2 and JNK pathway

Phytomedicine 23 (2016) 9–17

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Phytomedicine journal homepage: www.elsevier.com/locate/phymed

Vitexin reduces neutrophil migration to inflammatory focus by down-regulating pro-inflammatory mediators via inhibition of p38, ERK1/2 and JNK pathway Suellen Iara Guirra Rosa a, Fabrício Rios-Santos b, Sikiru Olaitan Balogun a, Domingos Tabajara de Oliveira Martins a,∗ a b

Pharmacology Area, Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, UFMT, 78060-900 Cuiabá, MT, Brazil Physiology Area, Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, UFMT, 78060-900 Cuiabá, MT, Brazil

a r t i c l e

i n f o

Article history: Received 23 August 2015 Revised 21 October 2015 Accepted 9 November 2015

Keywords: Cytotoxicity Flavonoid Leukocyte recruitment Immunomodulation

a b s t r a c t Background: Vitexin is a flavonoid found in plants of different genus such as Vitex spp. and Crataegus spp. Despite being an important molecule present in phytomedicines and nutraceuticals, the mechanisms supporting its use as anti-inflammatory remains unclear. Purpose: To investigate the cellular and molecular mechanisms involved in acute anti-inflammatory effect of vitexin with regard to neutrophil recruitment and macrophages activation. Methods: Anti-inflammatory properties of vitexin were evaluated in four models of neutrophil recruitment. The regulation of inflammatory mediators release was assessed in vivo and in vitro. Vitexin (5, 15 and 30 mg/kg p.o) effects on leukocytes migration to peritoneal cavity induced by zymosan (ZY), carrageenan (CG), n-formyl-methionyl-leucyl-phenylalanine (fMLP) and lipopolysaccharide (LPS) were evaluated in SwissWebster mice and the effects on the levels of TNF-α , IL-1β and IL-10 cytokines, and NO concentration were in the LPS-peritonitis. RAW 264.7 macrophages viability were determined by Alamar Blue assay as well as the capacity of vitexin in directly reducing the concentrations of TNF-α , IL-1β , IL-10, NO and PGE2 . Additionally, vitexin effects upon the transcriptional factors p-p38, p-ERK1/2 and p-JNK were evaluated by western blotting in cells activated with LPS. Results: Vitexin was not cytotoxic (IC50 > 200 μg/ml) in RAW 264.7 and at all doses tested it effectively reduced leukocyte migration in vivo, particularly neutrophils in the peritoneal lavage, independently of the inflammatory stimulus used. It also reduced TNF-α , IL-1β and NO releases in the peritoneal cavity of LPSchallenged mice. Vitexin had low cytotoxicity and was able to reduce the releases of TNF-α , IL-1β , NO, PGE2 and increase in IL-10 release by LPS activated RAW 264.7 cells. Vitexin was also able to regulate transcriptional factors for pro-inflammatory mediators, reducing the expression of p-p38, p-ERK1/2 and p-JNK in LPS-elicited cells. Conclusions: Vitexin presented no in vitro cytotoxicity. Inhibition of neutrophil migration and proinflammatory mediators release contributes to the anti-inflammatory activity of vitexin. These effects are associated with the inactivation of important signaling pathways such as p38, ERK1/2 and JNK, which act on transcription factors for eliciting induction of inflammatory response. © 2016 Elsevier GmbH. All rights reserved.

Introduction

Abbreviations: ZY, zymosan; CG, carrageenan; fMLP, N-formyl-methionyl-leucylphenylalanine; LPS, lipopolysaccharide; IL, interleukin; TNF-α , tumor necrosis factor α ; NF-kB, nuclear factor-kB; MAPKs, mitogen-activated protein kinase; p38, p38 MAP kinase; ERK1/2, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal protein kinase; DEXA, dexamethasone; PGE2 , prostaglandin E2 . ∗ Corresponding author. Universidade Federal de Mato Grosso, Faculdade de Medicina, Av. Fernando Corrêa da Costa, no. 2367, zip code: 78060-900, Cuiabá, MT, Brazil. Tel.: +55 65 3615 6231. E-mail address: [email protected], [email protected] (D.T.d.O. Martins). http://dx.doi.org/10.1016/j.phymed.2015.11.003 0944-7113/© 2016 Elsevier GmbH. All rights reserved.

Flavonoids are one of the most important and diverse phenolic groups distributed in the plant kingdom. Structurally, they have a characteristic C6 C3 C6 core biosynthesized from shikimic acid and acetic acid pathway. Changes to the central ring leads to the formation of subclasses like chalcones, flavanones, flavanonois, flavones, flavonols, isoflavones, flavan-3-ols and anthocyanidins (Cook and Samman 1996). Among these compounds, apigenin glycoside flavone identified as vitexin (8-β -D-glucopyranosyl-apigenin), originally isolated from

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Vitex lucens Kirk (formerly V. littoralis), stands out as an important secondary plants metabolites, such as Crataegus spp. (Edwards et al. 2012) and Vitex agnus-castus L. (Hajdu et al. 2007). Apart from its medicinal relevance, vitexin is also used as a marker of flavonoids ¯ e˙ et al. 2006), contents in a variety of phytomedicines (Urbonaviˇciut like Passiflora spp. based phyto-pharmaceuticals products, such as P. incarnata L. and P. foetida L., among others (Pongpan et al. 2007). Evidences of vitexin uses in human health are being supported by clinical trials that evaluated prescription of phytomedicines containing such compound in the treatments of generalized anxiety disorder (NCT00794456), insomnia (NCT01100645), and also as a nutraceuticals (NCT01647984). Besides, in experimental models, vitexin has shown activity as anticonvulsant (Abbasi et al. 2012), antitumoral (Yang et al. 2013), antinociceptive (Borghi et al. 2013; Demir Özkay and Can 2013), neuroprotector (Abbasi et al. 2013), cardioprotector (Dong et al. 2011) and as new anti-inflammatory molecule (Prabhakar et al. 1981). However, despite such evidences, little is known about the cellular mechanisms involved in its anti-inflammatory action. In regard to anti-inflammatory compounds, the discovery of new pharmacological strategies of immunomodulation is particularly important in diseases that involve multifactorial mechanisms of development, as observed in rheumatoid arthritis, Crohn’s diseases, sepsis, intestinal inflammatory diseases, just to mention but few (Nathan 2002). Part of limitations in controlling inflammatory conditions by well-known pharmaceutical products in market, such as NSAIDs, is due to their serious side effects on the gastrointestinal tracts, especially in some groups of patients with bleeding risk (Scheiman and Hindley 2010). Moreover, other classes of drugs, such as immunosuppressives (e.g. glucocorticosteroids), long term use may result in serious adverse effects, which includes infections susceptibility. It is worthwhile to mention that curiously, the success observed with some classes of NSAIDs, were due to other mechanisms of action of such compounds, and not merely inhibition of COX-2. In fact, indomethacin may also reduce directly leukocyte chemotaxis, acetylsalicylic acid may induce lipoxins (anti-inflammatory endogenous molecule), contributing to its overall pharmacological effect (Barton et al. 2000). In this context, new pharmacological strategies are being sought, like those that interfere with leukocyte recruitment and activation, pro-inflammatory mediators release (e.g. interleukin (IL)-1β , IL-10, tumor necrosis factor α (TNF-α ) and nitric oxide (NO) and more recently by regulation of transcriptional factors, such as nuclear factorkB (NF-kB) and mitogen-activated protein kinase (MAPKs) pathways (Alessandri et al. 2013). In fact, the intracellular pathways have received special attention, because they regulate the expression of inflammatory genes that can be activated by different inflammatory stimuli. For instance, it was demonstrated that LPS and fMLP (derived from Escherichia coli) and CG (phlogistic agent derived from algae Chondrus crispus), are capable of increasing cytokines release and migration of neutrophils to inflammatory sites by up-regulating the expressions of p38, ERK1/2 and JNK (Azuma et al. 2007). Furthermore, ZY (derived from Saccharomyces cerevisiae), also used as phlogistic agent in experimental models, is known to induce the recruitment of neutrophils and cytokines release into the inflammatory sites by increasing expressions of p38 and ERK1/2 (Di Paola et al. 2010). More recently, compounds such as quercetin, curcumin, (-)-epigallocatechin gallate (EGCG) and resveratrol were shown to present immunomodulatory effects by acting on alternative pathways such as inhibition of 5-lipoxygenase, protein kinases (MAPKs, PKC, Akt) and transcriptional factors, such as NF-kB (Koeberle and Werz 2014). Thus, this study investigated the effects of vitexin in acute inflammatory response after different phlogistic stimuli and the regulatory mechanisms upon pro-inflammatory mediators release and MAPK signaling pathways in macrophages.

Materials and methods Drugs and reagents ZY, CG, fMLP, LPS (E. coli, serotypes 055:B5 and 055:B8), dexamethasone acetate (DEXA), DMEM medium, RPMI-1640 phenol red free, fetal bovine serum, penicillin, streptomycin, doxorubicin, dimethyl sulfoxide, SB203580, PD98059, SP600125, NS-398 and Griess reagent were obtained from Sigma-Aldrich (USA). Vitexin (PubChem CID: 5280441), was obtained from Fluka (USA). Alamar Blue was obtained from Invitrogen (USA). All other chemicals were of reagent grade. Drugs were diluted in distilled water or 0.02% dimethyl sulfoxide as solubility same. Animals Male Swiss-Webster mice (25–30 g) from the Central Animal House of UFMT were used. The animals were maintained in propylene cages at 25 ± 1 °C with a 12 h light/dark cycle and had free access to standard pellet chow and water. Groups of six to eight animals were used for each experiment. The experimental protocol followed the International Principles for the Biomedical Research Involving Animal (CIOMS/OMS, 1985) and was approved by the Committee on the Use of Animal for Experimentation (CEUA/UFMT) with protocol no. 23108.028454/12-2. Cell culture The RAW 264.7 mouse macrophages cell line (ATCC TIB-71) was obtained from the Cell Bank of Rio de Janeiro. Cells were cultured in DMEM or RPMI-1640 supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml), and maintained at 37 ºC in an atmosphere of 5% of CO2 and 90% humidity. In vivo anti-inflammatory evaluation Peritonitis induced by different stimuli In order to assess the possible effect of vitexin on leukocyte recruitment into the peritoneal cavity, the mice were orally (p.o) pretreated (0.1 ml/10 g) with vehicle (2% Tween 80 in distilled water), vitexin (5, 15 and 30 mg/kg) or DEXA (0.5 mg/kg). After 1 h, it was injected by intraperitoneal (i.p.) route 0.2 ml/cavity of ZY (1 mg), CG (300 μg), fMLP (10 μg) and LPS (250 ng) dissolved in sterile saline for induction of peritonitis. A sham group received p.o the same volume of vehicle and sterile saline i.p. Four or six hours after the injection of stimulus, mice were anesthetized with 180 mg/kg ketamine and 30 mg/kg xylazine by i.p. route and the cells in the peritoneal cavities were collected through injecting of 3 ml of cold PBS 1x (pH 7.4) containing EDTA (3 mM). The abdomens were gently massaged, and the blood-free cell suspension was carefully aspirated with a syringe. The peritoneal lavage collected was used for cellular counting in Neubauer chamber, while an aliquot of this lavage was used to make smear for differential cell counting. Aliquots of peritoneal washing were stored in a freezer at –80 °C for posterior dosage of cytokines (Da Silva et al. 2014). Cytokine and nitrite quantification in the peritoneal lavage The cytokines concentrations (pg/ml) of TNF-α , IL-1β and IL-10 in peritoneal fluid of mice with LPS induced peritonitis was measured with commercially available ELISA kits according to manufacturer’s instructions (eBioscience, USA). Indirect estimation of NO through nitrite (NO2 − ) determination was performed by the colorimetric method based on the Griess reaction, employing the modified method of Ni et al. (2010). The lavage collected in LPS-induced peritonitis was incubated with an equal volume of Griess reagent complete at room temperature for 15 min and the absorbance (540 nm)

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was determined in ELISA microplate reader. The nitrite concentration was calculated by interpolation of the standard curve of sodium nitrite (NaNO2 ) and results were expressed as μM. In vitro anti-inflammatory assays Cell viability RAW 264.7 cells of density 2 × 104 cells per dish were plated on a 96-well microplate containing medium (growth control) with/without vitexin with concentration ranging from 3.12 to 200 μg/ml. Doxorubicin (0.0058–58 μg/ml) was used as a positive control. After incubation for 24 h at 37 °C and 5% CO2 , the treatments were removed and 200 μL of 10% Alamar Blue solution was added (Nakayama et al. 1997). After 5 h, the absorbance was read at 540 nm (oxidized state) and 620 nm (reduced state) with Multiskan® EX ELISA plate reader (Thermo Scientific, USA). Values of 50% inhibitory concentration (IC50 ) < 4 μg/ml were considered cytotoxic (Suffness and Pezzuto 1990). Inflammatory mediators release on RAW 264.7 In order to evaluate the direct effect of vitexin on NO, TNF-α , IL-1β , IL-10 and prostaglandin E2 (PGE2 ) release, RAW 264.7 cells (1 × 106 cells/wells) were plated in a 24-well plate overnight and stimulated with LPS. Cells were incubated with vitexin (25, 50 and 100 μg/ml) or DEXA (10 μM) (positive control) for 2 h, and incubated at 37 °C and 5% CO2 . Next, the cells were stimulated with LPS (1 μg/ml) for 24 h under the same condition. For negative control, the same amount of medium free of phenol red was used in the microplate well. After 24 h of incubation, 1000 μl of the supernatant of the cells were collected and stored at –20 °C posterior dosage of mediators inflammatory, as described in Section 2.4.2. Western blot analysis RAW 264.7 cells (2 × 106 cells/well) were seeded in 6-well plates and pretreated with vitexin (25, 50 and 100 μg/ml) and inhibitors specifics for p38 (SB203580), ERK1/2 (PD98059) and JNK (SP600125) at 10 mM for 2 h and stimulated with 1 μg/ml LPS for 30 min. After incubation, the cells were washed with DMEM and lyzed in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1%Triton X-100, and 0.1% SDS) supplemented with protease cocktail and phosphatase inhibitors (Sigma Fast, 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate and sodium fluoride 100 mM) for 30 min on ice and stirring. The cytosolic fraction was obtained by centrifugation (14000 x g) at 4 °C for 25 min. Protein concentration was estimated using the Bradford (1976) method. Samples (40 μg of protein) were subjected to SDS-PAGE analysis (10–12%) and acrylamide gels by semidry electrotransfer to nitrocellulose membranes (Biorad, USA). After transfer, membranes were blocked (20 mM Tris–HCl, pH 7.4, 125 mM NaCl, 0.2% Tween 20, 1% bovine serum albumin, 3% non-fat milk) for 1 h at room temperature and incubated for 4 h at 4 °C with specific primary antibodies: p-p38, p-ERK1/2, p-JNK and β -actin (Santa Cruz Biotechnology, USA). The blots were then incubated with secondary anti-rabbit or anti-mouse IgG conjugated to peroxidase (HRP) and immunoreactive bands were visualized by chemiluminescence (ECL Amersham, USA) and detected with ChemiDoc XRS systemTM software and subsequently analyzed with Image LabTM (Biorad, USA). Data analysis The results were expressed as mean ± standard error of mean (± SEM). Comparisons between means were analyzed by one-way analysis of variance (ANOVA), and when significant, it was followed by Student–Newman–Keuls test for multiple comparisons. Values of p < 0.05 were considered significant. The IC50 was determined from the linear regression relating the percentage of inhibition versus the

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logarithm of the concentrations tested and assuming a confidence level of 99% (p < 0.01) for the curve obtained. For in vitro assays that do not involve statistical analysis, we used the mean ± SEM of two or three independent experiments. All analyzes were performed using GraphPad Prism® 5.0 for Windows (USA). Results and discussion In vivo anti-inflammatory assays Neutrophil migration to peritoneal cavity induced by different phlogistic stimuli Firstly, our study evaluated if vitexin could exert its antiinflammatory effects by inhibiting leukocyte migration to an inflammatory focus. In this regard, four different phlogistic challenges were used, since ZY, CG, fMLP or LPS has upstream distinctive inflammatory mechanism of inflammation induction (Chen and Pan 2009). Vitexin doses were chosen based on pilot experiments from our lab and literature report (Can et al. 2013; Demir Özkay and Can 2013; Dos Reis et al. 2014). Oral pre-treatment of mice with vitexin reduced the total number of cells in all models, but since neutrophils represents up to 90% of cells, total cell counts are not shown. Our data shows that i.p. challenge with ZY, CG, fMLP or LPS induced significant increases (p < 0.001) in the number of neutrophils in the peritoneal cavity (1864%, 156%, 29% and 303%) compared to their sham groups (1.1 × 106 ; 5.8 × 106 ; 10.2 × 106 and 10.1 × 106 , respectively), and oral administration of vitexin significantly reduced neutrophil counts in all models (p < 0.001) (Fig. 1). The maximal effects were observed with 5 mg/kg (76.7%) in the ZY-challenged (Fig. 1A), at a dose of 30 mg/kg (47.7%) in the CG-induced (Fig. 1B), and at a dose of 15 mg/kg (49.8%) in LPS-challenged (Fig. 1D). Whereas in animals challenged with fMLP (Fig. 1C) the reduction in the number of neutrophils was dose-dependent, attaining a maximum value of 84.4% at the dose of 30 mg/kg, relative to the respective vehicle group. DEXA (0.5 mg/kg), used as a positive control, reduced the number of neutrophils in the peritoneal cavity in the four models of inflammation (p < 0.001) by 73.4% (ZY), 68.6% (CG), 68.8% (fMLP) and 47.9% (LPS). It has been stated that neutrophil recruitment is inherently governed by the nature of the inflammatory response (Batista et al. 2014). Our data demonstrated that vitexin exerts part of its antiinflammatory effect by reducing neutrophil migration to infectious focus, in a stimulus-independent manner. Our data are in accordance with others who demonstrated vitexin interference in the leukocytes trafficking in the LPS-induced acute pulmonary inflammation (De Melo et al. 2005) as well as with CG-induced pleurisy (Dos Reis et al. 2014). Cytokines and NO quantification in the peritoneal lavage Phlogistic challenges may have in common, the capability of priming and eliciting resident cells to synthesize pro-inflammatory mediators, which in turn regulates leukocytes trafficking. In this context, we choose LPS as our inflammatory challenge since it is a wellstudied model of induction of cytokines in endotoxemia (Borges et al. 2014). TNF-α and IL-1β play central role in inducing other proinflammatory mediators (such as chemokines and NO) and IL-10 may act in an opposite fashion, exerting an anti-inflammatory control upon neutrophil recruitment to inflammatory focus (Ajuebor et al. 1999). We demonstrate that i.p. administration of LPS caused significant increases (p < 0.001) in the liberation of TNF-α (720.8%) and IL-1β (6119.7%) when compared to the respective sham groups (34.9 pg/ml and 12.6 pg/ml, respectively). The maximal inhibitory effects of vitexin were observed in TNF-α levels at the dose of 30 mg/kg (69.9%, p < 0.001) and IL-1β at the dose of 15 mg/kg (56.4%, p < 0.001) (Fig. 2A–B). Vitexin did not interfere in the levels of IL-10 (p > 0.05)

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Fig. 1. Effect of vehicle (2% Tween), vitexin (5, 15 and 30 mg/kg) and dexa (0.5 mg/kg) administered orally on total number of neutrophils present in the peritoneal fluid of male mice with Zy (A), Cg (B), fMLP (C) and LPS (D) peritonitis. The sham group received only vehicle and i.p. injection of 0.9% saline (0.1 ml/10 g). The total number of cells was expressed as mean ± SEM for 7 animals. One-way analysis of variance, followed by Student–Newman–Keuls test. ††† p < 0.001 versus Sham; ∗ p < 0.05, ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus vehicle.

(data not shown). DEXA (0.5 mg/kg) significantly reduced (p < 0.001) TNF-α (84.3%) and IL-1β (97.3%) concentrations, respectively, when compared to the LPS group. Our data is in partial agreement with Borghi et al. (2013) who demonstrated the analgesic effect of vitexin by its reductions in TNFα and IL-1β and increasing IL-10 in the hyperalgesia model produced by intraplantar injection of CG in mice. This difference in the effect on IL-10 levels may be related to differences related to experimental model and the phlogistic agent employed. NO is a dual molecule, involved in physiological or pathological processes (Förstermann and Sessa 2012). Cytokines such as TNF-α and IL-1β may also induce NO formation by nitric oxide synthase 2 (NOS2), which has important contribution to tissue damage, oxidative stress and DNA damage when there is exacerbated secretion in the inflammatory focus (LeGrand et al. 2001). Furthermore, NO plays a critical role in the regulation of blood flow and vascular permeability, mucosal defense, leukocyte recruitment, neurotransmission and in modulation immune (Ni et al. 2010). Once released in biological fluids, NO is oxidized to inorganics anions such as NO2 − , which can be quantified as marker of NO production. We found that animals in the vehicle group with peritonitis-induced by LPS had NO concentration (indirect measure by nitrite) increased by about 7 folds (6.9 ± 1.2 μM) in relation to the sham group (0.7 ± 0.3 μM) (Fig. 3), while vitexin caused significant reduction (p < 0.05) in the NO concentration, attaining maximum effect at 15 mg/kg (70.9%). DEXA (0.5 mg/kg) was also able to inhibit NO by 54.2% (p < 0.05). It is interesting to note that vitexin effects, especially in the in vivo studies, are non-dose dependent. This non-linear relationship can be explained based on the following hypothesis: First, vitexin being a pleiotropic molecule is known to act on several target molecules within the living organisms. Thus, its activities have been linked to

its interactions with certain receptors amongst other mechanisms. Drug-receptor bindings have been shown to be extremely complex process, with varying degree of functional outputs that depends on several variables. It is therefore understandable to observe this type of non-dose dependent effects (Spedding 2011; Wenthur et al. 2014). In the case of in vivo studies, such effects are common, since the total observable effects depend on a milieu of physiological mediators of inflammatory homeostasis. In fact, similar non-dose dependent effects were observed for vitexin by studies done by Demir Ozkay and Can (2013) and Borghi et al. (2013). Second, it is also possible that higher dose of vitexin may provoke several physiological response mechanisms which may alter its effective concentration or several other factors too numerous to mention and thereby modulate its effect by compensatory mechanisms to maintain homeostasis. Investigation of in vitro anti-inflammatory activity of vitexin Cell viability assay Resident cells play a key role in the recognition of external stimuli and initiation of the inflammatory process (Ajuebor et al. 1999). Thus, we hypothesized that vitexin could play its anti-inflammatory effects by regulating the mononuclear cell response. In this sense, the RAW 264.7 macrophages lineage has often been used for the evaluation of anti-inflammatory drugs. Towards this end, we sought to exclude the toxic effects of vitexin and to determine the range of concentrations to be used in the study, RAW 264.7 cells were incubated for 24 h with increasing concentrations of vitexin, and our results show IC50 of vitexin was greater than 200 μg/ml. Whereas, doxorubicin, used as the positive control, presented IC50 = 4.8 ± 2.5 μg/ml. Based on these data concentrations of

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Fig. 2. Effect of oral pretreatment with vehicle (2% Tween), vitexin (5, 15 and 30 mg/kg) and dexa (0.5 mg/kg) on the concentration of TNF-α (A) and IL-1β (B), on peritoneal fluid of male mice with LPS-induced peritonitis (250 ng/0.2 ml/cavity). The sham group received only vehicle and injection i.p. of 0.9% saline (0.2 ml/cavity). The concentrations of TNF-α and IL-1β are expressed as mean ± SEM for 6–7 animals. One-way analysis of variance, followed by Student–Newman–Keuls test. ††† p < 0.001 versus Sham; ∗ p < 0.05; ∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus vehicle.

Fig. 3. Effects of oral pretreatment with vehicle (2% Tween), vitexin (5, 15 and 30 mg/kg) and dexa (0.5 mg/kg) on nitrite (NO2 – ) concentration quantified in the peritoneal lavage of male mice with LPS-induced peritonitis. Nitrite (NO2 − ) concentration was expressed as mean ± SEM for 6–7 animals. One-way analysis of variance, followed by Student–Newman– Keuls test. †† p < 0.01 versus Sham; ∗ p < 0.05 versus vehicle.

vitexin at 25, 50 and 100 μg/ml seem not to be toxic and were used in the subsequent in vitro experiments. Determination of cytokines release by macrophages Corroborating with the in vivo results, vitexin significantly reduced concentrations of TNF-α and IL-1β in RAW 264.7 cells

stimulated by LPS (Fig. 4A–B), reaching maximum inhibitions of 26.7% (p < 0.001) and 59.1% (p < 0.001) at concentration of 100 μg/ml respectively. DEXA (10 μM) reduced TNF-α concentrations by 38.4% and IL-1β by 98.8% (p < 0.001), but did not alter IL-10 levels. Interestingly, vitexin (100 μg/ml) caused a slight increase in the concentration of IL-10 by 22.8% (p < 0.05) in LPS-stimulated cells (Fig. 4C).

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Fig. 4. Effect of pretreatment with vitexin (25, 50 and 100 μg/ml) and dexa (10 μM) on the TNF-α (A); IL-1β (B) and IL-10 (C) concentration present in the supernatants of macrophages RAW 264.7 stimulated 24 h with LPS (1 μg/ml). The concentrations of TNF-α and IL-1β are expressed as mean ± SEM for two independent experiments. One-way analysis of variance, followed by Student–Newman–Keuls test. ††† p < 0.001 versus baseline; ∗ p < 0.05;∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus LPS.

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Fig. 5. Effect of treatment with vitexin (25, 50 and 100 μg/ml) and dexa (10 μM) on the nitrite (NO2 − ) production quantified in the supernatants of macrophages RAW 264.7 stimulated 24 h with LPS (1 μg/ml). Concentrations of nitrite were expressed as mean ± SEM for three independent experiments. One-way analysis of variance followed by Student–Newman–Keuls test was used. ††† p < 0.001 versus baseline; ∗ p < 0.05;∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus LPS.

These results indicate that in the anti-inflammatory action of vitexin, reductions of TNF-α and IL-β levels play a more important role when compared to the increase in the IL-10 as it was also observed in the in vivo assay. Determination of NO and PGE2 LPS may also markedly increases the release of PGE2 and NO in macrophages through expression of COX and NOS also activated by NF-kB and MAPKs (Yun et al. 2008). It is reported that cytokines such as IL-1β , TNF-α , IL-6 and others are also regulated by activation of these pathways signaling (Oh et al. 2015). Since neutrophils may be an important source of NO, and cytokines may have an autocrine mechanism in the induction NOS2, here, we confirmed that vitexin also reduced NO-release by macrophages. RAW 264.7 stimulated with 1 μg/ml of LPS caused increases of about 8 times (25.35 ± 2.69 μM) in the concentration of NO2 − in relation to the baseline value (3.6 ± 3.6 μM) (Fig. 5). This deleterious increase was attenuated by treatment with vitexin at 100 μg/ml reducing it by 95.1% (p < 0.001) and DEXA (10 μM) also attenuated the increase in the NO concentration (93.3%, p < 0.001). Based on this result, it is possible that vitexin might be acting directly

on NOS2 or another form of intracellular control. This hypothesis will have to be verified in future research. Our data are in accordance with other authors that have shown that vitexin reduced NO production in mice with pleurisy induced by CG (Dos Reis et al. 2014). On the other hand, Choi et al. (2014) failed to observe inhibitory effect of vitexin on NO production, possibly because they utilized concentrations that are 4 to 9 times lower than what was used in this study. In the similar manner, stimulation of RAW 264.7 with LPS resulted in significant increase (p < 0.001) in the PGE2 concentration (165.4 ± 39.4 pg/ml) in relation to the baseline group (1.2 ± 12.6 μM). Maximum inhibitory effect of vitexin on the production of PGE2 was noted at 25 μg/ml (75.2%, p < 0.001). In the same vein, NS398, used as a positive control, also inhibits it by 85.2% (p < 0.001) when compared to LPS group (Fig. 6). These results suggest that inhibition of PGE2 production is intimately involved in vitexin antiinflammatory mechanism of action. The inhibition of PGE2 might be explained judging by vitexin effect on NOS2, since NO is known to activate the COX enzymes, an event leading to overt production of PGs (Cuzzocrea and Salvemini 2007). In addition, PGE2 has been demonstrated to indirectly mediate neutrophil migration, or even potentiate the pro-inflammatory mediators such as cytokines, among others

Fig. 6. Effect of treatment with vitexin (25, 50 and 100 μg/ml) and dexa (10 μM) on the PGE2 concentration present in the supernatants of macrophages RAW 264.7 stimulated 24 h with LPS (1 μg/ml). The concentrations of PGE2 were expressed as mean ± SEM for two independent experiments. One-way analysis of variance, followed by Student–Newman– Keuls test. ††† p < 0.001 versus baseline; ∗∗∗ p < 0.001 versus LPS.

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Fig. 7. Effect of pretreatment with vitexin (25, 50 and 100 μg/ml) or 10 mM of specific kinases inhibitors (SB203580 for p38, PD98059 for MEK, SP600125 for JNK) on the phosphorylation of p38 (A), ERK1/2 (B) and JNK (C) in RAW 264.7 macrophages stimulated for 30 min with LPS (1 μg/ml). The levels of phosphorylation were estimated in relation to the relative amount of the endogenous β -actin control. Each line represents the mean of 3 independent experiments. One-way analysis of variance, followed by Student– Newman–Keuls test. ††† p < 0.001 versus baseline; ∗ p < 0.05;∗∗ p < 0.01 and ∗∗∗ p < 0.001 versus LPS.

(Kawahara et al. 2015). Although the expression of NOS2 and COX-2 were not evaluated in this study, the possibility of vitexin acting via inhibition of the expressions of these two potential targets for antiinflammatory therapeutics cannot be ruled out. Effects of vitexin on MAPKs signaling pathways in LPS stimulated RAW 264.7 cells Activation of MAPKs is intimately associated with the expression of multiple genes that together regulate the inflammatory response. Moreover, signal-transduction by MAPKs is related to the regulation of expression of the cytokines and recruitment of leukocytes, among others (Alessandri et al. 2013). Thus, compounds that down-regulate MAPKs pathway can attenuate the inflammatory response. In this context, selective inhibitors of MAPKs, such as SB203580, PD98059 and SP600125 are widely employed to identify highly specific compounds and advance its mechanism of action (Burkhard and Shapiro 2010). Therefore, we evaluated the effect of vitexin on LPS-induced phosphorylation of p38, ERK1/2 and JNK in RAW 264.7 cells using western blot analysis. As shown in Fig. 7A–C, LPS caused significant increases (by 10, 6 and 4 times, respectively) in the phosphorylation of p38, ERK1/2 and JNK, when compared to the baseline control values (0.1, 0.3 and 0.2). The maximal effects for vitexin were observed with 25 μg/ml for both p38 (39.2%, p < 0.01) and JNK (75.1%, p < 0.001) and at a concentration of 50 μg/ml (81.2%, p < 0.001) for ERK1/2, relative to the respective LPS group. All the standard inhibitors (SB203580, PD98059 and SP600125) were able to reduce the activities of p-p38, p-ERK and p-JNK (p < 0.001), respectively to the LPS group. The p38 MAPK is known to play an important role in cell migration and up-regulation of biosynthesis of TNF-α , IL-1β , IL-6 and IL-10 expression and transcriptional target enzymes (e.g., COX-2 and NOS2) (Yang et al. 2014). MAPK, JNK mediates the post-translational regulation of cytokine via AP-1. On its part, ERK1/2 regulates the cytokine expression by transcriptional and post-transcriptional mechanisms, as well as participating in the processes of growth, differentiation, and cell survival (Gaestel et al. 2007).

Vitexin attenuated the LPS-induced phosphorylation of all three MAPKs in RAW 264.7 cells with comparable intensity to the standard drug PD98059. Thus, the anti-inflammatory effect of vitexin may be related to inactivation of p38 kinases, ERK1/2 and JNK. As vitexin inhibited p-p38, and p-ERK1/2 it was expected that IL-10 concentration will be reduced, however, by contrast, there was an increase in IL-10 by treatment of RAW 264.7 with this flavonoid. Some authors have argued that such mechanism related to the inhibition of ERK and p38, did not always lead to reduction in the expression of IL-10 by the macrophages (Saraiva and O’Garra 2010), this being our observation. It is worthwhile to mention that its effect in controlling the induction of acute inflammatory response in a non-specific stimulus manner is probably due to the use of common pathways of transcription that are activated in the inflammatory process. In fact, fMLP, LPS, and CG are capable of activating p38, ERK1/2 and JNK pathways and thus increase the release of cytokines and the migration of neutrophils in vivo and in vitro (Azuma et al. 2007), while only p38 and ERK1/2 kinases have been implicated in the inflammatory action of ZY on the neutrophils (Di Paola et al. 2010). Until now, this is the first study to demonstrate the multi-target anti-inflammatory activity of vitexin. These findings are of utmost importance considering recent developments in biological systems and overall clinical experience, which have revealed the limitations of single-target drugs. Although, such drugs may successfully inhibit or activate a specific target, the desired effect to the entire biological system may not always be attainable. This might be due to the fact that organisms, in general, can affect effectiveness through compensatory mechanisms. For this reason coupled with the fact that development of diseases such as inflammatory ones is a complex process and it involves several aspects. Thus, the multi-target drug design concept is being embraced nowadays (Lu et al. 2012). Moreover, vitexin may represent a potential phytochemical or template molecule that should be investigated in different types of inflammation that involves neutrophils and macrophages activation. In conclusion, our data shows that vitexin anti-inflammatory actions are due to inhibition of leukocyte migration, particularly of the

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