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

Hiroshi Kawamata

Abstract: We have investigated the effect of Juzen-taiho-to (TJ-48) on inducible NO synthase (iNOS) expression and nitric oxide (NO) production in RAW264.7 cells, a murine macrophage cell line. TJ-48-lipopolysaccharide (LPS) combination induced iNOS mRNA expression earlier, stronger and remained longer that paralleled but with a higher NO production compared to LPS stimulation. TJ-48 itself showed no inducible effect either on NO production or iNOS mRNA expression. This phenomenon could be considerd to contribute, at least in part, to the beneficial effects of TJ-48 through the iNOS-mediated activation of biodefense mechanism.

Nitric oxide (NO), initially identified as an endothelium-derived relaxing factor (Palmer et al., 1987), is synthesized from L-arginine by NO synthase (NOS) in numerous mammalian cells and tissues (Nathan, 1992). To date, at least three major categories of the enzyme-mediated NO production have been clarified; the constituent and calcium-dependent isoforms principally present in endothelial and neuronal cells. These are referred to as ecNOS and ncNOS, respectively. The remaining one is the inducible and calcium-independent isoform (iNOS), which have been demonstrated in a wide variety of cells such as macrophages (Stuehr and Marletta, 1985; Nathan and Xie, 1994), hepatocytes (Curran et al., 1989), smooth muscle cells (Busse, 1990), endothelial cells (Gross et al., 1991), and cardiac myocytes (Schulz et al., 1992). The iNOS could produce high amounts of NO that are sustained for long periods when activated by various stimuli such as lipopolysaccharide (LPS), interferon-[Gamma], interleukin-1, and tumor necrosis factor (Lepoivre, 1989). The nanomolar concentrations of NO are considered to be sufficient for intracellular signaling and, especially, NO produced by iNOS in macrophages acts as a defense molecule with microcidal/static and tumor killing activities (Hibbs et al., 1990; Moncada et al., 1991; Keller et al., 1992).

Recent studies have shown that Juzen-taiho-to (TJ-48), one of the traditional Chinese medicines, has various biological activities: enhancement of phagocytosis (Maruyama et al., 1998), cytokine induction (Haranaka et al., 1985; Kubota et al., 1992) and antibody production (Hamada et al., 1998) and inhibitory effect on tumor progression or metastasis (Ohnishi et al., 1996). However, the mechanisms of these biological activities of TJ-48 have not yet been studied in detail. Thus, we focused this study on NO generation in vitro to obtain a better insight into the mechanism on these biological activities of TJ-48.

Materials and Methods


A murine monocyte/macrophage cell line, RAW264.7 cells, used in this study, was obtained from American Type Culture Collection and cultured in Dulbeccos’s modified Eagle’s minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. The cells were grown in the presence of 5% [CO.sub.2] in fully humidified air at 37 [degrees] C and passed every 4 days. For experiments, cells were detached with the aid of cell scrapers and plated in 96-well plates in fresh media.


Spray-dried hot-water extract of TJ-48 was kindly provided by Tsumura (Tokyo, Japan). The composition of TJ-48 is listed in Table 1. The extract was dissolved in distilled water at a concentration of 10 mg/ml. LPS from Eschrechia coli 0127:H8 (Difco, Detroit, MI, USA) was dissolved in serum-free DMEM at a concentration of 1 mg/ml. These reagents were sequentially passed through filters up to 0.3 [micro]m for sterilization and diluted with DMEM at appropriate concentrations.

Table 1. Composition of Juzen-taiho-to

Herbs Ratio

Astragali radix 3.0

Cinnamomi cortex 3.0

Rehmanniae radix 3.0

Paeoniae radix 3.0

Cnidii rhizoma 3.0

Atractylodis lanceae rhizoma 3.0

Angelicae radix 3.0

Ginseng radix 3.0

Hoelen 3.0

Glycyrrhizae radix 1.5

Quantification of Nitrite and Nitrate

The amounts of nitrite, an indicator of NO synthesis, in the conditioned medium (CM) of RAW cells were measured by the following method. Briefly, the confluent cells in a 96-well plate with 200 [micro]l of culture medium were further cultured in the presence of 1 [micro]g/ml of LPS or LPS (1 [micro]g/ml)-TJ-48 combination at various concentrations indicated in the text. At the appropriate times after stimulation, CM was collected and subjected to NO analysis using a NOx analyzer system (ENO-10, Eicom, Kyoto, Japan). Ten microliters of the CM was injected into an automated NO detector-HPLC system (ENO-10). [NO.sub.2] and [NO.sub.3], in a reduction column packed with copper-plated cadmium filings (NO-RED, Eicom), was mixed with Griess reagent (1.25% HCl with 5 g/L sulfanilamide, 0.25 g/L N-naphthylethylenediamine and 2.5% phosphoric acid) (Yamada et al., 1997) to form a purple azo dye in a reaction coil. The separation columns, the reduction columns, and the reaction coil were placed in an oven set at 35 [degrees] C. The absorbance of the product dye at 540 nm was measured using a flow-through spectrophotometer (NOD- 10, Eicom). The mobile phase, which was delivered by a pump at a rate of 0.33 ml/min, was 10% methanol containing 0.15 M NaCl/[NH.sub.4]Cl and 0.5 g/L EDTA4Na. The concentration of [NO.sub.2] and [NO.sub.3] in the Ringer’s solution and the reliability of the reduction column were examined during each experiment. All were conducted in triplicate.

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.


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.


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).

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).


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.



Recent studies have reported that TJ-48 exhibits various biological activities such as enhancement of phagocytosis, antibody production, cytokine induction, and inhibition of tumor progression or metastasis (Haranaka et al., 1985; Keller et al., 1992; Maruyama et al., 1998; Kubota et al., 1992; Ohnishi et al., 1996; Hamada et al., 1998). However, these studies have made no reference to the contribution of NO on the biological activities of TJ-48. In the present study, we measured the accumulation of nitrite/nitrate, a stable oxidation product of the unstable free radical NO, to quantify the induction of NO synthesis. The validity of the results shown in this study were ranged within those shown in a previous report (Keller et al., 1992). RAW264.7 cells used in this study showed no significant NO production in the resting state, but dramatically changed to exhibit NO synthesis in the presence of LPS in a time-dependent manner with a lag time of 6 hrs. Moreover, we have clearly demonstrated that TJ-48 enhances inducible NO synthesis in RAW264.7 cells when added in the presence of LPS, in spite of no inducible activity of this drug itself on these cells. RT-PCR analyses confirmed that enhanced induction of NO synthesis is closely related with the enhanced expression of iNOS mRNA at initiation and during the periods of expression of iNOS gene. This synergistic inductive effect of TJ-48 on NO production and expression of iNOS is also shown with INF-[Gamma], although INF-[Gamma] itself showed inducing activity in sharp contrast to TJ-48 (Kamijo et al., 1994). As for transcriptional induction of iNOS gene, most studies have dealt with LPS and INF-[Gamma] in cells from rodents (Nathan and Xie, 1994). These authors pointed out that a substantial number of different transcriptional factors might participate because of the complex structure of murine iNOS promoter-enhancer lesion. So far, two sets have been indentified. The action of LPS is dependent on nuclear factor [(NF)-.sub.[Kappa]] B heterodimers (Nathan and Xie, 1994), while INF-[Gamma] targets INF regulatory factor-1 at least in part via direct action on the iNOS promoter-enhancer (Kamijo et al., 1994). In this study, the most important is that the stimulation with combination TJ-48-LPS combination could induce the expression of iNOS at 4 hrs at which its expression was undetectable in the stimulation with LPS alone. When the enhancing effect of combined treatment of with TJ-48 (300 [micro]g/ml)-LPS on NO production was examined in hepatocarcinoma-derived Huh-1 cells, this effect was not observed (unpublished data).

Thus, it is suggested that TJ-48 exhibits mainly its enhancing effect on the iNOS-mediated NO production in LPS-activated macrophages, but not that on cNOS-mediated NO production in the cells other than macrophages. These data also indicate that TJ-48 is a unique enhancer for the expression of iNOS gene. However, futher studies are required to clarify whether TJ-48 acts simply as a helper for [NF-.sub.[Kappa]]B depending LPS or not.

Several authors pointed out that NO has a dual biological role (Hibbs et al., 1990; Moncada et al., 1992); the NO involved in pathogenesis of inflammatory disorders (eg., immune complex alveolitis and arthritis in rats), neurodegenerative diseases and certain viral infection models the induction of iNOS in macrophages is associated with nonspecific immunity such as microcidal/static activities and tumor killing effects. Some normal issues such as uterus in the pregnant rabbit (Sladek et al., 1993) and large airways in human (Nathan and Xie, 1994) express iNOS antigens. However, expression of iNOS, largely in macrophages, is more often related to infection or inflammation, and geared toward host defense. In summary, this study has demonstrated a synergistic inductive effect of TJ-48 on iNOS expression and NO production in LPS-activated RAW264.7 cells. This activity of TJ-48 might contribute at least in part to various biological activities of TJ-48 through iNOS-mediated activation of biodefense mechanism under specific conditions occurring synergistic combinations such as infection, inflammation and cancers.


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Hiroshi Kawamata(1), Hiroshi Ochiai(*)(2), Naoki Mantani(1) and Katsutoshi Terasawa(1)

(1) Departments of Japanese Oriental Medicine and (2) Human Science, Faculty of Medicine. Toyama Medical and Pharmaceutical University, Sugitani 2,630, Toyama 930-0194, Japan (*) Corresponding author

(Accepted for publication January 9, 2000)

COPYRIGHT 2000 Institute for Advanced Research in Asian Science and Medicine

COPYRIGHT 2000 Gale Group

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