KISEP Otology Korean J Otolaryngol 2000;43: 마우스대식세포주에서항산화제및 Dexamethasone 이유발형산화질소합성효소발현과산화질소생성에미치는효과 강준명 여상원 이흥엽 장기홍 서병도 Effect of Antioxidants

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1 KISEP Otology Korean J Otolaryngol 2000;43: 마우스대식세포주에서항산화제및 Dexamethasone 이유발형산화질소합성효소발현과산화질소생성에미치는효과 강준명 여상원 이흥엽 장기홍 서병도 Effect of Antioxidants and Dexamethasone on inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Murine Macrophage Cells Jun Myung Kang, MD, Sang Won Yeo, MD, Heung Youp Lee, MD, Ki Hong Chang, MD and Byung Do Suh, MD Department of Otolaryngology-Head and Neck Surgery, College of Medicine, The Catholic University, Seoul, Korea ABSTRACT Background and ObjectivesNitric oxide NO plays a role in a number of physiologic functions and may be cytotoxic in high concentrations. Some kinds of cells including macrophages are known to produce large quantities of NO in response to inflammatory stimuli such as cytokines and lipopolysaccharide LPS. Reactive oxygen species are also known to be important in the pathogenesis of cell and tissue injury such as otitis media with effusions. Using the macrophage RAW cell, we have examined the ability of oxidant hydrogen peroxide H 2 O 2 to stimulate NO production and inducible nitric oxide synthase inos mrna expression. Also, we have examined the effects of Dexamethasone DEX and antioxidants on H2O2 induced NO production. Materials and Method : Macrophages were cultured with LPS and H 2 O 2 in the presence or absence of DEX or antioxidants for 24 hr. The effect of DEX and antioxidants on NO production and inos mrna expression was examined by assaying the culture supernatant for oxidation product nitrite NO 2 and nitrate NO 3 content and Northern blot analysis. The effect of DEX on NO production when added at different stages of activation was determined. ResultsDEX significantly inhibited the formation of NO 2 and NO 3 and inos mrna expression in cells stimulated with LPS and H 2 O 2. Antioxidants significantly inhibited the H 2 O 2 -induced augmentation of LPS induced NO 2 and NO 3 formation and inos mrna expression. ConclusionH 2 O 2 contributes to the inflammatory process by augmenting the inos mrna expression and the NO synthesis induced by LPS, and DEX. On the other hand, antioxidants inhibit the NO synthesis by inhibition of inos mrna expression. Korean J Otolaryngol 2000;43: KEY WORDSiNOS Nitric oxide Dexamethasone Antioxidants. 136

2 Nitrite의측정 Nitrite+nitrate의측정 137

Fig. 1. Representative ethidium bromide-stained agarose gels of unlabeled RT-PCR products A and DIG digoxigenin labeled product B for inos in LPS treated macrophage cell.

3 Fig. 1. Representative ethidium bromide-stained agarose gels of unlabeled RT-PCR products A and DIG digoxigenin labeled product B for inos in LPS treated macrophage cell. Due to multiple incorporation of DIG-dUTP during the PCR process, the molecular weight of the DIG labeled PCR product is increased significantly compared to the unlabeled product 741 bp. Msize marker. 138 Korean J Otolaryngol 2000;43:136-42

4 Fig. 2. Effect of Dexamethasone DEXand N G -nitro-l-arginine methyl ester L-NAME on LPS induced NO2 +NO3 production depending on the time of addition. Cells were stimulated with 1 g/ml LPS for 24 hours. Results are means SEn=3 experiments. *p0.05 when compared the control no addition. Fig. 3. Dose-response relationship of NO2 +NO3 production depending on the H2O2 concentrations. Cells were stimulated with 1 g/ml LPS in the presence of the indicated concentrations of H2O2 for 24 hours. Results are means SE n=3 experiments. 139

Fig. 4. Effect of dexamethasone DEX on the production of NO2 plus NO3 production induced by LPS and H2O2. Cells were stimulated for 24 hours as below.

5 Fig. 4. Effect of dexamethasone DEX on the production of NO2 plus NO3 production induced by LPS and H2O2. Cells were stimulated for 24 hours as below. Column 1control unstimulated, Column 2 2 mm H2O2 without and with DEX, Column 31 g/ml LPS without and with DEX, Column 41 g/ml LPS plus 2 mm H2O2 without and with DEX. Results are means SEn=5 experiments. *p0.05 No DEX vs. DEX. A B Fig. 5. Effect of antioxidants on the production of NO2 plus NO3 production induced by LPS and H2O2. Cells were stimulated for 24 hours as below. Column 1control unstimulated, Clumn 22 mm H2O2 without antioxidants, with catalase and 2-mercaptoethanol, Column 31 g/ml LPS without antioxidants, with catalse and 2-mercaptoethanol, Column 41 g/ml LPS plus 2 mm H2O2 without antioxinants, with catalase and 2-mercaptoethanol. Results are means SEn=5 experiments. 2-Mercaptoethanol 20 mm pretreated for 3 hours before adding LPS plus H2O2. Catalase 3,000 U/ml. *p0.05 No antioxidants vs. antioxidants. Fig. 6. Effect of dexamethasone DEX and antioxidants on inos mrna by Northern analysis A. Cells were stimulated for 24 hours as below. Lane 1unstimulated, lane 21 g/ml LPS, lane 31 g/ml LPS plus 2 mm H2O2 with 2-mercaptoethanol, lane 41 g/ml LPS plus 2 mm H2O2 with DEX, lane 51 g/ml LPS plus 2 mm H2O2. TopNorthern hybridization with murine macrophage inos cdna as probe. BottomHybridization with-action cdna. In Panel B is the summary data n=3 with inos mrna on the vertical axis expressed as ratio to -actin mrna using scanning laser densitometry. 140 Korean J Otolaryngol 2000;43:136-42

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7 REFERENCES 1) Marletta MA, Yoon PS, Iyenger R, Leaf CD, Wishnok JS. Macrophage oxidation of L-arginine to nitritenitric oxide is an intermediate. Biochemistry ) Moncada S, Palmer PMJ, Higgs EA. Nitric oxide physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; ) Borm PJA, Henderson PT. Symposium on health effects of occupational exposure to inorganic dusts. Exp Lung Res ) Lorsbach RB, Murphy WJ, Lowenstein CJ, Synder SH, Russell SW. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. J Biol Chem ) Ball SS, Prazma J, Dais CGD, Rosbe KW, Pillsbury HC. Nitric oxide A mediator of endotoxin-induced middle ear effusion. Laryngoscope ) Heiss LN, Lancaster JR, Crobett JA, Goldman WE. Epithelial autotoxicity of nitric oxiderole in the respiratory cytopathology of pertussis. Proc Natl Acad Sci USA ) Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev ) Petruska JM, Marsh J, Bergeron M, Mossman BT. Brief inhalation of asbestos compromises superoxide production in cells from bronchoal-veolar lavage. Am J Respir Cell Mol Biol ) Barnes PJ. Reactive oxygen species and airway inflammation. Free Radical Biol Med ) Nathan CF, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem ) Chung HT. Trends in medical research Nitric oxide. Korean Society of Medical Biochemistry and Molecular Biology News ) Blough NV, Zafirio OC. Reaction of superoxide with nitric oxide to form peroxynitrite in alkaline aqueous solution. Inorg Chem ) Morris SM, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol Endocrinol Metab 29E ) Kinnula VL, Everitt JI, Mangum JB, Chang LT, Carpo JD. Antioxidant defense mechanism in cultured pleural mesothelial cells. Am J Respir Cell Mol Biol ) Geller DA, Nussler AK, Silvio MD, Lowenstein CJ, Shapiro RA, Wang ST, et al. Cytokines, endotoxins, glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA ) Barnes PJ. Molecular mechanisms of steroid action in asthma. J Allergy Clin Immunol ) Xie Q, Kashiwarbara Y, Nathan C. Role of transcription factor NF-B/Rel in induction of nitric oxide synthase. J Biol Chem ) Adcock IM, Brown CR, Kwon OJ, Barnes PJ. Oxidative stress induces NF-B DNA binding and inducible NOS mrna in human epithelial cells. Biochem Biophys Res Commun ) Rubanyi GM, Vonhoutte PM. Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle. Am J Physiol H ) Rhee SG. Redox signalling hydrogen peroxide as intracellular messenger. Experimental and Molecular Medicine Korean J Otolaryngol 2000;43:136-42

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,