Masateru Yamada

Inhibition by tectorigenin and tectoridin of prostaglandin E2 production and cyclooxygenase-2 induction in rat peritoneal macrophages

Tectorigenin and tectoridin, isolated from the rhizomes of Korean Belamcanda chinensis (Iridaceae) which are used as Chinese traditional medicine for the treatment of inflammation, suppressed prostaglandin E2 production by rat peritoneal macrophages stimulated by the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the endomembrane Ca2+-ATPase inhibitor, thapsigargin. Tectorigenin inhibited prostaglandin E2 production more potently than tectoridin. Neither compound inhibited the release of radioactivity from [3H]arachidonic acid-labeled macrophages stimulated by TPA or thapsigargin. In addition, activities of isolated cyclooxygenase (COX)-1 and COX-2 were not inhibited by the two compounds. Western blot analysis revealed that the induction of COX-2 by TPA or thapsigargin was inhibited by the two compounds in parallel with the inhibition of prostaglandin E2 production. These findings suggest that one of the mechanisms of the anti-inflammatory activities of the rhizomes of Belamcanda chinensis is the inhibition of prostaglandin E2 production by tectorigenin and tectoridin due to the inhibition of the induction of COX-2 in the inflammatory cells.

Antiinflammatory activity of hyperin from Acanthopanax chiisanensis roots.

The chloroform and the ethyl acetate fractions from the roots of Acanthopanax chiisanensis exhibited a significant inhibition of prostaglandin E2 (PGE2) production in rat peritoneal macrophages stimulated by the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). Hyperin was isolated as an active principle from the ethyl acetate fraction. It suppressed not only PGE2 production but also nitric oxide (NO) productionin vitro in a concentration dependent manner, their IC50, being 24.3 and 32.9 μM, respectively. Hyperin also caused a significant inhibition of increase in acetic acid-induced vascular permeability in micein vivo.

Dual effects of auranofin on prostaglandin E2 production by rat peritoneal macrophages

Incubation of rat peritoneal macrophages in medium containing various concentrations of auranofin (1, 3 and 10 microM) increased prostaglandin E2 production at 4 h in a concentration-dependent manner, in accordance with the increase in the release of [3H]arachidonic acid from membrane phospholipids. However, at 20 h, no stimulation of prostaglandin E2 production by auranofin was observed. When the peritoneal macrophages were incubated in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), thapsigargin or A23187, prostaglandin E2 production at 4 and 20 h was enhanced. The stimulator-induced prostaglandin E2 production at 20 h was suppressed by 10 microM of auranofin. Western blot analysis demonstrated that auranofin inhibited the induction of cyclooxygenase 2 by TPA, thapsigargin or A23187 at 4 and 20 h. The level of cyclooxygenase 1 did not change by treatment with these stimulators in the presence or absence of auranofin. These findings suggest that auranofin has dual effects on prostaglandin E2 production: without stimulation, auranofin increases prostaglandin E2 production at 4 h due to the increased release of arachidonic acid which is converted to prostaglandin E2 mainly by cyclooxygenase 1, but inhibits the stimulator-induced late-phase prostaglandin E2 production by inhibiting the induction of cyclooxygenase 2.

Enhancement by cyclo‐oxygenase inhibitors of platelet‐activating factor production in thapsigargin‐stimulated macrophages

1 Thapsigargin stimulated the accumulation of cell‐associated platelet‐activating factor (PAF) and extracellular prostaglandin E2 (PGE2) in rat peritoneal macrophages. PAF in the conditioned medium was less than the detectable amount. To obtain further insight into the mechanism of PAF accumulation, the role of PGE2 in PAF accumulation was investigated. 2 When macrophages were incubated in medium containing thapsigargin (30 ng ml−1, 46.1 nM) and cyclo‐oxygenase inhibitors such as indomethacin, naproxen or ibuprofen, the PAF content of the cells at 10 min was increased in a concentration‐dependent manner in accordance with inhibition of PGE2 production. The stimulation of PAF accumulation by cyclo‐oxygenase inhibitors was significant at 10 min. Without stimulation by thapsigargin, cyclo‐oxygenase inhibitors did not increase PAF accumulation. 3 In thapsigargin‐stimulated macrophages, when PGE2 (10−7 m) was added to the medium, the cyclo‐oxygenase inhibitor‐induced stimulation of PAF accumulation at 10 min was markedly inhibited. 4 The accumulation of PAF induced by thapsigargin alone at 10 min was also inhibited by exogenous PGE2 (10−8 and 10−7 m), or arachidonic acid (10−6 and 10−5 m) in accordance with the increase in PGE2 production. 5 The accumulation of PAF induced by thapsigargin alone or by thapsigargin and indomethacin (10−6M) was inhibited by dibutyryl cyclic AMP. 6 These results indicate that the concurrently produced PGE2 in thapsigargin‐stimulated macrophages down‐regulates PAF accumulation by increasing intracellular cyclic AMP levels, and that cyclo‐oxygenase inhibitors increase PAF accumulation by inhibiting PGE2 production.

Induction of nitric oxide synthase by lipopolysaccharide and its inhibition by auranofin in RAW 264.7 cells

In RAW 264.7 cells, a murine macrophage cell line, treatment with lipopolysaccharide (1 to 10 ng/ml) stimulated production of nitric oxide (NO), which was inhibited by L-N(G)-monomethyl-L-arginine acetate, an inhibitor of NO synthase. Auranofin, an orally active chrysotherapeutic agent, also inhibited the lipopolysaccharide-induced NO production in a concentration-dependent manner (0.3 to 3 microM). Other gold salts such as aurothioglucose and aurothiomalate had no effect. Western blot analysis demonstrated that the lipopolysaccharide (10 ng/ml)-induced expression of inducible NO synthase protein was inhibited by auranofin as well as by the protein synthesis inhibitor cycloheximide. In addition, the lipopolysaccharide-induced increase in the level of mRNA for inducible NO synthase was also lowered by auranofin. Furthermore, auranofin showed no direct effect on the conversion of [3H]arginine to [3H]citrulline by the cell lysate. These findings indicate that auranofin inhibits lipopolysaccharide-induced NO production by suppressing the expression of inducible NO synthase.

Inhibition of tumor necrosis factor-α production by SK&F 98625, a CoA-independent transacylase inhibitor, in cultured rat peritoneal macrophages

Abstract When rat peritoneal macrophages were incubated in medium containing thapsigargin, tumor necrosis factor-α (TNF-α) production was increased time-dependently. In the presence of SK&F 98625, a CoA-independent transacylase inhibitor, the thapsigargin-induced TNF-α production was inhibited dose-dependently. Platelet-activating factor (PAF) and prostaglandin E2 (PGE2) production were also inhibited by SK&F 98625. The SK&F 98625-induced inhibition of TNF-α production was not prevented by addition of PGE2. PAF antagonists such as E6123, L-652,731 and CV-6209 partially inhibited the thapsigargin-induced TNF-α production, suggesting that concurrently produced PAF in thapsigargin-stimulated macrophages up-regulates TNF-α production. The inhibition by SK&F 98625 of thapsigargin-induced TNF-α production might be partly due to the inhibition of PAF production.

Effects of JTE-522, a specific inhibitor of cyclooxygenase-2, on the recurrence of allergic inflammation in rats

JTE-522, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide , is a selective inhibitor of cyclooxygenase-2 at the enzyme level (IC50 is 6.4 x 10(-7) M for sheep cyclooxygenase-2, but it does not inhibit sheep cyclooxygenase-1 at concentrations up to 10(-4) M). In rat peritoneal macrophages in culture, it markedly inhibited cyclooxygenase-2-dependent prostaglandin E2 production and weakly inhibited cyclooxygenase-1-dependent prostaglandin E2 production, as did the selective cyclooxygenase-2 inhibitor NS-398 ([N-2(cyclohexyloxy-4-nitrophenyl)]-methanesulfonamide). In addition, the anti-inflammatory activity of JTE-522 was evaluated, using a model of recurrent air pouch-type allergic inflammation in rats. JTE-522, injected into the pouch just after a second antigen challenge, suppressed the accumulation of pouch fluid, the infiltration of leukocytes and the prostaglandin E2 content in the pouch fluid, as did NS-398 and indomethacin. These findings indicated that JTE-522 is a selective cyclooxygenase-2 inhibitor in cell culture systems and that the suppression by JTE-522 of the recurrence of allergic inflammation is due to the inhibition of cyclooxygenase-2.

Platelet-Activating Factor Production in the Stimulated Macrophages is Enhanced by the Cyclooxygenase Inhibitors

In inflammatory cells, PAF is considered to be synthesized via the remodeling pathway1). Activation of phospholipase A2 releases sn-2 fatty acids, predominantly arachidonic acid, which is metabolized to prostanoids by cyclooxygenase and lipoxygenase. The two different lipid mediators, PAF and prostanoids, are produced from a common precursor in membrane phospholipids when PAF is produced via the remodeling pathway. Because PAF and prostanoids are potent, biologically active mediators, we considered that their production is interdependent.

Determination of platelet activating factor using immunoaffinity mini-columns. Stimulation of platelet activating factor production by thapsigargin and effects of dexamethasone.

Thapsigargin, a hexaoxygenated tetraacylated sesquiterpene lactone isolated from the roots of Thapsia garganica L., is one of non-TPA type tumor promoters that stimulate prostaglandin E2 production at very low concentrations and induce inflammation when applied topically. Effects of thapsigar-gin on platelet activating factor (PAF) production by rat peritoneal macrophages were examined. Levels of PAF in the conditioned medium and in the cells were determined by radioimmunoassay after extraction of PAF using immunoaffinity mini-columns. It was demonstrated that thapsigargin at concentrations of 3 to 100 ng/ml stimulated production of cell-associated PAF when measured 10 min after incubation. Levels of PAF in the conditioned medium were less than the detectable amount.The stimulative effect by thapsigargin reached a plateau at a concentration of 30 ng/ml. Time course experiments revealed that the levels of cell-associated PAF showed a peak 10 to 15 min after incubation with thapsigargin at a concentration of 30 ng/ml. The levels were then declined until 40 min after incubation. When macrophages were preincubated for 3 h with dexamethasone at concentra-tions of 0.01, 0.1 and l μg/ml, thapsigargin (30 ng/ ml) -stimulated PAF production in the cells at 10 min were decreased. However, the maximum inhibition was about 50% even at 1μg/ml of dexamethasone. These date suggest that by treatment with thapsigargin, PAF is partly produced through formation of lyso-PAF by activated phospholipase A2 since dexamethasone shows partial inhibition of thapsigargin-induced PAF production, and thap-sigargin-stimulated PAF production may partly account for proinflammatory activity of thapsigar-gin.