title: Effect of notch1,2,3 genes silicing on NF-κB signaling pathway of macrophages in patients with atherosclerosis.
abstract: BACKGROUND
  Notch and NF-κB signaling pathways both play important roles in the regulation of atherosclerosis (AS). However, the mechanisms of notch and NF-κB signaling pathways on AS are still unclear. In this study, we aimed to investigate the effects of notch1,2,3 genes silicing by siRNA on notch and NF-κB signaling pathways of macrophages in patients with atherosclerosis (AS), so as to seek the treatment of AS from genetic perspective.
  
  
  METHODS
  Peripheral blood mononuclears of 31 patients with AS were isolated by density gradient centrifugation and transformed by PMA to macrophages. Then macrophages were transfected with notch1-siRNA (notch1-siRNA group), notch2-siRNA (notch2-siRNA group), notch3-siRNA (notch3-siRNA group), negative control siRNA (NC group) and none (control group). RT-PCR and Western blot analysis were applied to assess the expression level of Delta-like-4 (DLL4), Jagged-1 (JAG1), IκBα and P52. Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity. Subcellular distributions of NF-κB/P52 were detected through immunofluorescence. mRNA expression levels of TNF-α, IL-6 and IL-6 in macrophages were also determined with RT- PCR. The expression of 20S proteasome was detected by Western blot.
  
  
  RESULTS
  After transfected with siRNA, there was no difference in the expression of DLL4, JAG1, IκBα and P52 between NC group and control group (p>0.05). Compared with NC group and control group, the expression of DLL4, P52 and JAG1 in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group was significantly downregulated (p<0.05 or p<0.01, respectively), whereas the expression of IκBα was significantly increased (P<0.05 or p<0.01, respectively), especially in notch1-siRNA group. The binding activity of NF-κB DNA was lower in notch1- siRNA group, notch2-siRNA group and notch3-siRNA group compared with NC group and control group (p<0.05), especially in notch1-siRNA group. The fluorescence intensity of p52 was decreased significantly both in the nucleus and cytoplasm in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group compared with NC group and control group (p<0.05), which decreased more obviously in the nucleus, especially in notch1-siRNA group. The TNF-α, IL-1 and IL-6 expression of notch1-siRNA group, notch2-siRNA group and notch3-siRNA group was lower compared to NC group and control group (p<0.05 or p<0.01, respectively), also especially in notch1-siRNA group. 20S proteasome level was significantly lower in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group than in NC group and control group (p<0.05 or p<0.01, respectively), especially in notch1-siRNA group.
  
  
  CONCLUSIONS
  There was a positive regulation between Notch and NF-κB signaling pathway in patients with AS. Notch1 may play a more important role than notch2 and notch 3 in the regulation of NF-κB signaling pathway in AS.
authors.affiliation: Department of Cardiology, Taizhou People's Hospital, Taizhou 225300, PR China. Electronic address: tzcardiac@163.com.
authors.forename: Zhong-Bao
authors.initials: ZB
authors.lastname: Ruan
authors.affiliation: Health Science Center, Jiangsu University, Zhenjiang, 212001, PR China.
authors.forename: Xing-Li
authors.initials: XL
authors.lastname: Fu
authors.affiliation: Department of Cardiology, Taizhou People's Hospital, Taizhou 225300, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Institute of Clinical Medicine, Taizhou People's Hospital, Taizhou 225300, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Ye
authors.affiliation: Department of Cardiology, Taizhou People's Hospital, Taizhou 225300, PR China.
authors.forename: Ru-Zhu
authors.initials: RZ
authors.lastname: Wang
authors.affiliation: Department of Cardiology, Taizhou People's Hospital, Taizhou 225300, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D050197:Atherosclerosis / Q000235:genetics* / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000072100:Jagged-1 Protein / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D052003:NF-kappa B p52 Subunit / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D034622:RNA Interference; D051881:Receptor, Notch1 / Q000235:genetics* / Q000378:metabolism; D051883:Receptor, Notch2 / Q000235:genetics* / Q000378:metabolism; D000071656:Receptor, Notch3 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D016207:Cytokines; C424126:DLL4 protein, human; D018836:Inflammation Mediators; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D016328:NF-kappa B; D052003:NF-kappa B p52 Subunit; C498206:NFKB2 protein, human; C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; C499374:NOTCH3 protein, human; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000071656:Receptor, Notch3; D000072000:NF-KappaB Inhibitor alpha; D046988:Proteasome Endopeptidase Complex
keywords: Atherosclerosis; Macrophages; NF-κB signaling pathway; Notch signaling pathway; RNA interference