Effects of esculentoside A on turnour necrosis factor production by mice peritoneal macrophages

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments showed that it had strong anti-inflammatory effects. Tumour necrosis factor (TNF) is an important inflammatory mediator. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNF production from macrophages was altered by EsA under lipopolysaccharide (LPS) stimulated conditions. EsA was found to decrease both extracellular and cell associated TNF production in a dose dependent manner at concentrations higher than 1 μmol/l EsA. Previous studies have showed that EsA reduced the releasing of platelet activating factor (PAF) from rat macrophages. The reducing effects of EsA on the release of TNF and PAF may explain its anti-inflammatory effect.


Introduction
Esculentoside A (EsA) is a saponin isolated from the root of Phytolacca esculenta, and is identified as: 3-O-[/g-D--glucopyranosyl-(1 4)-/g-D--xylopyranosyl] phytolaccagenin. The structure of this compound is shown in Fig. 1. Experiments showed that it has strong anti-inflammatory effects. It is now known that tumour necrosis factor (TNF) possesses a number of properties of inflammatory response. 2 Platelet activating factor (PAF) is also an inflammatory mediator. 3 It has been shown that EsA inhibited the production of PAF by A23187 stimulated rat macrophages. 4 The aim of this work was to evaluate the effects of EsA on the production of TNF by LPS stimulated mice peritoneal macrophages. fresh culture medium, and frozen at -40C. After thawing, the cells were sonicated, and the lysates were centrifuged to remove the particles, and stored at -20C for activity assay. TNF production assay: TNF assay was performed essentially as described by Kunkel

Results
Effects of EsA on extracellular production of TNF from macrophages: The extracellar production of TNF in the supernatant after being dialysed was assessed by the killing of L929 cells. One unit of TNF was defined as the reciprocal of the dilution of a preparation that results in 50% survival of the cells. The results are presented in Table 1. It is observed that the decrease of TNF production was significant at the concentrations of 1 and 10/mol/1 EsA. Effects of EsA on intracellar production of TNF from peritoneal macrophages" The results in Table 1 demonstrate, that EsA reduced not only the extracellular production of TNF but also the intracellular production of TNF in a dose dependent manner. Intracellular TNF production is lower than that of extracellular production, and the concentration of EsA that significantly decreased TNF production is the same.
Eects of EsA on the kinetics of extracellular and cell associated TNF production: Peritoneal macrophages were cultured in LPS with or without EsA, and the supernatants were harvested at 2 h intervals.
Extracellular and intracellular TNF activity can both be detected after 2 h exposure to LPS. Figure 2 shows that intracellular and extracellular TNF production were both inhibited in a time dependent manner by EsA at a concentration of 5 #mol/1.   Table 2. Cell viability was confirmed by the trypan blue exclusion test (data not shown).

Discussion
In this study, EsA induced a dose dependent decrease in the extracellular and cell associated TNF concentrations measured in the supernatant and lysates of LPS stimulated macrophages. The kinetics of extracellular and cell associated TNF production were also changed.
TNF was initially identified as a factor that appeared in the circulation of animals following the injection of endotoxins. TNF is a product of stimulated monocytes and macrophages, but it is also produced by keratinocytes. 7 In addition to the cytotoxic activities of TNF in some types of transformed cells, recent data have shown that TNF mediated stimulation of collagenase synthesis and prostaglandin E2 (PgE2) production by synovial cells, 8 and stimulated bone resorption and inhibition, 9 suggesting that TNF might be an important mediator of inflammation. Platelet activating factor (PAF) is a mediator of anaphylaxis and inflammation; it plays an important role in inflammation, and there is cooperation between PAF and TNF in inflammatory reactions. 1 It has been found that EsA reduced release of PAF from rat macrophages. A previous study also showed that EsA can inhibit the inflammatory reaction induced by carrageenan. Thus, together with the present investigation it is suggested that the anti-inflammatory effects of EsA could be due to its reducing effects on release of TNF and PAF.