate plate reader (Tecan, Mannedorf, Switzerland) at 490 nm.

Cell culture and in vitro assay

THP-1 cells (monocyte isolated from peripheral blood from an acute monocytic leukemia patient), originally from ATCC (ATCC-TIB-202), were purchased from Banco de Células do Rio de Janeiro, Brazil. The cells were cultured in RPMI medium supplemented with 10% fetal bovine serum and were kept at 37 °C in a humidified atmosphere of 5% CO₂. For this, THP-1 cells (1 × 10⁶ cells/well) were plated in 24-well plates (500 µL) and differentiated in macrophages with PMA (phorbol myristate acetate, Sigma, 20 ng/mL) by 12 hours incubation at 37 °C with 5% CO₂. Next, cell supernatants were discarded, cells were starved by 12 hours with RPMI 1% serum. After wash with RPMI, cells were pretreated by 3 hours with ouratein D (1, 3, and 10 µM) at 37 °C with 5% CO₂, and then stimulated with MSU crystals (300 µg/mL for 6 h) as previously described [38].

Bone marrow-derived macrophages (BMDMs) were obtained as previously described [38], [39]. Wild-type C57BL/6 mice were euthanized and bone marrow cells were collected from tibias and femurs of mice and differentiated for 7 days in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 30% L929 cell-conditioned medium, plus antibiotics at 37 °C in a humidified atmosphere of 5% CO₂. Differentiated BMDMs were then detached, suspended in RPMI 1640 supplemented with complete differentiation medium, and plated (1 × 10⁶ cells/well) in 24-well tissue culture (500 µL) and incubated for further 12 h at 37 °C in a humidified atmosphere of 5% CO₂. After overnight incubation, BMDMs were pretreated by 3 hours with ouratein D (1, 3 and 10 µM) at 37 °C with 5% CO₂ and then pre-stimulated with lipopolysaccharide (LPS; serotype Escherichia coli O: 111: B4; 1 µg/mL; Sigma-Aldrich) for 1 hour and further stimulated with MSU crystals (300 µg/mL for 6 h) as previously described [39], [40].

The supernatants of both cell types were removed and the release of IL-1β was quantified by ELISA according to manufacturerʼs instructions (R&D Systems). The percentage of cytokine inhibition was calculated by the ratio between the amount of cytokine secreted by treated cells and the baseline level (pg/mL) observed for solvent control (0.1% DMSO).

Western blot analysis

Supernatants from the BMDM culture were subjected to SDS-PAGE analysis and Western blotting. Protein concentrations were quantified with the Bradford reagent (Biorad). Equal amounts of protein (20 µg) from each group were loaded and separated by electrophoresis on denaturing 12% polyacrylamide-SDS gels and electrotransferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with PBS containing 5% (w/v) nonfat dry milk and 0.1% Tween-20. Afterward, nitrocellulose membranes were washed three times (5 min each one) with PBS containing 0.1% Tween-20 and then incubated with the primary antibody at 4 °C overnight. The primary antibody used was mouse monoclonal against the p20 subunit of caspase-1 (1 : 1000; Adipogen). The membrane was washed three times for 5 min with PBS 0.1% Tween 20. Next, the membrane was incubated for 1 h at room temperature with the suitable HRP-conjugated secondary antibody (1 : 3000). Immunoreactive bands were visualized using the ECL detection system (GE Heathcare). The membrane was scanned and densitometry analysis of the bands was performed using ImageJ software (ImageJ, NIH). The results were expressed as arbitrary units (AU) and normalized to the values of total caspase-1 in the same sample [40].

Statistical analysis

Results are presented as the mean ± standard deviation (SD). Data were analyzed by one-way ANOVA, and differences among challenged untreated group and all groups were assessed by Dunnettʼs test. P values < 0.05 were considered significant. Calculations were performed using the Prism 5.0 software for Windows (GraphPad Software Inc., USA).

Contributorsʼ Statement

Conception and design of the work: P. R. V. Campana, F. A. Amaral, L. P. Sousa, M. M. Teixeira, F. C. Braga; data collection (MSU model): M. P. Rocha, D. P. Oliveira, V. L. S. Oliveira; (quantification of cytokines in MSU model): M. P. Rocha, D. P. Oliveira; (in vitro assay on THP-1): D. P. Oliveira, V. L. S. Oliveira; (in vitro assay on BMDMs): M. P. Rocha, I. Zaidan, L. C. Grossi, P. R. V. Campana; (Western blot): I. Zaidan, L. C. Grossi, L. P. Sousa; analysis and interpretation of data (MSU model): M. P. Rocha, D. P. Oliveira, V. L. S. Oliveira, F. A. Amaral, M. M. Teixeira; (in vitro assay on THP-1): V. L. S. Oliveira, I. Zaidan, L. C. Grossi, F. C. Braga; (in vitro assay on BMDMs): M. P. Rocha, I. Zaidan, L. C. Grossi, P. R. V. Campana; (Western blot): L. P. Sousa; statistical analysis: M. P. Rocha, D. P. Oliveira, F. A. Amaral, M. M. Teixeira; drafting the manuscript: M. P. Rocha, D. P. Oliveira, F. C. Braga; critical revision of the manuscript: P. R. V. Campana, F. A. Amaral, L. P. Sousa, M. M. Teixeira, F. C. Braga.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We thank CAPES (Brazil) for a masterʼs fellowship (MPR) and CNPq for post-doctoral (DPO) and research (FAA, FCB, LPS, MMT) fellowships.

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• 3 Dalbeth N, Choi HK, Joosten LAB, Khanna PP, Matsuo H, Perez-Ruiz F, Stamp LK. Gout. Nat Rev Dis Primers 2019; 5: 69
  CrossrefPubMedSearch in Google Scholar
• 4 Martillo MA, Nazzal L, Crittenden DB. The crystallization of monosodium urate. Curr Rheumatol Rep 2014; 16: 1-13
  CrossrefPubMedSearch in Google Scholar
• 5 Anderton H, Wicks IP, Silke J. Cell death in chronic inflammation: breaking the cycle to treat rheumatic disease. Nat Rev Rheumatol 2020; 16: 496-513
  CrossrefPubMedSearch in Google Scholar
• 6 Kingsbury SR, Conaghan PG, McDermott MF. The role of the NLRP3 inflammasome in gout. J Inflamm Res 2011; 4: 39-49
  PubMedSearch in Google Scholar
• 7 Xu Z, Zhang R, Zhang D, Yao J, Shi R, Tang Q, Wang L. Peptic ulcer hemorrhage combined with acute gout: Analyses of treatment in 136 cases. Int J Clin Exp Med 2015; 8: 6193-6199
  PubMedSearch in Google Scholar
• 8 Azevedo VF, Lopes MP, Catholino NM, Paiva ES, Araújo VA, Pinheiro GRC. Critical revision of the medical treatment of gout in Brazil. Rev Bras Reumatol Engl Ed 2017; 57: 346-355
  PubMedSearch in Google Scholar
• 9 Yamanaka HTG. Essence of the revised guideline for the management of hyperuricemia and gout. Japan Med Assoc J 2012; 55: 324-329
  PubMedSearch in Google Scholar
• 10 Pillinger MH, Mandell BF. Therapeutic approaches in the treatment of gout. Semin Arthritis Rheum 2020; 50: S24-S30
  CrossrefPubMedSearch in Google Scholar
• 11 Daoudi NE, Bouhrim M, Ouassou H, Bnouham M. Medicinal plants as a drug alternative source for the antigout therapy in Morocco. Scientifica (Cairo) 2020; 2020: 8637583
  PubMedSearch in Google Scholar
• 12 Wink M. Modes of action of herbal medicines and plant secondary metabolites. Medicines (Basel) 2015; 2: 251-286
  CrossrefPubMedSearch in Google Scholar
• 13 Chacon RG, Yamamoto K, Proença C, Cavalcanti TB, Graciano-Ribeiro D. A distinctive new species of Ouratea (Ochnaceae) from Jalapão Region, Tocantins, Brazil. Novon 2008; 18: 397-404
  CrossrefPubMedSearch in Google Scholar
• 14 Mecina GF, Santos VHM, Dokkedal AL, Saldanha LL, Silva LP, Silva RMG. Phytotoxicity of extracts and fractions of Ouratea spectabilis (Mart. Ex Engl.) Engl. (Ochnaceae). S Afr J Bot 2014; 95: 174-180
  CrossrefPubMedSearch in Google Scholar
• 15 Felício J, Gonçalez E, Braggio MM, Costantino L, Albasini A, Lins AP. Inhibition of lens aldose reductase by biflavones from Ouratea spectabilis . Planta Med 1995; 61: 217-220
  Thieme ConnectPubMedSearch in Google Scholar
• 16 Simoni IC, Felicio JD, Gonçalez E, Rossi MH. Avaliação da citotoxicidade de biflavonoides isolados de Ouratea spectabilis (Ochnaceae) em córnea de coelho SIRC. Arq Inst Biol 2022; 69: 95-97
  PubMedSearch in Google Scholar
• 17 Rocha MP, Campana PRV, Pádua RM, Souza Filho JD, Ferreira D, Braga FC. (3, 3″)-Linked biflavanones from Ouratea spectabilis and their effects on the release of proinflammatory cytokines in THP-1 cells. J Nat Prod 2020; 83: 1891-1898
  CrossrefPubMedSearch in Google Scholar
• 18 Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL, Gomez GA, Holley CL, Bierschenk D, Stacey KJ, Yap AS, Bezbradica JS, Schroder K. Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med 2018; 215 (03) 827-840
  CrossrefPubMedSearch in Google Scholar
• 19 Carbonari KA, Ferreira EA, Rebello JM, Felipe KB, Rossi MH, Felício JD, Filho DW, Yunes RA, Pedrosa RC. Free-radical scavenging by Ouratea parviflora in experimentally-induced liver injuries. Redox Rep 2006; 11: 124-130
  CrossrefPubMedSearch in Google Scholar
• 20 Campana PRV, Azevedo EPC, Amaral F, Pinho V, Ferreira D, Teixeira MM, Braga FC. In vitro and in vivo anti-inflammatory activity of Ouratea semisserrata . Planta Med 2016; 82: PB8
  PubMedSearch in Google Scholar
• 21 Zhang X, Liu Y, Deng G, Huang B, Kai G, Chen K, Li J. A purified biflavonoid extract from Selaginella moellendorffii alleviates gout arthritis via NLRP3/ASC/caspase-1 axis suppression. Front Pharmacol 2021; 12: 1-14
  PubMedSearch in Google Scholar
• 22 Silva CR, Frohlich JK, Oliveira SM, Cabreira TN, Rossato MF, Trevisan G, Froeder AL, Bochi GV, Moresco RN, Athayde ML, Ferreira J. The antinociceptive and anti-inflammatory effects of the crude extract of Jatropha isabellei in a rat gout model. J Ethnopharmacol 2013; 145: 205-213
  CrossrefPubMedSearch in Google Scholar
• 23 Lee YM, Shon EJ, Kim OS, Kim DS. Effects of Mollugo pentaphylla extract on monosodium urate crystal-induced gouty arthritis in mice. BMC Complement Altern Med 2017; 17: 447
  CrossrefPubMedSearch in Google Scholar
• 24 Jeong JH, Hong S, Kwon OC, Ghang B, Hwang I, Kim YG, Lee CK, Yoo B. CD14⁺ cells with the phenotype of infiltrated monocytes consist of distinct populations characterized by anti-inflammatory as well as pro-inflammatory activity in gouty arthritis. Front Immunol 2017; 8: 1260
  CrossrefPubMedSearch in Google Scholar
• 25 Jeong JH, Jung JH, Lee JS, Oh JS, Kim YG, Lee CK, Yoo B, Hong S. Prominent inflammatory features of monocytes/macrophages in acute calcium pyrophosphate crystal arthritis: a comparison with acute gouty arthritis. Immune Netw 2019; 19: e21
  CrossrefPubMedSearch in Google Scholar
• 26 Kadiyoran C, Zengin O, Cizmecioglu HA, Tufan A, Kucuksahin O, Cure MC, Cure E, Kucuk A, Ozturk MA. Monocyte to lymphocyte ratio, neutrophil to lymphocyte ratio, and red cell distribution width are the associates with gouty arthritis. Acta Med 2019; 62: 99-104
  PubMedSearch in Google Scholar
• 27 Cronstein BN, Terkeltaub R. The inflammatory process of gout and its treatment. Arthritis Res Ther 2006; 8: S3
  CrossrefPubMedSearch in Google Scholar
• 28 Amaral FA, Costa VV, Tavares LD, Sachs D, Coelho FM, Fagundes CT, Soriani FM, Silveira TN, Cunha LD, Zamboni DS, Quesniaux V, Peres RS, Cunha TM, Cunha FQ, Ryffel B, Souza DG, Teixeira MM. NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B (4) in a murine model of gout. Arthritis Rheumatol 2012; 64: 474-484
  CrossrefPubMedSearch in Google Scholar
• 29 Rauf SMA, Arvidsson PI, Albericio F, Govender T, Maguire GE, Kruger HG, Honarparvar B. The effect of N-methylation of amino acids (Ac-X-OMe) on solubility and conformation: A DFT study. Org Biomol Chem 2015; 13: 9993-10006
  CrossrefPubMedSearch in Google Scholar
• 30 Alam S, Khan F. Virtual screening, docking, ADMET and system pharmacology studies on Garcinia caged xanthone derivatives for anticancer activity. Sci Rep 2018; 8: 5524
  CrossrefPubMedSearch in Google Scholar
• 31 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001; 46: 3-26
  CrossrefPubMedSearch in Google Scholar
• 32 Ferraz CR, Carvalho TT, Manchope MF, Artero NA, Rasquel-Oliveira FS, Fattori V, Casagrande R, Verri WA. Therapeutic potential of flavonoids in pain and inflammation: mechanisms of action, pre-clinical and clinical data, and pharmaceutical development. Molecules 2020; 25: 762
  CrossrefPubMedSearch in Google Scholar
• 33 Ling X, Bochu W. A review of phytotherapy of gout: Perspective of new pharmacological treatments. Pharmazie 2014; 69: 243-256
  PubMedSearch in Google Scholar
• 34 Ruiz-Miyazawa KW, Staurengo-Ferrari L, Mizokami SS, Domiciano TP, Vicentini FTMC, Camilios-Neto D, Pavanelli WR, Pinge-Filho P, Amaral FA, Teixeira MM, Casagrande R, Verri WA. Quercetin inhibits gout arthritis in mice: Induction of an opioid-dependent regulation of inflammasome. Inflammopharmacology 2017; 25: 555-570
  CrossrefPubMedSearch in Google Scholar
• 35 Ruiz-Miyazawa KW, Borghi SM, Pinho-Ribeiro FA, Staurengo-Ferrari L, Fattori V, Fernandes GSA, Verri WA. The citrus flavanone naringenin reduces gout-induced joint pain and inflammation in mice by inhibiting the activation of NFκB and macrophage release of IL-1β . J Func Foods 2018; 48: 106-116
  CrossrefPubMedSearch in Google Scholar
• 36 Chen F, Hao L, Zhu S, Yang X, Shi W, Zheng K, Chen H. Potential adverse effects of dexamethasone therapy on COVID-19 patients: review and recommendations. Infect Dis Ther 2021; 10: 1907-1931
  CrossrefPubMedSearch in Google Scholar
• 37 IASP. 2022. Guidelines of the International Association for the Study of Pain. Accessed April 18, 2022 at: https://www.iasp-pain.org/resources/guidelines/
  PubMed
• 38 Galvão I, de Carvalho RVH, Vago JP, Silva ALN, Carvalho TG, Antunes MM, Teixeira MM. The role of annexin A1 in the modulation of the NLRP3 inflammasome. Immunology 2020; 160: 78-89
  CrossrefPubMedSearch in Google Scholar
• 39 Vago JP, Sugimoto MA, Lima KM, Negreiros-Lima GL, Baik N, Teixeira MM, Perretti M, Parmer RJ, Miles LA, Sousa LP. Plasminogen and the plasminogen receptor, Plg-R_KT, regulate macrophage phenotypic, and functional changes. Front Immunol 2019; 10: 1458
  CrossrefPubMedSearch in Google Scholar
• 40 Zaidan I, Tavares LP, Sugimoto MA, Lima KM, Negreiros-Lima GL, Teixeira LC, Miranda TC, Valiate BV, Cramer A, Vago JP, Campolina-Silva GH, Souza JA, Grossi LC, Pinho V, Campagnole-Santos MJ, Santos RA, Teixeira MM, Galvão I, Sousa LP. Angiotensin-(1–7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis. JCI Insight 2022; 7: e147819
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 30 August 2022

Accepted after revision: 10 January 2023

Accepted Manuscript online:
10 January 2023

Article published online:
15 March 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 3 Dalbeth N, Choi HK, Joosten LAB, Khanna PP, Matsuo H, Perez-Ruiz F, Stamp LK. Gout. Nat Rev Dis Primers 2019; 5: 69
  CrossrefPubMedSearch in Google Scholar
• 4 Martillo MA, Nazzal L, Crittenden DB. The crystallization of monosodium urate. Curr Rheumatol Rep 2014; 16: 1-13
  CrossrefPubMedSearch in Google Scholar
• 5 Anderton H, Wicks IP, Silke J. Cell death in chronic inflammation: breaking the cycle to treat rheumatic disease. Nat Rev Rheumatol 2020; 16: 496-513
  CrossrefPubMedSearch in Google Scholar
• 6 Kingsbury SR, Conaghan PG, McDermott MF. The role of the NLRP3 inflammasome in gout. J Inflamm Res 2011; 4: 39-49
  PubMedSearch in Google Scholar
• 7 Xu Z, Zhang R, Zhang D, Yao J, Shi R, Tang Q, Wang L. Peptic ulcer hemorrhage combined with acute gout: Analyses of treatment in 136 cases. Int J Clin Exp Med 2015; 8: 6193-6199
  PubMedSearch in Google Scholar
• 8 Azevedo VF, Lopes MP, Catholino NM, Paiva ES, Araújo VA, Pinheiro GRC. Critical revision of the medical treatment of gout in Brazil. Rev Bras Reumatol Engl Ed 2017; 57: 346-355
  PubMedSearch in Google Scholar
• 9 Yamanaka HTG. Essence of the revised guideline for the management of hyperuricemia and gout. Japan Med Assoc J 2012; 55: 324-329
  PubMedSearch in Google Scholar
• 10 Pillinger MH, Mandell BF. Therapeutic approaches in the treatment of gout. Semin Arthritis Rheum 2020; 50: S24-S30
  CrossrefPubMedSearch in Google Scholar
• 11 Daoudi NE, Bouhrim M, Ouassou H, Bnouham M. Medicinal plants as a drug alternative source for the antigout therapy in Morocco. Scientifica (Cairo) 2020; 2020: 8637583
  PubMedSearch in Google Scholar
• 12 Wink M. Modes of action of herbal medicines and plant secondary metabolites. Medicines (Basel) 2015; 2: 251-286
  CrossrefPubMedSearch in Google Scholar
• 13 Chacon RG, Yamamoto K, Proença C, Cavalcanti TB, Graciano-Ribeiro D. A distinctive new species of Ouratea (Ochnaceae) from Jalapão Region, Tocantins, Brazil. Novon 2008; 18: 397-404
  CrossrefPubMedSearch in Google Scholar
• 14 Mecina GF, Santos VHM, Dokkedal AL, Saldanha LL, Silva LP, Silva RMG. Phytotoxicity of extracts and fractions of Ouratea spectabilis (Mart. Ex Engl.) Engl. (Ochnaceae). S Afr J Bot 2014; 95: 174-180
  CrossrefPubMedSearch in Google Scholar
• 15 Felício J, Gonçalez E, Braggio MM, Costantino L, Albasini A, Lins AP. Inhibition of lens aldose reductase by biflavones from Ouratea spectabilis . Planta Med 1995; 61: 217-220
  Thieme ConnectPubMedSearch in Google Scholar
• 16 Simoni IC, Felicio JD, Gonçalez E, Rossi MH. Avaliação da citotoxicidade de biflavonoides isolados de Ouratea spectabilis (Ochnaceae) em córnea de coelho SIRC. Arq Inst Biol 2022; 69: 95-97
  PubMedSearch in Google Scholar
• 17 Rocha MP, Campana PRV, Pádua RM, Souza Filho JD, Ferreira D, Braga FC. (3, 3″)-Linked biflavanones from Ouratea spectabilis and their effects on the release of proinflammatory cytokines in THP-1 cells. J Nat Prod 2020; 83: 1891-1898
  CrossrefPubMedSearch in Google Scholar
• 18 Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL, Gomez GA, Holley CL, Bierschenk D, Stacey KJ, Yap AS, Bezbradica JS, Schroder K. Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med 2018; 215 (03) 827-840
  CrossrefPubMedSearch in Google Scholar
• 19 Carbonari KA, Ferreira EA, Rebello JM, Felipe KB, Rossi MH, Felício JD, Filho DW, Yunes RA, Pedrosa RC. Free-radical scavenging by Ouratea parviflora in experimentally-induced liver injuries. Redox Rep 2006; 11: 124-130
  CrossrefPubMedSearch in Google Scholar
• 20 Campana PRV, Azevedo EPC, Amaral F, Pinho V, Ferreira D, Teixeira MM, Braga FC. In vitro and in vivo anti-inflammatory activity of Ouratea semisserrata . Planta Med 2016; 82: PB8
  PubMedSearch in Google Scholar
• 21 Zhang X, Liu Y, Deng G, Huang B, Kai G, Chen K, Li J. A purified biflavonoid extract from Selaginella moellendorffii alleviates gout arthritis via NLRP3/ASC/caspase-1 axis suppression. Front Pharmacol 2021; 12: 1-14
  PubMedSearch in Google Scholar
• 22 Silva CR, Frohlich JK, Oliveira SM, Cabreira TN, Rossato MF, Trevisan G, Froeder AL, Bochi GV, Moresco RN, Athayde ML, Ferreira J. The antinociceptive and anti-inflammatory effects of the crude extract of Jatropha isabellei in a rat gout model. J Ethnopharmacol 2013; 145: 205-213
  CrossrefPubMedSearch in Google Scholar
• 23 Lee YM, Shon EJ, Kim OS, Kim DS. Effects of Mollugo pentaphylla extract on monosodium urate crystal-induced gouty arthritis in mice. BMC Complement Altern Med 2017; 17: 447
  CrossrefPubMedSearch in Google Scholar
• 24 Jeong JH, Hong S, Kwon OC, Ghang B, Hwang I, Kim YG, Lee CK, Yoo B. CD14⁺ cells with the phenotype of infiltrated monocytes consist of distinct populations characterized by anti-inflammatory as well as pro-inflammatory activity in gouty arthritis. Front Immunol 2017; 8: 1260
  CrossrefPubMedSearch in Google Scholar
• 25 Jeong JH, Jung JH, Lee JS, Oh JS, Kim YG, Lee CK, Yoo B, Hong S. Prominent inflammatory features of monocytes/macrophages in acute calcium pyrophosphate crystal arthritis: a comparison with acute gouty arthritis. Immune Netw 2019; 19: e21
  CrossrefPubMedSearch in Google Scholar
• 26 Kadiyoran C, Zengin O, Cizmecioglu HA, Tufan A, Kucuksahin O, Cure MC, Cure E, Kucuk A, Ozturk MA. Monocyte to lymphocyte ratio, neutrophil to lymphocyte ratio, and red cell distribution width are the associates with gouty arthritis. Acta Med 2019; 62: 99-104
  PubMedSearch in Google Scholar
• 27 Cronstein BN, Terkeltaub R. The inflammatory process of gout and its treatment. Arthritis Res Ther 2006; 8: S3
  CrossrefPubMedSearch in Google Scholar
• 28 Amaral FA, Costa VV, Tavares LD, Sachs D, Coelho FM, Fagundes CT, Soriani FM, Silveira TN, Cunha LD, Zamboni DS, Quesniaux V, Peres RS, Cunha TM, Cunha FQ, Ryffel B, Souza DG, Teixeira MM. NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B (4) in a murine model of gout. Arthritis Rheumatol 2012; 64: 474-484
  CrossrefPubMedSearch in Google Scholar
• 29 Rauf SMA, Arvidsson PI, Albericio F, Govender T, Maguire GE, Kruger HG, Honarparvar B. The effect of N-methylation of amino acids (Ac-X-OMe) on solubility and conformation: A DFT study. Org Biomol Chem 2015; 13: 9993-10006
  CrossrefPubMedSearch in Google Scholar
• 30 Alam S, Khan F. Virtual screening, docking, ADMET and system pharmacology studies on Garcinia caged xanthone derivatives for anticancer activity. Sci Rep 2018; 8: 5524
  CrossrefPubMedSearch in Google Scholar
• 31 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001; 46: 3-26
  CrossrefPubMedSearch in Google Scholar
• 32 Ferraz CR, Carvalho TT, Manchope MF, Artero NA, Rasquel-Oliveira FS, Fattori V, Casagrande R, Verri WA. Therapeutic potential of flavonoids in pain and inflammation: mechanisms of action, pre-clinical and clinical data, and pharmaceutical development. Molecules 2020; 25: 762
  CrossrefPubMedSearch in Google Scholar
• 33 Ling X, Bochu W. A review of phytotherapy of gout: Perspective of new pharmacological treatments. Pharmazie 2014; 69: 243-256
  PubMedSearch in Google Scholar
• 34 Ruiz-Miyazawa KW, Staurengo-Ferrari L, Mizokami SS, Domiciano TP, Vicentini FTMC, Camilios-Neto D, Pavanelli WR, Pinge-Filho P, Amaral FA, Teixeira MM, Casagrande R, Verri WA. Quercetin inhibits gout arthritis in mice: Induction of an opioid-dependent regulation of inflammasome. Inflammopharmacology 2017; 25: 555-570
  CrossrefPubMedSearch in Google Scholar
• 35 Ruiz-Miyazawa KW, Borghi SM, Pinho-Ribeiro FA, Staurengo-Ferrari L, Fattori V, Fernandes GSA, Verri WA. The citrus flavanone naringenin reduces gout-induced joint pain and inflammation in mice by inhibiting the activation of NFκB and macrophage release of IL-1β . J Func Foods 2018; 48: 106-116
  CrossrefPubMedSearch in Google Scholar
• 36 Chen F, Hao L, Zhu S, Yang X, Shi W, Zheng K, Chen H. Potential adverse effects of dexamethasone therapy on COVID-19 patients: review and recommendations. Infect Dis Ther 2021; 10: 1907-1931
  CrossrefPubMedSearch in Google Scholar
• 37 IASP. 2022. Guidelines of the International Association for the Study of Pain. Accessed April 18, 2022 at: https://www.iasp-pain.org/resources/guidelines/
  PubMed
• 38 Galvão I, de Carvalho RVH, Vago JP, Silva ALN, Carvalho TG, Antunes MM, Teixeira MM. The role of annexin A1 in the modulation of the NLRP3 inflammasome. Immunology 2020; 160: 78-89
  CrossrefPubMedSearch in Google Scholar
• 39 Vago JP, Sugimoto MA, Lima KM, Negreiros-Lima GL, Baik N, Teixeira MM, Perretti M, Parmer RJ, Miles LA, Sousa LP. Plasminogen and the plasminogen receptor, Plg-R_KT, regulate macrophage phenotypic, and functional changes. Front Immunol 2019; 10: 1458
  CrossrefPubMedSearch in Google Scholar
• 40 Zaidan I, Tavares LP, Sugimoto MA, Lima KM, Negreiros-Lima GL, Teixeira LC, Miranda TC, Valiate BV, Cramer A, Vago JP, Campolina-Silva GH, Souza JA, Grossi LC, Pinho V, Campagnole-Santos MJ, Santos RA, Teixeira MM, Galvão I, Sousa LP. Angiotensin-(1–7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis. JCI Insight 2022; 7: e147819
  CrossrefPubMedSearch in Google Scholar