Effect of Sasa senanensis Rehder Extract on NO and PGE₂ Production by Activated Mouse Macrophage-like RAW264.7 Cells

Materials and Methods

Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA); fetal bovine serum (FBS) (JRH Bioscience, KS, USA); lipopolysaccharide (LPS) from Escherichia coli (Serotype 0111:B4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chem Co., St Louis, MO, USA); dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA) (Wako Pure Chem. Ind., Osaka, Japan); 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy1-3-oxide (carboxy-PTIO; a spin trap agent), 1-hydroxy-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7; an NO generator) (Dojin, Kumamoto, Japan).

SE was prepared and supplied by Daiwa Biological Research Institute Co., Ltd., Kawasaki, Kanagawa, Japan. SE contained 58.2 mg solid materials/mL (5), and higher amounts of serine (839 μM), glycine (2606 μM), alanine (915 μM) and glutamic acid (452 μM), and lower amount of arginine (14 μM) than the culture medium (DMEM+10% FBS) (serine 390 μM, glycine 411 μM, alanine 96 μM, glutamic acid 106 μM, arginine 366 μM).

Cell culture. RAW264.7 cells (kindly supplied by Prof Ohmori, Meikai University, Japan) were cultured as an attached monolayer culture in DMEM supplemented with 10% heat-inactivated FBS under a humidified 5% CO₂ atmosphere (9, 10). When the cells were grown to confluency, they were detached by splashing the medium to them through the pipette, and seeded at lower cell density. The cells were not collected by scraping them out from the plate with a rubber policeman, since the scraping procedure damaged the cells (unpublished data).

Determination of viable cell number. RAW264.7 cells were inoculated at 1.5×10⁶/mL (100 μL) in a 96-microwell plate (Becton Dickenson, Labware, NJ, USA) and incubated for 1-2 hours. Near-adherent cells were replaced with fresh culture medium and treated for 24 hours with the indicated concentrations of SE in phenol red-free DMEM supplemented with 10% FBS in the presence or absence of LPS (0.1 μg/mL). Viable cell number was then determined by MTT methods. In brief, cells were incubated for 30 minutes with 0.2 mg/mL of MTT. After removal of the medium the cells were lysed with DMSO, and the absorbance at 540 nm was recorded using microplate reader (9, 10).

Assay for NO production. RAW264.7 cells were inoculated at 1.5×10⁶/mL (100 μL) in a 96-microwell plate and incubated for 1-2 hours. Near confluent cells were treated for 24 hours with the indicated concentrations of SE (0-25%) in phenol red-free DMEM supplemented with 10% FBS in the presence or absence of LPS (0.1 μg/mL). The NO production by RAW264.7 cells was quantified by Greiss reagent, using the standard curve of NO₂^-. To eliminate the interaction between sample and Greiss reagent, the NO concentration was also measured in the culture medium without the cells, and this value was subtracted from that obtained with the cells. Then concentration that inhibited the LPS-stimulated NO production by 50% (50% inhibitory concentration: IC₅₀) was determined from the dose-response curve (9). The efficacy of inhibition of NO production was evaluated by the selectivity index (SI), which was calculated using the following equation: SI=CC₅₀/IC₅₀.

Assay for prostaglandin E₂ (PGE₂) production. Raw264.7 cells were inoculated at 1.5×10⁶/mL in a 96-mirowell plate and incubated for 1-2 hours. Near-adherent cells were treated for 24 hours with various concentrations of SE (0-3.2%) in the presence of LPS (0.1 μg/mL). The culture supernatant was collected by centrifugation, and determined for the PGE₂ concentration by EIA kit (Cayman Chemical Co, Ann Arbor, MI, USA) (11).

Assay for iNOS protein expression. RAW264.7 cells were inoculated at 1.5×10⁶/mL in 96-well plates (Becton Dickinson) and incubated for 1-2 hours. Near-adherent cells were treated for 24 hours with various concentrations of SE (0-6.2%). The concentrations were selected by CC₅₀ in the presence or absence of LPS (100 ng/mL). The cell pellets were lysed with 50 μL of lysis buffer [10 mM Tris-HCl (pH 7.6), 1% Triton X-100, 150 mM NaCl, 5 mM EDTA-2Na and 2 mM PMSF] for 10 minutes on ice. The cell lysates were centrifuged at 16,000×g for 20 minutes at 4°C to remove the insoluble materials and the supernatant was collected. The protein concentrations of supernatant were measured by Protein Assay Kit (Bio Rad, Hercules, CA, USA). Equal amount of the protein from cell lysates (10 μg) was mixed with 2×sodium dodecyl sulfate (SDS)-sample buffer [0.1 M Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.01% bromophenol blue, 1.2% 2-mercaptoethanol], boiled for 10 minutes, and applied to the SDS-8% polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% non-fat skim milk in phosphate-buffered saline [PBS (-)] plus 0.05% Tween 20 for 90 minutes and incubated for 90 minutes at room temperature with anti-COX-2 or anti-iNOS (dilution: 1:2,000 and 1:1,000, respectively, Santa Cruz Biotechnology, Delaware, CA, USA) or anti-actin antibody (1:6,000, Sigma), and then incubated with horseradish peroxidase-conjugated anti-goat, anti-rabbit or anti-mouse IgG for 60 minutes at room temperature. Immunoblots were developed with a Western Lighting™ Chemiluminescence Reagent plus (9).

Assay for iNOS and COX-2 mRNA expression. RAW264.7 cells were inoculated at 1.5×10⁶/mL in 24-well plates and incubated for 1-2 hours. Near-confluent cells were treated for 24 hours with various concentrations of SE(0-3.1%) in the presence or absence of LPS (0.1 μg/mL). The expression levels of iNOS and COX-2 mRNA were measured by RT-PCR. The primer sequence and size is: (i) iNOS: PCR products size (496 bp), sense primer (5’-CCCTTCCGAAGTTTC TGGCAGCAGC-3’), antisense primer (5’-GGCTGTCAGAGCCTC GTGGCTTTGG-3’), (ii) COX-2: Product size (371bp), sense primer (5’-TTTGTTGAGTCATTCACCAGACAGAT-3’), antisense primer (5’-CAGTATTGAGGAGAACAGATGGGATT-3’), (iii) G3PDH: Products size (452 bp), sense primer (5’-ACCACAGTCCATG CCATCAC-3’), antisense primer (5’-TCCACCACCCTGTTGCT GTA-3’) (9).

NO radical scavenging activity. The radical intensity of benzocycloheptaoxazines was determined at 25°C, using electron spin resonance (ESR) spectroscopy (JEOL JES REIX Tokyo, Japan, X-band, 100 kHz modulation frequency) (9). The radical intensity of NO, produced from the reaction mixture of 20 μM carboxy-PTIO and 60 μM NOC-7, was determined in 0.1 M phosphate buffer, pH 7.4 in the presence of 30% DMSO (microwave power and gain were changed to 5 mW and 400, respectively). When NOC-7 and carboxy-PTIO were mixed, NO was oxidized to NO₂ and carboxy-PTIO was reduced to carboxy-PTI, which produces seven-line signals. Samples were added 3 minutes after mixing. The NO radical intensity was defined as the ratio of the signal intensity of the first peak of carboxy-PTI to that of MnO, and expressed as the ratio to the height of MnO, an external marker. The concentration that reduced the NO radical intensity by 50% (IC₅₀) was determined from the dose response curve (9).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Inhibition by SE of LPS-stimulated NO production. RAW264.7 cells were incubated for 24 hours with the indicated concentrations of SE in the presence (gray or black) or absence (white) of 0.1 μg/mL LPS and the viable cell number and extracellular concentration of NO were then measured. Each value represents mean ±SD from three independent experiments.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Inhibition by SE by LPS-stimulated PGE₂ production. RAW264.7 cells were incubated for 24 hours with various concentrations of SE in the presence of 0.1 μg/mL LPS, and then the viable cell number (circle) and extracellular PGE₂ concentration (bar) were determined. Each value represents mean SD from three independent experiments.