Activation of NF-κB in bronchial epithelial cells from children with asthma

Objectives To determine whether nuclear factor-κB (NF-κB) is activated in epithelial cells from children with asthma and to understand the role of NF-κB in airway inflammation in asthma. Methods Bronchial mucosa specimens were obtained from 9 children with asthma and 6 control subjects. NF-κB expression in epithelial cells were detected by immunohistochemical examination, and NF-κB-DNA binding was measured by electrophoretic mobility shift assay (EMSA). Results Nuclear expression of NF-κB in epithelial cells was observed in the 9 asthmatic children. NF-κB-DNA binding was found in 4 asthmatic children (EMSA was performed in 6 asthmatic children). In contrast, both nuclear expression and NF-κB-DNA binding were absent in the 6 control subjects. Conclusion These results indicated that NF-κB is activated in epithelial cells from asthmatic children and the NF-κB activation may be the basis for the increased expression of many inflammatory genes and for airway inflammation in asthma.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

Role of β2-adrenoceptor-β-arrestin2-nuclear factor-κB signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis

Objective:To investigate the β2-adrenoceptor(β2AR)-β-arrestin2-nuclear factor-κB(NF-κB) signal transduction pathway and the intervention effects of oxymatrine in a rat model of ulcerative colitis.Methods: Forty SD rats were randomly divided into four groups,which included the normal control group,the model group, the mesalazine group and the oxymatrine treatment group,with 10 rats per group.Experimental colitis induced with trinitrobenzene sulfonic acid(TNBS) was established in each group except the normal control group.The rats in the oxymatrine treatment group were treated with intramuscular injection of oxymatrine 63 mg/(kg·d) for 15 days and the rats in the mesalazine group were treated with mesalazine solution 0.5 g/(kg·d) by gastric lavage for 15 days. The rats in the normal control group and model group were treated with 3 mL water by gastric lavage for 15 days. Diarrhea and bloody stool were carefully observed.Histological changes in colonic tissue were examined on day 7 in 2 rats per group that were randomly selected.The expression of β2AR,β-arrestin2 and NF-κB p65 in colon tissue and spleen lymphocytes were detected with immunohistochemistry and Western immunoblotting techniques on day 16 after fasting for 24 h.Six rats died of lavage with 2 each in the normal control,the model group and the mesalazine group;and were not included in the analysis.Results:The rats in the model group suffered from looser stool and bloody purulent stool after modeling.But in the oxymatrine and mesalazine groups,looser stool and bloody purulent stool reduced after treatment.And the colonic wall in the model group was thickened and the colon length shortened.The colon mucosa was congested in multiple areas with edema,erosion,superficial or linear ulcer and scar formation,while the intestinal mucosa injury reduced in the mesalazine and oxymatrine groups(P<0.01).In colonic mucosa and in spleen lymphocytes,compared with the normal control group,the expression of NF-κBp65 were significantly increased(P<0.01) in the model group while the expressions ofβ2AR andβ-arrestin2 were significantly decreased(P<0.01).Compared with the model group,the expression of NF-κBp65 was significantly decreased in the mesalazine group(P<0.01) and oxymatrine treatment group(P<0.01) while the expressions of β2AR and β-arrestin2 were significantly increased(P<0.01).There were no statistically significant differences in the expression of β2AR,β-arrestin2 and NF-κBp65 between the mesalazine group and oxymatrine group(P>0.05).Conclusions:The β2AR-β-arrestin2-NF-κB signal transduction pathway participated in the pathologic course of ulcerative colitis.Oxymatrine attenuated ulcerative colitis through regulating the β2AR-β-arrestin2-NF-κB signal transduction pathway.     

Extract of Fructus Schisandrae chinensis Inhibits Neuroinflammation Mediator Production from Microglia via NF-κB and MAPK Pathways

Objective: To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis(EFSC) on lipopolysaccharide(LPS)-induced BV-2 cells and the possible involved mechanisms. Methods: Primary cortical neurons were isolated from embryonic(E17-18) cortices of Institute of Cancer Research(ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group(treated with 1 μg/mL LPS only) and EFSC groups(treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide(MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide(NO) and interleukin-6(IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence. Results: EFSC(200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase(iN OS) and cyclooxygenase 2(COX-2) expression in LPS-induced BV-2 cel s(P0.01 or P0.05). EFSC(200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia(P0.01). In addition, EFSC al eviated cel apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium(P0.01). The mechanistic studies indicated EFSC could suppress nuclear factor(NF)-κB phosphorylation and its nuclear translocation(P0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase(MAPK) pathway(P0.01 or P0.05). Conclusion: EFSC acted as an anti-inflammatory agent in LPS-induced glia cel s. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.     

Effect of Tiantai No.1(天泰1号) on β-Amyloid-induced Neurotoxicity and NF- к B and cAMP Responsive Element-binding Protein

To investigate the effect and molecular mechanism of Tiantai No.1 (天泰1号), a compound Chinese herbal preparation, for the prevention and reduction of neurotoxicity induced by beta-element-binding protein (CREB) pathways using the gene transfection technique. Methods: B104 neuronal cells were used to examine the effects of Tiantai No.1 on lowering the neurotoxicity induced by Abeta. The cells were pre-treated with Tiantai No.1 at doses of 50, 100, 150, or 200 μg/mL respectively for 3 days and co-treated with Tiantai No.1 and beta-amyloid peptidel-40 (Aβ 1-40, 10 μmol/L) for 48 h or post-treated with Tiantai No.1 for 48 h after the cells were exposed to beta-amyloid peptides25-35 (A β 25-35) for 8 h. In gene transfection assays, cells were treated with Tiantai No.1 at 50 μg/m/and 150 μg/mL for 5 days or co-treated with Tiantai No.1 and A β 1-40 (5 μ mo/L) for 3 days after electroporation for the with Tiantai No.1 lowered the neurotoxicity induced by Abeta, and post-treating with Tiantai No.1 reduced or blocked B104 neuronal apoptotic death induced by Abeta (P相似文献   

Ursolic Acid Inhibits T-Cell Activation through Modulating Nuclear Factor-κB Signaling

<正>Objective:To investigate the effects of ursolic acid(UA) on T-cell proliferation and activation,as well as to examine its effect on nuclear factor-κB(NF-κB) signaling pathway in T cells.Methods:T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30μmol/L in the presence of phorbol 12-myristate 13-acetate(PMA) or PMA plus ionomycin.The proliferation of T cells was measured by the MTT assay.The expressions of CD69,CD25,and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2(IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay(ELISA).The level of phosphorylated IκB-α(p-lκB-α) in total protein and p65,a subunit of NF-κB,nuclear translocation were measured by Western blot analysis.Results:UA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69,CD25,and CD71 in murine T lymphocytes upon in vitro activation(P0.01).Significant reduction of IL-2 production was found in activated T cells treated with UA(P0.01).The PMA-induced increase in p-lκB-αprotein was inhibited,and nuclear translocation of p65 from the cytoplasm was blocked by UA.Conclusion:UA is a potent inhibitor for T cell activation and proliferation;these effects are associated with the inhibition of NF-κB signaling pathway.     

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

Receptor activator of nuclear factor-kappaBligand(RANKL)promotes osteoclast formation,fusion,differentiation and activation.It plays akey role in osteoclast mediated bone erosion inrheumatoid arthritis(RA).Osteoprotegerin(OPG),as a soluble decoy receptor of RANKL,can prevent bone erosion in RA.Our previousstudies have shownthat Triptolide(TP),an activecompoundidentifiedin a traditional Chinese herb--Tripterygium wilfordii Hook F(TWHF),can ef-fectively inhibit bone destruction in ex…     

Expression of NF-κB in hRPE Cells Stimulated by PDTC and IL-1β

The constitutive expression of nuclear-factor-κB (NF-κB) in human pigment epithelial (hRPE) cells cultivated in vitro and the possible changes when incubated with PDTC and IL-I were investigated. The synchronized hRPE cells in vitro were divided into two groups. In nonPDTC group, hRPE cells were exposed respectively to IL-1β and NS (for detecting the constitutive expressions of NF-κB in hRPE cells) ; In PDTC group, PDTC-pretreated hRPE cells were exposed respectively to IL-1β?Aand NS. (for detecting the constitutive expression of NF-κB in PDTC-pretreated hRPE cells). The expression of NF-κB in hRPE cells in two groups was detected by immunofluorescence stain and flow cytometry. The results showed that the constitutive expression of NF-κB in hRPE cells in vitro was 8.05 %, and increased to 30.26 % by IL-1β. After PDTC pretreatment, the constitutive expression of NF-κB in hRPE cells was decreased to 3.74%, and 3.66 % by IL-l,respectively. It was concluded that the expressions of NF-κB in hRPE cells could be increased significantly by IL-1βand depressed effectively by PDTC. Also, PDTC could significantly inhibit the activation of NF-κB induced by IL-1β.     

Pro-apoptotic role of NF-κB pathway inhibition in lipopolysaccharide stimulated polymorphonuclear neutrophils

Objective To investigate the role of nuclear factor kappa B (NF-κB) pathway inhibition in lipopolysaccharide (LPS)-stimulated apoptosis of polymorphonuclear neutrophils (PMNs).Methods Rats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate (PDTC) and gliotoxin were used as NF-κB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-κB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling and DNA fragmentation. IκBα degradation was analyzed by Western blot. NF-κB DNA binding activity was detected by an electrophoretic mobility shift assay.Results (1) The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS (100μg per rat) intratracheal instillation were alleviated by PDTC (50, 100, or 200mg/kg, i. p. ) in a dose-dependent manner. (2) PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment. (3) IκBα degradation and increased NF-KB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment.Conclusion Inhibition of either NF-κB itself or the upstream signals in NF-κB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role.     

核转录因子κB和糖皮质激素受体在慢性阻塞性肺疾病患者稳定期的相互作用

目的 探讨慢性阻塞性肺疾病(COPD)稳定期外周血和气道黏膜核转录因子κB(NF-κB)和糖皮质激素受体(GCR)的表达.方法 应用Werstern blot方法检测COPD稳定期患者和相匹配的肺功能正常对照组各8例外周血淋巴细胞和支气管黏膜核蛋白核转录因子NF-κB和GCR的表达.结果 COPD患者稳定期外周血淋巴细胞和支气管黏膜核蛋白GCR表达均显著低于对照组(P<0.05,P<0.01),两组支气管黏膜表达显著高于外周血淋巴细胞(P<0.05,P<0.01);COPD患者稳定期外周血淋巴细胞和支气管黏膜核蛋白NF-κB表达均显著高于对照组(P<0.05),两组支气管黏膜表达高于外周血淋巴细胞(P<0.05,P<0.01).结论 COPD患者稳定期与对照组NF-κB和GCR表达差异性提示COPD患者即使在稳定期仍存在慢性炎症,尤其是气道局部炎症更为明显.     

Effects of andrographolide on the activation of mitogen activated protein kinases and nuclear factor-κB in mouse peritoneal macrophage-derived foam cells

Objective:To observe the effect of andrographolide on the activation of mitogen-activated protein kinases(MAPKs) and expression of nuclear factor-κB(NF-k B) in macrophage foam cells.Methods:The mouse peritoneal macrophages were cultured in the media in the presence of oxidized low-density lipoprotein (ox-LDL),ox-LDL+andrographolide,or neither(control).The phosphorylation of MAPK molecules(p38MAPK, JNK,ERK1/2) and the expressions of NK-κB p65 were examined by Western blot.Results:As compared with cells in the control group,the expressions of phospho-p38 and NF-κB p65 were increased in the cells cultured with either ox-LDL or ox-LDL+andrographolide(P<0.01),but attenuated significantly in the presence of ox-LDL+ andrographolide when compared with ox-LDL(P<0.05).The phospho-JNK increased in the presence of either ox-LDL or ox-LDL+andrographolide when compared with control cells(P<0.01),but no significant difference existed between ox-LDL and ox-LDL+andrographolide(P>0.05).The expression of phospho-ERK1/2 was increased in the presence of ox-LDL compared with the control cells(P<0.01),but no significant differences existed between the cells cultured in the presence of ox-LDL+andrographolide and the control medium(P>0.05). Conclusions:Andrographolide could inhibit the activation of ERK1/2,p38MAPK and NK-k B induced by ox-LDL in macrophage foam cells,which might be one of its mechanisms in preventing atherosclerosis.     

LMP1 activates NF- κB via degradation of IκBα in nasopharyngeal carcinoma cells

Objective To elucidate the mechanisms by which Epstein- Barr virus- encoded latent membran e protein 1 activates NF- κB in nasopharyngeal carcinoma cells. Methods A tetracycline- regulated LMP1- expressing nasopharyngeal carcinoma cell line, T et- on- LMP1- HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting . The subcellular localization of NF- κB (p65) was detected by indirect immuno fluorescence assay. The NF- κB transactivity was studied by transient transfec tion and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, a lthough the total protein levels remained stable. The steady- state level of to tal IκBβ protein may have resulted from the initiation of an autoregulation lo op after the activation of NF- κB. No change in the IκBβ level was detected . NF- κB (p65) was translocated from the cytoplasm to the nucleus following de gradation of IκBα. After the introduction of the dominant- negative mutant of IκBα (Del 71) into Tet- on- LMP1- HNE2 cells, both nuclear translocation and transactivation of NF- κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF - κB via phosphorylation and degradation of IκBα, but not IκBβ. The do minant- negative mutant of IκBα (Del 71) could completely inhibit both the nuc lear translocation and transactivation of NF- κB induced by LMP1.     

Adenovirus-mediated overexpression of novel mutated IκBα inhibits nuclear factor κB activation in endothelial cells

Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.     

The role of NF-κB in hepatocellular carcinoma cell

Objective To evaluate the role of nuclear factor-kappaB (NF-κB) and IκBα in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IκBα (mIκBα) plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin.Methods Western blot was used to determine the expression of NF-κB and IκBα in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem κB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mIκBα plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of κB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.Results Western blot analysis for nuclear extract showed more P50 (NF-κB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IκBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P&lt;0.01）.Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappaB (IκBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIκBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.