Oleocanthal Modulates LPS-Induced Murine Peritoneal Macrophages

Article Cite This: J. Agric. Food Chem. 2019, 67, 5552−5559

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Oleocanthal Modulates LPS-Induced Murine Peritoneal Macrophages Activation via Regulation of Inflammasome, Nrf-2/HO1, and MAPKs Signaling Pathways Tatiana Montoya,† Maria L. Castejón,† Marina Sánchez-Hidalgo,† Alejandro González-Benjumea,§ José G. Fernández-Bolaños,‡ and Catalina Alarcón de-la-Lastra*,†

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Department of Pharmacology, Faculty of Pharmacy and ‡Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Seville 41012, Spain § Department of Plant Biotechnology, Institute of Natural Resources and Agrobiology of Seville, CSIC, Seville, Spain ABSTRACT: The present study was designed to investigate the role of the canonical and noncanonical inflammasome, MAPKs and NRF-2/HO-1, signaling pathways involved in the antioxidant and anti-inflammatory activities of oleocanthal in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. Isolated cells were treated with oleocanthal in the presence or absence of LPS (5 μg mL−1) for 18 h. Oleocanthal showed a potent reduction of reactive oxygen species (ROS) (25 μM, 50. 612 ± 0.02; 50 μM, 53. 665 ± 0.09; 100 μM, 52. 839 ± 0.02), nitrites (25 μM, 0.631 ± 0.07; 50 μM, 0.652 ± 0.07; 100 μM, 0.711 ± 0.08), and pro-inflammatory cytokines levels when compared with LPS−DMSO-treated control cells. In terms of enzymes protein expression, oleocanthal was able to downregulate iNOS (25 μM, 0.173 ± 0.02; 50 μM, 0.149 ± 0.01; 100 μM, 0.150 ± 0.01;p < 0.001), COX-2 (25 μM, 0.482 ± 0.08; 50 μM, 0.469 ± 0.05; 100 μM, 0.418 ± 0.06; p < 0.001), and mPGES-1 (25 μM, 0.185 ± 0.11; 50 μM, 0.218 ± 0.13; 100 μM, 0.161 ± 0.15; p < 0.001) as well as p38 (25 μM, 0.366 ± 0.11; 50 μM, 0.403 ± 0.13; 100 μM, 0.362 ± 0.15; p < 0.001), JNK (25 μM, 0.443 ± 0.11; 50 μM, 0.514 ± 0.13; 100 μM, 0.372 ± 0.15; p < 0.001), and ERK (25 μM, 0.294 ± 0.01; 50 μM, 0.323 ± 0.01; 100 μM, 0.274 ± 0.01; p < 0.001) protein phosphorylation, which was accompanied by an upregulation of Nrf-2 (25 μM, 1.57 ± 0.01; 50 μM, 1.54 ± 0.01; 100 μM, 1.63 ± 0.05; p < 0.05) and HO-1(25 μM, 2.12 ± 0,03; 50 μM, 2.24 ± 0.01; 100 μM, 1.92 ± 0.05; p < 0.01) expression in comparison with LPS− DMSO cells. Moreover, oleocanthal inhibited canonical and noncanonical inflammasome signaling pathways. Thus, oleocanthal might be a promising natural agent for future treatment of immune-inflammatory diseases. KEYWORDS: inflammasome, macrophages, MAPKs, Nrf-2/HO-1, oleocanthal

1. INTRODUCTION Nonsteroideal anti-inflammatory drugs (NSAIDs) are taken in an abusive manner, being the most prescribed drugs among the population. NSAIDs are associated with both self-consumption and treatment of chronic diseases, which make progress with inflammation and edema sing. However, the long-term and high-dose use of these drugs (∼2400 mg) needed to relieve inflammatory or immune pathologies (rheumatoid arthritis or osteoarthritis) can promote side effects such as renal, gastrointestinal, or cardiovascular damage.1,2 Therefore, it is for this reason that the use of nutraceuticals has become quite important. They allow the control of key inflammatory parameters in chronic diseases without triggering these adverse effects and improving the lifestyle quality of the patient.3,4 In the search of bioactive natural compounds, oleocanthal has attracted research attention increasingly since 2003, when it was identified by Andrewes and colleagues for first time.5 Oleocanthal is one of the major secoiridoids that is composed of around 10% of whole polyphenolic content in extra virgin olive oil (EVOO) and may partly promote the health benefits of following a Mediterranean-style diet.6 This secoiridoid is known as a natural NSAID, not only due to perceptual similarity with anti-inflammatory drug ibuprofen7 but also due to its demonstrated relevant pharmacological properties and its involvement in pathogenic processes. Many © 2019 American Chemical Society

studies have reported their beneficial role as chemopreventive,8−12 in neurodegenerative diseases,13−17 and in exerting anti-inflammatory activities.7,18−20 In this way, oleocanthal has demonstrated its anti-inflammatory and antioxidative properties on lipopolysaccharide (LPS)-induced chondrocytes downregulated nitrite production and inducible nitric oxide synthase (iNOS) protein expression.7,19 Accordingly, recent studies have reported that this compound could inhibit other proinflammatory factors such as interleukin (IL)-6, tumor necrosis factor alpha (TNF)-α, or macrophages inflammatory protein (MIP)-1α.20 On the other hand, macrophages are one of the most important components of the immune system, participating in innate and adaptive immune responses to realize a critical role in controlling infections and inflammatory processes. In particular, Toll-like Receptor 4 (TLR4), which is primarily found on macrophages and plays a central role in their activation, recognizes different signals or stimulus, such as the bacterial endotoxin LPS. Received: Revised: Accepted: Published: 5552

January 31, 2019 April 16, 2019 April 21, 2019 May 1, 2019 DOI: 10.1021/acs.jafc.9b00771 J. Agric. Food Chem. 2019, 67, 5552−5559

Article

Journal of Agricultural and Food Chemistry

The purity of the extracted oleocanthal was based on the 1H NMR and 13C NMR spectra and HPLC analyses. NMR spectra were registered in CDCl3 in a Bruker Avance-300 spectrometer. For HPLC analysis, a Waters 600 HPLC system equipped with a Waters 2996 PDA detector, set to 244 nm, was used; analysis was performed on a reverse-phase C18 column (Nova-Pak, 4 μm, 3.9 × 150 mm column). Mobile phase consisted of a gradient of 2% v/v acetic acid in water (A) and acetonitrile (B). The elution program at a flow rate of 1.0 mL/min was as follows: 0−2 min from 100% to 95% of A; 2−10 min from 95% to 75% of A; 10−20 min from 75% to 60% of A; 20−30 min from 60% to 50% of A. The analyses showed that the isolated oleocanthal had a purity higher than 95% (Data not shown). 2.2. Isolation and in Vitro Culture of Peritoneal Macrophages. Female Swiss mice (8−10 weeks of age) were purchased from Harlan Interfauna Ibérica (Barcelona, Spain) and housed at 20− 25 °C with 40−60% humidity, a 12 h light/dark cycle, and ad libitum food standard rodent chow (Panlab A04, Seville, Spain) and water access. All experiments were performed in our Animal Laboratory Center (Faculty of Pharmacy, University of Seville, Spain) in accordance with the Guidelines of the European Union regarding animal experimentation (Directive of the European Counsel 2010/ 630/EU) and followed a protocol approved by the Animal Ethics Committee of the University of Seville. Isolation of peritoneal macrophages was performed as previously described.35 A 1 mL amount of 3.8% (w/v) sterile thioglycollate medium (BD Difco, Le Pont de Claix, France) was injected, and 3 days later, macrophages were collected from the peritoneum in cold phosphate-buffered saline (PBS). The collected cells were centrifuged, resuspended in RPMI supplemented with 10% FBS, penicillin, and streptomycin, and incubated 2 h in a humidified CO2 incubator. The medium was replaced with fresh medium without FCS but serially diluted oleocanthal (25−100 μM) to remove nonadherent cells. Cells were stimulated with LPS from Escherichia coli (5 μg mL−1) (SigmaAldrich, St. Louis, MO, US) for 18 h. Viability was always ⩾95%. 2.3. Cell Viability Assay. Cells viability was measured using the sulforhodamine B (SRB) assay.29 Macrophages were seeded in 96well plates and treated with different concentrations of oleocanthal for 18 h, as the protocol described by Montoya et al.35 The absorbance values were measured at 510 nm using a microplate reader (BioRad, Madrid, Spain) and expressed as percentage of viability with respect to 100% from control untreated cells 2.4. Measurement of Nitrites Production. According to our previous work,35 nitrites levels was measured by Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) at 540 nm using an ELISA reader (BioTek, Bad Friedrichshall, Germany). DMSO−LPS absorbance values were regarded as 100% of nitrite production percentage using as reference a standard curve. 2.5. DCFDA Cellular Reactive Oxygen Species Detection. Intracellular ROS concentrations was measured using a DCFDA assay kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. There were 2.5 × 104 cells well−1 seeded on a 96-well black plate and LPS-stimulated in the presence or absence of pretreatment with oleocanthal (25, 50, and 100 μM). DCFDA (25 μM) was added to each well at 37 °C for 45 min. Excitation and emission wavelengths (485 and 535 nm, respectively) were read using a fluorescence microplate reader (Biotek, Bad Friedrichshall, Germany). 2.6. Determination of Pro-Inflammatory Cytokines. Proinflammatory cytokines were quantified on supernatants using IL-6 and interferon (IFN)-γ (Diaclone, Besancon Cedex, France), IL-1β and TNF-α (R&D System, Minneapolis, Cánada, USA), and IL-17 (Peprotech, London, UK) ELISA kits. 2.7. Western Blotting Analysis. Cells were lysed in ice-cold PBS containing a cocktail of protease and phosphatase inhibitors as Cárdeno et al.23 described in order to isolate proteins. Bradford’s method was used to determinate the total protein content.29 Aliquots of 25 μg of protein were separated on 10% acrylamide gel by sodium dodecyl sulfatepolyacryamide gel electrophoresis, transferred to nitrocellulose membrane, and incubated overnight with antibodies against COX-2, mPGES1, and iNOS (Cayman, Ann Arbor, MI,

In the activated state, macrophages express enzymes such as iNOS and cyclooxygenase (COX)-2 that overproduce nitric oxide (NO) and prostaglandin (PG) E2, respectively, and several pro-inflammatory cytokines such as TNF-α, IL-1β, IL6, IL-17, and IL-18, among others.21,22 Mitogen-activated protein kinases (MAPKs), nuclear transcription factor κB (NFκB), or inflammasome signal transduction pathways have been associated with gene expression of previously cited proinflammatory mediators. Current studies have reported that the inflammasome pathway induces pyroptosis and the activation and secretion of pro-inflammatory cytokines (IL-1β and IL-18)23 in a caspase-1-dependent manner.24,25 The inflammasome typically contains as essential components, as sensor protein, the adapter protein apoptosis-associated speck-like protein containing a CARD domain (ASC), the inflammatory protease caspase-1, and a nucleotide binding domain (NOD)-like receptor (NLR) member (NLRP1, NLRP3, NLRC4).26 The activated sensor recruits ASC to induce caspase activation, generating IL-1β and IL-18 mature forms from their biologically inactive precursors. Once activated, they are eventually secreted.24−26 Importantly, a key transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), is implicated in the induction of haem oxygenase-1 (HO-1) enzyme, and thus, both of them may regulate the cellular antioxidant response against reactive oxygen species (ROS), modulating acute inflammatory responses.22 Although the anti-inflammatory properties of oleocanthal have been already elucidated in LPS-activated murine macrophages J774, ATDC5 murine chondrogenic cells, and human primary osteoarthritis chondrocytes, nothing is known about the role of the inflammasome and Nrf-2/HO-1 signaling pathways associated with its anti-inflammatory and antioxidant properties. Therefore, the current study aims at identifying signaling pathways involved in this process focusing on activation of canonical and noncanonical inflammasome, MAPKs and Nrf-2/HO-1, signaling pathways.

2. MATERIALS AND METHODS 2.1. Chemicals. Oleocanthal was isolated from extra virgin olive oil, Arbequina cultivar (400 g). The oil was mixed with cyclohexane (200 mL) and H2O−MeOH (6:4 v/v, 200 mL) and vigorously shaken for 30 s with an electric mixer. The mixture was centrifuged (4000 rpm for 5 min) before allowing the two phases to separate in a separating funnel. The process was repeated with extra H2O−MeOH (6:4 v/v, 200 mL) to ensure complete extraction of phenolic compounds. The combined polar fractions were concentrated to dryness using a rotary evaporator to afford a residue, which was purified by column chromatography using silica gel 60, Merck (40−63 mm), as the stationery phase and a gradient EtOAc/cyclohexano (0:1 → 1:3) as eluent. The purity of oleocanthal (30 mg), above 95%, was determined by NMR spectroscopy. 1H NMR (300 MHz, CDCl3): δ (ppm) 9.63 (m, 1H, H-3), 9.21 (d, 1H, J1,8 = 2.0 Hz, H-1), 7.02 (d, 2H, J4′,5′ = 8.5 Hz, H-4′, H-8′), 6.75 (d, 2H, J5′,7′ = 8.5 Hz, H-5′, H7′), 6.61 (c, 1H, J8,10 = 7.1 Hz, H-8), 4.24 (m, 2H, H-1′), 3.61 (m, 1H, H-5), 2.97 (ddd, 1H, J4,3 = 1.1 Hz, J4,5 = 8.5 Hz, J4a,4b = 18.3 Hz, H-4a), 2.89 (t, 2H, J2′,1′ = 6.9 Hz, H-2′), 2.74 (dd, 1H, J5,4 = 8.5 Hz, J4b,4a = 18.3 Hz, H-4b), 2.70 (dd, 1H, J6,5 = 8.4 Hz, J6a,6b = 15.8 Hz, H6a), 2.60 (dd, 1H, J5,6 = 8.4 Hz, J6b,6a = 15.8 Hz, H-6b), 2.05 (d, 3H, J10,8 = 7.1 Hz, H-10). 13C NMR (75.5 MHz, CDCl3): δ (ppm) 200.5 (C-3), 195.3 (C-1), 172.0 (C-7), 154.4 (C-8), 149.5 (C-6′), 143.4 (C-9), 135.5 (C-3′), 130.0 (C-4′, C-8′), 115.5 (C-5′, C-7′), 64.9 (C1′), 46.4 (C-4), 37.0 (C-6), 34.5 (C-2′), 27.4 (C-5), 15.3 (C-10). 5553

DOI: 10.1021/acs.jafc.9b00771 J. Agric. Food Chem. 2019, 67, 5552−5559

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Journal of Agricultural and Food Chemistry USA), pJNK, JNK, pp38, and p38 (Cayman Chemical, Ann Arbor, MI, USA), pERK and ERK (Cell Signaling, Danvers, MA, USA), HO1 and Nrf-2 (Cayman Chemical, Ann Arbor, MI, USA), NLRP-3 and ASC (Cell Signaling Technology, Danvers, MA, USA), Caspase-1 and Caspase-11 (Novus Biologicals, Littleton, CO, USA), and IL-18 (Abcam, Cambridge, UK). Blots were incubated for 2 h in blocking solution with either horseradish peroxidase-labeled (HRP) secondary antibody antirabbit (Cayman Chemical, Ann Arbor, MI, USA) or antimouse (Dako, Atlanta, GA, USA). β-Actin antibody (SigmaAldrich, MO, USA) was used to prove equal loading of blots. The immunosignals were captured using Amersham Imager 600 from GE Healthcare (Buckinghamshire, UK) and analyzed and quantified by Image Processing and Analysis in Java (ImageJ, Softonic). 2.8. Statistical Evaluation. Results are expressed as means ± standard error of the mean (SEM) from three independent experiments. Statistical significance was analyzed by one-way analysis of variance (ANOVA) and evaluated using Tukey’s multiplecomparisons test as a post hoc test with Graph Pad Prism Version 5.01 software (San Diego, CA, USA). Differences were considered significant when P < 0.05.

iNOS implication on NO secretion was regulated by oleocanthal in LPS-activated cells. Nitrites measured were used as an indicator of NO synthesis due to their half-life. In comparison with control cells, nitrite levels were induced in LPS-stimulated cells. Figure 3 B shows a dose-dependent reduction of NO generation in treated cells, relating to reduction of iNOS protein expression confirmed by Western blot (Figure 3). In these results, oleocanthal inhibited significantly the expression of iNOS compared with LPS− DMSO-treated cells. 3.4. Oleocanthal Suppressed COX-2 and mPGES-1 Overexpression Induced by LPS. All assayed doses of oleocanthal were able to downregulate COX-2 protein expression significantly in comparison with LPS-stimulated macrophages. Similarly, LPS incubation showed a significant overexpression of mPGES-1 protein. Nevertheless, oleocanthal treatment (25−100 μM) resulted in a potent inhibition of mPGES-1 protein expression in comparison with LPS−DMSO control cells (Figure 4). 3.5. Effect of Oleocanthal on LPS-Activated MAPKs in Murine Peritoneal Macrophages. We observed that oleocanthal was able to inhibit the phosphorylation of p38, JNK, and ERK after LPS incubation. Figure 5 shows that downregulation of the MAPKs signaling pathway is a possible mechanism to explain the anti-inflammatory effects of this EVOO secoiridoid. All concentrations tested (25, 50, and 100 μM) prevented significantly JNK, p38, and ERK activation after 18 h of exposure to stimulus. 3.6. Nrf-2/HO-1 Signaling Pathways Is Upregulated by Oleocanthal in Murine Peritoneal Macrophages. To investigate whether oleocanthal modulates the Nrf-2 signaling pathway, we determined the Nrf-2 and HO-1 protein expressions by Western blotting. Oleocanthal treatment caused a marked increase in Nrf-2 and HO-1 expression after LPS induction compared with LPS−DMSO control cells (Figure 6). 3.7. Canonical and Noncanonical Inflammasome Expression Is Inhibited by Oleocathal. The inflammatory response induced by canonical inflammasome is processing by a chain of activations and assemblies. NLRP-3 depends on ASC adaptor, which activates caspase-1 through pro-caspase-1. Finally, the autocatalytic action of caspase-1 triggers pro-IL-1β and pro-IL-18 transformation into their active forms.30 Therefore, we examined the effects of oleocanthal on the protein levels of via canonical components, including NLRP-3, ASC, and pro- and cleaved caspase-1. After LPS, expressions of these proteins were significantly induced in comparison with control cells in the absence of stimulus. Although ASC protein levels did not significantly downregulate, NLRP-3 and both forms of caspase-1 were markedly reduced after oleocanthal treatment (Figure 7A, 7B, and 7D). In the same way, we investigated the effect of this secoiridoid via noncanonical inflammasome on peritoneal macrophages. The expression of pro-, partially cleaved, and cleaved caspase-11 was overexpressed after 18 h of LPS exposure. Nevertheless, oleocanthal was able to prevent this activation in a dose-dependent manner. Therefore, as shown in Figure 7, Western blot analysis revealed parallel results in IL-1 β (Figure 2A) and IL-18 levels diminutions after incubation with oleocanthal when compared to stimulated control cells.

3. RESULTS 3.1. Effect of Oleocanthal on Cell Viability. To evaluate the effects of this secoiridoid on the mouse peritoneal macrophages, we performed the SRB assay. After 18 h, the cell viability of cells treated with oleocanthal was not significantly altered at a concentration of 1.6−200 μM (>90%) (Figure 1).

Figure 1. Effect of oleocanthal on cell viability. Macrophages survival was major to 90% after oleocanthal treatment (1.6−200 μM) for 18 h. Cell viability was measured following the Materials and Methods description.

3.2. Effect of Oleocanthal on Cytokines Secretion. To assess whether oleocanthal was able to inhibit IL-1β, IL-6, IL17, INF-γ, and TNF-α secretion, specific ELISA assays were used to measured their levels in cell culture supernatant. Murine peritoneal macrophages stimulated with LPS increased levels of cytokines when compared with macrophages unstimulated (Figure 2). Thirty minutes of secoiridoid pretreatment showed a significant inhibition of pro-inflammatory cytokines (Figure 2A, 2B, 2C, 2D, and 2E, respectively). 3.3. Intracellular ROS, Nitrite Production, and iNOS Expression Are Downregulated by Oleocanthal in LPSStimulated Murine Peritoneal Macrophages. DCFDA penetrated into cells, where it is hydrolyzed by esterases and with ROS is oxidized to the highly fluorescent 2′,7′dichlorofluorescein (DCF). As shown in Figure 3, the fluorescence signal of DCF was significantly incremented in positive control cells, such as LPS−DMSO-induced cells. However, oleocanthal pretreatment could demonstrate a potent antioxidant activity, and it significantly attenuated ROS production (Figure 3). 5554


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Figure 2. Effects of oleocanthal on IL-1β (A), IL-6 (B), IL-17(C), INF-γ (D), and TNF-α (E) levels. Peritoneal macrophages were pretreated for 30 min with oleocanthal and LPS-stimulated (5 μg mL−1) for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Data are expressed as means ± SEM (n = 5). (+) p < 0.05; (+++) p < 0.001 vs control cells (no stimulated); (**) p < 0.01; (***) p < 0.001 vs LPS−DMSO control-treated cells.

Figure 3. ROS production, nitrite production, and iNOS expression are downregulated by oleocanthal. Cells were exposed to different concentrations of compound (25−100 μM) and 30 min later stimulated with LPS and incubated for 18 h: (A) ROS production; (B) nitrite production; (C) Western blot analysis of iNOS protein expression. Band intensity was measured by ImageJ software. Bottom gel represents β-actin showing the equal loading of cell lysates. Experimental results are means ± SEM (n = 4) (++) p < 0.01; (+++) p < 0.001 vs control cells; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 vs LPS−DMSO-treated control cells.

4. DISCUSSION

inflammatory response in activated murine peritoneal macrophages by LPS. In vitro studies have demonstrated previously the beneficial effects on the inflammatory response of this polyphenol

In this study, we reported that oleocanthal, a secoiridoid from EVOO, attenuated the oxidative effects and prevented the 5555


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Figure 6. Nrf-2/HO-1 signaling pathway is upregulated by oleocanthal on LPS-activated peritoneal macrophages. Murine macrophages were pretreated with oleocanthal for 30 min, followed by LPS stimulation for 18 h. Nrf-2 and HO-1 protein levels were Western blot detected. Densitometry was performed following normalization to the control (β-actin housekeeping gene). Data are represented as the means ± SEM (n = 4). (*) p < 0.05; (***) p < 0.001 vs LPS−DSMO-treated cells.

Figure 4. Oleocanthal suppressed COX-2 and mPGES-1 overexpression induced by LPS. Peritoneal macrophages were pretreated with indicated concentrations of oleocanthal and then LPS stimulated for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Densitometry was performed following normalization to the control (β-actin housekeeping gene). Data are represented as the means ± SEM (n = 4). (+++) p < 0.001 vs control cells (no stimulated); (***) p < 0.001 vs LPS−DSMO-treated cells.

been observed in LPS-activated macrophages,22,32 and has been supported by diverse studios.19,20,23,27,28,31,33−35 Our data showed that LPS induced a significant increase of these proinflammatory and oxidative mediators on murine macrophages. However, oleocanthal pretreatment inhibits significantly the levels of intracellular ROS, NO production, and upregulation of iNOS protein expression. Under LPS exposition, Th1 and Th17 induced the secretion of different pro-inflammatory cytokines.32 This observation corresponds with our previously works23,27,33−35 and the present study, where after 18 h of LPS incubation cytokines levels were strongly increased. However, treatment of cells with oleocanthal inhibited the secretion of TNF-α, IL-1β, IL-6, IFN-γ, and IL-17 in a concentration-dependent manner. Accordingly, Scotece et al. (2012), using LPS-stimulated J774 macrophages, found that oleocanthal reversed LPSinduced changes in levels of MIP-1α, IL-6, TNF-α, and IL1β.20 PGE2 is generated in macrophages by pro-inflammatory mediators, such as LPS. This prostanoid is an important cause of inflammation pathogenesis related to cardiovascular diseases, arthritis, and inflammatory bowel disease, among others.36 The generation of PGE2 is mediated by COX-2, which catalyzes the conversion of arachidonic acid into PGs. Also, mPGES-1, another inducible enzyme, takes place. mPGES-1’s striking induction in tissues and cells related to the inflammatory response suggests its key role in chronic diseases in which COX-2 is involved.37,38 According to previous studies where LPS enhanced the expression of COX-2 and mPGES-1 in murine peritoneal macrophages,27,33,34 our results show that after incubation with oleocanthal COX-2 and mPGES-1 overexpressions were efficiently reduced. Nrf-2/HO-1 has attracted major attention in recent years because it is involved in a potent cytoprotective response against inflammation in activated macrophages. HO-1 is closely linked to Nrf-2 activation and catalyzes the degradation of heme to produce biliverdin, iron, and carbon monoxide.39 This degradation product downregulated the expressions of

Figure 5. Effect of oleocanthal on LPS-activated MAPKs. Peritoneal macrophages were pretreated with indicated concentrations of oleocanthal for 18 h and then LPS stimulated for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Densitometry was performed following normalization to the control (p38, JNK, and ERK housekeeping genes, respectively). Data are represented as the means ± SEM (n = 4). (+++) p < 0.001 vs control cells (no stimulated); (**) p < 0.01; (***) p < 0.001 vs LPS−DMSO-treated control cells.

present in olive trees.7,19,20,47 However, for first time, our findings demonstrate that oleocanthal inhibits pro-inflammatory factors, such as NO, ROS, cytokines, and protein expression (iNOS, COX-2, mPGES-1), by preventing activation of MAPKs, Nrf-2/HO-1, and inflammasome pathways. Balance disruption of the intracellular reduction−oxidation state leads to oxidative stress mainly characterized by ROSmediated damage as a result of deregulated cellular redox balance. The oxidative stress induces stimulation of the iNOS gene, which mediates the generation of NO. This process has 5556


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Figure 7. Inflammasome signaling pathway was downregulated by oleocanthal in murine peritoneal macrophages. Protein expressions were analyzed by immunoblot for (A) NLRP-3, (B) ASC, (D) pro-capase-1 and cleaved caspase-1, (E) pro-caspase-11, partially cleaved and cleaved caspase-11, (C) IL-18. Murine cells were pretreated for 30 min with oleocanthal (25−100 μM) followed by stimulation with 5 μg/mL LPS for 18 h. Densitometry was performed following normalization to the control (β-actin housekeeping gene). Data are represented as the means ± SEM (n = 4). (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 vs LPS−DSMO-treated cells.

inflammasome pathways in LPS-activated murine peritoneal macrophages. In spite of the fact that NLRP-3 protein activation has not been elucidated yet, many cellular mechanism are accepted for decreasing intracellular potassium, increasing ROS production,49,50 nuclear transcription factor NF-kB,23 or MAPKs51,52 signaling pathways, among others. Qiao et al. (2012) reported that in C57BL/6j and RAW264.7 macrophages, TLR-induced NF-kB activation is involved in the regulation of NLRP3 transcription through binding to the conserved NF-kB binding sites in the NLRP3 promoter.25 This fact was supported by Fan et al. (2018), whose work described that the activation of either NF-kB and MAPKs signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects could be attenuated using pharmacological inhibitors of these two pathways.51 It is well known that LPS exposure promotes NLRP3 and IL-1β upregulation mediated by the p38 MAPK signaling pathway because blocking this pathway using an inhibitor reversed the LPSinduced upregulation of TLR2, NLRP3, caspase-1, IL-1β, and IL-6 in NR8383 cells.52 Taking this into account, NLRP-3 becomes activated and recruits ASC, which joins up with procaspase-1 to form a multiprotein complex named the “canonical” inflammasome. This complex activates caspase-1, which processes pro-IL-1β and pro-IL-18 inactive forms into mature secreted cytokines and induces pyroptosis.24−26 On the other hand, caspase-11 mediates the inflammatory response termed noncanonical inflammasome. After LPS recognition, oligomerization of intracellular LPS−pro-caspase-11 complexes to activation of the caspase-11 inflammasome result in pyroptosis as well as leading to processing of the mature pro-inflammatory cytokines IL-1β and IL-18.50 Recently, numerous works have explained that caspase-11 does not need the adaptor molecule ASC for activation. However, active caspase-11 cooperates with components of the

iNOS, COX-2, NO, PGE2, TNF-α, IL-1β, INF-γ, IL-6, among other pro-inflammatory mediators.40−42 Our data, in accord with previous studies, showed Nrf-2/HO-1 signaling pathway expression was decreased in murine peritoneal macrophages23,27,33,35 or RAW 264.743 after incubation with LPS. By contrast, oleocanthal treatment reversed LPS effects and sustained a significant dual upregulation of both proteins, demonstrating a parallel action on Nrf-2 and HO-1 enzymes. In agreement with the literature, the MAPKs pathway plays an essential role in induction and propagation of inflammation on LPS-activated macrophages.44 There are three major subfamilies of MAPK, ERK, JNK, and p38, which play a critical role for the regulation of pro-inflammatory genes such as cytokines, COX-2, and iNOS.45,46 LPS can induce phosphorylated forms of JNK, p38, and ERK. Accordingly, our data demonstrated that JNK, p38, and ERK phosphorylations were reduced by oleocanthal treatment. Although there are few studies about the effects of this polyphenol in the MAPK pathway, a recent report by Iacono et al.19 also demonstrated that treatment with oleocanthal (1−25 μM) downregulated p38 phosphorilation in a culture of ATDC-5 and LPS-activated human primary osteoarthitis chondrocites.47,48 Similarly, it has been observed that oleocanthal (10, 25, and 50 μM) was able to inhibit p38 and ERK phosphorylation in ARH-77 cells treated for 3 h.11 Maybe these discrepancies could be related in terms of different concentrations assayed, time of exposition, and changes in cell experimental lines and conditions. Actually, inflammasome multimeric protein complexes are involved in the development of cancer, autoinflammatory, metabolic, and neurodegenerative diseases and in the host defense. To date, no work has described previously a downregulation action of oleocanthal in this signaling pathway. However, our work has shown for the first time that oleocanthal modulated both canonical and noncanonical 5557


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NLRP3 inflammasome to induce caspase-1-dependent maturation of pro-IL-1 β and pro-IL-18.30,50 In this line, our data reported that LPS was able to induce a significant increase of NLRP3 protein expression, while oleocanthal treatment reverted this activation on peritoneal macrophages. However, changes in ASC adapter protein expression were not observed in treated cells. Finally, our data show like immunosignals of both pro-caspase-1 and caspase-1 and pro-caspase-11 and caspase-11 were induced by LPS. Nonetheless, pro- and cleaved caspases and just like that IL-18 protein expression were significantly reduced after treatment with this secoiridoid compound in a dose-dependent matter. Accordingly, to establish a possible mechanism of action of oleocanthal in the inflammatory response is a difficult point. However, in this study we reported that there is an evident connection between regulation of ROS levels, MAPKs and Nrf-2/HO-1 expressions, and regulation of inflammasome. We could define that downregulation of expression and activation of inflammasomecanonical and noncanonicalis mediated by the effects of olecanthal on MAPKs and Nrf-2/HO-1 signaling pathways, such as ROS levels, and not the opposite. In conclusion, our study demonstrated for fist time the antioxidant and anti-inflammatory effect of oleocanthal in LPSstimulated murine peritoneal macrophages which was related to modulation of pro- inflammatory mediators such as IL −1β, TNF-α, INF-γ, IL6, IL-17, and IL-18 and the protein expression of inflammatory enzymes including iNOS, COX2, and mPGES-1. The mechanism underlying these protective effects could be related to the enhanced of Nrf-2/HO-1 and inhibition of intracellular ROS production, MAPKs, and canonical and noncanonical inflammasome cascade signaling pathways. Thus, oleocanthal might be a promising natural agent for future treatment of immune-inflammatory diseases.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +34 954 55 98 77. E-mail: [email protected]. ORCID

Tatiana Montoya: 0000-0002-8638-8418 Funding

This study was supported by research grant AG2017-89342-P (Ministerio de Economiá y Competitividad, Spain). We thank Dirección General de Investigación de Investigación of Spain (CTQ2016-78703-P), Junta de Andaluci á (FQM134, CTS259), and the European Regional Development Fund (FEDER) for funding. A.G.B. is thankful for the FPU fellowship. T.M. gratefully acknowledges support from a Postgraduate Program of the PIF fellowship and financial sponsorship from VI Plan Propio de Investigación y Transferencia, University of Seville. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful for the assistance of the Center for Technology and Innovation Research, University of Seville (CITIUS).

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REFERENCES

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Oleocanthal Modulates LPS-Induced Murine Peritoneal Macrophages

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