Anti-inflammatory effect of Papenfussiella kuromo in lipopolysaccharide or peptidoglycan-induced macrophage cells

1 College of Pharmacy and Wonkwang-Oriental Medicines Research Institute, Wonkwang University, Iksan, Jeonbuk, 570-749, Korea. 2 Department of Oriental Medicine Resources, College of Bio Industry Science, Sunchon National University, Sunchon, Jeonnam, 540-742, Korea. 3 Faculty of Biological Science and Institute of Biotechnology, Wonkwang University, Iksan, Jeonbuk, 570-749, Korea. 4 Department of Oriental Medical Food & Nutrition, Semyung University, Jecheon, Chungbuk, 390-711, Korea.


INTRODUCTION
The ocean, which is called 'the mother of origin of life', is the source of structurally unique natural products that are mainly accumulated in living organisms.Several of these *Corresponding author.E-mail: sssimi@wonkwang.ac.kr.compounds show pharmacological activities and are helpful in the invention and discovery of biologically active compounds.Especially, algal are known to produce a lot of secondary metabolites including bioactive compounds with different activities (González et al., 2001).Now, the screening of algal extracts for biologically active compounds makes rapid progress with simple antibiotic assays and expanded to include testing for biologically active products.Compounds with antiviral, antibacterial, antifungal, anti-mitotic, and antitumorigenic activities have been detected in green, brown and red algae (Zhu et al., 2006;Oh et al., 2008;Bennamara et al., 1999).Papenfussiella kuromo is a brown alga belonging to the family Chordariaceae (order Ectocarpales, genus Papenfussiella).It is widely distributed in Japan and the eastern seaboard, southern coast, Jeju island in Korea.However, there is yet no report regarding P. kuromo inducing anti-inflammatory action.
Bacteria stimulate the innate immune system of a host and the release of inflammatory molecules such as cytokine and chemokines as a response to infections (Medzhitov and Janeway, 1998;Hoffmann et al., 1999).Lipopolysaccharide (LPS) is a well-known activator of the innate immune system in Gram-negative infections (Dziarski et al., 2000).Peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, is an alternating βlinked N-acetylmuramyl and N-acetylglucosaminyl glycan whose residues are cross-linked by short peptides (Bone, 1994;Ulevitch, 1995).Like LPS as a cell wall component of Gram-negative bacteria, PGN induces most of the clinical manifestations of bacterial infections, including inflammation, fever, septic shock, etc (Schleifer and Kandler, 1972).Most of these effects are due to the activation of macrophages and generation of proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 (Wang et al., 2001;Xu et al., 2001;Bhakdi et al., 1991;Mattson et al., 1993).An inflammatory response implicates macrophages, which secrete a number of mediators (oxidants, cytokine, lytic enzymes, etc.) responsible for initiation, progression and persistence of acute or chronic state of inflammation (Kim et al., 2004).These inflammatory mediators are involved in the pathogenesis of many inflammation-associated human diseases (Simons et al., 1996;Glombitza and Koch, 1989;Ritchlin et al., 2003).
The aim of this study was to examine P. kuromo antiinflammatory effect in PGN-induced or LPS-induced RAW 264.7 macrophage cells activation.Understanding the underlying mechanism related to the inhibition of inflammatory reaction by P. kuromo will be of benefit by adding to the potential new source of drug for the treatment of inflammatory diseases.

Plant and extract
P. kuromo was collected by Dr. H.G. Choi.P. kuromo was air-dried in the dark at room temperature and then ground into a powder using a mechanical grinder.Approximately 500 g of the powdered materials was then extracted in 1500 ml of ethanol for 7 day (room temperature).EtOH extract yield is 42.9858 g.The extract was filtered (pore size, 0.45 μm), lyophilized, and kept at 4°C.The dried extract was then dissolved in phosphate buffered saline (PBS) in preparation for use.A sample of the P. kuromo has been deposited at the Herbarium of the College of Pharmacy, Wonkwang University, Iksan.

Cell culture
The murine macrophage cell line, RAW 264.7, was obtained from the Korea Research Institute of Bioscience and Biotechnology (Seoul, Republic of Korea) and grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 100 U/ml of penicillin/streptomycin sulfate.The cells were incubated in a humidified 5% CO2 atmosphere at 37°C.To stimulate the cells, the medium was replaced with fresh RPMI 1640 medium, LPS or PGN were added in the presence or absence of P. kuromo for the indicated periods.

MTS assay for cell viability
The cell viability was determined by MTS assay.RAW 264.7 cells were plated at a density of 5 × 10 4 cells/well in 96-well plates (Nunc, Denmark).Each experiment included a non-treated group as control.P. kuromo (10, 50 and 100 μg/ml) was then added to each well, after which the plates were incubated for 24 h at 37°C under 5% CO2.MTS solutions (5 mg/ml) were added to each well and the cells were cultured for another 2 h, after which the optical density was read at 490 nm.Cytotoxicity was then calculated using the formula: 1 -(mean absorbance value of treated cells / mean absorbance value of untreated cells).

Enzyme-linked immunosorbent assay (ELISA)
Cells were seeded at 5 × 10 5 ml -1 per well in 24-well tissue culture plates and pretreated with various concentrations of P. kuromo (100 or 50 μg/ml) for 30 min before LPS (200 ng/ml) or PGN (30 μg/ml) stimulation for 24 h.ELISA plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) were coated overnight at 4°C with anti-mouse IL-6 antibody diluted in coating buffer (0.1 M carbonate, pH 9.5) and were then washed three times with PBS containing 0.05% Tween 20.The nonspecific protein binding sites were blocked with assay diluent (PBS containing 10% FBS, pH 7.0) for at least 1 h.Immediately, each sample and IL-6 standard were added to the wells.After incubation for 2 h, a working detector (biotinylated anti-mouse IL-6 monoclonal antibody and streptavidin-horseradish peroxidase reagent) was added and incubated for 1 h.Accordingly,

cDNA
Primer sequence

Measurement of PGE2 production
The RAW 264.7 cells were cultured in 24-well culture plates (5 × 10 5 ml -1 ).P. kuromo (100 or 50 μg/ml), LPS (200 ng/ml) and PGN (30 μg/ml) were added to the culture medium and incubated at 37°C for 24 h.The medium was collected in a microcentrifuge tube and was centrifuged.The supernatant was decanted into a new microcentrifuge tube, and the amount of PGE2 was determined by a PGE2 Enzyme Immuno-Assay Kit (Amersham Biosciences, Little Chalfont, UK) according to the procedure described by the manufacturer.

Measurement of nitrite oxide (NO) production
NO production was assayed by measuring the nitrite in the supernatants of cultured RAW 264.7 cells.The cells were seeded at 1 × 10 5 ml -1 in 96-well culture plates.After preincubation of the RAW 264.7 cells for 18 h, the cells were pretreated with P. kuromo (100 or 50 μg/ ml) and stimulated with LPS (200 ng/ml) or PGN (30 μg/ml) for 24 h.The supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid) and it was incubated at room temperature for 5 min.The concentrations of nitrite were measured by reading at 570 nm.Sodium nitrite (NaNO2) was used to generate a standard curve.

Western blot analysis
Protein expression was assessed by western blot analysis according to standard procedures.The RAW 264.7 cells were cultured in 60-mm-diameter culture dishes (3 × 10 6 ml -1 ) and were pretreated with various concentrations of P. kuromo (100 or 50 μg/ml).After 30 min or 1 h, LPS (200 ng/ml) or PGN (30 μg/ml) was added to the culture medium, and the cells were incubated at 37°C.After incubation, the cells were washed twice in ice-cold PBS (pH 7.4).The cell pellets were re-suspended in lysis buffer on ice for 15 Kang et al. 2793 min, and the cell debris was removed by centrifugation.The protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions.Equal amounts of protein (20 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were then transferred onto a polyvinylidene membrane (Millipore, Bedford, MA).
The membrane was blocked with 5% nonfat milk in Tris buffered saline with Tween 20 buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.05% Tween 20, pH 7.4).After blocking, the membrane was incubated with primary antibodies for 18 h.The membrane was then washed with Tris buffered saline with Tween 20 and was incubated with anti-mouse or anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies.Immunoreactivity was detected using enhanced chemiluminescence (Amersham, Milan, Italy).
The conditions for amplification were as follows: denaturation at 94°C for 3 min for the first cycle and for 30 s starting from the second cycle, annealing of IL-6 at 57°C for 45 s, annealing of COX-2 at 53°C for 30 s and annealing of iNOS at 56°C for 30 s, and extension at 72°C for 90 s for 35 cycles.Final extension was performed at 72°C for 7 min.PCR products were electrophoresed on 2% agarose gel and were stained with ethidium bromide.The primers used are tabulated in Table 1.

Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett's t-test for multiple comparisons (Lee et al., 2006).The data from the experiments are presented as means ± standard error of mean (SEM) (n=5).The numbers of independent experiments assessed are given in the figure legends.

Effects of P. kuromo on cytotoxicity in RAW 264.7 cells
The cytotoxicity of P. kuromo was evaluated in the RAW264.7 cells by an MTT assay.The results of P. kuromo were found not to affect viability of the RAW 264.7 cells for 24 h (Figure 1).

Effects of P. kuromo on LPS-induced or PGN-induced NO production
To assess the effects of P. kuromo on the LPS-induced or PGN-induced NO production in RAW 264.7 cells, cell culture media were harvested and NO levels were measured.P. kuromo inhibited NO production dosedependently in LPS stimulated RAW 264.7 cells.Also, P. kuromo inhibited NO production in PGN-stimulated RAW 264.7 cells in a dose-dependent (Figure 2).

Effects of P. kuromo on LPS-induced or PGN-induced PGE
2 production To assess the effects of P. kuromo on the LPS-or PGNinduced PGE 2 production in RAW 264.7 cells, cell culture media were harvested and PGE 2 levels were measured.To examine whether P. kuromo inhibits PGE 2 production, cells were pre-incubated with P. kuromo for 1 h and then activated with LPS (200 ng/ml) or PGN (30 μg/ml) for 24 h.As shown in Figure 3, P. kuromo inhibited the production of PGE 2 in a dose-dependent manner.

Effects of P. kuromo on LPS-induced or PGN-induced iNOS mRNA and COX-2 mRNA expression
To assess the effects of P. kuromo on LPS-induced or PGN-induced mRNA expression of iNOS and COX-2, RAW 264.7 cells were evaluated to identify the antiinflammatory mechanism.iNOS was strongly expressed in cells that were treated by LPS or PGN, and RT-PCR analysis showed that iNOS mRNA expression was related to the nitrite levels (Figure 4A).Also, as shown in Figure 4B, COX-2 mRNA expression was undetectable in unstimulated RAW 264.7 cells (control).However, in response to LPS or PGN, COX-2 is strongly expressed and P. kuromo significantly inhibited COX-2 expression in a dose-dependent manner.

Effects of P. kuromo on LPS-induced or PGN-induced IL-6 production and IL-6 mRNA expression
Since P. kuromo was found to inhibit the proinflammatory mediators, we investigated the effects of P. kuromo on LPS-or PGN-induced IL-6 production by ELISA.Pretreatment of P. kuromo reduced IL-6 production in a dose-dependent manner (Figure 5A).Since P. kuromo was found to inhibit the proinflammatory mediators, we investigated the effects of P. kuromo on LPS-or PGN-induced IL-6 expression by RT-PCR.Pretreatment of P. kuromo reduced IL-6 mRNA expression in a dose-dependent manner (Figure 5B).

Effects of P. kuromo on the phosphorylation of mitogen-activated protein kinase (MAPKs) in LPSinduced or PGN-stimulated RAW 264.7 cells
MAPKs are essential for LPS-induced iNOS expression to occur in RAW 264.7 cells.Therefore, the effects of P. kuromo on the activation of MAPKs in LPS-or PGNstimulated RAW 264.7 cells were evaluated.As shown in Figure 6, P. kuromo markedly inhibited the activating phosphorylation of ERK 1/2, whereas phosphorylation of JNK 1/2 and p38 MAPK was unaffected by treatment with P. kuromo (data not shown).Taken together, these results indicate that ERK 1/2 phosphorylation was Kang et al. 2795 inhibited by P. kuromo pretreatment.

DISCUSSION
Recently, many studies have evaluated the inhibitory effects of plant-derived anti-inflammatory agents in vitro.However, marine bio-resources are not sufficiently investigated in term of their full therapeutical option.Marine algae produce various metabolites and have been recognized as promising targets in the search for biologically active compounds.So far, we have performed the screening studies on marine edible algae which could serve as important resources in the discovery of antiinflammatory activities.P. kuromo is a brown alga belonging to Chordariaceae, and is widely distributed along the shore of Korea, Japan.However, no studies conducted to date have reported the bioactivity by which the anti-inflammatory action of ethanol extract of P. kuromo occurs.In this study, we evaluated the pharmacological basis for P. kuromo for the treatment of various inflammatory diseases.The effects of P. kuromo on macrophage functions related to inflammation were investigated to verify possible mechanisms underlying its beneficial effects.LPS and PGN, the toxicants from bacteria, are potent inducers of inflammatory cytokines, such as TNF-α, IL-6 and IL-8.Although, PGN is much less investigated in comparison to LPS, PGN is regarded as the Grampositive equivalent to LPS in some aspects.We investigated PGN-induced signal transduction and biological effects, as well as compare the effect of PGN with that of LPS.We demonstrated that P. kuromo inhibited LPS-or PGN-induced pro-inflammatory mediators, including NO and PGE 2 .Especially, NO is an important mediator in the inflammatory process and is produced at inflamed sites by iNOS.High levels of NO have been reported in a variety of pathological processes including various forms of inflammation, circulatory shock, and carcinogenesis (MacMicking et al., 1997;Ohshima and Bartsch, 1994;Szabó, 1995).The activity of iNOS can be controlled by regulating its synthesis via activating intracellular signaling transduction.The effect of LPS signaling on iNOS gene expression has been investigated (Vallance and Leiper, 2002;Bronte and Zanovello, 2005).Therefore, an inhibitor of iNOS might be effective as a therapeutic agent for inflammatory diseases (Koo et al., 2001).The results of this study showed that P. kuromo inhibited LPS-or PGN-induced NO production in RAW 264.7 macrophages.To further investigate the mechanism underlying these inhibitions by P. kuromo, the expression of iNOS mRNA levels was examined by RT-PCR, respectively, which revealed that P. kuromo reduced iNOS mRNA expression.Taken together, these results indicate that P. kuromo has a potent antiinflammatory effect that occurs through the inhibition of the expression of iNOS and NO production.To explore the mechanism of inhibition of NO and PGE 2 production production in RAW 264.7 cells, the effects of P. kuromo on the iNOS and COX-2 gene were examined.P. kuromo inhibited the expression of COX-2 and iNOS mRNA in a dose-dependent manner, as assessed by RT-PCR, respectively.These results imply that P. kuromo exerts its effects through the inhibition of the iNOS and COX-2 transcription.
Pharmacological studies using MAPK-specific inhibitors experimented to show that the MAPKs (ERK, p38 and ERK) pathway is required for LPS-induced NO production and iNOS expression in RAW 264.7 macrophages.According to published reports, MAPKs have been implicated in the transcriptional regulation of the iNOS gene and specific MAPK inhibitors suppress the expression of the iNOS gene (Chen et al., 1999;Chan and Riches, 1998).Also, MAPKs play a critical role in the regulation of cell growth and differentiation, particularly in response to cytokine and stress (Johnson and Lapadat, 2002).Several studies have demonstrated that MAPKs are involved in LPS-induced iNOS expression (Kang et al., 2007;Chen et al., 1999;Kim et al., 2004).However, the role of MAPK activation in PGN-induced iNOS/NO production is still unclear.So, the effects of P. kuromo on the LPS-and PGN-induced phosphorylation of MAPKs were evaluated in this study.Interestingly, PKpretreatment macrophages inhibited ERK 1/2 phosphorylation, but not JNK 1/2 and p38 MAPK phosphorylation in both LPS-and PGN-stimulation.This suggests that P. kuromo inhibition of iNOS/NO production might not be mediated by blocking ERK signals for iNOS gene expression in RAW 264.7 cells.In addition, the results show similarity and difference between Gram-negative bacteria and Gram-positive bacteria, and mediated signal transduction and induction of inflammatory cytokines in macrophages.

Conclusion
To conclude, we have shown that P. kuromo inhibited LPS-or PGN-induced IL-6, NO and PGE 2 productions, as well as iNOS and COX-2 expressions in macrophages and inhibited phosphorylation of ERK 1/2.Thus, from this study, data have been generated to show that the antiinflammatory effect of P. kuromo can possibly be used as a therapeutic agent against Gram-negative and Grampositive bacteria.The fact that P. kuromo has better effect on PGN, its anti-inflammatory activity will be greater against pathogens like methicillin-resistant Staphylococcus aureus (MRSA).Therefore, it is possible after further research, that P. kuromo could be used against septicemia.Taken together, these findings indicate that P. kuromo may represent a potential new source of drugs for the treatment of inflammatory disease.

Figure 1 .Figure 2 .
Figure 1.Effects of P. kuromo on cell viability in RAW 264.7 cells.Cell viability was evaluated using the MTS assay.Data represent the mean (SE) of duplicate measurements from 5 separate experiments (n = 5).

Figure 3 .Figure 4 .
Figure 3. Effects of P. kuromo on LPS-induced or PGNinduced PGE2 production in RAW 264.7 cells.RAW 264.7 cells were pretreated with the indicated concentration of PK for 1 h before being incubated with LPS (200 ng/ml) or PGN (30 μg/ml) for 24 h.Control cells were incubated with the vehicle alone.*P < 0.05 compared with the LPS or PGN treated group.Data shown were representative of a total of six experiments (n = 5).

Figure 5 .Figure 6 .
Figure 5. Effects of P. kuromo on LPS-induced or PGN-induced IL-6 production and IL-6 mRNA expression.(A) RAW 264.7 cells were pretreated with the indicated concentrations of PK for 1 h before being incubated with LPS (200 ng/ml) or PGN (30 μg/ml) for 24 h.Production of IL-6 was measured by ELISA.The experiment was repeated three times and similar results were obtained.Results are mean ± SE.Statistical significance: *P < 0.05 compared with the LPS or PGN treated group.(B) IL-6 mRNA was assessed by RT-PCR in RAW 264.7 cells.Cells were pretreated with the indicated concentrations of P. kuromo for 1 h before being incubated with LPS (200 ng/ml) or PGN (30 μg/ml) for 24 h.The β-actin mRNA was carried out in parallel to confirm equivalency of cDNA preparation.The experiment was repeated five times and similar results were obtained (n = 5).

Table 1 .
Sequences of oligonucleotide primers designed for RT-PCR.