(LO) is a member of the Lythraceae family (10,11). This tree species has commercial value. It is used for medium-heavy construction (door, window frames), bridge and boat building, various agriculture implements and tool handles. LO has traditionally been used as an herbal medicine in Java without distinction of related species (e.g., L. speciosa) without distinction of species; the bark for diarrhea, the leaves for malaria and dermatosis (12,13). The effects and biological activity of the methanolic extract of LO (LOME) on inflammation, as well as the mechanisms of action responsible for these effects remain largely unknown. To date, this plant has shown few and unclear effects to the best of our knowledge. In the present study, we examined the anti-inflammatory effects of LOME in various experiments. We used an LPS-stimulated, murine macrophage cell line, RAW264.7, as a model of inflammation to determine the effects of LOME. The binding of LPS to downstream signaling cascades, including mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways (14), led to the production of inflammatory mediators from macrophages, such as TNF-α, IL-1β, IL-6, PGE₂ and NO. Our results indicated that LOME inhibited NO, PGE₂ and the pro-inflammatory cytokines in LPS-stimulated macrophages through NF-κB signaling, as well as the MAPK, extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK)1/2 pathways. These results provide evidence for the use of LOME as an anti-inflammatory treatment.

LO was collected from the Ujung Kulon National Park, in the province of Banten, Indonesia. Plant samples were collected and identified by staff at the Center for Pharmaceutical and Medical Technology (PTFM; Tangerang, Indonesia), and verified at the Herbarium Bogoriense (LIPI; Bogor, Indonesia). Voucher specimens recorded as KRIB 0038535 and PMT 537, have been deposited in the herbarium (KRIB) of the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) as well as in the Center for Pharmaceutical and Medical Technology (PTFM) and the Herbarium Bogoriense. After drying and grinding the leaves of LO, the powder (10.8 kg) was added to methanol (100 liters). The extraction was performed using the method of repercolation at room temperature. The extract was filtered and concentrated by a rotary evaporator (Rotavapor 4000; Heidolph, PT AbadiNusa Usahasemesta, Jakarta) under reduced pressure, to obtain 1,030 g LOME. In subsequent experiments, LOME was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/ml, and then diluted to various concentrations prior to use.

The murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% antibiotic-antimycotic solution (Invitrogen, Grand Island, NY, USA). The RAW264.7 cells were maintained by weekly passage, and the cells were utilized for experimentation at 60–80% confluence. Following the incubation of the RAW264.7 cells for 4 h, 0–30 µg/ml LOME was added.

Cell viability was determined by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco, LLC, Solon, OH, USA) to purple formazan. Briefly, the RAW264.7 cells were seeded into each well of 96-well plates at a density of 1×10⁴ cells/well. After 4 h, the cells were treated with LOME for 24 h. MTT solution (5 µl; 5 mg/ml) was then added to each well. After a 4 h incubation at 37°C with 5% CO₂, the supernatant was removed and the formazan crystals formed by the viable cells were dissolved in 100 µl DMSO. The absorbance of each well was read at 570 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Based on the results of the cytotoxicity assay, we used a nontoxic concentration of LOME on the RAW264.7 cells in subsequent experiments.

Nitrite concentration in the medium was measured as an indicator of NO production according to the Griess reaction method, as previously described (15). The RAW264.7 cells were plated at a density of 2.5×10⁵ cells/ml in 96-well plates, and then incubated with or without LPS (0.5 µg/ml) in the absence or presence of various concentrations of LOME for 24 h. The nitrite accumulation in the supernatant was assessed by a Griess reaction. Each 100 µl of the culture supernatant was mixed with an equal volume of the Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine and 1% sulfanilamide in 5% phosphoric acid] and incubated at room temperature for 10 min. The absorbance was measured at 540 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc.) and a series of known concentrations of sodium nitrite were used as a standard.

The RAW264.7 cells were plated at a density of 2.5×10⁵ cells/ml in a 96-well culture plate. The cells were incubated with various concentrations (5, 10, 20 or 30 µg/ml) of LOME for 1 h, followed by LPS treatment (0.5 µg/ml) for 24 h. Following a 24 h incubation, the PGE₂ concentration in the culture media was quantified using an enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to manufacturer's instructions.

The levels of IL-6, IL-1β and TNF-α were determined using commercial ELISA kits. IL-6, IL-1β and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The assay procedure for each kit was conducted according to the manufacturer's instructions. The concentrations of the mediators were determined at 450 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc.).

RT-PCR was performed to detect the mRNA expression of iNOS, COX-2, IL-6, IL-1β, TNF-α and β-actin. Briefly, after LPS (0.5 µg/ml) stimulation of the RAW264.7 cells for 6 h, total RNA was isolated using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. An RT reaction was perormed using a kit for producing cDNA (Qiagen GmbH, Hilden, Germany). PCR was performed using specific forward and reverse primers, and a premix according to the manufacturer's instructions (Bioneer Corporation, Daejeon, Korea). The PCR conditions for each PCR reaction were as follows: 94°C for 5 min (1 cycle), 94°C for 30 sec, 60°C for 30 sec, and 72°C for 45 sec (for 30 cycles); and then a final extension phase at 72°C for 10 min. The primer sequences were as follows: TNF-α sense, 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ and antisense, 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′; IL-1β sense, 5′-GTG TCT TTC CCG TGG ACC TT-3′ and antisense; 5′-TCG TTG CTT GGT TCT CCT TG-3′; IL-6 sense, 5′-AAC GAT GAT GCA CTT GCA GA-3′ and antisense, 5′-GAG CAT TGG AAA TTG GGG TA-3′; iNOS sense, 5′-CAA GAG TTT GAC CAG AGG ACC-3′ and antisense, 5′-TGG AAC CAC TCG TAC TTG GGA-3′; COX-2 sense, 5′-GAA GTC TTT GGT CTG GTG CCT G-3′ and anti-sense, 5′-GTC TGC TGG TTT GGA ATA GTT GC-3′; and β-actin sense, 5′-TGT TTG AGA CCT TCA ACA CC-3′ and antisense, 5′-CGC TCA TTG CCG ATA GTG AT-3′. β-actin expression was included as an internal, housekeeping gene control. The reaction products were separated by electrophoresis on a 1.5% agarose gel, stained with EtBr, and visualized by UV transillumination (CoreBioSystem, Seoul, Korea). The images were captured using an Olympus C4000 zoom camera system (C4000; Olympus America Inc., Melville, NY, USA).

The RAW264.7 macrophages were pretreated with the indicated concentrations of LOME or vehicle for 1 h and stimulated with LPS (0.5 µg/ml) for 20 min (phosphor) and 18 h (COX-2 and iNOS). Proteins in each sample (30 µg of total protein) were resolved on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel by SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and exposed to the appropriate antibodies. Each membrane was incubated for 1 h in 5% skim milk in TBS-T buffer (0.1 M Tris-HCl, pH 7.4, 0.9% NaCl and 0.1% Tween-20) to block non-specific binding, and then incubated with primary antibodies that recognized p65 (cat. no. sc-372, 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); iκBα (cat. no. 9242, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), iNOS (cat. no. ADI-905-431, 1:1,000; obtained from Enzo Life Sciences, Inc., Farmingdale, NY, USA); COX-2 (cat. no. sc-1747, 1:1,000; Santa Cruz Biotechnology, Inc.); β-actin (cat. no. 4967, 1:2,000) and PARP (cat. no. 9542) (both from Cell Signaling Technology, Inc.); the total forms of ERK2 (sc-154), p38 MAPK (sc-7149), JNK1/3 (sc-474) (1:1,000, all from Santa Cruz Biotechnology, Inc.); the phosphorylated forms of p38 MAPK (ADI-KAP-MA022) and JNK1/2 (KAP-SA011 (1:1,000; both from Enzo Life Sciences, Inc.); and the phosphorylated forms of ERK (9106, 1:1,000; Cell Signaling Technology, Inc.). Each protein was detected using an enhanced chemiluminescence (ECL) detection system (Bio-Rad Laboratories, Inc.).according to manufacturer's instructions

The RAW264.7 cells were cultured on Permanox plastic chamber slides (Nalge Nunc International, Rochester, NY, USA) and fixed in methanol at 4°C for 20 min. The slides were washed three times with phosphate-buffered saline (PBS) and blocked with 3% (w/v) BSA in PBS for a further 30 min. The slides were then incubated for 24 h at 4°C with anti-iNOS (rabbit polyclonal IgG, 1:200 dilution), and anti-NF-κB p65 subunit (rabbit polyclonal IgG, 1:200 dilution) (both from Santa Cruz Biotechnology, Inc.) antibodies. After washing to remove excess primary antibody, the slides were further incubated with anti-rabbit Alexa Fluor 488-conjugated antibody (Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature, washed with PBS, and mounted using ProLong Gold Antifade reagent containing 4′,6-diamidino-2-phenylin-dole (DAPI; Invitrogen, Carlsbad, CA, USA) for 5 min, prior to the localization and quantification of the nuclei. Subsequently, the slides were cover-slipped and visualized using a confocal laser scanning microscope (LSM510m; Carl Zeiss, Inc., Oberkochen, Germany). Images of the samples were captured under the same exposure conditions, and the nuclei were quantified from the images obtained.

For the statistical analyses, the values are expressed as the means ± SEM of the sample determinations. The statistical significance was determined using a two-tailed Student's t-test for independent means, and a P-value <0.05 was considered to indicate a statistically significant difference.

A prerequisite to studying the biological activity of LOME was to ensure that it did not exert a detrimental effect on cell metabolism. To determine whether LOME affected cell viability, the RAW264.7 cells were incubated for 24 h with the extract at a wide range of concentrations (0–30 µg/ml), and cell viability was evaluated using an MTT assay. A statistically significant decrease in cell survival was observed at a concentration of 30 µg/ml (Fig. 1).

As it is important to regulate the overproduction of NO, which induces pro-inflammatory responses in inflammatory disorders (16), we examined the effect of LOME on the regulation of NO production in LPS-stimulated macrophages. Prior to evaluating the regulatory effect of LOME in LPS-stimulated macrophages, we first confirmed that LOME did not induce NO production in the RAW264.7 cells. However, the pre-incubation of cells with LOME for 1 h prior to LPS stimulation resulted in reduced NO production compared with that in the LPS-stimulated RAW264.7 cells. Furthermore, NO production was gradually attenuated by increasing the concentration of LOME up to 30 µg/ml (Fig. 2A). To determine whether LOME reduces NO production by regulating protein expression, we measured the expression of iNOS in the RAW264.7 cells by RT-PCR and western blot analysis. According to this analysis, iNOS expression was downregulated in a dose-dependent manner (Fig. 2C and D). We further examined the effect of LOME, which inhibits iNOS, using immunocytochemistry. The iNOS levels were markedly increased in LPS-stimulated cells (Fig. 2D); however, pre-incubation with the LOME prevented the expression of these proteins. Thus, we suggest that LOME inhibits iNOS production.

The unstimulated RAW264.7 cells secreted basal levels of PGE₂ and LPS stimulation induced an increase in PGE₂ production (Fig. 3A). In a dose-response study, LOME treatment significantly diminished LPS induction of COX-2, which corresponded with a decreased accumulation of PGE₂ in the culture media (Fig. 3). The LPS-stimulated RAW264.7 cells overexpressed COX-2 mRNA, whereas the LOME-treated RAW264.7 cells stimulated with LPS, exhibited decreased expression of COX-2 mRNA. COX-2 protein expression was consistent with the mRNA results (Fig. 3B and C). LOME inhibited COX-2 protein expression in the RAW264.7 cells stimulated with LPS.

The levels of TNF-α, IL-1β and IL-6 were measured in the media of RAW264.7 cells that were treated with varying concentrations of LOME, and then treated with 0.5 µg/ml of LPS (Fig. 4A–C). It was found that LOME significantly reduced the production of TNF-α, IL-1β and IL-6 in a dose-dependent manner (Fig. 4A–C). LOME alone had no effect on TNF-α, IL-1β and IL-6 production in the RAW264.7 cells (data not shown). Additionally, the mRNA levels of TNF-α, IL-1β and IL-6 were evaluated using RT-PCR (Fig. 4D). Changes in the mRNA expression of TNF-α, IL-1β and IL-6 were similar to those observed in their protein expression levels.

Three MAPKs, namely ERK, p38 and JNK, are known to be activated by LPS. MAPKs play an important role in the transcriptional regulation of the LPS-induced expression of iNOS and COX-2 by activating the transcription factor NF-κB (17). As shown in Fig. 5, LOME clearly decreased the phosphorylation of ERK1/2, JNK and p38. These results indicate that the inhibitory effect of LOME on TNF-α, IL-1β, IL-6, NO and PGE₂ may have been mediated by the downstream MAPK pathways.

NF-κB plays a pivotal role in regulating the expression of iNOS, COX-2 and inflammatory cytokines such as TNF-α, IL-1β and IL-6 (18). As a plausible molecular mechanism for the inhibition of the inflammatory response, the effect of LOME on the NF-κB signaling pathway was explored (Fig. 6A). LPS exposure (0.5 µg/ml) for 30 min facilitated the degradation of IκB-α, the phosphorylation of IκB-α, and the nuclear accumulation of NF-κB. However, pre-treatment with LOME inhibited the degradation of IκB-α. Furthermore, LOME decreased the LPS-mediated phosphorylation of IκB-α and nuclear NF-κB accumulation in a dose-dependent manner. We further investigated the effect of the LOME on NF-κB activation using immunocytochemistry. The LPS-stimulated translocation of the NF-κB p65 subunit from the cytosol to the nucleus in the RAW264.7 cells (Fig. 6B), was inhibited by LOME. Thus, the anti-inflammatory effect of LOME may be due to inhibition of the NF-κB signaling pathway.

In the present study, Bay 11-7082 was used to inhibit the NF-κB pathway. Bay 11-7082, also known as (E)-3-(4-methylphenylsulfonyl)-2-propenenenitrile, has a molecular weight of 207.25 kDa and was provided at ≥98% purity (HPLC). It is an irreversible inhibitor of IκB-α phosphorylation, which increases the stabilization of IκB-α and specifically blocks NF-κB signaling. Bay 11-7082 has been shown to be a potent inducer of apoptosis in a number of cancer cell lines. Although Bay 11-7082 has not undergone clinical development, prior in vivo animal experiments have demonstrated its limited toxicity and therapeutic effectiveness (19,20).

LOME significantly suppressed the LPS-induced activation of NF-κB in the RAW264.7 cells, as shown by western blot analysis. Bay 11-7082 (Calbiochem-Merck, Darmstadt, Germany), a potential anti-inflammatory agent, is an irreversible inhibitor of cytokine-inducible IκB-α phosphorylation (IC₅₀=10 µM) (21). Bay 11-7082 blocked the LPS-induced increases in the activation of NF-κB. As expected, LOME (20 µg/ml) inhibited the LPS-induced activation of NF-κB (Fig. 7A). These results suggest that LOME reduces inflammation by inhibiting the LPS-induced activation of NF-κB in the RAW264.7 cells. In addition, LOME and Bay 11-7082 also affected NO expression. The inhibition of NO expression by each of the compounds was clearly effective (Fig. 7B).