Lipopolysaccharide-treated RAW Research Articles

Effect of wogonin, a plant flavone from Scutellaria radix, on the suppression of cyclooxygenase-2 and the induction of inducible nitric oxide synthase in lipopolysaccharide-treated RAW 264.7 cells

Plant flavonoids show anti-inflammatory activity both in vitro and in vivo. Some flavonoids, such as flavone derivatives, have been reported previously to inhibit nitric oxide (NO) production by suppressing inducible nitric oxide synthase (iNOS) expression. In this investigation, the effects of wogonin, a potent inhibitor of NO production among the flavonoids tested, on cyclooxygenase-2 (COX-2) induction and activity were elucidated further in connection with iNOS, using a mouse macrophage cell line, RAW 264.7. Wogonin inhibited NO and prostaglandin E 2 (PGE 2) production from lipopolysaccharide-induced RAW cells with ic 50 values of 31 and 0.3 μM, respectively. When added after the induction of iNOS and COX-2, wogonin inhibited the formation of PGE 2 ( ic 50 = 0.8 μM), but not the production of NO. Wogonin inhibited COX-2 activity directly ( ic 50 = 46 μM) from the homogenate of aspirin-pretreated RAW cells, as determined by measuring [ 14C]PGE 2 formation from [ 14C]arachidonic acid. However, it did not inhibit iNOS or phospholipase A 2 activity. Western blotting showed that wogonin suppressed the induction of both iNOS and COX-2. Prednisolone also suppressed the induction of iNOS and COX-2. Whereas RU-486 (a steroid receptor antagonist) reversed the suppressive activity of prednisolone, it did not affect the suppressive activity of wogonin, suggesting that the suppressive activity of wogonin is not mediated by binding to a steroid receptor. Results from the present study demonstrated that wogonin is a direct COX-2 inhibitor, as well as an inhibitor of iNOS and COX-2 induction. Wogonin may be a potential agent for use in the treatment of inflammatory diseases.

Quenching of Nitric Oxide by an Oral Carbonaceous Adsorbent

The ability of carbonaceous particles (AST-120), originally developed as an enteral adsorbent of uremic toxins, to quench nitric oxide (NO) was tested. NO in solutions prepared by two methods [NO gas bubbling and NO generating system, i.e., decomposition of l-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene] were determined by a NO-specific reduction of carboxy-2-phenyl-4,4,5,5-tetramethyl-imidaz-oline-l-oxyl-3-oxide using an electron paramagnetic resonance spectrometry. NO concentrations were less in samples containing increasing concentrations of AST-120. In a separate study, nitrite concentrations in lipopolysaccharide-treated RAW264 cells were significantly less in incubation medium containing AST-120. Thus, AST-120 may be applicable as an enteral anti-NO agent.

Quenching of nitric oxide by an oral carbonaceous adsorbent.

The ability of carbonaceous particles (AST-120), originally developed as an enteral adsorbent of uremic toxins, to quench nitric oxide (NO) was tested. NO in solutions prepared by two methods [NO gas bubbling and NO generating system, i.e., decomposition of 1-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene] were determined by a NO-specific reduction of carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide using an electron paramagnetic resonance spectrometry. NO concentrations were less in samples containing increasing concentrations of AST-120. In a separate study, nitrite concentrations in lipopolysaccharide-treated RAW264 cells were significantly less in incubation medium containing AST-120. Thus, AST-120 may be applicable as an enteral anti-NO agent.

Extracellular ATP Enhances mRNA Levels of Nitric Oxide Synthase and TNF-α in Lipopolysaccharide-Treated Raw 264.7 Murine Macrophages

Extracellular ATP potentiates, by activation of P2y-type purinergic receptors, the production of NO induced by low doses of lipopolysaccharide (LPS) in the murine macrophagic cell line RAW 264.7 (Tonetti et al. (1994) Biochem. Biophys. Res. Commun. 203, 430-434). Release of TNF-α, known to be an autocrine factor for iNOS expression, was enhanced, too, following exposure of either LPS-induced or uninduced cells to externally added micromolar ATP. Reverse transcription-PCR experiments showed that extracellular ATP increases mRNA levels of both inducible NO synthase (iNOS) and of TNF-α to extents comparable to those of enzymatic and biological activities, respectively. These data demonstrate that activation of purinergic receptors by extracellular ATP results in an enhanced expression of the iNOS and TNF-α genes.

Lipopolysaccharide-treated RAW 264.7 cells produce a mediator that inhibits lipoprotein lipase in 3T3-L1 cells.

RAW 264.7 cells upon stimulation with lipopolysaccharide secrete a protein mediator(s) that suppresses lipoprotein lipase activity in differentiated 3T3-L1 cells. The mediator(s), which is absent from unstimulated culture supernatants, is nondialyzable and thermolabile. Preliminary characterization suggests that this mediator(s) may be the same as that previously found in medium from lipopolysaccharide-treated thioglycollate-elicited mouse peritoneal macrophage cultures.