NF-κB pathways and COX-2 in LPS-activated RAW 264.7 cells

Author’s Accepted Manuscript Dilodendron bipinnatum Radlk. inhibits proinflammatory mediators through the induction of MKP-1 and the down-regulation of MAPKp38/JNK/NF-κB pathways and COX-2 in LPS-activated RAW 264.7 cells Ruberlei Godinho de Oliveira, Geovane Roberto de Campos Castilho, André Luiz da Cunha, Fábio Miyajima, Domingos Tabajara de Oliveira Martins

PII: DOI: Reference:

www.elsevier.com/locate/jep

S0378-8741(16)31323-X http://dx.doi.org/10.1016/j.jep.2017.02.026 JEP10730

To appear in: Journal of Ethnopharmacology Received date: 14 October 2016 Revised date: 13 February 2017 Accepted date: 14 February 2017 Cite this article as: Ruberlei Godinho de Oliveira, Geovane Roberto de Campos Castilho, André Luiz da Cunha, Fábio Miyajima and Domingos Tabajara de Oliveira Martins, Dilodendron bipinnatum Radlk. inhibits pro-inflammatory mediators through the induction of MKP-1 and the down-regulation of MAPKp38/JNK/NF-κB pathways and COX-2 in LPS-activated RAW 264.7 c e l l s , Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.02.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dilodendron bipinnatum Radlk. inhibits pro-inflammatory mediators through the induction of MKP-1 and the down-regulation of MAPKp38/JNK/NF-κB pathways and COX-2 in LPS-activated RAW 264.7 cells ab

Ruberlei Godinho de Oliveira, a Geovane Roberto de Campos Castilho, cAndré Luiz da Cunha, d,eFábio Miyajima, a*Domingos Tabajara de Oliveira Martins a

Department of Basic Health Sciences, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Coxipó, Cuiabá, Mato Grosso,78060-900, Brazil. b

Faculdade de Farmácia, Universidade de Cuiabá, Avenida Beira Rio, no. 3100, CEP: 78025-190. Cuiabá-MT, Brazil. c

Departamento de Bioquímica, Escola Paulista de Medicina, Unifesp, São Paulo, SP 04044-020, Brazil. d

Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3GL, UK. e

Group of Neuropharmacology, Drug Research and Development Center, Federal University of Ceara (NPDM-UFC), Fortaleza-CE, Brazil. *

Corresponding author. Tel.: +55 653615623;

fax: +55 6536156231.

E-mail address: [email protected] (D.T.O. Martins). 1

Present address: Federal University of Mato Grosso, Av. Fernando Corrêa da Costa,

Boa Esperança, Cuiabá, Mato Grosso, 78060-900, Brasil.

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Abstract Ethnopharmacological relevance: The stem bark of Dilodendron bipinnatum Radlk. (Sapindaceae), a tree native to Pantanal of Mato Grosso, Brazil, popularly known as “mulher-pobre” (poor woman), has been historically used by the locals, after decoction and maceration, for the treatment of inflammatory conditions. We have recently shown that these preparations indeed possess anti-inflammatory properties, which are mediated by the inhibition of cell migration and the modulation of Th1 and Th2 cytokines. The NO pathway was not affected. Aim of the present study: The aim of the present study was to further investigate the mechanisms responsible for the anti-inflammatory properties of the hydroethanolic extract of the stem bark of Dilodendron bipinnatum (HEDb). Materials and methods: HEDb was obtained by maceration of the stem bark of D. bipinnatum as previously described. The corresponding effects on a macrophage-like cell line, RAW 264.7, were investigated. The apoptosis of RAW 264.7 upon treatment with LPS, HEDb and N-acetyl-L-cysteine (NAC) was assessed by flow cytometry, using an Annexin V-PE kit. The production of inflammatory cytokines (TNF-α, IL-1β and IL-10) and PGE2 were evaluated by ELISA, after cell challenge with LPS. The intracellular redox state and changes in mitochondrial membrane potential were also assessed by flow cytometry, using DCFH-DA and JC-1 as probes. The protein expression levels of MAPK p-p38, p-ERK, p-JNK, MKP-1 and COX-2 were analysed by western blotting. Nuclear translocation of NF-қB was assessed by immunofluorescence microscopy. The quantified results are presented as a nuclear:cytoplasmic ratio. Results: LPS, HEDb and NAC did not appear to decrease the number of viable cells in comparison to control treatment. HEDb attenuated the production of pro-inflammatory cytokines (IL-1β and TNF-) and PGE2 induced by LPS but did not affect IL-10. The production of ROS was also inhibited by HEDb (1, 5 or 20 µg/mL), even at the lowest concentration; at 20 µg/mL, HEDb was more effective than NAC, which was used as a positive control (74.1% and 66.2% inhibition of LPS's effect, respectively). LPS induced an increase in ΔΨm (19.2%, p <0.001), while HEDb inhibited the change in ΔΨm (7.7% at 20 µg/mL, p<0.001). The results of western blotting showed that HEDb inhibited the expression of MAPK p-p38, p-JNK and COX-2, while the expression of MKP-1 was increased. p-ERK was not affected. LPS promoted the nuclear translocation of NF-қB p65 (67%, p<0.01) in RAW 264.7 cells, in comparison to baseline (33%). Pre-treatment with HEDb inhibited this translocation of NF-κB p65 (58% at 20 µg/mL, p<0.001). Conclusion: HEDb has a potent anti-inflammatory activity and acts on multiple targets and biological pathways of potential therapeutic relevance.

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1. Introduction Inflammation is a defensive response of the organism to infection, physical injury, chemical insult, or radiation (White and Mantovani, 2013). Macrophages play a key role in the initiation of immune and inflammatory responses and are versatile cells that play diverse functions, such as phagocytosis, production of core molecules and antigen presentation (Ginhoux et al., 2016). In response to many inflammatory stimuli, macrophages are activated via Tolllike receptors (TLRs), for example, TLR4 binds to LPS triggering a subsequent phosphorylation of cytoplasmic proteins, such as mitogens activated protein kinases (MAPK) p38, ERK and JNK (Hou et al., 2015), which together causes the activation of the nuclear factor kappa B (NF-қB) and downstream transcriptional mediators that exacerbate inflammation (Park et al., 2015). These coordinated events, amongst several other effects, increase the production of pro-inflammatory mediators like adhesion molecules, growth factors, proinflammatory cytokines (TNF-α, IL-1β, IL-6), lipid eicosanoids (PGE2 and LTB4), as well as activation of inducible enzymes (COX-2 and iNOS) and intracellular reactive oxygen species (ROS). These cytoplasmic and nuclear alterations promote inflammation, augmented innate immune response, vasodilation, cell proliferation and survival (Lawrence, 2009). Although a natural response to harmful stimuli aiming to deal with the tissues damaged and eradicate the initial cause of cell injury, inflammation is a generic process that can be detrimental for the functioning of organism if not limited. Inflammation is typically managed with non-steroidal anti-inflammatory drugs (NSAIDs), but this approach may not slow disease progression. Currently, there are no treatments that effectively suppress chronic inflammation. Moreover, their continuous use may lead to clinically significant gastrointestinal kidney damage, higher risk of cardiovascular events, and liver injury, whether in tandem with inhibition of the COX-1 enzyme (Diaz–Gonazales and Sánchez–Madrid, 2015). In pursuit of safer and more effective compounds of less side effects, the medicinal plants have been (and remain) a vital source of therapeutics for various illnesses and disease processes, including inflammation (Dzoyem and Eloff, 2015). However, the road leading to the discovery and validation of novel compounds remains locked inside the floristic biodiversity of our lands, with Brazil particularly amongst the countries with the largest botanic wealth (RBG Kew, 2016). Page 3of 24

Dilodendron bipinnatum Radlk., Sapindaceae, is a native plant from South America, but not endemic to Brazil. It is a tree characterized by outer margin leaflet teeth convex, noticeable ciliate leaflet margin, the lower midrib and lateral nerves possess spreading hairs; flowers are typically small well-developed petals with apically rounded calyx lobes (Gentry and Steyermark, 1987). It is widely distributed in the Amazon, Cerrado (Brazilian Savannah) and Atlantic rain forest basins of Brazil and neighbouring South American countries Peru, Bolivia and North of Paraguay (de Azevedo Neta Mahon et al., 2014; Somner et al., 2016). It is colloquially known in the native language as “mulher-pobre” - poor-woman, “maria pobre” - poor-Mary, “correiro”, “dry-flour” or “poor-castorbean” (Bieski et al., 2012; Gentry; Steyermark, 1987). Previously, we reported in a landmark ethnopharmacological study of the traditional communities from the Pantanal basin in the Mato Grosso State, that the stem bark has historically been used in the form of decoction and maceration for the treatments of various disease conditions. Particularly, the inner stem bark of Dilodendron bipinnatum is used by the population for the treatments of uterine inflammation, bone fractures, general pain and dermatitis, among others (Bieski et al., 2012). It was on this basis that we previously investigated the anti-inflammatory effects of this plant to corroborate its use in popular medicine (de Oliveira et al., 2014). Considering the importance of safety in any form of medicine destined for human and animal use, we also investigated its potential toxicity, and demonstrated, using in vitro and in vivo acute and chronic toxicological models, its high safety margin, when used at the doses stipulated in the popular medicine (de Azevedo Neta Mahon et al., 2014). A study by Santos et al. (2010) reported that the ethanol extract of the leaves and bark of D. bippinatum presented in its composition, a combination of steroids (βsitosterol, stigmasterol, campesterol and 3-O-β-D-citostenone) and triterpenoids (cicloeucalenol and 24-methylene), whereas we have previously described the standardization of the hydroethanolic extract of the D. bipinnatum stem bark (HEDb) by means of phytochemical analysis (de Oliveira et al., 2014), which revealed the presence of flavonoids and tannins as major metabolic classes, particularly epigallocatechin gallate, catechin, gallocatechin, and gallic acid. The claims for significant antiinflammatory activity of HEDb are certainly of scientific relevance, and therefore the present study was aimed at providing further evidence and mechanistically investigate signalling pathways and the mode of action in established in vitro models of inflammation. We sought to further evaluate the anti-inflammatory activity of HEDb Page 4of 24

and its mode of action in established in vitro models of inflammation with murine macrophages RAW 264.7 stimulated with LPS. We hypothesize that HEDb exerts its effects through significant alterations of key pro-inflammatory pathways: MAPKs, cyclooxygenase and NF-ҡB. 2. Material and Methods 2.1. Botanic material Stem bark of D. bipinnatum was harvested at Poconé, Mato Grosso, Brazil, coordinates S 15°56´528 and W 05°70´567. Authorized access to the traditional indigenous knowledge and genetic heritage for research was obtained from the Council on Genetic Heritage of the Ministry of Environment (CGEN/MMA) under reference number 045/2009, as reported by Bieski et al. (2012). Botanical identification was done at the Herbario UFMT by Prof. Germano Guarim Neto, and a voucher specimen (no. 20.529) was deposited at the herbarium. This plant is currently not listed as either threatened, or endangered species in Brazil.

2.2 Cell culture Murine macrophage-like RAW 264.7 (Code: 0212) cell lines were obtained from the Cell Bank of Rio de Janeiro. The cells were maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL), at 37ºC in a humidified incubator containing 5% CO2.

2.3 Drugs and reagents Bradford reagent, Escherichia coli lipopolysaccharide (LPS, serotype 055:B8), dexamethasone acetate, ethylenediaminetetraacetic acid (sodium-EDTA), bovine serum albumin (BSA), DAPI, Dulbecco's modified Eagle's medium (DMEM), sodium fluoride, sodium orthovanadate, sodium pyrophosphate cocktail protease inhibitor tablets, N-Acetyl-L-cysteine (NAC), ammonium persulfate, triton X-100 and Trizma base were purchased from Sigma–Aldrich (USA). JC-1 Kit was from BD™ MitoScreen (Heidelberg, Germany) and Annexin V-PE Apoptosis Detection Kit was from BD Pharmingen™ (Heidelberg, Germany), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from Invitrogen (USA), and foetal bovine serum (FBS) was from Cultilab (São Paulo, Brazil). ELISA Kits to quantify TNF-α, IL-1β and IL-10 were purchased from eBioscience (USA), and to quantify PGE2 from Cayman Chemical Page 5of 24

(Ann Arbor, MI, USA). Nitrocellulose membrane and precision plus protein kaleidoscope standards were purchased from Bio-Rad (Bio-Rad Laboratories, Inc.). ECL Prime western blot detection reagent was purchased from GE Healthcare Life Sciences (Brazil). The antibodies here used were β-actin (sc-81178), phospho-p38 (sc398305), phospho-ERK1/2 (sc-7383), phospho-JNK (sc-6254), COX-2 (sc-376861) and MKP-1 (sc-271684) and horseradish peroxidase-labelled secondary antibody (sc358914), all of them from Santa Cruz Biotechnology Inc. JNK inhibitor (SP600125), the p38 inhibitor (SB203580) and the ERK1/2 inhibitor (PD98025) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents and drugs used were of analytical grade. Glass coverslips (13 mm) were from Ted Pella Inc. (Redding, CA, USA). Paraformaldehyde 20% solution was from Electron Microscopy Sciences (Hatfield, PA, USA). Anti-NF-κB p65 antibody was from Abcam (Cambridge, MA, USA). Alexa Fluor® 488 donkey anti-rabbit IgG, DAPI and Prolong® Gold antifade mounting medium were from Invitrogen (Carlsbad, CA, USA) - Molecular Probes (Eugene, OR, USA).

2.4 Extract preparation The stem bark of D. bipinnatum was cleaned, dried at room temperature, and grinded in an electric mill (model TE-625 Tecnal, São Paulo, Brazil) with mesh sieve size 40. The dried powder thus obtained was macerated in 70% hydroethanolic solution (1:3, w/v) for 7 days, filtered and concentrated in a rotary evaporator (Marconi MA 120, São Paulo, Brazil) under pressure of 600 mm Hg, at 40°C. The residual solvent was eliminated in an oven at 45°C, for 24 h. So, HEDb was obtained, which was stored in an amber bottle, at 4°C. At the time of use, HEDb was dissolved in distilled water to obtain the desired solutions.

2.5 Cell viability assay Cell viability was assessed by the Annexin V-PE kit, using fluorescenceactivated cell sorting (FACS). In brief, RAW 264.7 cells (3x105 cells per dish) were plated on a 12-well microplate containing culture medium either with or without HEDb (1; 5 or 20 µg/mL). After 24 h, the cells were harvested, washed twice with cold phosphate buffered saline (PBS), and resuspended in binding buffer (0.1 M Hepes, pH 6

7.4, 1.4 M NaCl, 25 mM CaCl2), at a concentration of 1 x 10 cells/mL. To 100 μL of this cell suspension, 5 μL of Annexin V-PE (BD Biosciences, San Diego, CA, USA) and 5 μL of 7-amino-actinomycin D (7-AAD; included in the kit) were added and Page 6of 24

incubated for 15 min in the dark. Then 400 μL of binding buffer was added. Within 1 h, FACS analysis was performed at an excitation wavelength of 650 nm. At the beginning of apoptosis, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the membrane. Annexin V (conjugated with the fluorochrome PE) binds PS with high affinity, and identifies apoptosis at an early stage. In addition, viable cells with intact membranes exclude 7-AAD, whereas the membrane of damaged cells is permeable to 7-ADD. Viable cells are both Annexin V-PE and 7ADD negative. Data were analysed with CellQuest software (Becton Dickinson, USA). For each sample, 20,000 events were counted. Values of IC50

>

30 µg/mL were

considered non-cytotoxic (Suffness and Pezzuto, 1990).

2.6

Anti-inflammatory assays

2.6.1 Quantification of cytokines by ELISA RAW 264.7 cells were plated in a 12-well culture plate and submitted to pretreatment with HEDb (1; 5 or 20 µg/mL), or dexamethasone (10 µg/mL). After 1 h, control (no pretreatment) and pretreated cells were stimulated by LPS (1 µg/mL) for 24 h, in culture medium supplemented with 10% FBS. The levels of the cytokines (pg/mL) TNF-α, IL-1β and IL-10 were determined in the culture media using a commercial ELISA kit, and absorbance was read in a Microplate reader Multiskan® (Thermo Scientific, USA) at 450 nm.

2.6.2 Measurement of PGE2 RAW 264.7 (5×105 cells/well) were seeded onto a 12-well culture plate, and maintained at 37 °C overnight, in culture medium supplemented with 10% FBS. The cells were pre-incubated with HEDb (1; 5 or 20 µg/mL) or indomethacin (15 µg/mL) for 1 h, and then stimulated for 24 h with LPS. Controls were maintained under the same culture conditions, but were not pretreated or stimulated. The PGE2 concentration in the conditioned culture media was measured using a commercial Prostaglandin E2 ELISA Kit – Monoclonal (Cayman Chemical, Company Ann Arbor, MI, USA). 2.6.3 Determination of intracellular reactive oxygen species (ROS) The intracellular redox state of the cells was assessed by incubation with 2′,7′dichlorofluorescein diacetate (DCFH-DA), which freely permeates cells, and upon incorporation, is converted, by oxidation, to the fluorescent 2,7-dichlorofluorescein (DCF). The fluorescence was quantified by flow cytometry. In brief, RAW 264.7 cells

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(3x105 cells per dish) were plated on a 12-well microplate containing culture medium either with or without HEDb (1; 5 or 20 µg/mL), and NAC (10 µg/mL). After 24 h incubation, cells were harvested, washed twice with cold PBS, and incubated with 3 μM DCFH-DA, for 30 min, at 37°C, in the dark. After washing with cold PBS, the fluorescence was measured by a FACS Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). For each sample, 20,000 events were counted. DCF fluorescence was measured at an excitation wavelength of 488 nm (FL-1) and emission at 515–540 nm. The results were analysed by CellQuest software program (Becton Dickinson, USA).

2.6.4 Detection of mitochondrial membrane potential The mitochondrial membrane potential (ΔΨm) was used to estimate apoptosis, using

the

JC-1

Kit

(BDTM

MitoScreen

Kit,

USA).

JC-1

Kit

contains

tetraethylbenzimidazolylcarbocyanine iodide, a cationic dye that accumulates in energized mitochondria. At low mitochondrial membrane potential, JC-1 is at low concentration, and remains predominantly a monomer that yields green fluorescence (emission 530 ± 15 nm). At high mitochondrial membrane potential, the dye is at high concentration and aggregates, yielding a red-orange emission (590 ± 17.5nm). Therefore, a decrease in the aggregate fluorescent count is indicative of depolarization. In brief, RAW 264.7 cells (3x105 per dish) were plated on a 12-well microplate containing medium either with or without HEDb (1, 5 or 20 µg/mL). After 24 h incubation, cells were collected, washed twice with cold PBS, resuspended in cold PBS supplemented with 0.5 µM JC-1, and incubated for 15 min., at 37 ºC. The cells were washed according to the kit protocol, and 20,000 events were counted for each sample on a BD Accuri C6 flow cytometer using the kit template.

2.6.5 Western blot analysis RAW 264.7 were seeded overnight at a starting density of 2 x 106 cells/mL to a 6-well plate. The cells were incubated in the medium (growth control), HEDb (1; 5 and 20 µg/mL) and controls SB203580, PD98025 and SP600125 for MAPKs and indomethacin for COX-2 for 1 h. After, the cells were incubated to LPS challenge to evaluated to response of MAPK (p38, JNK e ERK) and nuclear factor kappa B (NFKBp65), for Mitogen-activated protein kinase phosphatase 1 (MKP-1) for 4 h and 24 h for COX-2. Cells were harvested in ice-cold lysis buffer containing (0.5 mM sodium orthovanadate, 1 mM EDTA, protease inhibitor mixture and phosphate-buffered saline, pH 7.4). The cell lysate was obtained from the supernatant after centrifugation at 14.000 Page 8of 24

rpm 4° C 30 min. The protein concentration was determined using a Bradford assay kit (Sigma–Aldrich, USA). Equal amounts of proteins (40 µg/lane) were blotted onto a membrane nitrocellulose (Bio-Rad, Hercules, CA, USA). After the proteins were separated on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to nitrocellulose membranes (Bio-rad Laboratories, Inc.). The transferred proteins were incubated 4 h at 4 °C with a dilution of primary antibody for the pERK1/2 (1:1000), MAPK p-p38 (1:2500), p-JNK (1:800), MKP-1 (1:1000), COX-2 (1:2500), and β-actin (1:2500), and then washed four times with Tris-buffered saline (TBS) containing 0.5% Tween-20 (TBS-T, pH 7.4) for 10 min. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h and again washed four times in TBS-T. Appropriate horseradish peroxidase-conjugated secondary antibody was used for 2 h at room temperature in 1:10,000 dilutions (Santa Cruz Biotechnology- USA). The membranes were developed with chemiluminescent detection ECL Prime (GE Healthcare Life Sciences, Brazil). The intensity of each band was quantitatively determined using Chemidoc (Bio-rad Laboratories, Inc.) and quantitatively determined using Image J software (http://rsb.info.nih.gov).

2.6.6 Immunofluorescence staining for NF-κB nuclear translocation The effect of HEDb on nuclear translocation of NF-κB was assessed by immunofluorescence microscopy. RAW 264.7 macrophages were seeded overnight on to 13 mm glass coverslips placed at a 24-well plate (5 x 104 cells/coverslip). After 12 h the medium was removed and cells were pre-treated with HEDb (1; 5 or 20 µg/mL) or dexamethasone (10 µg/mL) for 1 h, followed by activation with LPS (10 ng/mL). After 30 min, cells were rinsed twice with Hank's Balanced Salt Solution (HBSS++, HBSS containing Ca2+ and Mg2+), fixed with 4% paraformaldehyde (15 min) and washed thrice with HBSS++. Cells were permeabilized with methanol at -20ºC for 10 min, sequentially washed twice with HBSS++ and 0.1 M glycine prepared in HBSS++, and blocked for 1 h in blocking buffer (1% BSA, 0,1% Triton X-100 in HBSS++). Cells were then stained with rabbit anti-p65 NF-κB antibody (1:800 dilution in blocking buffer) overnight at 4º C followed by incubation with Alexa Fluor 488 donkey antirabbit Igg (1:250 dilution in blocking buffer). Nuclei were stained with DAPI (1 μg/mL in blocking buffer). After each staining cells were washed with HBSS ++ to remove unbound antibody. Slices were then mounted using Prolong Gold antifade mounting medium and fluorescence images were captured on a Leica TCS SP8 confocal microscope. A negative control prepared by omission of primary antibody was used for Page 9of 24

background correction. A minimum of five fields of each experiment, performed in duplicate, were examined. The nuclear and cytoplasmic staining intensities were compared according to Noursadeghi et al. (2008), to give the nuclear:cytoplasmic ratio as a relative measure of NF-κB nuclear localization.

2.7 Statistics The results were expressed as mean ± standard error medium (X ± SEM). Comparisons between means were analysed by one-way analysis of variance (ANOVA). Where significant, this was followed by a multiple comparison of StudentNewman-Keuls test. P-values below 0.05 were considered statistically significant. The assays were performed in triplicates and all values were analysed with GraphPad Prism® software version 5.01. Inc. La Jolla, CA 92037 USA (www.graphpad.com).

3. Results 3.1 HEDb did not alter cell viability Fig. 1 shows that RAW 264.7 cells stimulated with LPS (1 µg/mL) presented cell viability levels comparable to the basal (80%), and pretreatment with either HEDb (1; 5 or 20 μg/mL), or NAC (10 μg/mL) did not cause cytotoxicity, since cell viability was also equivalent to the basal (80%) (Fig. 1A-B). A

Page 10of 24

100

B % surviving cells

80 60 40 20 0 Basal

1

5 HEDb

20

10

g/mL

NAC

LPS 1 g/mL

Fig. 1. Histogram of flow cytometry analysis of from RAW 264.7 macrophages (A). Q2LL represents viable cells. B: Effects of 70% hydroethanolic extract of Dilodendron bipinnatum stem bark (HEDb – 1; 5 and 20 μg/mL), and Nacetyl cysteine (NAC - 10 μg/mL) on cell viability of cell RAW 264.7 with the Annexin V-PE and 7-aminoactinomycin D (7-AAD) kit after 24 h of treatments and LPS (1 μg/mL). Cells (20,000). Results are expressed as mean ± standard error mean.

3.2 HEDb attenuates LPS-induced IL-1β and TNF-α production in RAW 264.7 macrophages Fig. 2A shows that the basal concentration of IL-1β in RAW 264.7-conditioned culture medium was 8.7 ± 5.07 pg/mL; this value increased 94.8% upon LPS challenge (p < 0.001). Pretreatment with HEDb significantly reduced IL-1β concentrations in RAW 264.7 culture medium, in a dose dependent manner, with the highest inhibitory effect (61.5%, p < 0.001) observed at the highest concentration (20 μg/mL). Similarly, the basal TNF-α concentration in culture medium was 368.0 ± 150.4 pg/mL, which increased 152% upon LPS treatment (930.2 ± 258.8, p < 0.001) (Fig. 2B). Again, pretreatment with the HEDb (20 μg/mL) prior to LPS challenge reduced TNF-α levels (727.2 pg/mL, which represents a 27.9% decrease p < 0.05). Comparatively, a 48% decrease (p < 0.01) was observed for 10 μg/mL dexamethasone. The IL-10 concentration in the culture medium of RAW 264.7 cells was 123.4 ± 0.81 pg/ml. LPS had no effect on the IL-10 levels, and pre-treatment with either HEDb (1; 5, or 20 µg/mL) or 10 μg/mL dexamethasone did not significantly affect the IL-10 levels) (data now show). B

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80

A

## 1500

†††

†††

#

TNF- (pg/mL)

IL-1 (pg/mL)

60

***

40

*** *** 20

0

Basal

1

5

20

HEDb

10

1000

* ** 500

0

g/mL

Basal

1

Dexa

5

20

HEDb

10

g/mL

Dexa

LPS 1 g/mL

LPS 1 g/mL

Fig. 2. Effect of HEDb (1; 5 or 20 μg/mL) and dexamethasone (Dexa – 10 μg/mL) on the concentration of IL-1β (A) and TNF-α (B) by RAW 264.7 cells. Cells were pre-treated with either HEDb or Dexa for 1 h, and then incubated with LPS (1 µg/mL) for 24 h. The conditioned media were collected, and the concentration of TNF-α and IL-1β were measured in culture medium by a commercial ELISA kit. Results are expressed as mean ± standard error mean. Statistical significance was evaluated by one-way analysis of variance, followed by Student-Newman-Keuls test. ††† p < 0.001 vs. basal; *** p < 0.001 vs. LPS. ** p < 0.01. *p < 0.05 vs. LPS. #p < 0.05 and ##p < 0.01 vs. 20 μg/mL.

3.3 HEDb attenuates LPS-induced PGE2 production in RAW 264.7 macrophages The basal concentration of PGE2 in the culture medium of RAW 264.7 cells was 31.87 ± 3.9 pg/mL. LPS treatment significant increased PGE2 production and release (94.8%, p < 0.001). Pre-incubation with HEDb reduced PGE2 concentration in culture medium (significant only at 20 μg/mL, 61.48%, p < 0.001), but not to the basal level, while indomethacin (positive control) decreased PGE2 to the basal levels (99.6%, p < 0.001) (Fig. 3). 1500

PGE2 (pg/mL)

1250

†††

1000

750

* 500

250

*** 0

Basal

LPS

1

5

20

HEDb

15 Indo

g/mL

LPS

Fig. 3. Effect of HEDb (1; 5 or 20 μg/mL) and indomethacin (Indo – 15 μg/mL) on the on the concentration of PGE2 by RAW 264.7 cells. Cells were pre-treated with either HEDb or Indo for 1 h, and then incubated with LPS (1 μg/mL) for 24 h. The conditioned media were collected, and the concentration of PGE2 was measured in culture medium by a commercial ELISA kit. Results are expressed as mean ± standard error mean. Statistical significance was evaluated by one-way analysis of variance, followed by Student-Newman-Keuls test. ††† p < 0.001 vs. basal; * p < 0.05 vs. LPS. *** p < 0.001 vs. LPS.

3.4 HEDb attenuates LPS-induced intracellular ROS production in RAW 264.7 macrophages The level of ROS in response to LPS was increased in about 248.6% (p < 0.001) relative to the baseline (222.6 ± 66.3). HEDb inhibited, in a non-concentration– dependent fashion, the ROS production (highest effect with 20 μg/mL, 67.5%, p < 0 .001). NAC (10 μg/mL), used as positive control in this assay, inhibited ROS production by 76.8% (p < 0.001) relative to the LPS treated cells (Fig. 4A-B). A

Page 12of 24

Basal and LPS

HEDb 5 µg/mL

NAC

HEDb 20 µg/mL

Cell counts

HEDb 1 µg/mL

FL-1 1000

ROS generation (IMF)

††† 800 600

*** 400

*** *** ***

200 0 Basal

1

5

20

EHDb

10

g/mL

N-Acet

LPS 1 g/mL

Fig. 4. Histogram of flow cytometry analysis of ROS production induced by LPS in RAW 264.7 cells (A). Effects of HEDb (1; 5 or 20 μg/mL) and N-acetyl cysteine (NAC - 10 μg/mL) on the intracellular production of reactive oxygen species (ROS) induced by LPS (B). The concentration of ROS was measured in the cells by exposure to 2′,7′dichlorofluorescein diacetate (DCFH-DA - 3 μM) for 30 min. Afterwards, the cells were analysed by flow cytometry. Results are expressed as mean ± standard error mean. Statistical significance was evaluated by one-way analysis of variance, followed by Student-Newman-Keuls test. ††† p < 0.001 vs. basal; *** p < 0.001 vs. LPS.

3.5 HEDb protects ΔΨm from LPS induced depolarization in RAW 264.7 macrophages LPS treatment for 24 h caused a marked increase (19.2%, p < 0.001) in mitochondrial membrane depolarization compared to untreated cells (3.1%). All concentrations of HEDb (1; 5 or 20 µg/mL) prevented the mitochondrial membrane depolarization induced by LPS, reaching the maximal value with 5 µg/mL (6.6 ± 1.0 %, p < 0.001) relative to LPS treated cells (19.2 ± 0.2%) (Fig. 5A-B). A

HEDb 1 µg/mL

HEDb 5 µg/mL

HEDb 20 µg/mL

NAC

Cell counts

Basal and LPS

FL-1

Page 13of 24

1000

B ROS generation (IMF)

††† 800 600

***

***

400

*** ***

200 0 Basal

1

5

20

10

HEDb

g/mL

NAC

LPS 1 g/mL

Fig. 5. Histogram of flow cytometry analysis of mitochondrial membrane potential from LPS induced depolarization in RAW 264.7 macrophages (A). Effect of HEDb (1; 5 or 20 μg/mL) and N-acetyl cysteine (NAC - 10 μg/mL) on LPS-induced changes of mitochondrial membrane potential (ΔΨm) of RAW 264.7 macrophages (B). The mitochondrial membrane potencial was measured by the JC-1 assay for flow cytometry (0.5 μM, 30 min). Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence ratio. Results are expressed as mean ± standard error mean. Statistical significance was evaluated by one-way analysis of variance, followed by Student-Newman-Keuls test. †††p < 0.001 vs. basal; *** p < 0.001 vs. LPS.

3.6 HEDb inhibits LPS-induced MAPK p-p38 and p-JNK activation in RAW 264.7 macrophages LPS stimulation of the RAW cells significantly increased (94.6%, p < 0.001; 72.1%, p < 0.001; 60.2%, p < 0.001) the expression of p-p38, p-ERK 1/2 and p-JNK, respectively, in comparison to baseline. HEDb at the three concentrations tested inhibited in a non-concentration dependent manner the phosphorylation of p-JNK, causing maximum effect at 1 μg/mL (57.6, p < 0.001). At 5 and 20 μg/mL HEDb attenuated in a nonconcentrated fashion, the p-p38 phosphorylation, producing maximum effect at 5 μg/mL (52%, p < 0.001) when compared with LPS group. None of the three concentrations HEDb tested produced inhibition of p-ERK phosphorylation. The positive controls SB203580 (0.1 μM), PD98025 (10 μM) and SP600125 (10 μM) inhibited p-p38 (86%, p < 0.001), p-ERK (72%, p < 0.05) and p-JNK (75%, p < 0.001) phosphorylation respectively, compared to LPS group (Fig. 6A-C).

Basal LPS (µg/mL) HEDb (µg/mL) SB203580 (µM)

-

1 -

1 1 -

1 5 -

1 20 -

1 0.1

p-p38 β-actin A

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††† 100

(%) p-p38 / -actin

80

** 60

*** 40

***

20

0

Basal

LPS

1

5

20

g/mL

0.1

Figure 6. Effect of HEDb (1; 5 or 20 μg/mL) and SB203580 (0.1 μM - used as positive control) on the expression of MAPK p-p38 in RAW 264.7 cells stimulated with LPS (1 μg/mL) after 30 min. The MAPK p-p38 levels were normalized against β-actin, used as internal control. The relative intensity (%) was calculated by the ratio of p-p38/βactin and expressed as percentage inhibition compared to LPS. Each column represents the mean of 3 independent experiments. Results are expressed as mean ± standard error mean, and one-way analysis of variance, followed by Student-Newman-Keuls test was performed. †† p < 0.01 versus basal and **p < 0.01, ***p < 0.001 versus LPS treated.

Basal LPS µg/mL HEDb µg/mL PD980125 (µM)

-

1 -

1 1 -

1 5 -

1 20 -

1 10

††

B

100

(%) p-ERK / -actin

80

†† 60

40

*

20

0

Basal

LPS

1

5

20

PD980125

Figure 6. Effect of HEDb (1; 5 or 20 μg/mL) and PD980125 (10 μM - used as positive control) on the expression of p-ERK 1/2 in RAW 264.7 cells stimulated with LPS (1 μg/mL) after 30 min. The p-ERK 1/2 levels were normalized against β-actin, used as internal control. The relative intensity (%) was calculated by the ratio of p-ERK 1/2/β-actin and expressed as percentage inhibition compared to LPS. Each column represents the mean of 3 independent experiments. Results are expressed as mean ± standard error mean, and one-way analysis of variance, followed by Student-Newman-Keuls test. †† p < 0.01 versus basal and * p < 0.05 versus treated with LPS.

Page 15of 24

Basal LPS µg/mL

-

1

1

1

1

1

HEDb µg/mL SP600125 (µM)

-

-

1 -

5 -

20 -

10

p-JNK β-actin †††

C

100

(%) p-JNK /  - actina

80

*** 60

***

***

40

*** 20

0

Basal

LPS

1

5

20

g/mL

SP600125

Figure 6. Effect of HEDb (1; 5 or 20 μg/mL) and SP600125 (10 μM - used as positive control) on the expression of p-JNK in RAW 264.7 cells stimulated with LPS (1 μg/mL) after 30 min. The p-JNK levels were normalized against β-actin, used as internal control. The relative intensity (%) was calculated by the ratio of p-JNK/β-actin and expressed as percentage inhibition compared to LPS. Each column represents the mean of 3 independent experiments. Results are expressed as mean ± standard error mean, and one-way analysis of variance, followed by Student-Newman-Keuls test was performed. †††p<0.001 versus basal and ***p < 0.001 versus LPS treated.

3.7 HEDb inhibits LPS-induced COX-2 activation in RAW 264.7 macrophages COX-2 levels increased by 54% in response to LPS (p < 0.001) in comparison to baseline. At 20 µg/mL, HEDb inhibited by 30% (p < 0.05) the expression of COX-2. Indomethacin, 10 µg/mL, inhibited COX-2 expression by 45% (p < 0.001), relative to LPS (Fig. 7). Basal LPS µg/mL

-

1

1

1

1

1

HEDb µg/mL Indo (µM)

-

-

1 -

5 -

20 -

10

COX-2 β-actin

††† 100

*

β

(%) COX-2 / -actin

80

†††

*** 60

40

20

0

Basal

LPS

1

5

20

10

g/mL

Fig.7. Effect of HEDb (1; 5 or 20 μg/mL) and indomethacin (Indo – 10 μg/mL) on the expression of cyclooxygenase2 (COX-2) by RAW 264.7 cells. Cells were pre-treated with either HEDb or Indo for 1 h, and then incubated with LPS (1 μg/mL) for 24 h. The COX-2 levels were normalized against β-actin, used as internal control. The relative intensity (%) was calculated by the ratio of COX-2/β-actin and expressed as percentage inhibition compared to LPS.

Page 16of 24

Each column represents the mean of 3 independent experiments. Results are expressed as mean ± standard error mean, and one-way analysis of variance, followed by Student-Newman-Keuls test was performed.††† p < 0.001 versus basal. ** p < 0.01 and *** p < 0.001 versus LPS treated.

3.8 HEDb induced MKP-1 expression in RAW 264.7 macrophages LPS increased MKP-1 activation by 80% (p < 0.05) in comparison to baseline (20%). Pretreatment with HEDb markedly up regulated the expression of MKP-1, reaching maximum response with 20 μg/mL (171%, p < 0.001) in comparison to LPS (Fig. 8). Basal LPS (µg/mL) HEDb (µg/mL)

-

1 -

1 1

1 5

1 20

MKP-1 β-actin

MKP-1/-actina (%)

400

***

300

***

***

1

5

200

† 100

0 Basal

LPS

20

µg/mL

Fig. 8. Effect of HEDb (1; 5 or 20 μg/mL) on the expression of MKP-1 by RAW 264.7 cells. Cells were pre-treated with either HEDb for 1 h, and then incubated with LPS (1 μg/mL) for 4 h. The MKP-1 levels were normalized against β-actin, used as internal control. The relative intensity (%) was calculated by the ratio of MKP-1/β-actin and expressed as percentage inhibition compared to LPS. Each column represents the mean of 3 independent experiments. Results are expressed as mean ± standard error mean, and one-way analysis of variance, followed by StudentNewman-Keuls test was performed. † p < 0.05 versus basal, *** p < 0.001 versus LPS treated.

3.9 HEDb inhibits LPS-induced NF-қB nuclear translocation The ability of HEDb to inhibit translocation of the transcription factor NF-κB was investigated. As seen in Figure 9, non-stimulated RAW 264.7 cells exhibit low constitutive nuclear translocation of NF-κB, demonstrated by low nuclear:cytoplasmic ratio (0.51). HEDb (20 µg/mL) alone was not able to produce NF-κB translocation (0.50). A robust activation was seen upon stimulation with LPS (10 ng/mL) for 30 min, leading to significant increase in NF-κB in nucleus (1.73). Pre-treatment of cells with HEDb inhibited the translocation of NF-κB to the nucleus in LPS-stimulated cells at the Page 17of 24

three concentrations tested, with higher effect at 5 µg/mL (29.5% inhibition compared to LPS).

NF-kB (nucleus:cytoplasm)

2.0

###

*** ***

1.5

*

**

1.0 0.5 0.0

Basal

20

1

5

20

Dexa g/mL

HEDb LPS 10 ng/mL

Fig. 9. Effect of HEDb (1; 5 or 20 μg/mL) and dexamethasone (Dexa – 10 μg/mL) on the nuclear translocation of NF-қB p65 induced by LPS (10 ng/mL) on RAW 264.7 cells. The experiment was performed as described in Methods, and images, from left to right, are stained with DAPI, for NF-қB p65 protein, and merged images. Each column represents the mean of 3 independent experiments. Results are expressed as mean ± standard error mean, and one-way analysis of variance, followed by Student-Newman-Keuls test was performed. Each column represents the mean of 3 independent experiments. ††† p < 0.001 vs. basal; * p < 0.05, * p < 0.01 and *** p < 0.001 versus treated with LPS.

4. Discussion Macrophages play a central role in regulating inflammation and both the innate and adaptive immune responses. To exhibit their functions, macrophages must undergo a series of cellular processes including activation, proliferation, differentiation and cytokine production when stimulated by LPS, representing important targets for antiinflammatory drugs (Chenga et al., 2014).

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This study focused on anti-inflammatory effects of HEDb on LPS-stimulated RAW 264.7 cells. Noncytotoxic concentrations of HEDb (1, 5 and 20 µg/mL) were selected based on Alarmar blue assay (de Oliveira et al., 2014). To verify whether apoptosis and/or necrosis were induced at these concentrations, cell viability tests were carried out with Annexin-V and 7-AAD staining (Zimmermann and Meyer, 2011). In this assay, pretreatment of the RAW 264.7 cells stimulated with LPS with HEDb did not alter cell viability, indicating that the tested concentrations of HEDb did not results in cellular apoptosis and necrosis, corroborating the findings of de Oliveira et al. (2014). Given that, macrophages are the principal immunological cells responsible for host defence, in addition to being the principal target of LPS, the anti-inflammatory effect of HEDb due to the direct effect on the macrophage. We demonstrated that in RAW 264.7 macrophage stimulated with LPS, HEDb significantly reduces the activation of p-p38MAPK, p-JNK and COX-2 protein expressions, NF-қB nuclear translocation, generation of ROS and mitochondrial membrane depolarization. Moreover, HEDb caused an increase in the expression of MKP-1 and a reduction in the concentrations of pro-inflammatory cytokines (IL-1β and TNF-α), as well as PGE2 in the cell supernatant. No changes were observed in the expression of p-ERK 1/2 and IL10 levels. The MAPKs p38, JNK and ERK play key roles in inflammatory process. It is reported that blockage of macrophage p38, ERK and JNK pathways causes down regulation of COX-2 expression, TNF-α and IL-1β production during inflammation (Park et al., 2015). Because of the broad pro-inflammatory role of MAPKs in several in vitro systems, inhibition of this pathway has been advocated as a novel therapeutic strategy for inflammatory diseases (Kaminska et al., 2005; Guma et al., 2012; Astolfi et al., 2015; Shia et al., 2015; Yokota et al., 2015). The direct inhibition of the phosphorylation of p38, JNK and NF-қB in RAW 264.7 may respond, at least in part, for the anti-inflammatory effect of HEDb previously described by Oliveira et al. (2014), which is independent of ERK 1/2 inhibition. The MKP-1, a phosphatase that participates in phosphorylation of the three MAPK, particularly p38 and JNK, represents a novel target for anti-inflammatory drugs (Tucsek et al., 2011). It is critically involved in inflammatory signalling of macrophages, and is responsible for switching off pro-inflammatory cytokine production in vitro and in vivo (Bognar et al., 2013). HEDb induces activation of MKP-

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1 and hence caused inhibition of p38 and JNK activation of pro-inflammatory signalling pathways (Manetsch et al., 2012). Several studies have reported that LPS activates the MAPK pathways and the translocation of NF-KBp65 fraction to the cell nucleus, which induces the transcription of inflammatory mediators such as IL -1β, TNF-α and COX-2 (Park et al., 2013). IL-1β and TNF-α cytokines are the principal proinflammatory mediators of the macrophages after stimulation with LPS. HEDb at the three concentrations tested inhibited concentration-dependently IL-1β levels in the cell supernatant, whereas TNF-α concentration was only lowered at the highest concentration. However, no changes were observed in the levels of IL-10 by pre-treatment of RAW 264.7 with HEDb. The reduction in the concentration of these two cytokines in vitro by HEDb is in line with the results in the test of acute peritonitis by LPS in mice (de Oliveira et al., 2014), but differ as regards to effect on IL-10 an important pleiotropic immunoregulatory cytokine produced mainly by macrophages and which controls the degree and duration of the inflammatory response. These results indicate that inhibition of IL-1β and TNF-α by HEDb stems from a direct action on macrophages and that this effect contributes, at least in part, to the anti-inflammatory mechanism of HEDb. It is not known why HEDb increased levels of IL-10 only in vivo. However, some hypotheses may be considered, such as pharmacokinetic interference, neuronal modulations and / or hormonal, communication between different cell types and even release IL-10 by cells other than macrophages (T and B lymphocytes, dendritic cells, monocytes and mast cells) (Veranth et al., 2006; Trifunovic et al., 2015). Generation of ROS in large quantities has been implicated in the propagation of inflammation to trigger the release of proinflammatory mediators, which stimulate the recruitment of additional macrophages to the inflammatory site. It also plays a key role in phagocytosis by macrophages (Ledesma et al., 2009; Mittal et al., 2014). LPS rapidly stimulates mitochondrial generation and intracytoplasmic ROS in RAW 264.7 cells, resulting in activation of the pathways of MAPKs and cytokine secretion, mainly TNFα and IL-1β (Kasahara et al., 2011). HEDb inhibited mitochondrial and intracytoplasmic levels of ROS in RAW 264.7, indicating that the inhibition of ROS is an important part of the anti-inflammatory mechanism of action HEDb. The expression of pro-inflammatory cytokines depends on COX-2 expression during inflammation (Yamamoto and Gaynor et al, 2001). COX-2 is an inducible isoform of prostaglandin synthase found in activated phagocytes responsible for generating prostanoids during inflammation (Ermert et al., 1998). HEDb inhibited Page 20of 24

COX-2 expression as well as PGE2 concentration in the supernatant of RAW 264.7 activated by LPS, suggesting that PGE2 reduction results from the inhibition of COX-2 and that this pathway plays an important role in anti-inflammatory effect HEDb. Based on previous and present results, we concluded that the HEDb has a potent anti-inflammatory activity, acting on multiple targets of biological relevance but whose precise mechanistic routes warrant further investigations. However, to our work HEDb seemed to exert its anti-inflammatory effects particularly by augmenting MPK-1 expression and attenuating mitochondrial destabilization, ROS production as well as JNK and p38 MAPKs, resulting eventually in diminished activation of NF-kB. This in turn downregulated the transactivation of target genes, including IL-1β and TNF-α, and COX-2 (with consequent reduction of PGE2) in a model of LPS-activated RAW 264.7 cells. Our findings suggest that HEDb provides new insights into a novel mechanism of anti-inflammatory action, thus highlighting its potential as a phytotherapeutic agent.

5. Acknowledgements

The present research was funded by Coordenação de Aperfeiçoamento de Pessoal

de

Nível

Superior

(Pró-Amazônia

–

Biodiversidade

e

Sustentabilidade/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support (proc. no. 23038.000731/2013-56), Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT – proc. no. 157552/2014) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/BIONORTE – proc. no. 551737/2010-7).

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Park, H.H., Kim, M.J., Li, Y., Park, Y.N., Lee, J., Lee, Y.J., Kim, S.G., Park, H.J., Son, J.K., Chang, H.W., Lee, E., 2013. Britanin suppresses LPS-induced nitric oxide, PGE2 and cytokine production via NF-κB and MAPK inactivation in RAW 264.7 cells. Int Immunopharmacol. 15, 296-302. Park, J., Min, J.S., Kim, B., Chae, U.B., Yun, J.W., Choi, M.S., Kong, I.K., Chang, K.T.; Lee, D.S., 2015. Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways. Neurosci Lett. 584, 191-196. RBG Kew (2016). The State of the World’s Plants Report – 2016.Royal Botanic Gardens, Kew. Santos, J.C., Oliveira, C.A.N., Varella, L., Matos, A.P., 2010. Constituintes químicos e atividade antimicrobiana dos extratos de Dilodendron bipinnatum (Sapindaceae). Quim. Nova. 33, 2080-2082. Shia, X., Zhenga, Z., Li, J., B, Xiao, Z., Qia, W., Zhanga, A., Wua, Q., Fanga, Y., 2015. Curcumin inhibits Aβ-induced microglial inflammatory responses in vitro: Involvement of ERK1/2 and p38 signaling pathways. Neurosci Lett. 594, 105-110. Somner, G.V., Ferrucci, M.S., Acevedo-Rodríguez, P., 2016. Dilodendron in Lista de Espécies da Flora do Brasil., Jardim Botânico do Rio de Janeiro. Disponível em: . Acesso em: 15 março 2016. Suffness, M., Pezzuto, J.M., 1990. Assays related to cancer drug discovery. In: Hostettmann K, (ed.). Methods in plant biochemistry: assays for bioactivity. London: Academic Press. 71-133. Trifunović, J., Miller, L, Debeljak, Ž., Horvat, V., 2015. Pathologic patterns of interleukin 10 expression--a review. Biochem Med (Zagreb). 25, 36-48. Tucsek, Z., Radnai, B., Racz, B., Debreceni, B., Priber, J.K., Dolowschiak, T., Palkovics, T., Gallyas, F.Jr., Sumegi, B., Veres, B., 2011. Suppressing LPS-induced early signal transduction in macrophages by a polyphenol degradation product: a critical role of MKP-1. J Leukoc Biol. 89, 105-111. Veranth, J.M., Moss, T.A., Chow, J.C., Labban, R., Nichols, W.K.; Walton, J.C., Watson, J.G., Yost, G.S., 2006. Correlation of in vitro cytokine responses with the chemical composition of soil-derived particulate matter. Envir Health Perspect. 114, 341-349. Wancket, L.M., W. Frazier, J., Liu, Y., 2012. Mitogen-activated protein kinase (MKP)1 in immunology, physiology, and disease. Life Sci. 90, 237-248. Yokota, T., Wang, Y., 2016. p38 MAP kinases in the heart. Gene. 575, 369-376. Zimmermann, M., Meyer, N., 2011. Annexin V/7-AAD staining in keratinocytes. Methods Mol Biol. 740, 57-63. White, E.S., Mantovani, A.R., 2013. Inflammation, wound repair, and fibrosis: reassessing the spectrum of tissue injury and resolution. J Pathol. 229, 141-144. Page 23of 24

Flow cytometry

Protein expression

ROS

MAPK p-p38 and p-JNK

MKP-1

Immunofluorescence

HEDb

NF-kBp65

ELISA (pg/mL) IL-1β TNF-α COX-2 PGE-2

Intensidad relative (%) p-p38 / p38

150

125

††† 100

75

**

50

***

25

***

0

Basal

1

5

EHDb (g/mL)

20

0,1 SB203580 (M)

LPS 1 g/mL

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