Effect of Scutellariae Radix Ingredients on Prostaglandin E₂ Production and COX-2 Expression by LPS-activated Macrophage

Materials and Methods

Materials. The following chemicals and reagents were obtained from the indicated companies: Sairei-to (No. 114, Tsumura Corp., Tokyo, Japan); baicalin, baicalein, wogonin (Wako Pure Chemical Industries, Ltd., Osaka, Japan); Dulbecco's modified Eagle's medium (DMEM) (GIBCO BRL, NY, Grand Island, USA); fetal bovine serum (FBS), LPS from Escherichia coli (serotype 0111:B4) (Sigma Chem. Ind., St. Louis, MO, USA).

Cell culture. RAW264.7 cells that had been established from the peritoneal fluid of BALB/c mice and shown the phenotype characteristics of monocytes and macrophages (7) (supplied by Professor Ohmori, Meikai University) were cultured in DMEM supplemented with 10% heat-inactivated FBS, under a humidified 5% CO₂ atmosphere.

Measurement of PGE₂ production. RAW264.7 cells were subcultured in 24-well plates and incubated for 24 hours without or with Scutellariae radix (75 μg/ml) or its ingredients (baicalin, baicalein or wogonin; 1, 5, 10 μg/ml) in the presence or absence of LPS (100 ng/ml). The culture medium supernatant was collected by centrifugation and determined for the PGE₂ concentration by EIA kit (Cayman Chemical Co, Ann Arbor, MI, USA).

HPLC analysis. RAW264.7 cells were incubated for 3 hours with each flavonoid. The cells were then washed three times with phosphate-buffered saline without Ca²⁺ and Mg²⁺ [PBS(-)], lysed with lysis buffer [10 mM Tris-HCl (pH 9.6), 1% Triton® X-100 150 mM NaCl, 5 mM EDTA-2Na] and deproteinized with an equivalent amount of acetonitrile. After centrifugation for 5 minutes at 10,000×g, the supernatant was collected and stored at -40°C until HPLC determination. Ten μl of supernatant were injected into an HPLC system (ODS-HG-5; 4.5 mm × 150 mm, Develosil, Nomura Chemical Co., Ltd., Aichi, Japan), eluted with a mobile phase of 50% acetonitrile in water containing 0.2% phosphate and at a flow rate of 1 ml/min, and then detected with UV at 274 nm.

Western blot analysis. RAW264.7 cells were treated under the same condition for the measurement for PGE₂ production. The cell pellets were suspended in lysis buffer and mixed with an equal volume of 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], and boiled for 10 minutes. The protein in the cell lysate was determined by Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and aliquots equivalent to 20 μg protein were applied to 8% SDS polyacrylamide gel electrophoresis and then transferred to PVDF membrane (Immobilon-P; Millipore Corp, Bedford, MA, USA). The membranes were then blocked with 5% skimmed milk in PBS(-) containing 0.05% Tween 20 overnight at 4°C, incubated with antibodies against COX-2 (1:2,000) (Santa Cruz Biotechnology, Delaware, CA, USA) or Actin (1:2,000) (Sigma) for 90 minutes at room temperature and then incubated with horseradish peroxidase-conjugated anti-goat IgG (1:2,000) (GE Healthcare UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire, England) for 60 minutes at room temperature. Immunoblots were developed with a Western Lightning™ Chemiluminescence Reagent Plus system (Perkin Elmer Life Sciences, Boston, MA, USA).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1.

Chemical structures of baicalin, baicalein and wogonin.

Assay for mRNA expression. Total RNA was isolated by ISOGEN (NIPPON GENE CO., Tokyo, Japan) protocol. RAW264.7 cells were treated under the same condition for the measurement for PGE₂ production. The cells were lysed in 500 μl ISOGEN, and mixed with 100 μl chloroform. After centrifugation at 12,000×g for 15 minutes, the supernatant was collected and mixed with 250 μl isopropanol. After centrifugation at 12,000×g for 10 minutes, the pellet was washed in 500 μl 75% ethanol, air dried and dissolved in DEPC-treated H2O. A reverse transcriptase (RT) reaction was performed with 1.0 μg of total RNA, using Rever Tra Ace (Toyobo Co., LTD, Osaka, Japan) and oligo (dT)₂₀ primer. Single strand cDNA obtained by RT reaction was amplified, using KOD plus (Toyobo), COX-2 specific primer or β-actin specific primer. RT-PCR products were applied to 2% agarose gel, and after separation, the gels were stained with ethidium bromide and then photographed under UV light.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Inhibition of LPS-stimulated PGE₂ production by Scutellariae radix extracts. RAW264.7 cells were incubated for 24 hours with or without 100 ng/ml LPS in the presence of Scutellariae radix (75 μg/ml), baicalin, baicalein or wogonin (1, 5, 10 μg/ml), and the PGE₂ concentration in the culture medium was then determined. Each value represents mean±SD from three independent experiments.