Inhibitory effect of esculentoside A on tumour necrosis factor α production by human monocytes

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong anti-inflammatory effects. Tumour necrosis factor (TNF) is a very important inflammatory mediator. It is known that there are two types of TNF—TNFα is from macrophages/monocytes and TNFβ is from activated lymphocytes. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNFα production from human peripheral monocytes was altered by EsA under lipopolysaccharide (LPS)-stimulated conditions. EsA was found to decrease TNFα production in a dose-dependent manner at concentrations higher than 1 μmol/l EsA. Recent studies have shown that EsA has a curative effect on chocolate cyst and other inflammatory diseases. Our previous studies have shown that EsA could reduce the release of platelet activating factor (PAF) from rat macrophages, and inhibit interleukin-1 and interleukin-6 production from routine macrophages. The reducing effects of EsA on the release of TNFα, IL-1, IL-6 and PAF may explain its anti-inflammatory effect.

. Experiments have shown that it has strong anti-inflammatory effects. It is now known that tumour necrosis factor (TNF) possess a number of properties of inflammatory response. 2'3 Platelet activating factor (PAF), interleukin-1 and interleukin-6 are also inflammatory mediators. It has been shown that EsA inhibited the production of PAF by A23187 stimulated rat macrophages, 4'5 and the production of TNFa, IL-1, IL-6 of LPS stimulated murine peritoneal macrophages. <8 The aim of this work was to evaluate the effects of EsA on the production of TNFa by LPS stimulated human peripheral monocytes.
Human peripheral monocyte preparation: Human monocytes were isolated by a combina-tion of Ficoll-Hypaque gradient centrifugati0n and centrifugal elutriation. This procedure yielded populations of monocytes with greater than 95% purity. The cells were washed three times in RPMI-1640 and suspended at 1 x 10 cells per 1 ml in freshly prepared standard culture medium. The cells were plated at 1 ml of cells per well. TNF production: Two hours later, the medium containing non-adherent cells was decanted and the non-adherent cells in supernatant were counted to measure the adherence ratio. TNF0t activity was expressed as U/10 monocytes. The adherent cells were rinsed twice with Hanks's solution. Then the adherent cells were incubated with Aisv (1 t.tmol/1) for 6 h. After incubation, the medium was discarded, and the cells were washed three times with RPMI-1640 to remove Ai8r. Fresh culture medium without serum was added to every well with lipopolysaccharide (LPS, 10 lag/ml) in the presence or absence of EsA. The cultures were incubated in a humidi-cells. One unit of TNFa was defined as the recified atmosphere of 5% CO2 and at 37C for procal of the dilution of a preparation that another 6 h. The supernatants were harvested results in 50% survival of the cells. The results and centrifuged. The cell-free supernatants were are presented in Table 1. It is observed that the collected, dialysed in phosphate-buffer solution decrease of TNFa production was significant at for 24h, and stored at-20C prior to activity the concentrations of I and 10 l.tmol/1 EsA.

assay.
Effects of EsA on the kinetics of TNF production TNF production assay: TNFa assay was per-from human monocytes: Monocytes were culformed essentially as described by Kunkel et al. 5 tured in LPS (10 l.tg/ml) with or without EsA, and with minor modifications. Briefly, L929 cells the supernatants were harvested at 2h intervals.
(50000/well) were dispensed into 96-well fiat-TNFa activity can be detected after 2 h exposure bottomed microtitre plates in a volume of 0.1 to LPS. Figure 2 shows that TNFa production by ml/well. The following day, the cells were incu-human monocytes were inhibited in a time bated for 18 h in the presence of 1 l.tg of actino-dependent manner by EsA at a concentration of mycin D and serial 1:2 dilutions of test sample. 5 I.tmol/1.
Media were then decanted, and the remaining cells in each well were stained with crystal violet Ejects of Ex4 on cellular activi{y: In order to for about 15 min, washed with tap water, and eliminate the possibility that EsA was toxic for dried at 40C. Absorbance of the cells in each the cells tested, cell viability was monitored by well was read using a microenzyme linked immumeasuring LDH activity in the supernatant and by nosorbent autoreader. Units of TNF0t activity the trypan blue exclusion test performed at the were defined as described by Kunkel Table 2. Cell viability was confirmed by the trypan blue exclusion test. The cell viability ratio in test samples was more than 99% ( Table 2). The result was the same as previously reported.

Discussion
In this study, EsA induced a dose-dependent decrease in the TNFa concentrations measured in the supernatant of LPS-stimulated human monocytes. The kinetics of TNFa 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. In addition to the cytotoxic activities of TNF in some types of transformed cells, recent data have shown that TNFa mediated stimulation of collagenase synthesis and prostaglandin E2 (PGE2) production by synovial cells, 7 and stimulated bone resorption and inhibition, 8 suggesting that TNFa 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 TNFa in inflammatory reactions. 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. Recent clinical trials showed that a Chinese herb containing EsA had a significant curative effect on chocolate cyst. Our recent test showed that EsA could significantly inhibit It-1 and IL-6 production from murine macrophages. 2 Thus, together with the present investigation it is suggested that the anti-inflammatory effects of EsA might be due to its reducing effects on the release of TNFa, PAF, IL-1, IL-6 and other inflammatory mediators.