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

Consecutively, the efficacy of a 48-hr combined stimulation on NO production was studied using a fixed dose of LPS (1 [micro]g/ml) and various doses of TJ-48 (50 to 500 [micro]g/ml).

As shown in Figure 2, a low but recognizable NO level (15.4 [ or -] 0.34 [micro]M) was detectable in the culture supernatant even in the absence of both drugs at 48 hrs. Thus, this background level was referred to as stimulation index (SI) of 1.0. When the cells received a 48-hr treatment with TJ-48 alone at a dose of 300 [micro]g/ml, NO level (15.6 [ or -] 0.36 [micro]M) was almost the same as that of the unstimulated cells, giving an SI of 1.03. Increased SIs were obtained with the combined stimulations in a dose-dependent manner, compared with that of stimulation with LPS alone yielding SI of 2.5. Although the effect of 50 [micro]g/ml of TJ-48 was negligible (SI 3.7 versus 2.5), SIs of 4.3, 5.3 and 7.3 for 100, 300 and 500 [micro]g/ml of TJ-48 in the combined stimulations, respectively, were significantly higher than that of the LPS simulation alone. None of the TJ-48 dose tested affected cell viability evaluated by trypan blue exclusion and MTT tests, compared with that of untreated cells (data not shown).

[Figure 2 ILLUSTRATION OMITTED]

Effect of Juzen-taiho-to on iNOS mRNA Expression in RAW264.7 Cells

Time-dependent changes of iNOS-mRNA levels in response to the stimulation with either LPS alone or TJ-48-LPS combination were studied (Figure 3). In this study, the expression of iNOS mRNA was also undetectable in the unstimulated cells (data not shown). In cells receiving the stimulation with LPS alone, iNOS mRNA was undetectable at 4 hrs, but became abruptly detectable attaining its peak at 6 hrs, thereafter gradually decreased and finally disappeared at 14 hrs (Figure 3-A). On the other hand, iNOS mRNA in response to TJ-48-LPS stimulation became detectable at 4 hrs, 2 hrs earlier than that of stimulation with LPS alone. Thereafter, this level reached its peak at 6 hrs and then decreased more gradually to 14 hrs when a faint band of iNOS mRNA could be detectable (Figure 3-B). The expression of iNOS mRNA was not induced in the presence of with TJ-48 alone throughout the experiment (data not shown). Control RT-PCR analyses showed almost equivalent expression of the GAPDH gene in all experimental points, indicating that the expression of iNOS mRNA is specifically induced by these stimulations. Comparing iNOS/GAPDH ratio based on densitometric analyses between two kinds of stimulation receiving with LPS alone and TJ-48-LPS combination, the levels of iNOS mRNA in the latter group were 2.0-fold higher than that of the former group at the peak period of 6 hrs (Figure 3-C). These data suggest that TJ-48 could enhance the LPS-mediated expression of iNOS gene at initiation and during the periods of iNOS gene expression. To examine dose-dependent effect of TJ-48 on the iNOS mRNA expression, the same analyses were carried out using various doses of TJ-48 with the fixed dose of LPS at 6 hrs. Data presented in Figure 4 show that iNOS mRNA levels became 1.1-, 1.6- and 1.9-fold higher at doses of 30, 300 and 500 [micro]g/ml, respectively, compared with that of the stimulation with LPS alone. These data indicated that TJ-48 could enhance the expression of LPS-mediated iNOS mRNA in a dose-dependent manner in RAW 264.7 cells.