Enhanced Expression of Inducible Nitric Oxide Synthase by Juzen-Taiho-To in LPS-Activated RAW264.7 Cells, a Murine Macrophage Cell Line

American Journal of Chinese Medicine, Spring, 2000 by Hiroshi Kawamata, Hiroshi Ochiai, Naoki Mantani, Katsutoshi Terasawa

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from cells at the indicated time after stimulation by the acid guanidium thiocyanate-phenol-chloroform method using RNAzol[TM] (Cinna/Biotex, Houston, TX, USA). Total RNA (1 [micro]g) was reverse-transcribed with reverse transcriptase (Superscript2, Gibco BRL, Gaithersburg, MD, USA) and oligo [(dT).sub.16] primer. The reverse transcription products from total RNA served as a templates for PCR, which was composed of 30 cycles of denaturation (94 [degrees] C for 1 min), annealing (58 [degrees] C for 1 min), and extension (72 [degrees] C for 1 min), using a thermal cycler (Perkin-ElmerCetus) and oligonucleotide primers (Hattori et al., 1995). The parallel expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was tested under the same PCR conditions as an internal standard. The sequences of primers were as follows: iNOS,5'-CAA CCA GTA TTA TGG CTC CT-3' (sense); 5'-GTG ACA GCC CGG TCT TTC CA-Y (antisense); GAPDH, 5'-TCC CTC AAG ATT GTC AGC AA-3' (sense); 5'-AGA TCC ACG GAT ACA AA-3' (antisense) (Fort et al., 1985; Terada et al., 1992; Lyons, C.R., 1992).

A portion (10 [micro]l) of PCR mixture was electrophoresed in a 1.6% agarose gel containing 0.2 [micro]g/ml ethidium bromide in Tris borate/EDTA buffer. The gel was then photographed under ultraviolet transillumination. For quantification, PCR bands on the photograph of the gel were scanned using a computer analysis system (H.P. Scan Jet 4P and ATTO Densitograph ver. 4) and normalized the iNOS signal relative to the corresponding GAPDH mRNA signal from the same sample. Data were expressed as the iNOS/GAPDH ratio.

Statistical Analysis

Values were expressed as mean [ or -] standard deviation (SD) of three observations. Significance of difference was tested by Student's t-tests, with the Bonferroni-correction for the comparison of multiple means. A p value less than 0.05 was considered to be statistically significant.

Results

Effect of Juzen-taiho-to on the Production of Nitrite/Nitrate in LPS-stimulated RAW264. 7 Cells

Initially, we studied time-dependent NO production after LPS stimulation by determining the cumulative production of nitrite/nitrate in the CM as shown in Figure 1. The concentrations of NO in LPS-treated cells increased in a time-dependent manner. After a lag phase of 6 hrs, at which the NO levels were less than 15 [micro]M, NO synthesis was induced and progressively increased up to the experimental period of 48 hrs.

[Figure 1 ILLUSTRATION OMITTED]

Based on this finding, the effect of combined stimulation on the NO production was investigated using a fixed concentration of TJ-48 at 300 [micro]g/ml and LPS 1 [micro]g/ml. The time-dependent NO production with a lag phase of 6 hrs was almost the same as that of stimulation with LPS alone.

However, the levels of NO production were higher than those of stimulation with LPS alone at 24 and 48 hrs (71.9 [ or -] 7.20 [micro]M versus 58.3 [ or -] 5.15 [micro]M and 96.2 [ or -] 2.16 [micro]M versus 84.5 [ or -] 4.20 [micro]M, at 24 hrs and 48 hrs, respectively). TJ-48 alone did not induce NO production (data not shown).