Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation
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1 : Acta Physiologica Sinica August June (3): (4): * ; : (macrophage) (% O 2 5% CO 2 ) (2 7 - dichlorofluorescein diacetate DCFH-DA) Western blotting (reverse transcription polymerase chain reaction RT-PCR) (reactive oxygen species ) IκBα (Tyr) P65 mrna (nuclear factor kappa B) IκBα Tyr (P<0.05) N- (N-acetylcysteine NAC 500 µmol/l) Tyr genistein (200 µmol/l) IκBα Tyr (P<0.0); P65 mrna (P<0.0) IκBα Tyr ; IκBα : ; ; ; κb : Q29 Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation ZHANG Cui-Ping * XIE Yin-Zhi CHEN Peng 2 HONG Xin XIAO Zhong-Hai MA Yan LU Yong-Da Institute of Health and Environmental Medicine Academy of Military Medical Sciences Tianjin China; 2 Institute of Radioactive Medicine Academy of Military Medical Sciences Beijing China Abstract: The effects of hypoxia on the level of reactive oxygen species () IκBα tyrosine phosphorylation transcription of P65 mrna and activation in isolated rat peritoneal macrophages were investigated by DCFH-DA fluorescence spectrophotometry Western blotting and RT-PCR. The results obtained are as follows. () During hypoxia the levels of intracellular began to increase at h then reached a peak at 2 h and began to decrease after 3 h. IκBα tyrosine phosphorylation began to rise after 2 h hypoxia and was the highest after 3 h hypoxia. After 4 h hypoxia it decreased gradually. activation began to increase after 3 h hypoxia and reached a peak after 4 h hypoxia. (2) When antioxidant NAC (500 µmol/l) was added into the medium the level of IκBα phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 µmol/l) activation induced by hypoxia was blocked significantly. (3) The expression of p65 mrna was also elevated markedly during hypoxia. These results suggest that hypoxia may lead to IκBα phosphorylation and activation through intracellular and that the regulation of activity may involve IκBα phosphorylation and the expressions of each subunit gene of. Key words: hypoxia; macrophages; reactive oxygen species; nuclear factor kappa B Received Accepted Corresponding author. Tel: ; Fax: ; zcp666666@sohu.com
2 56 Acta Physiologica Sinica August (4): [2] / Tyr [] 999 Beraud (Pervanadate pv) IκBα Tyr NFκB (macrophage) (reactive oxgen species ) IκBα P65 mrna. ( ) (phytohemagglutinin PHA 0 mg/ml) 0. ml 3 d 4 d 70% ~2 min ( ) PBS 0 min g 0 min 0 6 / ml.2 %.3 DCFH-DA (2 0 6 ml >95%) DCFH-DA (0 µmol/l) 30 min DCFH- DA DCFH- DA DCFH DCFH-DA Hepes 488 nm 535 nm.4 Western blotting [3] P-Tyr IκBα µg/µl 30 µl % (bovine serum protein BSA) TBST.5 h p-tyr NF-kB (p65) ( :2 000) 4 TBST 3 IgG IgG ( :2 000) h TBST 3 ECL min X min 6 min 3 min IBAS.5 RT-PCR P65 mrna TRIzol (Gibco) RNA RNA RNA RNA : p65: 5 GGA AAT GTA TGT TCC TAA GCT CGA CAG3 5 CGT GTC GCC GCC CGA CCG AAT CGA CGG C3 640 bp; β-actin: 5 ACC TCT ATG CCA ACA CAG3 5 GTA ACA GTC CGC CTA GAA G3 266 bp ( ) Titan TM RT-PCR (BOEHRINGER MANNHEIM ) 50 µl RT-PCR 8 µl 2 µl 0.5 µg/ml.5% 80 V 45 min GDS.6 5 mean SD SPSS t P< ( ) 98% ( 2) h h
3 : h IκBα Tyr 2 h IκBα Tyr 3 h (P<0.0) 5 h. Fig.. Cultured mice peritoneal macrophages. 2. Fig.2. Identification of macrophages with non-specificesterase dye method. 4. IκBα Fig. 4. Effect of hypoxia on the level of IκBα tyrosine phosphorylation in macrophages. Lane control; lane 2 h; lane 3 2 h;lane 4 3 h; lane 5 4 h; lane 6 5 h. * P<0.05 ** P<0.0. A: Expression of Tyr- P (IκBα) in macrophages during hypoxia by Western blotting. B: Expression of Tyr-P (IκBα) in macrophages during hypoxia. 3. Fig. 3. Effect of hypoxia on the level of in macrophages. * P< 0.05 ** P<0.0 compared with control group; ## P<0.0 compared with 2 h group. 2 h (P<0.0) 5 h ( 3) NF-kB 2.3 IκBα Tyr 5. NAC IκBαTyr-P Fig. 5. Antioxidant NAC blocked the hypoxia-induced IκBα tyrosine phosphorylation. Lane control; lane 2 hypoxia; lane 3 NAC+hypoxia. ** P<0.0 compared with control group; ## P<0.0 compared with hypoxia group. A: Expression of Tyr-P(IκBα) in cultured macrophages by Western blotting. B: Expression of Tyr-P (IκBα) in cultured macrophages.
4 58 Acta Physiologica Sinica August (4): ( 4) 3 h IκBα Tyr (P < 0.0) NAC (500 µmol/ L) 3 h IκBα Tyr ( 5) NAC Tyr IκBα Tyr 2.4 (% O 2 ) h 2 h 4 h (P<0. 0) 5 h ( 6) 7. genistein NFκB Fig. 7. Protein tyrosine kinase inhibitor blocked the hypoxia-induced NF-kB activation in macrophage nuclei. Lane control; lane 2 hypoxia; lane 3 genistein+hypoxia. * P<0.05 ** P<0.0 compared with control group; ## P<0.0 compared with hypoxia group. A: Concentration of in macrophage nuclei by Western blotting. B: Concentration of in macrophage nuclei. 6. Fig. 6. Effect of hypoxia on concentration of activated in macrophage nuclei. Lane control; lane 2 h; lane 3 2 h; lane 4 3 h; lane 5 4 h; lane 6 5 h. * P<0.05 ** P<0.0 compared with control group; # P<0.05 compared with 4 h group. A: Concentration of in macrophage nuclei during hypoxia by Western blotting. B: Concentration of in macrophage nuclei during hypoxia. 4 h (P< 0.0) Tyr genistein (200 µmol/l) IκBα Tyr 4 h 7 IκBα Tyr 2.5 p65 mrna 6 h p65 8. p65 mrna Fig.8. Effect of hypoxia on p65 mrna expression in macrophages. Upper photo: electrophoresis afte RT-PCR. M marker: and bp. ** P<0.0 compared with control group.
5 : 59 (P<0.0) ( 8) 3 p50/p65 IκBα p50/p65 (nuclear localization signal NLS) [5] (IκB kinase IKK) [4] IκBα IκB IκBα N IκBα N 2 22 (ubiquitin) IκBα ATP 26 S [7] Src p56 lck IκBα Tyr IκBα NF-kB IκBα Tyr H 2 O 2 : (tumor necrosis factor TNF-α) - (interleukin- IL-) (phorbol 2-myristate 3- acetate PMA) [89] (glutathione GSH) [8] [6] Vitamin C (catalase CAT) [0] [] (lipopolysaccharides LPS) IκBα NAC genistein IκBα IκBα Tyr TPK(tyrosine protein kinase)-ras-mapk (mitogen-activated protein kinase) JAKs (just another kinase)-stat (sigal transductors and activator of transcription) P65 mrna IkBa Kieran p50 cdna Ten p50 p05 DNA p05 IκBα p05 p00 c-rel Bcl-3 p65 mrna NF-kB : IκBα ; ( Tyr-P ) [56] [34] Barnes PJ Karin M. Nuclear factor-κb: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 997;336(5): Beraud C Henzel WJ Baeuerle PA. Involvement of regulation and catalytic subunits of phosphoinositide kinase in NFkappa B activation.proc Natl Acad Sci USA 999;96(2): EF J Henkel T Machleidt T Alkalay I Kronke M Ben-Neriah. [2]
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