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Evaluation of miR-181b and miR-126-5p expression levels in T2DM patients compared to healthy individuals: Relationship with NF-κB gene expression.
Dehghani, M, Aghaei Zarch, SM, Vahidi Mehrjardi, MY, Nazari, M, Babakhanzadeh, E, Ghadimi, H, Zeinali, F, Talebi, M
Endocrinologia, diabetes y nutricion. 2020;(7):454-460
Abstract
BACKGROUND Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder whose prevalence is rising very fast across the world. Diagnosis of this disease in early stages (pre-diabetic stage) plays an important role in reducing mortality associated with this disorder. miRNAs, as key players in the pathogenesis of T2DM, have been investigated in several studies. Furthermore, their expression profile changes in the early stages of diabetes mellitus in body fluids such as serum, peripheral blood, and peripheral blood mononuclear cell (PBMC) have been studied. Due to their high stability and the presence of non-invasive sensitive methods for their measurement, such as real-time PCR, they can be used for early diagnosis of T2DM as a biomarker. In this experimental study, the expression levels of miR-181b, miR-126-5p, and NF-κB were measured in patients with T2DM, pre-diabetic subjects, and healthy controls in a Yazd population. MATERIAL AND METHOD Ninety asymptomatic subjects including 30 T2DM, 30 pre-diabetic, and 30 healthy subjects (diagnosis based on WHO criteria) were included in this study. Real-time PCR was used to measure the expression levels of miR-181b and miR-126-5p. Moreover, the NF-κB expression level was also measured to determine its relationship with these two microRNAs. RESULT In this study, the expression level of miR-181b and miR-126-p decreased gradually in pre-diabetic as well as T2DM subjects compared to healthy controls. Furthermore, our study showed a significant negative correlation between these two miRNAs and NF-κB for the first time. CONCLUSION These results introduce these anti-inflammatory miRNAs as powerful tools for early diagnosis of T2DM.
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Regulation of iNOS expression by NF-κB in human lens epithelial cells treated with high levels of glucose.
Jia, J, Liu, Y, Zhang, X, Liu, X, Qi, J
Investigative ophthalmology & visual science. 2013;(7):5070-7
Abstract
PURPOSE To explore the regulation of inducible nitric oxide synthase (iNOS) expression by nuclear factor kappa B (NF-κB) in human lens epithelial cells (LECs) treated with high levels of glucose, and to elucidate the impact of this in the pathogenesis of cataracts associated with diabetes. METHODS LECs (SRA01/04) were cultured in vitro. NF-κB nuclear translocation and iNOS expression were measured at different glucose concentrations and at various time points, and the optimal concentration for detecting changes in the patterns of NF-κB nuclear translocation and iNOS expression was chosen. As a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) was used to assess the effect of inhibiting NF-κB. Western blotting and inverted fluorescence microscopy were used to monitor the nuclear translocation of NF-κB. PCR and Western blotting were used to measure iNOS expression. Using the University of California, Santa Cruz database and the TFSEARCH program, we searched the DNA sequence upstream of iNOS for the core binding sequence for NF-κB. Chromatin immunoprecipitation (ChIP) was used to measure the binding of NF-κB. RESULTS The nuclear translocation of NF-κB was measured upon glucose treatment, and the concentration of NF-κB in the nucleus was found to peak at 25 to 30 minutes of treatment with 25 mM glucose. iNOS mRNA and protein levels also increased significantly in a time- and concentration-dependent manner and iNOS mRNA and protein reached their peak values after 8 hours of treatment with 25 mM glucose. The binding of NF-κB to the promoter of the iNOS gene was enhanced in the 25 mM glucose group compared with the 5.5 mM glucose group or the 25 mM glucose + 100 μL PDTC group, and this difference was statistically significant (P < 0.05). CONCLUSIONS NF-κB regulates iNOS expression in a time- and concentration-dependent manner. Under high glucose conditions, NF-κB is activated and rapidly translocates to the nucleus, leading to increased binding to the iNOS promoter and a consequent increase in iNOS expression. The findings of this study provide important experimental evidence that clarifies the pathogenesis of cataracts associated with diabetes and contributes to the search for therapeutic targets of these cataracts.
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Identical LDL-cholesterol lowering but non-identical effects on NF-κB activity: High dose simvastatin vs combination therapy with ezetimibe.
Rudofsky, G, Reismann, P, Groener, JB, Djuric, Z, Fleming, T, Metzner, C, Grafe, IA, Bierhaus, A, Nawroth, PP
Atherosclerosis. 2012;(1):190-6
Abstract
OBJECTIVE Lowering LDL-cholesterol by statins has been proven to be associated with reduction of proinflammatory regulators e.g. activation of the transcription factor NF-κB. To our knowledge, anti-inflammatory potential of newer cholesterol lowering agents such as ezetimibe is less intensively studied. Therefore we analyzed the effects of equipotent LDL-lowering therapy with simvastatin alone compared to a combination with ezetimibe on NF-κB activation in peripheral blood mononuclear cells (PBMCs) of patients with type 2 diabetes. METHODS Thirty-one patients with type 2 diabetes were included in a double-blind, randomized trial receiving either 80 mg simvastatin (sim80; n = 10) or a combination of 10 mg simvastatin and 10 mg ezetimibe (sim10eze10; n = 11) or placebo (n = 9) for eight weeks. NF-κB binding activity and inflammatory markers (IL-6, hsCRP) were analyzed at baseline and after eight weeks of treatment. NF-κB binding activity was analyzed by electrophoretic mobility shift assay. IL-6 and hsCRP were measured by ELISA. RESULTS After eight weeks of treatment LDL-cholesterol was lowered to the same extent in both treatment groups (p = 0.40) but not in placebo. However, patients taking sim80 showed a significant reduction of mononuclear NF-κB binding activity compared to baseline (p = 0.009) while no effect was observed in the sim10eze10 group (p = 0.79). Similar differences in anti-inflammatory effects were also observed when analyzing hsCRP (sim80: p = 0.03; sim10eze10: p = 0.40) and IL-6 levels (sim80: p = 0.15; sim10eze10: p = 0.95). CONCLUSION High dose simvastatin therapy reduces proinflammatory transcription factor NF-κB binding activity and hsCRP levels, while combination of low dose simvastatin with ezetimibe resulting in a similar LDL-reduction does not affect these inflammatory markers.
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Nuclear factor-kappaB activity in peripheral blood mononuclear cells in cachectic and non-cachectic patients with chronic heart failure.
Siednienko, J, Jankowska, EA, Banasiak, W, Gorczyca, WA, Ponikowski, P
International journal of cardiology. 2007;(2):111-6
Abstract
BACKGROUND Inflammatory immune mechanisms are involved in the pathogenesis and progression of chronic heart failure (CHF), and also promote the development of generalised body wasting seen in this syndrome. We examined the activity of nuclear factor kappa-B (NF-kappaB), the major mediator of immune response, in peripheral blood mononuclear cells (PBMC) isolated from cachectic and non-cachectic patients with CHF. METHODS Using electromobility shift assay, NF-kappaB activity was assessed in nuclear fractions of PBMC isolated from 43 patients with systolic CHF (88% men, age: 64 years [median], left ventricular ejection fraction [LVEF]: 30%, ischaemic CHF aetiology: 79%, NYHA class [I/II/III/IV]: 2/21/19/1, 10 patients with cardiac cachexia) and 12 healthy adult subjects. RESULTS As compared to healthy controls, NF-kappaB activity in PBMC was increased in patients with CHF (P<0.05), in particular in those with severe CHF (NYHA class III-IV) (P<0.05). NF-kappaB activity in PBMC in CHF patients was not related either to age, sex, CHF aetiology, LVEF, or any clinical parameters reflecting disease severity (haemoglobin, LDL cholesterol, sodium and creatinine levels) (all P>0.1). Regardless of the severity of CHF expressed as NYHA class, patients with cardiac cachexia demonstrated significantly reduced NF-kappaB activity in PBMC as compared to both non-cachectic CHF patients (P<0.001) and healthy controls (P<0.05). CONCLUSION The activity of NF-kappaB system in peripheral immune cells is augmented in patients with advanced CHF, whereas it is diminished in those with cardiac cachexia. The significance of derangements within NF-kappaB system in PBMC for immune phenomena seen in cachectic and non-cachectic CHF patients remains further studies.
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Dysregulated interleukin-8 secretion and NF-kappaB activity in human cystic fibrosis nasal epithelial cells.
Carrabino, S, Carpani, D, Livraghi, A, Di Cicco, M, Costantini, D, Copreni, E, Colombo, C, Conese, M
Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society. 2006;(2):113-9
Abstract
BACKGROUND It is not clear whether cystic fibrosis (CF) airway inflammation is a consequence of bacterial infection or is intrinsically dysregulated. The aim of this study was to investigate IL-8 secretion and NF-kappaB activity in primary respiratory epithelial cells cultured from nasal polyps obtained from CF and non-CF subjects. METHODS NF-kappaB activity was studied by electrophoretic mobility-shift and quantitative colorimetric assays in nuclear extracts. Immunoreactive IL-8 levels were assessed by ELISA in cell culture supernatants. Both parameters were studied at baseline and following challenge with Pseudomonas aeruginosa or stimulation with pro-inflammatory cytokines. RESULTS Under basal conditions, CF cells presented a significant higher activity of NF-kappaB than non-CF cells (P=0.0004). P. aeruginosa challenge and IL-1beta/H2O2 co-stimulation caused four and two fold induction of NF-kappaB activity in non-CF and CF cells, respectively. IL-8 levels in unstimulated CF cells were significantly higher than in non-CF cells (P=0.0025). Upon incubation with P. aeruginosa and IL-1beta/H2O2, non-CF cells produced 6.3 times more IL-8 than unstimulated cells, whereas IL-8 secretion increased only of 1.4 times in CF cells. CONCLUSIONS CF respiratory epithelial cells exhibit a basal dysregulated production of IL-8 that partially correlates to enhanced NF-kappaB activity. Our data corroborate the hypothesis of a basal exaggerated inflammatory response in the CF respiratory epithelium.
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15-deoxy-delta (12,14)-PGJ2 inhibits astrocyte IL-1 signaling: inhibition of NF-kappaB and MAP kinase pathways and suppression of cytokine and chemokine expression.
Zhao, ML, Brosnan, CF, Lee, SC
Journal of neuroimmunology. 2004;(1-2):132-42
Abstract
We studied the role of 15-deoxy-delta (12,14)-PGJ2 (15d-PGJ2), a macrophage inhibitor with reported therapeutic effects on experimental allergic encephalomyelitis, in human astrocyte activation in vitro. 15d-PGJ2 inhibited a broad range of astrocyte inflammatory gene expression induced by IL-1, including cytokines (TNFalpha and IL-6), chemokines (RANTES/CCL5 and IP-10/CXCL10) and inducible nitric oxide synthase. 15d-PGJ2 inhibited transactivation of NF-kappaB-dependent promoters, as well as p38 and JNK MAPK phosphorylation induced by IL-1, while having no inhibitory effect on IFN-induced Stat signaling pathways. Our results demonstrating 15d-PGJ2-mediated astrocyte deactivation through inhibition of NF-kappaB are similar to those described for macrophages, and add astrocytes as additional targets for this prostaglandin (PG).
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Comparative methodologic study of NFkappaB activation in cultured endothelial cells.
Mercié, P, Belloc, F, Bihlou-Nabera, C, Barthe, C, Pruvost, A, Renard, M, Seigneur, M, Bernard, P, Marit, G, Boisseau, MR
The Journal of laboratory and clinical medicine. 2000;(5):402-11
Abstract
The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.