Curcumin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against IκB-α, STAT1 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA, USA), while antibodies against NF-κB p65, NF-κB p50, and PARP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

P. intermedia ATCC 25611 was used throughout. It was grown anaerobically on the surface of enriched Trypticase soy agar containing 5% (v/v) sheep blood, or in general anaerobic medium broth (Nissui, Tokyo, Japan) supplemented with 1 µg/mL menadione and 5 µg/mL hemin. Culture purity was assessed by gram staining and plating on solid medium.

LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method as described previously [17]. The protein content of the purified LPS was less than 0.1%. Coomassie blue staining of overloaded sodium dodecyl sulfate (SDS)-polyacrylamide gels did not reveal any visible protein bands in the purified LPS, confirming the purity of the preparation (data not shown).

The murine macrophage cell line RAW 264.7 (American Type Culture Collection, Rockville, MD, USA) was in Dulbecco's modified Eagle's medium as described previously [17]. The cells were seeded into 24-well culture plates at a density of 5×10⁵ cells/well and incubated for at least 12 hours to allow them to adhere to the plates. After washing three times with medium, various concentrations of P. intermedia LPS and curcumin were added and the cells were cultured for 24 hours, after which culture supernatants were collected and assayed for IL-6.

The cellular toxicity of curcumin was assessed by the MTT assay. Cells were incubated with various concentrations of P. intermedia LPS and curcumin for 24 hours, and MTT was added to the cultures to a final concentration of 0.5 mg/mL. After incubation at 37℃ in 5% CO₂ for 2 hours, the supernatant was removed and the cells were solubilized in dimethyl sulfoxide. The extent of reduction of MTT to formazan within the cells was quantified by measuring absorbance at 570 nm with a Spectra Max 250 ELISA Reader (Molecular Devices Inc., Sunnyvale, CA, USA).

The amount of IL-6 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (OptEIA, BD Biosciences Pharmingen, San Diego, CA, USA) according to protocols recommended by the manufacturer.

Cells were plated in 100 mm tissue culture dishes at a density of 1×10⁷ cells/dish and treated with various concentrations of P. intermedia LPS and curcumin for 24 hours. Total RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. cDNA was prepared from 1 µg of the total RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). A real-time PCR was performed using the CFX96 real-time PCR detection system (Bio-Rad Laboratories Inc.) with a specific primer for mouse IL-6. As an endogenous control, β-actin primer was used. PCR was conducted with SsoFast EvaGreen Supermix (Bio-Rad Laboratories Inc.) according to the manufacturer's instruction. Thermal cycler conditions were as follows: After denaturing at 98℃ for 30 seconds, PCR was performed for 45 cycles, each of which consisted of denaturing at 95℃ for 1 second, annealing/extending at 60℃ for 5 seconds. The following PCR primers for IL-6 (162 bp), and β-actin (149 bp) were used: IL-6 sense, 5'-GCCAGAGTCCTTCAGAGAGATACAG-3' and antisense, 5'-GAATTGGATGGTCTTGGTC-CTTAGC-3'; β-actin sense, 5'-TGAGAGGGAAATCGTGCGTGAC-3' and antisense, 5'-GCTCGTTGCCAATAGTGATGACC-3'. Each assay was normalized to β-actin mRNA.

Cells were plated in 60-mm tissue culture dishes, at a density of 4×10⁶ cells per dish, and treated with various concentrations of P. intermedia LPS and curcumin for the indicated periods of time. Cell lysates were prepared as described previously [17]. The nuclear fraction was prepared from cells using the ActiveMotif nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. Protein concentrations were determined with the bicinchoninic acid protein assay reagents (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The same amount of protein (30 µg) was then subjected to SDS-polyacrylamide gel electrophoresis on 10 to 12% acrylamide gels with 3% stacking gels. The resolved proteins were transferred to a nitrocellulose membrane by electroblotting, and the blots were blocked for 1 hour in PBST (Phosphate Buffered Saline with 0.1% Tween-20) containing 3% nonfat dry milk, followed by incubation with specific primary antibodies. They were then washed three times for 10 minutes each with PBST, incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour and visualized by enhanced chemiluminescence (Cell Signaling Technology) as recommended.

Cells were plated in 60-mm tissue culture dishes, at a density of 4×10⁶ cells per dish, and incubated with curcumin (20 µM) in the absence or presence of P. intermedia LPS (10 µg/mL) for the indicated periods of time. After extracting the nuclear protein as described above, the DNA-binding activity of NF-κB in nuclear extract was assayed by using a TransAM NF-κB p65/NF-κB p50 transcription factor assay kits (Active Motif) according to the manufacturer's recommended procedures. Oligonucleotide with the NF-κB consensus binding site (5'-GGGACTTTCC-3'), to which the activated NF-κB contained in nuclear extracts specifically binds, has been immobilized on a 96-strip well plate. The activated NF-κB p65 and p50 specifically bound to this oligonucleotide was detected using specific antibodies to NF-κB p65 and p50, respectively. A Spectra Max 250 ELISA Reader was used to read the sample absorbance, with results expressed as optical density emitted at 450 nm.

Data are expressed as means±SD and statistical analysis was performed using Student's t-test with P<0.05 considered statistically significant.

Stimulation of RAW 264.7 cells with P. intermedia LPS induced significant increases in IL-6 (Fig. 1). Curcumin inhibited IL-6 production dose-dependently in P. intermedia LPS-treated cells. Treatment of cells with 20 µM curcumin reduced IL-6 production by 83%. Curcumin did not affect the cell viability as determined by MTT assay (data not shown), indicating that inhibitory effects of curcumin on IL-6 production was unrelated to its cytotoxicity. As shown in Fig. 2, curcumin also reduced P. intermedia LPS-induced IL-6 mRNA expression in a dose-dependent manner.

The previous study [17] showed that the NF-κB and JAK2/STAT1 pathways are involved in IL-6 production induced by P. intermedia LPS. First, the effect of curcumin on P. intermedia LPS-induced degradation of IκB-α, upstream signaling pathway of NF-κB, was determined by immunoblotting. The degradation of IκB-α induced by LPS was not inhibited when cells were co-treated with curcumin (Fig. 3). Next, the effects of curcumin on the nuclear translocation of NF-κB subunits were examined. Nuclear fractions were immunoblotted with antibodies against NF-κB p65 and p50. As shown in Fig. 4A, curcumin did not affect P. intermedia LPS-induced p65 nuclear translocation. Instead, translocation of p50 subunit was dose-dependently hampered in the presence of curcumin (Fig. 4A). Finally, DNA-binding activity of NF-κB in nuclear extract was analyzed by using the ELISA-based NF-κB p65/NF-κB p50 transcription factor assay kits. DNA-binding activities of NF-κB p65 and p50 subunits induced by P. intermedia LPS were attenuated by treatment with curcumin (Fig. 4B).

It was examined whether curcumin regulates P. intermedia LPS-induced IL-6 production through inhibiting the STAT1 pathway. STAT1 phosphorylation induced by P. intermedia LPS was dose-dependently inhibited by curcumin (Fig. 5).