The Effect of Hydrogen Peroxide on inducible Nitric Oxide Synthase Expression in Murine Macrophage RAW264.7 Cells / 결핵

BACKGROUND: Nitric oxide is a short-lived effector molecule derived from L-arginine by the nitric oxide synthase(NOS). Nitric oxide plays a role in a number of physiologic and pathophysiologic functions including host defense, edema formation, and regulation of smooth muscle tone. Some kinds of cells including macrophage are known to produce large quantities of nitric oxide in response to inflammatory stimuli such as interleukin-1beta(IL-1beta), tumor necrosis factor-alpha(TNF-alpha), interferon-gamma(IFN-gamma) and lipopolysaccharide(LPS). Reactive oxygen species are also known to be important in the pathogenesis of acute cell and tissue injury such as acute lung injury model. METHODS: Using the RAW264.7 cells, we have examined the ability of oxidant hydrogen peroxide(H2O2) to stimulate nitric oxide production and inducible NOS mRNA expression. Also, we have examined the effects of NOS inhibitors and antioxidants on H2O2 induced nitric oxide production. RESULTS: Stimulation of RAW264.7 cells with combinations of 100 ng/ml IL-1beta, 100 ng/ml TNF-alpha, and 100 U/ml IFN-gamma or 100 U/ml IFN-gamma and 1 microg/ml LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite(NO2-) and nitrate(NO3-). Addition of 250 microM- 2 mM H2O2 to the cytokines significantly augmented the synthesis of NO2- and NO3-(p<0.05). When cells were incubated with increasing concentrations of H2O2 in the presence of IL-1beta, TNF-alpha and IFN-gamma at constant level, the synthesis of NO2- and NO3- was dose-dependently increased(p<0.05). 3. NG-nitro-L-arginine methyl ester(L-NAME), dose dependently, significantly inhibited the formation of NO2- and NO3- in cells stimulated with LPS, IFN-gamma and H2O2 at constant level(p<0.05). 4. Catalase significantly inhibited the H2 O2-induced augmentation of cytokine- induced NO2- and NO3- formation(p<0.05). But, boiled catalase did not produce a significant inhibition in comparison with the native enzyme. Another antioxidant 2-mercaptoethanol and orthophenanthroline dose-dependently suppressed NO2- and NO3- synthesis(p<0.05). Northern blotting demonstrated that H2O2 synergistically stimulated the cytokine-induced iNOS mRNA expression in RAW264.7. CONCLUSION: These results suggest that H2O2 contributes to inflammatory process by augmenting the iNOS expression and nitric oxide synthesis induced by cytokines.

Acute pulmonary responses in vivo to silica complexed with H+, Zn2+, or Fe3+

This investigation is to determine whether the surface complexation of iron influence acute pulmonary responses induced by silica. For this study, three varieties of cation complexed silica were used: silica-H+, -Zn2+, and -Fe3+, since the first two are not active in the transport of electrons and generate little free radicals relative to the dust with the surface iron. Rats (270 to 280 g) were intratracheally (IT) instilled with saline, silica-H+, -Zn2+, or -Fe3+ (5 mg in 0.5 ml saline). After 4 h, cell number, type, and differentiation were analysed in the bronchoalveolar lavage cells, and the levels of lactate dehydrogenase (LDH) and total protein were determined in the lavage fluid. In addition, bronchoalveolar lavage cells were cultured, and nitric oxide production was measured using nitrate assay. Inducible nitric oxide synthase (iNOS) mRNA in the bronchoalveolar lavage cells was also determined by northern blot analysis. Differential counts of the lavage cells showed that red blood cells were increased by 9-, 8-, and 13-fold and total leukocytes (lymphocytes plus polymorphonuclear neutrophils) by 48-, 36-, and 33-fold, following IT silica- H+, -Zn2+, and -Fe3+, respectively compared with the saline group. Meanwhile, there were no significant differences in red blood cells and total leukocytes among any of the cation complexed silica groups. The levels of LDH and total protein in the lavage fluid were significantly increased by 3- to 4-fold. However, compared among these silica groups, Fe3+ complexation did not significantly change the LDH activity and total protein. NO production in cultured bronchoalveolar lavage cells was elevated by 2-fold, following IT any of the silica treatments compared with the saline group. Furthermore, the steady-state levels of iNOS mRNA in the lavage cells were greatly increased. There were any differences in iNOS mRNA expression among the silica-treated groups as with NO production. These findings suggest that surface complexed iron may not influence the acute pulmonary responses resulted from 4h exposure to silica.

Inducible nitric oxide synthase mRNA expression and nitric oxide production in silica-induced acute inflammatory lung injury

Stimulated alveolar macrophages and neutrophils produce nitric oxide, a free radical by an inducible nitric oxide synthase (iNOS), which reacts with superoxide anion to form peroxynitrite, a more highly reactive toxic species. The objectives of the present study were to evaluate acute inflammatory lung injury and to determine iNOS mRNA induction and nitric oxide production by rat broncho-alveolar lavage cells following intratracheal treatment of silica. After 4 h exposure to silica, differential counts of bronchoalveolar lavage cells and lactate dehydrogenase (LDH) activity as well as total protein in the broncho-alveolar lavage fluid were determined. Broncho-alveolar lavage cells were also assayed for iNOS mRNA and the productions of nitrite and nitrate measured in the cells cultured. Differential analysis of broncho-alveolar lavage cells showed that the number of alveolar macrophages slightly decreased following silica treatment; however, red blood cells, lymphocytes, and neutrophils significantly were increased by 9-, 14-, and 119-fold following silica treatment, respectively, compared with the saline control. It was also found significant increases in the LDH activity and total protein in the lavage fluid obtained from silica-treated rats, indicating silica-induced acute lung injury. Northern blot analysis demonstrated that the steady state levels of iNOS mRNA in broncho-alveolar lavage cells were increased following silica treatment. The productions of nitrite and nitrate in the cultured cells were significantly increased by 2-fold following silica treatment, respectively, which were attenuated by the NOS inhibitor Nomega-nitro-L-arginine-methyl ester(L-NAME) and partially reversed by L-arginine. These findings suggest that nitric oxide production in alveolar macrophages and recruited neutrophils is increased in response to silica. Nitric oxide may contribute in part to acute inflammatory lung injury.

Inhibition by higenamine of lipopolysaccharide-induced iNOS and mRNA expression and NO production in rat aorta

Higenamine was widely used as traditional remedy for the treatment of rheumatoid arthritis. Nitric oxide (NO) may be a critical mediator in this inflammatory disease. Synovial tissue from humans with inflammatory arthritis expresses NOS2 (iNOS) mRNA and protein, and generates NO in vitro. We therefore, investigated the effect of higenamine on the induction of nitric oxide synthase (NOS) promoted by lipopolysaccharide (LPS). Prophylactic application of higenamine selectively prevented LPS-primed initiation of L-arginine-induced relaxation and restored phenylephrine(PE)-induced contraction in rat aorta. LPS-stimulated nitrite production in the incubation medium was reduced by higenamine. Furthermore, RT-PCR and Northern analysis indicated that higenamine reduced iNOS expression primed by LPS in rat aorta. These results suggest that higenamine prevents LPS-promoted induction of NOS in vascular smooth muscle.