Marine-Derived Secondary Metabolite, Griseusrazin A, Suppresses

Article pubs.acs.org/jnp

Marine-Derived Secondary Metabolite, Griseusrazin A, Suppresses Inflammation through Heme Oxygenase‑1 Induction in Activated RAW264.7 Macrophages Dong-Sung Lee,†,‡,⊥ Chi-Su Yoon,†,⊥ Yong-Taek Jung,§ Jung-Hoon Yoon,§ Youn-Chul Kim,† and Hyuncheol Oh*,† †

College of Pharmacy, Wonkwang University, Iksan, 54538, Republic of Korea Department of Biomedical Chemistry, College of Health and Biomedical Science, Konkuk University, Chung-Ju 27478, Republic of Korea § Department of Food Science and Biotechnology, Sungkyunkwan University, Jangan-gu, Suwon 16419, Republic of Korea ‡

S Supporting Information *

ABSTRACT: A new secondary metabolite, named griseusrazin A (1), was isolated from the marine-derived bacterium Streptomyces griseus subsp. griseus. The structure of the compound was determined by analysis of spectroscopic data including MS, COSY, HSQC, HMBC, and 15N-HMBC data. Griseusrazin A (1) inhibited the production of inflammatory mediators, such as prostaglandin E2 and nitric oxide, which was mediated through the suppression of the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α, in the LPS-stimulated cells was also effectively blocked by griseusrazin A (1). Furthermore, this anti-inflammatory activity of 1 was linked to its inhibitory effects against the nuclear translocation of NF-κB p50 and p65, as wells as NF-κB binding activity. In the further study to elucidate the anti-inflammatory mechanism, 1 was shown to induce heme oxygenase-1 (HO-1) expression through the enhancement of nuclear translocation of nuclear factor E2-related factor 2. Furthermore, the anti-inflammatory activity of 1 in the LPS-stimulated cells was partially reversed by an HO inhibitor, tin protoporphyrin. These results indicate that the anti-inflammatory effect of 1 is associated with Nrf2-mediated HO-1 expression.

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In heme catabolism, heme oxygenase 1 (HO-1), known as a rate-limiting enzyme, produces equimolar amounts of carbon monoxide (CO), bilirubin/biliverdin, and free iron.7 The degradation of heme is considered critical to cell survival under conditions of oxidative stress and inflammation. HO-1 and its product, CO, are known to suppress the expression of proinflammatory cytokines (TNF-α, IL-1β, and macrophage inflammatory protein-1β) and simultaneously to increase the expression of the anti-inflammatory cytokine IL-10 in lipopolysaccharide (LPS)-stimulated macrophages and endothelial cells.8 In addition, the induction of HO-1 suppresses the production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 through inhibitory effects on the expression of iNOS and COX-2.9,10 It was also shown that the effect of HO-1 on the expression of iNOS is associated with the inactivation of the nuclear factor (NF)-κB pathway.11,12 Marine microbes are regarded as a valuable resource for the discovery of novel secondary metabolites.13,14 In our continuing search for bioactive secondary metabolites from marine micro-

acrophages play a vital role in biophylaxis and are extensively used as an in vitro model to investigate various natural resources for the identification of potential antiinflammatory compounds. Pro-inflammatory cytokines and mediators are associated with biophylaxis, and their overproduction leads to the pathogenesis of various disorders, which include autoimmune diseases, bacterial sepsis, rheumatoid arthritis, otitis media, periodontitis, hearing loss, and chronic inflammation.1,2 Inflammation is a complex biological feedback to harmful stimuli, which can cause cell damage. In addition, it is a defensive mechanism by an organism to eliminate harmful stimuli and to initiate the body’s healing process.3 Acute or chronic inflammation is a complicated biological process mediated by the activation of immune cells including macrophages.4 Macrophages modulate various physiological immune responses, including the upregulation of pro-inflammatory cytokines and enzymes. Under inflammatory conditions, these pro-inflammatory cytokines and enzymes, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), lead to cell damage, tissue injury, and the activation of cells associated with rheumatoid arthritis, atherosclerosis, and chronic hepatitis.5,6 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 5, 2016

A

DOI: 10.1021/acs.jnatprod.6b00009 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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the case of structure A: two from the pyrazine ring and one from the N-methylformamide moiety. For compound 1, 3 J NH correlations of H2-1′ and H3-1″ with a single nitrogen atom resonating at 333 ppm were consistent with structure 1. Additional 15NH-HMBC correlations of H-8′, H-4′, and H3-9′ with a nitrogen atom resonating at 126 ppm were also observed. Therefore, the gross structure of griseusrazin A (1) was assigned as shown in Figure 1. Effects of Griseusrazin A (1) on the Production of Proinflammatory Mediators and Cytokines via the NF-κB Pathway in LPS-Induced RAW264.7 Macrophages. NO has broad physiological and pathological effects on many tissues, including immune cells. Macrophages appear to be a major source of iNOS,15−17 which differs from other NOS isoforms, because it is not constitutively expressed. Instead, iNOS is induced by cytokines, such as interferon (IFN)-γ or TNF-α, or other immunological stimuli, including LPS. iNOS is a crucial pro-inflammatory enzyme stimulating the production of NO in immune-activated macrophages.18 Prostaglandins are also important mediators of inflammatory symptoms causing fever and pain. In the production of these mediators, the inducible enzyme COX-2 and prostaglandin endoperoxide H synthase (PGHS) play an important role in converting arachidonic acid (AA) to PGH2, which is then transformed to prostaglandins such as PGD2, PGE2, and PGF2α or thromboxane A2.19,20 As an initial readout for the anti-inflammatory activity of 1, the effect of 1 in LPS-induced RAW264.7 macrophages was evaluated by estimating the concentrations of NO and PGE2, as well as the levels of iNOS and COX-2 protein expression. In these initial assays, griseusrazin A (1) was shown to dose-dependently suppress the production of NO and PGE2 and reduce the iNOS and COX-2 protein expression (Figure 2). These results suggested that griseusrazin A (1) possesses anti-inflammatory activity that is mediated through the inhibition of proinflammatory enzyme expression. The pro-inflammatory activities of cytokines, such as TNF-α, IL-1β, and IL-6, are well known to play major roles in controlling tumor progression and inflammation.21 Because our initial data indicated that griseusrazin A (1) decreased the production of pro-inflammatory enzymes (iNOS and COX-2) as well as the corresponding inflammatory products (NO and PGE2) in LPSstimulated cells, further anti-inflammatory activity of 1 was assessed by evaluating the mRNA and protein levels of TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW264.7 macrophages. When the cells were preincubated with 1 for 6 h and treated with LPS for 24 h, griseusrazin A (1) caused concentration-dependent decreases in TNF-α, IL-1β, and IL-6 production (Figure 3A, B, and C), as well as concentration-dependent decreases in mRNA expression (Figure 3D, E, and F). These results suggested that griseusrazin A (1) can suppress pro-inflammatory cytokine gene expression at the transcriptional level. NF-κB is a key molecule in an important signaling pathway involved in inflammatory diseases, where it regulates various proinflammatory genes and cytokines.22 NF-κB, a p65/p50 heterodimer, exists as an inactive complex bound to IκB-α in the cytoplasm. During inflammation, IκB-α is phosphorylated and subsequently degraded, thereby dissociating from the complex to release activated NF-κB, which translocates in the nucleus to activate the NF-kB pathway.23 Therefore, the effect of 1 against the NF-κB pathway in the LPS-stimulated cells was evaluated, and our results showed that LPS-induced nuclear p50 and p65 protein levels significantly decreased upon pretreatment with 1 for 6 h prior to LPS stimulation (Figure 4A). Furthermore,

organisms found in Korea, we initiated the study of the chemical constituents of extracts obtained from cultures of the bacterium Streptomyces griseus subsp. griseus. This study led to the isolation of a pyrazine-type metabolite, designated as griseusrazin A (1), which displayed anti-inflammatory activity in the LPS-stimulated murine macrophage-like cell line RAW264.7. We further showed that the anti-inflammatory activity of 1 is mediated through a mechanism involving HO-1 expression. Compound 1 may be of use to explore inflammatory mechanisms in cellular models and, thus, inspire the design of novel anti-inflammatory drugs.

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RESULTS AND DISCUSSION Structure Determination of Griseusrazin A (1). The positive HRESIMS spectrum of 1 exhibited an [M + H]+ ion at m/z 403.2116, suggesting the molecular formula C24H26N4O2. The 13C NMR spectrum revealed the presence of only 10 resonances, suggesting that griseusrazin A (1) has a symmetrical structure. The 13C NMR and DEPT data indicated the presence of a carboxyl carbon, a 1,4-disubstituted benzene ring, an sp3 methylene, and two methyl groups. Analysis of 1H NMR and HSQC data for 1 readily indicated the presence of a formyl group, an N-methyl, a methyl group, and an isolated methylene group. Connection of these structural units was established by analysis of HMBC data. HMBC correlations of the N-methyl to a formyl carbon and a formyl proton to the N-methyl carbon allowed the identification of an N-methylformamide group, and this group was then found to be attached to C-5′ of the benzene ring by an HMBC correlation of the N-methyl with C-5′. HMBC correlations of H-1′ with C-2′ and C-3′ and of H-3′ with C-1′ indicated the connection of C-1′ to C-2′. The carbon sequence of C-1′ → C-2 → C-3 → C-1″ was established by HMBC correlations of H2-1′ with C-2 and C-3 and of CH3-1″ with C-2 and C-3. Consideration of the chemical shift values of two aromatic carbons (C-2: 151.6 ppm, C-3: 149.4 ppm) and the fact that there is no available oxygen atom required by the molecular formula suggested that these carbons must be connected to nitrogen. On the basis of this result and the symmetric nature of griseusrazin A (1), two possible symmetric structures (1 and A) satisfying the molecular formula could be suggested (Figure 1).

Figure 1. Chemical structure of griseusrazin A (1) and an alternative symmetrical combination that could apply to the structure of griseusrazin A (A).

Inspection of these two possible structures indicated that the exact positions of the substituents in the pyrazine ring could be determined by analysis of 15NH-HMBC data. In the case of structure 1, the 15NH-HMBC experiment would provide correlations for two different nitrogen atoms, namely, the nitrogen atoms of the pyrazine ring and the N-methylformamide moiety. On the other hand, the 15NH-HMBC correlations due to three different nitrogen atoms were expected to be observed in B



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Figure 2. Effects of griseusrazin A (1) on nitrite (A) and PGE2 (B) production and iNOS (C) and COX-2 (D) expression in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were pretreated for 6 h at the indicated concentrations of 1 and stimulated for 24 h with LPS (500 ng/mL). The concentrations of nitrite (A) and PGE2 (B) were determined. Western blotting analysis was performed, and the representative blots of three independent experiments are shown. The data represent the mean ± SD of three experiments. *p < 0.05 compared with the LPS-treated group.

Figure 3. Effects of griseusrazin A (1) on LPS-induced production and mRNA expression of pro-inflammatory cytokines in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were pretreated with the indicated concentrations of 1 for 6 h and then treated with LPS (500 ng/mL) for 24 h. The concentrations of TNF-α (A), IL-1β (B), and IL-6 (C) production were determined as described in the Experimental Section. TNF-α (D), IL-1β (E), and IL-6 (F) mRNA expression levels were determined by real-time PCR. Data represent mean ± SD of three experiments. *p < 0.05 compared with the LPS-treated group.

to suppress the induction of pro-inflammatory enzymes, mediators, and cytokines. Effect of Griseusrazin A (1) on HO-1 Expression via the Nrf2 Signaling Pathway. It has been reported that HO-1 has both antioxidative and anti-inflammatory activities in various experimental models.10,12 The anti-inflammatory activity of HO1 in activated macrophages is associated with the inhibition of

a significant increase in the DNA binding activity of NF-κB was observed in the cells treated with LPS alone (Figure 4B). However, pretreatment with griseusrazin A (1) dose-dependently suppressed the increase of the DNA binding activity induced by LPS treatment. These results suggested that the NFkB pathway would be the upstream target for griseusrazin A (1) C



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mediated suppression of pro-inflammatory responses, RAW264.7 cells were pretreated with 1, and the expression levels of HO-1 mRNA and protein were evaluated. When the cells were pretreated with 1 (5−40 μM) for 6 h, significantly enhanced expressions of HO-1 mRNA and protein were observed (Figure 5A and B). Nuclear-factor-E2-related factor 2 (Nrf2) is a crucial modulator for the induction of phase II enzyme genes, including HO-1. During induction, Nrf2 is detached from Keap1 protein and subsequently translocates into the nucleus. In the nucleus, Nrf2 binds to cis-acting antioxidant response elements and upregulates the transcription of inducible protective genes.26−28 Therefore, we assessed the effect of 1 on the nuclear translocation of Nrf2. The cells were incubated with 1 for 0 to 120 min with 20 min increments at the level of 40 μM, and the results of Western blot analysis indicated that the level of nuclear Nrf2 was gradually increased, with a concomitant decrease in the cytoplasmic levels (Figure 5C). This supports the hypothesis that Nrf2-mediated HO-1 expression is closely related to the antiinflammatory activity of 1 that was shown to inhibit the production of pro-inflammatory mediators derived from the NFκB pathway. To verify the correlation between HO-1 induction and the anti-inflammatory activity of 1, we tested the influence of tin protoporphyrin (SnPP), a competitive inhibitor of HO-1 activity, on the suppressive effects of 1 against the production of proinflammatory mediators and cytokines in LPS-induced cells. Therefore, macrophages were pretreated with 1 (40 μM) for 6 h in the presence or absence of SnPP followed by LPS stimulation. As shown in Figure 6, pretreatment of SnPP partially reversed the suppressive effects of 1 against the production of proinflammatory mediators and cytokines in the LPS-stimulated cells. These results further suggested that the anti-inflammatory activity of 1 is linked to the finding that 1 effectively stimulates the HO-1 expression pathway.

Figure 4. Effects of griseusrazin A (1) on the translocation of NF-κB p50 and p65 (A) and NF-κB DNA binding activity (B) in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were pretreated for 6 h at the indicated concentrations of 1 and stimulated for 30 min with LPS (500 ng/mL). Western blotting analysis was performed for NF-κB in nuclear fractions (A). A commercially available NF-κB ELISA (Active Motif) was used to determine the degree of NF-κB binding in nuclear extracts (B). The data represent mean ± SD of three experiments. *p < 0.05 compared with the LPS-treated group.

pro-inflammatory chemokine and cytokine productions.24 HO-1 and CO can also suppress the production of NO and PGE2 by reducing the expression of corresponding pro-inflammatory enzymes (i.e., iNOS and COX-2).25 Therefore, the upregulation of HO-1 expression could be regarded as one of advantageous approaches for the control of oxidative injury and macrophage stimulation in inflammatory diseases. To evaluate whether induction of HO-1 plays a key role in the griseusrazin A (1)-

Figure 5. Effects of griseusrazin A (1) on HO-1 mRNA (A), protein expression (B), and Nrf2 nuclear translocation (C). RAW264.7 macrophages were incubated for 12 h with the indicated concentrations of 1. Murine peritoneal macrophages were treated with 40 μM 1 for 20, 40, 80, 100, and 120 min. The nuclei were separated from the cytosol using PER-Mammalian Protein Extraction buffer. Representative blots from three independent experiments are shown. The data represent the mean ± SD of three experiments. *p < 0.05 compared with the control group. D



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Figure 6. Effects of SnPP on the inhibition of nitrite (A), PGE2 (B), TNF-α (C), IL-1β (D), and IL-6 (E) production and iNOS and COX-2 (F) expression by griseusrazin A (1) pretreatment on LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were pretreated for 6 h with 1 (40 μM), in the presence or absence of SnPP (50 μM), and subsequently stimulated for 24 h with LPS (500 ng/mL). The nitrite (A), PGE2 (B), TNF-α (C), IL-1β (D), and IL-6 (E) concentrations were determined as described in the Experimental Section. The data represent the mean ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.05 compared with the group treated with LPS alone; #p < 0.05 compared with the group treated with 1 and LPS. previously,29 except that ribonuclease T1 was used in addition to ribonuclease A. The 16S rRNA gene was amplified by PCR using two u n iv e rs a l p r i m e rs , fo rw a r d p r i m e r ( 5 ′ - G A G T T T G A T CCTGGCTCAG-3′) and reverse primer (5′-AGAAAGGAGGTGATCCAGCC-3′), as described previously.30 Sequencing of the amplified 16S rRNA gene was performed as described previously.31 Alignment of sequences was carried out with CLUSTAL W software.32 Gaps at the 5′ and 3′ ends of the alignment were omitted from further analysis. The phylogenetic tree was inferred by using the neighborjoining33 method in the PHYLIP package.34 Evolutionary distance matrices for the neighbor-joining method were calculated using the algorithm of Jukes and Cantor35 with the DNADIST program. The stability of relationships was assessed by a bootstrap analysis based on 1000 resamplings of the neighbor-joining data set using the programs SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE of the PHYLIP package. The almost complete 16S rRNA gene sequence of the strain 09-0144 determined in this study comprised 1442 nucleotides, corresponding to approximately 96% of the Escherichia coli 16S rRNA sequence. In the phylogenetic tree constructed using the neighborjoining algorithm, the strain 09-0144 fell within the clade comprising Streptomyces species, joining the type strain of Streptomyces griseus subsp. griseus by a bootstrap confidence value of 61.9%. The strain 09-0144 exhibited the highest 16S rRNA gene sequence similarity value (99.6%) to Streptomyces griseus subsp. griseus KCTC 9080T and a 16S rRNA gene sequence similarity value of 98.7−99.5% to the type strains of the other Streptomyces species used in the phylogenetic analysis. The 16S rRNA gene sequence of strain 09-0144 has been deposited in the GenBank database under accession number KU668703. Fermentation, Extraction, and Isolation. The 09-0144 strain was cultured on 60 Petri dishes (43 mm), each containing 20 mL of the culture media (soluble starch 10.0 g, peptone 4.0 g, yeast extract 2.0 g, calcium carbonate 1.0 g, agar 15.0 g) with 1.0 L of artificial seawater. Plates were individually inoculated with 2 mL of seed cultures of the bacterial strain. Plate cultures were incubated at 25 °C for a period of 14 days. Extraction of the agar media with EtOAc (4 × 1 L) provided an organic phase, which was then concentrated in vacuo to yield 379.4 mg of an extract. The EtOAc extract was subjected to C18 flash column chromatography (4 × 25 cm), eluting with a stepwise gradient of 20%, 40%, 60%, 80%, and 100% (v/v) MeOH in H2O (400 mL each). The

In summary, our results demonstrate that griseusrazin A (1) possesses anti-inflammatory activity that is associated with its inhibitory effect against the activation of the NF-κB pathway in LPS-stimulated cells. Furthermore, it was shown that the antiinflammatory activity of 1 is correlated with its inductive effect on the expression of HO-1 through the Nrf2 signaling pathway.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on a Biochrom 1300 UV/visible spectrophotometer. IR spectra were recorded on a Spectrum GX FT-IR system (PerkinElmer). NMR spectra (1D and 2D) were recorded in acetone-d6 using a JEOL JNM ECP-400 spectrometer (400 MHz for 1H and 100 MHz for 13C), and the chemical shifts were referenced relative to the residual solvent peaks (δH/δC = 2.05/29.8). HSQC and 13C-HMBC experiments were optimized for 1 JCH = 140.0 Hz and nJCH = 8.0 Hz, respectively. 15N-HMBC experiments was optimized for 1JNH = 90.0 Hz and nJNH = 8.0 Hz, respectively. ESIMS data were obtained using a Q-TOF micro LC-MS/ MS instrument (Waters) at Korea University, Seoul, Korea. Flash column chromatography was carried out using YMC octadecylfunctionalized silica gel (C18). HPLC separations were performed on a preparative-C18 column (21.2 × 150 mm; 5 μm particle size) with a flow rate of 5 mL/min. Compounds were simultaneously detected by UV absorption at 210 and 254 nm. Solvents for extractions and flash column chromatography were reagent grade and used without further purification. Solvents used for HPLC were analytical grade. Strain Isolation and Identification. Streptomyces griseus subsp. griseus strain 09-0144 was isolated from tidal flat sediments on Hwangdo in the Yellow Sea, South Korea, in April 2010. The sample was serially diluted by using sterile 0.85% (w/v) NaCl, and 1 mL of the diluted sample was spread on ISP4 agar (Difco) with artificial seawater (NaCl 23.6 g, KCl 0.64 g, MgCl2·6H2O 4.53 g, MgSO4·7 H2O 5.94 g, CaCl2· 2H2O 1.3 g). The agar plate was incubated at 30 °C for 10 days, and colonies that appeared were streaked onto fresh agar plates to obtain pure cultures. The isolates were suspended in 20% (w/v) glycerol and preserved at −70 °C. The strain 09-0144 was identified based on phylogenetic analysis of the 16S rRNA gene sequence. Chromosomal DNA was isolated and purified according to the method described E



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fractions that eluted with 60% MeOH (36.6 mg) were combined and purified by semipreparative reversed-phase HPLC, eluting with a gradient from 55% to 100% MeOH in H2O (0.1% formic acid) over 60 min to yield griseusrazin A (1, 2.3 mg, tR = 28 min). Griseusrazin A (1): yellow solid; UV (MeOH) λmax (log ε) 239 nm (4.3), 282 nm (4.0); IR (KBr) νmax 3428, 2923, 1670, 1604, 1515, 1414, 1384, 1348, 1115 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 403.2116 [M + H]+ (calcd for C24H27N4O2, 403.2134).

analysis was performed using GraphPad Prism software, version 3.03 (GraphPad Software Inc.).

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* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00009. 1D- and 2D-NMR and HRESI mass spectra for compound 1 (PDF)

Table 1. NMR Data for Griseusrazin A (1) in Acetone-d6 δC,a type

position

HMBC (H → C#)

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c

151.6, C 149.4,c C 40.7, CH2 137.3, C 130.6, CH 122.8, CH 141.8, C 162.3, CH 31.5, CH3 21.5, CH3

2/5 3/6 1′ 2′ 3′/7′ 4′/6′ 5′ 8′ 9′ 1″ a

δH, mult. (J in Hz)b

b

4.16, s

2, 3, 2′, 3′/7′

7.32, d (8.4) 7.22, d (8.4)

1′, 3′/7′, 5′ 2′, 3′/7′, 5′

8.44, s 3.22, s 2.48, s

9′ 5′, 8′ 2, 3

ASSOCIATED CONTENT

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +82-63-852-8837. Tel: +82-63-850-6815 (H. Oh). Author Contributions ⊥

D.-S. Lee and C.-S. Yoon contributed equally to this work.

Notes

The authors declare no competing financial interest.

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c

100 MHz. 400 MHz. Interchangeable.

ACKNOWLEDGMENTS This work was financially supported by a grant from Wonkwang University (2014). The strain used in this study was obtained from the Program for Collection, Management and Utilization of Biological Resources (grant NRF-2013M3A9A5075953) from the Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea.

Cell Culture. RAW264.7 macrophages were maintained at a density of 5 × 105 cells/mL in DMEM medium with supplement and were incubated according to the method described previously.36 Preparation of Nuclear and Cytosolic Fractions. Cells were homogenized (1:20, w:v) in PER-Mammalian Protein Extraction buffer (Pierce Biotechnology) containing freshly added protease inhibitor cocktail I (EMD Biosciences) and 1 mM phenyl methyl sulfonyl fluoride. The details of procedures for the preparation of nuclear and cytosolic fractions are described elsewhere.10 Western Blot Analysis. Cells were harvested by centrifugation at 200g for 3 min. Subsequently, the cells were washed with PBS and lysed using RIPA lysis buffer containing 25 mM Tris-HCl buffer (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS. The details of Western blot analysis are described elsewhere.10 Quantitative Real-Time Polymerase Chain Reaction (qRTPCR). Total RNA was isolated from the cells using Trizol (Invitrogen), in accordance with the manufacturer’s recommendations, and quantified spectrophotometrically at a wavelength of 260 nm. Total RNA (4 μg) was reverse-transcribed using a High Capacity RNA-to-cDNA kit (Applied Biosystems). The cDNA was then amplified using a SYBR Premix Ex Taq kit (TaKaRa Bio Inc.) and a StepOnePlus real-time PCR system (Applied Biosystems). The primer sequences were as follows: IL-1β (forward 5′-AATTGGTCATAGCCCGCACT-3′, reverse 5′AAGCAATGTGCTGGTGCTTC-3′), IL-6 (forward 5′-ACTTCACAAGTCGGAGGCTT-3′, reverse 5′-TGCAAGTGCATCATCGTTGT3′), TNF-α (forward 5′-CCAGACCCTCACACTCACAA-3′, reverse 5′- ACAAGGTACAACCCATCGGC-3′), HO-1 (forward 5′CTCTTGGCTGGCTTCCTT-3′, reverse 5′-GGCTCCTTCCTCCTTTCC-3′), and GAPDH (forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′). Determination of Nitrite, PGE2, TNF-α, IL-1β, and IL-6. The production of nitrite in conditioned media was determined according to the method described previously.24 Levels of PGE2, TNF-α, IL-1β, or IL-6 present in each sample were determined using a commercially available kit from R&D Systems. The assay was performed according to the manufacturer’s instructions as described previously.24 DNA Binding Activity of NF-κB. The DNA binding activity of NFκB in nuclear extracts was measured using a TransAM kit (Active Motif) according to the manufacturer’s instructions as described previously.12 Statistical Analysis. The data were expressed as the mean ± standard deviation (SD) of at least three independent experiments. To compare three or more groups, one-way analysis of variance (ANOVA) followed by the Newman−Keuls post hoc test was used. Statistical

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