Oleuropein Suppresses LPS-Induced Inflammatory Responses in

Article pubs.acs.org/JAFC

Oleuropein Suppresses LPS-Induced Inflammatory Responses in RAW 264.7 Cell and Zebrafish Su-Jung Ryu,†,¶ Hyeon-Son Choi,‡,#,¶ Kye-Yoon Yoon,† Ok-Hwan Lee,§ Kui-Jin Kim,∥ and Boo-Yong Lee*,⊥ †

Department of Biomedical Science, CHA University, Kyonggi 463-836, South Korea Department of Food Science and Technology, Seoul Women’s University, 621 Hwarang-ro, Nowon-gu, Seoul 139-774, South Korea § Department of Food Science and Biotechnology, Kangwon National University, Chunchenon 200-701, South Korea ∥ Laboratory for Lipid Medicine & Technology, Department of Medicine, Harvard Medical School - Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, United States ⊥ Department of Food Science and Biotechnology, CHA University, Kyonggi 463-836, South Korea ‡

ABSTRACT: Oleuropein is one of the primary phenolic compounds present in olive leaf. In this study, the anti-inflammatory effect of oleuropein was investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 and a zebrafish model. The inhibitory effect of oleuropein on LPS-induced NO production in macrophages was supported by the suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In addition, our enzyme immunoassay showed that oleuropein suppressed the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6). Oleuropein inhibited the translocation of p65 by suppressing phosphorylation of inhibitory kappa B-α (IκB-α). Oleuropein also decreased activation of ERK1/2 and JNK, which are associated with LPS-induced inflammation, and its downstream gene of AP-1. Furthermore, oleuropein inhibited LPS-stimulated NO generation in a zebrafish model. Taken together, our results demonstrated that oleuropein could reduce inflammatory responses by inhibiting TLR and MAPK signaling, and may be used as an anti-inflammatory agent. KEYWORDS: oleuropein, anti-inflammation, RAW 264.7 cell, zebrafish, NF-κB(p-65)

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INTRODUCTION The olive, Olea europaea, is an evergreen tree that grows in the Mediterranean, Asia, and Africa.1 Its fruit is commonly used as a source of olive oil, which is important for the Mediterranean diet. Olive leaf is also known to be a natural resource of various beneficial polyphenols. The olive leaf is commonly used as a traditional medicine for malaria and fever in Mediterranean countries.2 Many studies have been performed to examine the phytochemicals in olive leaf; compounds such as tyrosol, kaempterol, hydroxytyrosol, and oleuropein have been identified.1 Oleuropein is one of the major phytochemicals found in olive leaf and is known to have biological effects such as antioxidant, antiobesity, and antimicrobial activity.3−5 In addition, Drira et al. reported that this olive leaf-derived compound inhibits adipocyte differentiation by suppressing the cell cycle.4 Inflammation is a physiological response against harmful stimuli, such as pathogens, in the body.6 It exerts protective effects by inducing release of signaling molecules, which neutralize injurious pathogens.7 However, chronic inflammation has detrimental effects. These inflammatory processes can interfere or destroy healthy cells, even causing cancer or the formation of a plaque on the artery wall.8 Recent studies have shown that chronic inflammation is also associated with diseases such as diabetes, high blood pressure, and obesity.9−11 The immune system recognizes a variety of pathogens, which trigger production of pro-inflammatory cytokines such as interleukin-6 (IL-6), nitric oxide (NO), inducible nitric oxide © XXXX American Chemical Society

synthase (iNOS), and cyclooxygenase-2 (COX-2). The activation of Toll-like receptors (TLRs) is related to the production and activation of these cytokines.12−14 Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a signaling molecule in TLR pathways, plays a major role in inflammatory responses by stimulating the expression of proinflammatory genes. The activation of NF-κB as a transcription factor requires the degradation of IκBα by phosphorylation.15,16 In addition, activation of MAPK pathways, including p-38, JNK, and ERK, leads to the activation of NF-κB.17 MAPK pathway also regulated another inflammatory key gene named AP-1 by phosphorylation.18 The constant activation of these signaling pathways can cause excessive inflammatory responses. In the current study, researchers used zebrafish as an in vivo model to assess the anti-inflammatory effect of oleuropein. Zebrafish are a useful vertebrate model in biological research due to their physiological similarity to mammals, availability in large quantities, transparent body, and low cost.19,20 Recent studies have used zebrafish as a model for drug discovery.21,22 Zebrafish also have innate and acquired immune systems similar to those of mammals,23 with dynamic and vivid embryo images. In this report, we examined the inhibitory effect of Received: August 5, 2014 Revised: January 22, 2015 Accepted: January 22, 2015

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DOI: 10.1021/jf505894b J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structure of oleuropein (A) and its effect on cell viability (B). RAW 264.7 cells (1 × 104/well) were treated with oleuropein (100−400 μg/mL) for 12, 24, 48, and 72 h. Cell viability was determined using the XTT assay (B). Data are presented as means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p < 0.05). obtained from BioLegend (San Diego, CA). Maxime PT Premix KIT was purchased from iNtRON (Gyeonggi-do, Korea). iNOS, COX-2, IL-1β, IL-6, and GAPDH oligonucleotide primers were obtained from Bioneer (Seoul, Korea). Compound 2, 3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT), was purchased from WEL GENE (Daegu, Korea). Oleuropein (>98.0%), TRIzol reagent, diaminofluorophore 4-amino-5-methylanino-2,7-difluorofluoroescein diacetate (DAF-FM DA), Griess reagent, and lipopolysaccharide (LPS) (Escherichia coli, serotype 0111:04) were obtained from Sigma Chemical Co. (St. Louis, MO).

oleuropein on inflammatory responses and signaling in LPSinduced RAW 264.7 macrophages and a zebrafish model.

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MATERIALS AND METHODS

Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), and phosphatebuffered saline (PBS) were purchased from Gibco (Gaithersburg, MD). iNOS, COX-2, p65, p-IκB-α, IκB-α, p-ERK, ERK, p-p38, p38, pJNK, JNK, and GAPDH monoclonal antibodies and secondary antibody were obtained from Cell Signaling Technology (Boston, MA). The enzyme immunoassay (EIA) kits for IL-1β and IL-6 were B


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Figure 2. Inhibition by oleuropein of NO production and iNOS and COX-2 expression in LPS-stimulated RAW 264.7 cells. LPS-stimulated RAW 264.7 cells were treated with oleuropein (100, 200, or 300 μM) for 4 h, followed by oleuropein (100, 200, or 300 μM) and/or LPS for 24 h. Nitric oxide concentrations (A) in the culture media were determined by Griess assay. mRNA (B) and protein (C) levels were determined by RT-PCR and Western blot, respectively. The results were quantified using the ImageJ software (D). Data are presented as means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p < 0.05). Cell Culture. RAW 264.7 macrophage cells (American Type Culture Collection, CL-173, and passage 5−7) were cultured in DMEM with 1.5 g/L sodium bicarbonate, 1% P/S, and 10% FBS at 37 °C and in a humidified chamber with a 5% CO2 atmosphere. Cells were incubated with 100, 200, and 300 μM oleuropein, and then stimulated with LPS at 1 μg/mL for the indicated times. XTT Assay. RAW 264.7 cells (∼1 × 104 per well) were seeded in a 96-well plate and incubated in a CO2 incubator at 37 °C for 12, 24, 48, and 72 h. Cells were treated with various concentrations (100−400 μg/mL) of oleuropein and incubated for 12, 24, 48, and 72 h, after which 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carbox-anilide (XTT) was added to the culture medium. The cytotoxicity of oleuropein was determined on the basis of the absorbance at 450 and 690 nm measured using an ELISA plate reader. Nitrite Determination in RAW 264.7. RAW 264.7 cells (∼1 × 104 per well) were plated in 96-well plates, treated with various concentrations of oleuropein and then incubated with or without LPS (1 μg/mL) for 24 h. Nitrite levels, which reflect NO levels, in culture media were determined using the Griess reaction. Cell culture medium (100 μL) was mixed with 100 μL of Griess reagent and incubated at room temperature for 15 min. Absorbance was then measured at 540 nm using an ELISA reader. Nitrite levels in samples were determined using a standard sodium nitrite curve. Enzyme-Linked Immunosorbent Assay (ELISA). RAW 264.7 cells were pretreated with various oleuropein concentrations for 1 h and then further stimulated with LPS (1 μg/mL) for 24 h. The supernatants were collected and stored at −80 °C until cytokine analysis. IL-1β and IL-6 levels in supernatants were determined using

ELISA MAX Kits (BioLegend, San Diego, CA), according to the manufacturer’s instructions. RNA Isolation and Reverse Transcription Polymerase Chain Reaction. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA), and 1 μg of the total RNA was used to produce cDNA using an RT-PCR system. Amplification of the target genes was performed using specific oligonucleotide primers by PCR. The primers used were as follows: iNOS, forward (5′-CCCTTCCGAAGTTTCTGGCAGCAG-3′) and reverse (5′-GGCTGTCAGAGCCTCGTGGCTTTG-3′); COX-2, forward (5′-ATGCTCCTGCTTGAGTATGT-3′) and reverse (5′-CACTACATCCTGACCCACTT-3′); IL-6, forward (5′CCATCTCTCCGTCTCTCACC-3′) and reverse (5′AGACCGCTGCCTGTCTAAAA-3′); IL-1β, forward (5′C AG GAT GAGG AC AT GAGC ACC -3 ′) a n d r e v er s e ( 5 ′CTCTGCACACTCAAACTCCAC-3′); GAPDH, forward (5′AACTTTGGCATTGTGGAAGG-3′) and reverse (5′ACACATTGGGGGTAGGAACA-3′). The PCR products were separated on 1.0% agarose gels, stained with ethidium bromide, and photographed. The expression levels were quantified by scanning using a gel documentation and analysis system (ImageJ, SAS). Western Blot Analysis. RAW 264.7 cells were washed with PBS buffer, lysed with lysis buffer, and then centrifuged to remove cell debris. The total protein content of the supernatant was determined using the Bradford assay. Protein extracts (50 μg) were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were immunoblotted with primary antibodies specific for iNOS, COX-2, p65, p-IκB-α, IκB-α, p-ERK, ERK, p-p38, p38, pJNK, JNK, and GAPDH overnight. Secondary antibodies conjugated to horseradish peroxidase (1:1000) were then applied for 1 h. The C


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Figure 3. Effect of oleuropein on IL-6 and IL-1β mRNA and protein levels in LPS-stimulated RAW 264.7 cells. Cells were treated with oleuropein (100, 200, or 300 μM), followed by LPS. Cells were then incubated for a further 4 or 24 h. IL-6 and IL-1β mRNA levels were determined by RTPCR and visualized on a gel. (A) Cytokine levels in culture media were determined using an enzyme immunoassay kit. (B) Data are presented as means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p < 0.05). intensity of individual zebrafish larvae was quantified using an ECLIPSE E600 (Nikon, Tokyo, Japan). Statistical Analysis. All experiments were performed in triplicate. The results were analyzed statistically using an analysis of variance (ANOVA) and Duncan’s multiple range test. A p value < 0.05 was considered to indicate statistical significance (SAS Instititue, NC).

bands were visualized using enhanced chemiluminescence (ECL) and detected using the LAS imaging software (Fuji, New York, NY). Nitrite Determination in Zebrafish. Synchronized zebrafish embryos were collected and rearranged using a pipet at 20 embryos/ well in six-well plates containing 2 mL of egg water. After 7−9 h postfertilization (hpf), embryos were incubated with or without various concentrations of oleuropein for 1 h. Zebrafish were stimulated by LPS (5 μg/mL) for 24 h at 28.5 °C. The zebrafish embryos were then transferred into fresh embryo medium. NO levels in the inflammatory zebrafish model were measured using a fluorescent probe dye, diaminofluorophore 4-amino-5-methylanino-2,7-difluorofluoroescein diacetate (DAF-FM DA). Transformation of DAF-FM DA by NO generates highly fluorescent triazole derivatives. Following stimulation by LPS, the zebrafish larvae were transferred into 96-well plates and treated with DAF-FM DA solution (1 μM) for 1 h in the dark at 28.5 °C. After incubation, the zebrafish larvae were rinsed in fresh zebrafish embryo medium and anesthetized with tricaine methanesulfonate solution before observation. The fluorescence

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RESULTS AND DISCUSSION Effect of Oleuropein on RAW 264.7 Cell Viability. There is the chemical structure of oleuropein in Figure 1A. Oleuropein was nontoxic to the RAW 264.7 cells at the indicated range of concentrations (Figure 1B) and also has no toxicity at serial time −12, 24, 48, and 72 h. No morphological changes in the cells were observed on the basis of microscopic analysis (data not shown). This result shows that oleuropein has no effect on cell apoptosis. Accordingly, all of the following experiments were performed using oleuropein concentrations of 100, 200, and 300 μM. D


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Figure 4. Effect of oleuropein on the phosphorylation of IκB-α and the nuclear translocation of NF-κB. Cells were pretreated with oleuropein (100, 200, or 300 μM) for 1 h and then with LPS (1 μg/mL) for 15 min. Protein levels were determined by Western blotting. NF-κb p65 levels in the cytosol and nucleus were quantified using the ImageJ software and normalized to Lamin B and β-actin, respectively. (A) The results were quantified using the ImageJ software and normalized to IκBα (B). Data are presented as means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p < 0.05).

Effect of Oleuropein on LPS-Induced NO Production and Expression of iNOS and COX-2. Overproduction of iNOS-mediated nitric oxide is a representative inflammatory reaction, and may be involved in other negative cellular physiologies such as mutagenesis, DNA damage, and the formation of N-nitrosoamine.24−26 Cyclooxygenase-2 (COX2), another inflammatory marker, is also associated with production of proinflammatory substances such as prostaglandins, and is upregulated during inflammation. In particular, COX-2-activated pathways, which are responsible for the conversion of arachidonic acid to prostaglandin and other eicosanoids, are of clinical importance as major targets for nonsteroid anti-inflammatory drugs such as aspirin, which is commonly used for inflammation and pain.27 However, these drugs have several side effects such as gastrointestinal bleeding, swelling of skin tissue, and allergy. We examined the effect of oleuropein on LPS-induced NO production, a mediator of the inflammatory response. As shown in Figure 2A, the NO level in culture medium was reduced by oleuropein treatment in a dose-dependent manner. This decrease in NO production was due to the downregulation of iNOS, a major pro-inflammatory enzyme that produces NO, at the mRNA and protein levels (Figure 2B,C). In addition, COX-2 (also an inflammatory marker) mRNA and protein levels were decreased by oleuropein treatment in a dose-dependent manner (Figure

2B,C). Expression of iNOS and COX-2 in mRNA was reduced by 42% and 43%, respectively, and in protein levels was reduced by 72% and 45%, respectively, by 300 μM oleuropein (Figure 2D). Our data showed that oleuropein, an olive compound, decreased LPS-induced NO production dose-dependently by downregulating iNOS (Figure 2A,B), which is closely associated with the synthesis of NO and COX-2 expression. This result indicated that oleuropein could be a potential antiinflammatory phytochemical. Recent studies identified various phytochemicals with anti-inflammatory effects. Resveratrol, EGCG, and tyrosol are also known to downregulate COX-2 and iNOS.28−30 Effect of Oleuropein on LPS-Induced IL-1β and IL-6 Release and Their mRNA Expressions. Inflammation generally involves the abnormal regulation of cytokines. Cytokines such as IL-6 and IL-1β are pro-inflammatory in vitro and in vivo.31 Inflammation generally involves the abnormal regulation of cytokines. Cytokines such as IL-6 and IL-1β are pro-inflammatory in vitro and in vivo, and play important roles in the extent of inflammation and recruit other immune cells implicated in the pathogenesis of diverse inflammatory conditions, such as rheumatoid arthritis and septic shock.32 We further examined the anti-inflammatory effect of oleuropein by assaying levels of pro-inflammatory cytokines. RT-PCR analysis showed that expression of IL-1β E


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Figure 6. Effect of oleuropein on LPS-induced NO production in zebrafish embryo. Zebrafish embryos were pretreated with oleuropein for 1 h and then exposed to LPS (5 μg/mL) for 24 h (A). The NO level was measured after staining with DAF-FM-DA (A). The results were quantified using the ImageJ software. (B) Data are presented as means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p < 0.05).

and IL-6 (inflammatory cytokines) was reduced by oleuropein treatment (Figure 3A). Levels of these cytokines in culture medium were decreased by oleuropein treatment in a dosedependent manner. IL-6 and IL-1β levels were decreased, respectively, compared with the LPS-induced group at 300 μM oleuropein (Figure 3B). In particular, IL-1β in the presence of 300 μM oleuropein was reduced to the level of the control (Figure 3B). Therefore, oleuropein suppressed the LPSinduced release of pro-inflammatory cytokines. Our results showed that LPS induced release of cytokines (Figure 3B) was effectively reduced by oleuropein treatment at the mRNA (Figure 3A). However, we did not explore whether other cytokines, such as TNF-α and IL-10, were also regulated by

Figure 5. Inhibitory effect of oleuropein on LPS-induced activation of MAP kinases and AP-1 in RAW 264.7 cells. RAW 264.7 were pretreated with oleuropein (100, 200, or 300 μM) for 1 h and then with LPS (1 μg/mL) for 15 min. Total proteins (50 μg) were subjected to Western blotting (A and C). The protein levels were quantified using the ImageJ software, and those of the phosphorylated forms were normalized to total protein levels (B). The data of AP-1 were quantified using the ImageJ software and normalized to GAPDH (D). Data are presented as means with standard deviations of three replicates. Results were analyzed by ANOVA and Duncan’s multiple range test (p < 0.05). F


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indicated that inhibition of ERK and JNK rather than p38 contributes to the anti-inflammatory effect of oleuropein in the LPS-induced inflammatory response. This result suggested that the anti-inflammatory effect of oleuropein might be due at least in part to the inhibition of ERK and JNK. Effect of Oleuropein on LPS-Induced NO Production in Zebrafish Model. We used a zebrafish model to investigate the anti-inflammatory effect of oleuropein in vivo. Nitric oxide (NO) production in zebrafish was determined using a fluorescent probe dye. As shown in Figure 6A, the nitric oxide level in zebrafish was elevated after LPS treatment compared with the positive control. Therefore, LPS increased nitric oxide production in zebrafish, similar to that in RAW 264.7 cells. LPS-induced increases in NO production were significantly suppressed in the presence of oleuropein (Figure 6A). In particular, 300 μM oleuropein inhibited LPS-induced NO production in zebrafish by 62% compared with the LPS control group (Figure 6B). Our data showed that oleuropein effectively reduced LPS-induced NO production in zebrafish. This result was correlated with the in vitro cell culture data, suggesting that oleuropein exerts anti-inflammatory effects. To our knowledge, this is the first report of an anti-inflammatory effect of oleuropein in zebrafish. However, we did not explore the genetic regulation of inflammatory factors at the gene and protein levels. Thus, further studies on the inflammatory responses of zebrafish in the presence of oleuropein are required. Oleuropein is a relatively abundant phenolic compound in olive leaf compared with other components, such as tyrosol and hydroxytyrosol, which have been suggested to possess various biological activities; also, our data showed that oleuropein inhibited inflammatory responses in RAW 264.7 cells and zebrafish by suppressing NF-κB translocation and the MAP kinase pathway. Doses of oleuropein in the range 100−300 uM are very high so we think that concentrations were supraphysiological. Our study also provides information important for the development of anti-inflammatory agents containing oleuropein.

oleuropein. Analysis of these cytokines would provide more information on the anti-inflammatory mechanism of oleuropein. Effect of Oleuropein on LPS-Induced IκB Phosphorylation and NF-κB Translocation. Regulation of inflammatory cytokines and inflammatory responses is transcriptionally governed by NF-κb transcription factors. These transcription factors regulate inflammatory related genes in the nucleus, where they bind to the DNA of pro-inflammatory mediators to induce their transcription.33,28 In addition, transcriptional activity of NF-κB is dependent on IκBα phosphorylation,34 by which p65, a subunit of NF-κB, translocates into the nucleus to promote the expression of inflammatory genes including iNOS, COX-2, and cytokines such as IL-6 and IL-1β.35,36 Our results showed that oleuropein-mediated inhibition of IκBα phosphorylation (Figure 4B) blocks the translocation of p65 from the cytosol to the nucleus (Figure 4A), suggesting that oleuropein exerts its anti-inflammatory effects by suppressing NK-κB, a major component of the TLR pathway. The nuclear NF-κB level increased significantly after LPS treatment compared with the normal control (Figure 4A). Oleuropein (300 μM) inhibited the translocation of p65, a subunit of NFκB, into the nucleus by 40% compared with the LPS-induced control. However, lower concentrations of oleuropein exerted no significant effects on the translocation of p65 into the nucleus. Cytosolic NF-κB levels decreased with LPS treatment, but increased in the oleuropein-treated group (300 μM). This result was correlated with the nuclear NF-κB pattern, i.e., a reduction in the presence of oleuropein (300 μM) (Figure 4A). Translocation of NF-κB into the nucleus is associated with phosphorylation of IκB-α in the TLR4 pathway, a major inflammation pathway. Accordingly, we examined the phosphorylation of IκB-α, a mediator of NF-κB activation. Oleuropein inhibited IκB-α phosphorylation, suggesting that NF-κB translocation into the nucleus was inhibited by suppression of IκB-α phosphorylation (Figure 4B). Therefore, the inhibitory effect of oleuropein on LPS-induced inflammatory responses was due to the deactivation of NF-κb and IκB-α, major components in the TLR4 pathway. Effect of Oleuropein on LPS-Induced Activation of Mitogen-Activated Protein (MAP) Kinase. MAP kinases are another signaling pathway that plays a critical role in inflammation through activation of NFκB.17 This kinase family is composed of several subgroups, such as JNK, ERK, and p38. The activation of these kinase groups mediates various inflammatory responses in vitro and in vivo.37−39 The inflammatory response can be activated through the MAP kinase pathway. Thus, we determined whether MAP kinase signaling is involved in oleuropein-mediated inhibition of LPSinduced inflammatory responses. MAP kinase plays a critical role in LPS-induced inflammation signaling at the transcriptional level. Since the MAP kinase pathway is phosphorylationdependent, we examined the phosphorylation status of components of this pathway. Oleuropein significantly decreased LPS activated ERK and JNK by inhibiting their phosphorylation (Figure 5A). Phosphorylation of ERK and JNK induced by LPS was reduced by 20% and 62%, respectively, by 300 μM oleuropein (Figure 5B). However, phosphorylation of P38 induced by LPS, also a component of the MAP kinase pathway, was not decreased in the presence of oleuropein. Our data also showed that oleuropein decreased AP-1, downstream gene related with MAP kinase (Figure 5C). AP-1 is another inflammatory key gene by regulation NO.18 Our results

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82-31-725-8371. Fax: +82-31-725-8282. E-mail: [email protected]. Author Contributions ¶

These authors contributed equally to this work.

Author Contributions #

Co-first author.

Notes

The authors declare no competing financial interest.

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REFERENCES

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