LPS (Escherichia coli 026:B6), Griess reagent, Tween-20, bovine serum albumin and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and other tissue culture reagents were purchased from Gibco-BRL (Grand Island, NY, USA). Other chemicals were obtained from Sigma-Aldrich unless otherwise indicated.

The RAW 264.7 cell line, which was derived from murine macrophages, was obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in DMEM medium, supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, at 37°C in a 5% CO₂ humidified air environment. Previously purified 5HHMF (19) was used in this study. The 5HHMF was dissolved in dimethyl sulfoxide (DMSO) to produce a 10 mg/ml stock solution and then adjusted to final concentrations using complete DMEM.

Cell viability was evaluated by the MTT assay. RAW 264.7 cells were seeded into 96-well plates at a density of 4×10³ cells/well and maintained at 37°C for 24 h. The cells were exposed to various concentrations of 5HHMF (0, 5, 10 and 15 μg/ml) for 1 h and stimulated with LPS (100 ng/ml). After 24 h of incubation, the MTT (0.5 mg/ml in phosphate-buffered saline, PBS) solution was added to each well and incubated for another 3 h. The formazan crystals were dissolved in 200 μl DMSO and the cell viability was determined subsequent to measuring the absorbance at a wavelength of 540 nm with a microplate reader (Dynatech MR-7000; Dynatech Laboratories Inc., Chantilly, VA, USA).

The nitrite concentration in the medium was measured according to the Griess reaction and the calculated concentration was taken as an indicator of NO production. The supernatant of cell cultures was mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water). The optical density at 540 nm was measured and calculated against a sodium nitrite standard curve. The accumulated PGE₂ in the culture medium was measured using a PGE₂ enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s instructions.

The inhibitory effect of 5HHMF on the production of pro-inflammatory cytokines, TNF-α and IL-1β, from LPS-treated RAW 264.7 cells was determined using a mouse ELISA kit (R&D Systems, Minneapolis, MN, USA), as described previously (22).

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The total RNA (1 μg) obtained from the cells was reverse-transcribed using M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI, USA) to produce cDNA. RT-generated cDNA encoding iNOS, COX-2, TNF-α, IL-1β and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was amplified by PCR using selective primers (Table I). Subsequent to amplification, aliquots of the PCR reaction mixture were electrophoresed on an agarose gel.

The cells were washed with PBS three times and lysed with a lysis buffer (1% Triton X-100, 1% deoxycholate and 0.1% NaN₃) containing protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany). Equal quantities of protein were separated on 10% sodium dodecyl sulfate-polyacrylamide mini-gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Milford, MA, USA). Subsequent to incubation with the appropriate primary antibody, the membrane was hybridized with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Following three washes with Tris-buffered saline with Tween-20, immunoreactive bands were visualized using the Enhanced Chemiluminescence Detection system (Pierce Biotechnology Inc., Rockford, IL, USA). In a parallel experiment, nuclear protein was prepared using nuclear extraction reagents (Pierce Biotechnology Inc.), according to the manufacturer’s instructions.

NF-κB p65 nuclear localization was detected by indirect immunofluorescence assays using confocal microscopy. RAW 264.7 cells were cultured directly on glass coverslips in 6-well plates for 24 h. Subsequent to stimulation with 100 ng/ml LPS and/or 15 μg/ml 5HHMF, the cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and blocked with 1.5% normal donkey serum (Sigma-Aldrich). A polyclonal antibody against NF-κB p65 (1 μg/well) was applied for 1 h followed by a 1 h incubation with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). After washing with PBS, the coverslips were mounted in Fluoromount-G™ (Southern Biotechnology Associates Inc., Birmingham, AL, USA) and fluorescence was visualized using a Zeiss LSM 510 laser scanning confocal device attached to an Axiovert 100 microscope using a Plan-Apochromat ×100 Oil DIC objective (Carl Zeiss, Oberkochen, Germany) (23).

Data are presented as the mean ± SD. Statistical significance was determined using analysis of variance followed by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The protective effect of 5HHMF was initially investigated based on LPS-induced cytotoxicity of RAW 264.7 cells using an MTT assay. As shown in Fig. 1C, LPS (100 ng/ml) treatment significantly induced cytotoxicity compared with that observed in the unstimulated control cells. However, the growth of LPS-stimulated RAW 264.7 cells was significantly enhanced by 5HHMF in a dose-dependent manner. Moreover, 5, 10 and 15 μg/ml concentrations of 5HHMF exhibited a protective effect on LPS-stimulated cytotoxicity in RAW 264.7 cells. However, 5HHMF alone did not show any obvious cytotoxic effect at the concentrations of 5–15 μg/ml (Fig. 1B).

Pro-inflammatory mediators such as NO and PGE₂ are important in the inflammatory response. To determine the level of NO production, nitrite released into the culture medium was measured using Griess reagent. As shown in Fig. 2A, LPS alone markedly induced NO production in the cells compared with that in the control. However, pre-treatment with 5HHMF (up to 15 μg/ml) significantly repressed the levels of NO production in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner (Fig. 2A). The effects of 5HHMF on the production of PGE₂, another important inflammatory mediator, was also investigated in LPS-stimulated RAW 264.7 cells. As shown in Fig. 2B, treatment of RAW 264.7 cells with LPS resulted in a marked increase in PGE₂ release compared with that in the untreated control after 24 h of exposure to LPS. However, 5HHMF inhibited LPS-mediated PGE₂ production in a concentration-dependent manner at the concentrations tested. These results suggest that pretreatment with 5HHMF results in significant suppression of the expression of LPS-mediated pro-inflammatory mediators.

To elucidate the mechanism involved in the inhibition of NO and PGE₂ by 5HHMF in LPS-stimulated RAW 264.7 cells, the effect of 5HHMF on iNOS and COX-2 protein and gene expression levels was investigated by western blot and RT-PCR analyses (Fig. 3). iNOS and COX-2 protein and mRNA expression in unstimulated RAW 264.7 cells was marginally detectable. However, iNOS and COX-2 expression increased markedly in response to LPS, and 5HHMF significantly inhibited the iNOS and COX-2 proteins in a dose-dependent manner (Fig. 3A). Under the same conditions, the levels of iNOS and COX-2 mRNA expression were correlated with their protein levels (Fig. 3B). These results indicated that reduced expression of iNOS and COX-2 by 5HHMF was responsible for inhibiting NO and PGE₂ production.

As 5HHMF potently inhibited the pro-inflammatory mediators NO and PGE₂, its effects on LPS-stimulated pro-inflammatory cytokines, such as TNF-α and IL-1β, were investigated by an enzyme immunoassay and RT-PCR analysis. As shown in Fig. 4A and B, TNF-α and IL-1β levels increased significantly in the culture media of LPS-stimulated RAW 264.7 cells. However, pre-treatment with 5HHMF significantly decreased the release of these pro-inflammatory cytokines in a concentration-dependent manner. In addition, the TNF-α and IL-1β mRNA levels induced by LPS decreased significantly in a concentration-dependent manner following 5HHMF treatment (Fig. 4C). These results suggested that 5HHMF is effective in suppressing pro-inflammatory cytokine production by altering TNF-α and IL-1β transcription levels in activated RAW 264.7 cells.

Previous studies have suggested that NF-κB is an important transcription factor that regulates iNOS, COX-2 and inflammatory cytokine expression (24,25). A number of the predominant mechanisms involving the activation of NF-κB include the phosphorylation of IKK and degradation of IκB-α, which allow the release of free NF-κB and its translocation into the nucleus (26). To investigate whether 5HHMF regulates the NF-κB pathway, we investigated whether 5HHMF prevented the translocation of the NF-κB p65 subunit to the nucleus. Western blot analysis showed that the quantity of NF-κB p65 in the nucleus increased markedly following exposure to LPS alone; however, the LPS-induced p65 level in the nuclear fractions decreased following 5HHMF pre-treatment. In addition, western blot analysis was used to investigate whether 5HHMF blocked LPS-stimulated degradation of IκB-α. As shown in Fig. 5A, IκB-α was markedly degraded 15 min after LPS treatment. This LPS-induced IκB-α degradation was significantly reversed by 5HHMF. Furthermore, the translocation of NF-κB to the nucleus in RAW 264.7 cells was analyzed using immunofluorescence staining and confocal microscopy to clearly understand the effect of 5HHMF on NF-κB p65 nuclear translocation (Fig. 5B). The confocal images revealed that NF-κB p65 was normally sequestered in the cytoplasm (Fig. 5B, middle panel), and that nuclear accumulation of NF-κB p65 was markedly induced following the stimulation of RAW 264.7 cells with LPS (Fig. 5B, LPS panel). The LPS-induced translocation of NF-κB p65 was completely eliminated subsequent to pre-treating the cells with 5HHMF (Fig. 5B, LPS+5HHMF panel). Nuclear translocation of NF-κB p65 was not induced in the cells following pre-treatment with 5HHMF alone, in the absence of LPS stimulation (Fig. 5B, 5HHMF panel). Thus, these results demonstrated that the anti-inflammatory effect of 5HHMF in LPS-stimulated RAW 264.7 cells involves the NF-κB pathway.