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

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

Cells

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.

Reagents

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.