Inhibitory effect of esculentoside A on prostaglandin E2 production from murine peritoneal macrophages and rabbit synovial cells in vitro

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong antiinflammatory effects. To investigate the mechanism of anti-inflammatory effects of esculentoside A (EsA),[3H] arachidonic acid (AA) prelabelled murine macrophage and radioimmunoassay were used to test the effect of EsA on the total release of AA and prostaglandin E2 in culture supernatants. The results showed that EsA had no significant effect on the total release of AA from murine macrophages. EsA (2.5-10 μmol/l), from unstimulated murine peritoneal macrophages and rabbit synovial cells, could decrease the production of prostaglandin E2. In A23187 and LPS-treated macrophages and synovial cells, EsA (10 μmol/l) could significantly decrease the prostaglandin E2 production. These results confirmed that EsA exerted an inhibitory effect on prostaglandin E2 production from murine macrophages and rabbit synovial cells.


Introduction
Esculentoside A (EsA) is a saponin isolated from the root of Phytol acc a esculent a, and was identi ed as 3-0-[b-D-glucopyranosyl-(1-4)-b-Dxylopyranosyl] phytolaccagenin. The structure of this compound is shown in Fig. 1. Previous experiments have shown that it has strong antiin ammatory effects, 1 signi cantly decreasing the production of tumour necrosis factor (TNF) from LPS stimulated murine macrophages 2 and platelet activating factor (PAF) from A 23187 stimulated rat macrophages. 3 EsA also inhibited IL-1 production and phagocytic activity in murine macrophages. 4 Prostaglandin E 2 was an important in ammatory mediator and was found at the site of in ammation. PGE 2 caused vasodilatation leading to redness. 5 It has been implicated in the angiogenesis required for the spread of the pannus in the synovial hyperplasia component of rheumatoid arthritis. 6 PGE 2 is an arachidonic acid (AA) metabolite. In order to study the mechanism of the anti-in ammatory effect of EsA, this paper studied the effect of EsA on AA release from macrophages and the production of prostaglandin E 2 from murine peritoneal macrophages and rabbit synovial cells.

Preparation of murine macrophages
Thioglycolate medium (1 ml, 3%) was injected i.p. into the C 57 BL/6 mice. Four days later the cells in the peritoneal cavity were harvested with D-Hank's solution, washed twice in RPMI-640 and adhered for 2 h at 378 C in a culture bottle in a CO 2 incubator. The nonadherent cells were decanted and the remaining adherent cells were digested with trypsin (0.25%) for about 3± 4 min. RPMI-1640 containing 10% FCS was added to the culture bottle and the cell suspension was adjusted to 10 6 ml with RPMI-1640 containing 10% FCS and disposed at 1 ml/ well in 24-well plates.

Rabbit synovial cells culture
Synovial cells were prepared from a rabbit (weighing 2500± 3000 g). Brie y, the rabbit was killed by bleeding. Synovium was taken out under ascetic conditions and cut in 1± 2 mm 3 . The synovium was directly adhered on to the bottle, MEM (including 20% FCS, 200 mg/ml glutamin) was added to the bottle. The medium was refreshed after 2± 3 days. The synovium was taken out when the synovial cells conuenced.
[ 3 H]arachidonic acid uptake by murine macrophages The experiment was carried out as previously reported. 7 Brie y, 10 7 macrophages in 1 ml medium (containing 10% FCS) were added to 35 mm culture dishes for 2 h. Nonadherent cells were washed away by D-Hank's solution.
[ 3 H]AA, 18500 Bq in 1 ml RPMI-1640 were added to each well for 4 h. The supernatant was decanted and the cells were washed twice. Zymosan, 400 mg/ml was added to each well after EsA in 1 ml RPMI-1640 at different concentrations was co-cultured with macrophages for 20 min. The supernatants were collected at 2, 5 and 15 h and radioactivity was counted in a bscintillator.

Measurement of prostaglandin E 2
The experiment was carried out as in Ref. 6. The rabbit's synovial cells (2 3 10 5 /ml) and murine peritoneal macrophages (1 3 10 6 /ml) in 1 ml medium (MEM containing 10% FCS) was seeded in wells respectively and incubated for 24 h. The supernatants were decanted and the cells were washed with MEM three times. A 23187 and LPS were added in the presence and absence of EsA. After 24 h incubation the supernatants were adjusted to pH 3.5 with 10% HCOOH and extracted with ethylacetate twice (2 ml each time). The organic section was evaporated and the residual was reconstituted with 200 ml RIA assay buffer. The PGE 2 content was tested and expressed as ng/2 3 10 5 synovial cells and ng/10 6 macrophages respectively.

Statistics
Each experiment was carried out three times and the results presented here were representative of the three experiments. The same tendencies occurred in the parallel experiments. The results were expressed as the arithmetic mean SEM. The differences between the control group and treatment groups were analysed by Student's t-test; P , 0.05 was regarded as signi cant.

PGE 2 production from rabbit synovial cells
The same results as for murine peritoneal macrophages were observed in rabbit synovial cells. EsA (2.5± 10 mmol/l) inhibited PGE 2 production from unstimulated rabbit synovial cells.

Discussion
The present study demonstrated that EsA suppressed the production of PGE 2 from murine peritoneal macrophages and rabbit synovial cells. EsA at lower concentrations could inhibit PGE 2 production from unstimulated macrophages and synovial cells. In LPS and A 23187 treated groups, the inhibitory concentration reached 10 mmol/l. That is to say, the inhibitory concentration of EsA on PGE 2 production in stimulated cells is higher than that in unstimulated cells. Prostaglandins are products of oxidation of arachidonic acid. The cyclooxygenase (COX) enzyme is the rst dedicated enzyme in prostaglandin synthesis. At present, there appear to be at least two COX isozymes: a constitutive enzyme denoted COX-I which is responsible for the physiological synthesis of prostaglandins in tissue, and an inducible form termed COX-II which appears to be the major enzyme responsible for in ammatory prostaglandin synthesis. Our studies in the present paper have shown that EsA could inhibit both physiological and in ammatory prostaglandins' production from macrophages and synovial cells. PGE 2 is a metabolite of AA. We assumed that EsA inhibited PGE 2 production through its inhibitory effect on the release of AA. The results in Fig. 2 con rmed that EsA had no signi cant effect on the release of AA. Previous experiments proved that released AA could be re-uptake by cell membrane. 8 In the present research paper, our experiments con rmed that EsA had no effect on AA release in [ 3 H]AA prelabelled murine macrophages. We did not know whether EsA could affect AA uptake by cell membrane or not. Further experiments about the effect of EsA on AA uptake by cell membrane, activity of cyclooxygenase, lipoxygenase or other pathways are needed to clarify the anti-in ammatory mechanism of EsA.  3. Effect of EsA on the production of PGE 2 (ng) from 10 6 macrophages on various states. n 4, mean SEM, P , 0.05, P , 0.01 vs control group.