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Effect of notch1,2,3 genes silicing on NF-κB signaling pathway of macrophages in patients with atherosclerosis.
- Source :
-
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie [Biomed Pharmacother] 2016 Dec; Vol. 84, pp. 666-673. Date of Electronic Publication: 2016 Sep 30. - Publication Year :
- 2016
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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.<br />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.<br />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.<br />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.<br /> (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)
- Subjects :
- Adaptor Proteins, Signal Transducing
Atherosclerosis metabolism
Calcium-Binding Proteins
Cells, Cultured
Cytokines genetics
Cytokines metabolism
Humans
Inflammation Mediators metabolism
Intercellular Signaling Peptides and Proteins genetics
Intercellular Signaling Peptides and Proteins metabolism
Jagged-1 Protein genetics
Jagged-1 Protein metabolism
NF-KappaB Inhibitor alpha genetics
NF-KappaB Inhibitor alpha metabolism
NF-kappa B p52 Subunit genetics
NF-kappa B p52 Subunit metabolism
Proteasome Endopeptidase Complex metabolism
Receptor, Notch1 metabolism
Receptor, Notch2 metabolism
Receptor, Notch3 metabolism
Signal Transduction
Transfection
Atherosclerosis genetics
Macrophages metabolism
NF-kappa B metabolism
RNA Interference
Receptor, Notch1 genetics
Receptor, Notch2 genetics
Receptor, Notch3 genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1950-6007
- Volume :
- 84
- Database :
- MEDLINE
- Journal :
- Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
- Publication Type :
- Academic Journal
- Accession number :
- 27697639
- Full Text :
- https://doi.org/10.1016/j.biopha.2016.09.078