低氧激活巨噬細胞內(nèi)NF-κB 信號轉(zhuǎn)導通路的機制

1、Acta Physiologica Sinica, June 25, 2004, 56 (3: 269-274 : Acta Physiologica Sinica, August 25, 2004, 56 (4: 515-520 NF-B 515 NF- B 1,* 1 , 1 , 2 , , 1 , 1 , 1 , 1 300050; 2 , 100850 : (macrophage dichlorofluorescein diacetate, DCFH-DA chain reaction, RT-PCR P65 mRNA , (200 µmol/L ; : , NF-B ; :

2、 Q291 ; ; , (P<0.01 , ROS , (1% O 2 , 5% CO 2 , Western blotting 2,7(2,7(reverse transcription polymerase IB NF-B (Tyr (P<0.05, genistein , , P65 mRNA NF-B (reactive oxygen species, ROS NF-B(nuclear factor kappa B , IB Tyr NIB Tyr , IB B NF-B (N-acetylcysteine, NAC, 500 µmol/L Tyr NF-B RO

3、S, , IB NF-B (P<0.01; Tyr Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation ZHANG Cui-Ping1,*, XIE Yin-Zhi1, CHEN Peng2, HONG Xin1, XIAO Zhong-Hai1, MA Yan1, LU Yong-Da1 1 Institute of Health and Environmental Medicine, Academy of Militar

4、y Medical Sciences, Tianjin 300050, China;2Institute of Radioactive Medicine, Academy of Military Medical Sciences, Beijing 100850, China Abstract: The effects of hypoxia on the level of reactive oxygen species (ROS, IB tyrosine phosphorylation, transcription of P65 mRNA and NF-B activation in isola

5、ted rat peritoneal macrophages were investigated by DCFH-DA fluorescence spectrophotometry, Western blotting and RT-PCR. The results obtained are as follows. (1 During hypoxia, the levels of intracellular ROS began to increase at 1 h, then reached a peak at 2 h, and began to decrease after 3 h. IB t

6、yrosine phosphorylation began to rise after 2 h hypoxia and was the highest after 3 h hypoxia. After 4 h hypoxia it decreased gradually. NF-B 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

7、level of IB phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 µmol/L, NF-B activation induced by hypoxia was blocked significantly. (3 The expression of p65 mRNA was also elevated markedly during hypoxia. These results su

8、ggest that hypoxia may lead to IB phosphorylation and NF-B activation through intracellular ROS, and that the regulation of NF-B activity may involve IB phosphorylation and the expressions of each subunit gene of NF-B. Key words: hypoxia; macrophages; reactive oxygen species; nuclear factor kappa B

9、, NF-B , , Received 2003-09-22 Accepted 2004-01-14 Corresponding author. Tel: +86-10-66931315; Fax: +86-10-66931315; E-mail: zcp666666 516 1 2 Acta Physiologica Sinica, August 25, 2004, 56(4: 515-520 1999 Beraud NFNF-B , BSA 1% TBST p-Tyr 1:2 000 4 , 30 µl (bovine serum protein, / Tyr IB Tyr 1.

10、5 h NF-kB (p65 , TBST IgG IgG ( TBST IBAS 1.5 RT-PCR 10 6 (Pervanadate, pV B ( , (macrophage species, ROS P65 mRNA , , IB , (reactive oxgen NF-B ROS NF-B NF-B 3 3 1 min, 1:2 000 ECL 6 min, 1 h, 1 min X 3 min, 4 P65 mRNA TRIzol , RNA : p65: (Gibco , 1 1.1 ( (phytohemagglutinin, PHA, 10 mg/ml, 0.1 ml,

11、 1 , 3 d, 4d PBS, , 4 , ml, 1.2 1%, 1.3 DCFH-DA (2 10 DCFH-DA (10 µmol/L DA DA ROS , , nm 1.4 Western blotting NF- B 10 6 3 6 RNA RNA , , , , RNA , 5 GGA AAT GTA TGT TCC TAA GCT CGA CAG3, 5CGT GTC GCC GCC CGA CCG AAT CGA CGG C3, 640 bp; -actin: ACA CAG3, G3, Titan MANNHEIM TM , 70% ( 10 min , ,

12、 1 12 min, 5ACC TCT ATG CCA 5GTA ACA GTC CGC CTA GAA 266 bp ( RT-PCR RT-PCR GDS 5 , SPSS , (BOEHRINGER , 8 µl, 1.5% 2 µl , 250 g, 10 min, , 10 6 / , 50 µl 0.5 µg/ml , 80 V, 45 min, , ROS ml, 30 min, ROS >95%, DCFHDCFH- 1.6 mean t SD , P<0.05 , , DCFH, 2 2.1 , , ( , , , , RO

13、S 1 2 3 ROS 98% ( 2 4 5 h 1h , , DCFH-DA Hepes 488 nm, P-Tyr IB 4 , 2 µg/µl, 2.2 , 535 1 : NF-B 517 2 3 4 2h 5 h, , IB Tyr (P<0.01, IB , Tyr ,5h 1 ,3h 1. Fig.1. Cultured mice peritoneal macrophages. 4. IB Fig. 4. Effect of hypoxia on the level of IB tyrosine phosphory2. Fig.2. Identific

14、ation of macrophages with non-specificesterase dye method. lation in macrophages. Lane 1, control; lane 2, 1 h; lane 3, 2 h;lane 4, 3 h; lane 5, 4 h; lane 6, 5 h. *P<0.05,*P<0.01. A: Expression of TyrP (IB in macrophages during hypoxia by Western blotting. B: Expression of Tyr-P (IB in macroph

15、ages during hypoxia. 3. ROS Fig. 3. Effect of hypoxia on the level of ROS in macrophages. *P< 0.05, *P<0.01 compared with control group; #P<0.01 compared with 2 h group. ROS , ,2h 5h (P<0.01, ( ROS 3 NF-kB 5. NAC IB Tyr-P Fig. 5. Antioxidant NAC blocked the hypoxia-induced IB tyrosine ph

16、osphorylation. Lane 1,control; lane 2, hypoxia; lane 3, NAC+hypoxia. *P<0.01 compared with control group; #P<0.01 compared with hypoxia group. A: Expression of Tyr-P(IB in cultured macrophages by Western blotting. B: Expression of Tyr-P 2.3 I B Tyr (IB in cultured macrophages. 518 Acta Physiol

17、ogica Sinica, August 25, 2004, 56(4: 515-520 ( 0.01, L, 4 3h 3h IB Tyr (P < NAC (500 µmol/ , IB Tyr ( 5 NAC Tyr Tyr ROS NF- B (1% O 2 1 NF-B NF-B 4h ( 2 3 4 5 2h , (P<0. 6 , ROS IB 2.4 h , 01, 5 h 7. genistein NF- B Fig. 7. Protein tyrosine kinase inhibitor blocked the hypoxia-induced NF-

18、kB activation in macrophage nuclei. Lane 1, control; lane 2, hypoxia; lane 3, genistein+hypoxia. *P<0.05, *P<0.01 compared with control group; # P<0.01 compared with hypoxia group. A: Concentration of NF-B in macrophage nuclei by Western blotting. B: Concentration of NF-B in macrophage nucl

19、ei. 6. NF-B Fig. 6. Effect of hypoxia on concentration of NF-B activated in macrophage nuclei. Lane 1, control; lane 2, 1 h; lane 3, 2 h; lane 4, 3 h; lane 5, 4 h; lane 6, 5 h. *P<0.05, *P<0.01 compared with control group; # P<0.05 compared with 4 h group. A: Concentration of NF-B in macrop

20、hage nuclei during hypoxia by Western blotting. B: Concentration of NF-B in macrophage nuclei during hypoxia. 4h , NF-B Tyr IB Tyr 7 NF-B (P< , IB , 8. p65 mRNA 0.01, genistein (200 µmol/L 4h , Tyr NF-B 2.5 6h , p65 , NF-B Fig.8. Effect of hypoxia on NF-B p65 mRNA expression in macrophages.

21、Upper photo: electrophoresis afte RT-PCR. M, marker: 100, 250, 500, 750, 1 000, and 2 000 bp. * P<0.01 compared with control group. NF-B p65 mRNA : NF-B 519 (P<0.01 ( 8 NF-B NF-B ROS , ROS , NF-B ROS IB Tyr , 3 NF-B NF-B p50/p65 IB NF-B 5 NF-B TPK(tyrosine protein kinase-Ras-MAPK (mitogen-acti

22、vated protein kinase , , JAKs (just another kinase-STAT (sigal transductors and activator of transcription NF-B P65 mRNA , IkBa , IB (IB kinase, IKK 32 22 36 , (ubiquitin IB ATP 6 p50/p65 (nuclear localization signal, NLS, 4 IB , NF-B , NF-B NF-B , Kieran NF-B Ten NF-B p50 , DNA, p105 12 cDNA, p105

23、IB N IB N , 21 p50 NF-B NF-B p100, c-Rel, Bcl-3 NF-kB , : IB NF-B NF-B , NF-B , , -1 NF-B 15,16 IB, p105, , p65 mRNA , NF-B ROS, ROS , NF-B ; , 26 S 7 , NF-B lck , Src p56 IB IB Tyr NF-B , NF-kB, NF-B , ROS H 2O 2, 8 IB Tyr ROS , , ROS (interleukin-1, IL-1 acetate, PMA NF-B 8,9 NF-B : (phorbol 12-my

24、ristate 13ROS Vitamin C (catalase, ( Tyr-P NF-B 13,14 (tumor necrosis factor, TNF- , , , NF-B , (glutathione, GSH CAT10, , ROS 11 , NF-B NF-B , , (lipopolysaccharides, LPS NF-B ROS , NF-B ROS NF-B , ROS NF-B , genistein IB NF-B ROS 1 Barnes PJ, Karin M. Nuclear factor-B: a pivotal transcription fact

25、or in chronic inflammatory diseases. N Engl J Med 1997;336(15: 1066-1071. 2 Beraud C, Henzel WJ, Baeuerle PA. Involvement of regulation and catalytic subunits of phosphoinositide kinase in NFkappa B activation.Proc Natl Acad Sci USA 1999;96(2:429434. IB NAC 3 4 , , EF . ,J , , 1992, 785-906. . Henke

26、l T, Machleidt T, Alkalay I, Kronke M, Ben-Neriah. 520 Rapid proteolysis of I kappa B- is necessary for activation of transcription factor NF-kappa B. Nature 1993;365(6442: 182-185. 5 Greene EL, Velarde V, Jaffa AA. Role of reactive oxygen species in bradykinin induced mitogen activated protein kina

27、se and c-fos induction in vascular cells. Hypertension 2000;35(4: 942-947. 6 Flohe L, Brigelius-Flohe R, Saliou C, Traber MG, Packer. Redox regulation of NF-kappa B activation. Free Radic Biol Med 1997;22(6:1115-1126 7 Rudolf A, Heike L, Patrick. Tyrosine phosphorylation of IkBa activates NF-B witho

28、ut proteolytic degradation of IB. Cell 1996;86(5: 787-798. 8 Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species and intracellular messengers during cell growth and differention. Cell Physiol Biochem 2001;11(4:173-186. 9 Mercurio F, Manning AM. NF kappa-B as a primary regulator of the stress

29、 response. Oncogene 1999;18(45: 6163-6171. 10 Karin M, Lin A. NF kappa-B at the crossroads of life and deat. Nat Immunol 2002;3(3:221-227. 11 Chandel NS, Trzyna WC, Mcclintock DS, Schumacker PT. Role of oxidants in NF-B activation and TNF- gene transcription induced by hypoxia and endotoxin. J Immunol 2000; Acta Physiologica Sinica, August 25, 2004, 56(4: 515-520 165(2:1013-1021. 12 Ten RM, Paya CV, Israel N, Le Bail O, Mattei MG. The characterization