OBJECTIVETo observe the effect of TSP-2, the antibody of Toll-like receptor 2 extracellular domain, on the expression and DNA-binding activity of nuclear factor-κB (NF-κB) p65 protein in mice with ulcerative colitis (UC).

METHODSSixty BALB/c mice were randomized equally into normal control group, UC model group, TSP-2 treatment group, and rabbit IgG treatment group. In the latter 3 groups, the mice were fed with 5% DSS (C6H7Na3O14S3) solution for 7 days to induced UC, followed then by treatment with daily injections of TSP-2 or rabbit IgG as appropriate for 7 days. The disease activity index was recorded during the treatment. The colitis tissues were collected after the treatments for HE staining and detecting the expression and DNA-binding activity of NF-κB p65 in the colon mucosa by Western blotting and ELISA.

RESULTSThe DNA binding activity and expressions of NF-κB P65 protein increased significantly in UC model group (P<0.05). TSP-2 treatment group significantly decreased the disease activity index (P<0.05) and lowered the DNA-binding activity and expression of NF-κB P65 protein (P<0.05) in the UC mouse models, while rabbit IgG produced no such effects (P>0.05).

CONCLUSIONTSP-2 can suppress the DNA-binding activity and protein expressions of NF-κB P65 and regulate excessive immune response in the intestines to ameliorate ulcerative colitis in mice.

Animals ; Colitis, Ulcerative ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Rabbits ; Thrombospondins ; pharmacology ; Transcription Factor RelA ; genetics ; metabolism

The purpose of this paper was to study the transcriptional regulation of nuclear respiratory factor 1 (NRF1) on nuclear factor kappa B (NF-κB), a key molecule in lipopolysaccharide (LPS)-induced lung epithelial inflammation, and to clarify the mechanism of NRF1-mediated inflammatory response in lung epithelial cells. In vivo, male BALB/c mice were treated with NRF1 siRNA, followed with LPS (4 mg/kg) or 0.9% saline through respiratory tract, and sacrificed 48 h later. Expression levels of NRF1, NF-κB p65 and its target genes were detected by Western blot and real-time PCR. Nuclear translocation of NRF1 or p65 was measured by immunofluorescent technique. In vitro, L132 cells were transfected with NRF1 siRNA or treated with BAY 11-7082 (5 μmol/L) for 24 h, followed with treatment of 1 mg/L LPS for 6 h. Cells were lysed for detections of NRF1, NF-κB p65 and its target genes as well as the binding sites of NRF1 on RELA (encoding NF-κB p65) promoter by chromatin immunoprecipitation assay (ChIP). Results showed that LPS stimulated NRF1 and NF-κB p65. Pro-inflammatory factors including interleukin-1β (IL-1β) and IL-6 were significantly increased both in vivo and in vitro. Obvious nuclear translocations of NRF1 and p65 were observed in LPS-stimulated lung tissue. Silencing NRF1 resulted in a decrease of p65 and its target genes both in vivo and in vitro. In addition, BAY 11-7082, an inhibitor of NF-κB, significantly repressed the inflammatory responses induced by LPS without affecting NRF1 expression. Furthermore, it was proved that NRF1 had three binding sites on RELA promoter region. In summary, NRF1 is involved in LPS-mediated acute lung injury through the transcriptional regulation on NF-κB p65.
Acute Lung Injury/genetics* ; Animals ; Lipopolysaccharides/pharmacology* ; Male ; Mice ; NF-kappa B/metabolism* ; Nuclear Respiratory Factor 1/genetics* ; RNA, Small Interfering ; Transcription Factor RelA/metabolism*

This study was purpose to compare and analyze the chronic lymphocytic leukemia human bone marrow stromal cells (CLL-hBMSC) and normal hBMSC (N-hBMSC) so as to provide theoretical evidence for establishment of CLL-hBMSC interaction model to imitate CLL microenvironment. Mononuclear cells (MNC) were isolated from bone marrow of CLL patients and healthy donors and then were cultured, hBMSC were established by expanding for at least five passages. The mRNA expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), was analyzed by real-time PCR. The mRNA and protein expression of lymphotoxin beta receptor (LTβR) were determined by real-time PCR and Western blot, respectively. The individual NF-κB members at protein level of CLL-hBMSC and N-hBMSC were examined by Western blot. The effect of LTα1β2 on individual NF-κB family members at protein level in CLL-hBMSC and N-hBMSC was also examined by Western blot. The death of CLL cells was determined by flow cytometry with PI staining when cultured with or without CLL-hBMSC and N-hBMSC at different time points. The results showed that the hBMSC could be established successfully from bone marrow of CLL patients, which were similar to N-hBMSC. Adhesion molecules, such as VCAM-1 and ICAM-1, were found to be expressed at similar mRNA levels in CLL-hBMSC and N-hBMSC. LTβR expressions at mRNA and protein levels were comparable between CLL-hBMSC and N-hBMSC. The protein expression of the individual NF-κB family members could be detected in CLL-hBMSC and N-hBMSC with similar expression levels. LTα1β2 stimulation activated both the classical ( RelA/p50 ) and alternative ( RelB/p52 ) NF-κB complexes in CLL-hBMSC and N-hBMSC. The capacities of CLL-hBMSC and N-hBMSC to protect CLL cell survival were similar. It is concluded that there is no statistical difference between bone marrow from healthy donors and CLL patients in the efficiency of generating of hBMSC. LTβR-NF-κB signaling molecules are expressed and activated on hBMSC with a similar pattern.
Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; pathology ; Lymphotoxin beta Receptor ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Transcription Factor RelB ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo study the effect of chemokine receptor 7 (CCR-7) small interfering RNA (siRNA) on proliferation and invasion of squamous cell carcinoma of head and neck (SCCHN).

METHODSCCR-7 siRNA was co-transfected into SCCHN cell line PCI-4B using Lipofectamine 2000. CCR-7 protein level was detected by western blotting. SCCHN cell proliferation was detected by MTT, and the change of actin cytoskeleton observed by confocal laser scanning microscope. Transwell assays were used to determine chemotaxis and invasion of SCCHN cells. The activity and nuclear translocation of nuclear factor-kappa B (NF-kappa B) were detected by TransAM NF-kappa B p65 kit and fluorescence microscope respectively.

RESULTSAfter CCR-7 siRNA transfection, the protein level of CCR-7 was significantly decreased. The changes induced by CCL-19, including increased proliferation rate, polarized actin polymerization, increased chemotaxis rate and invasion rate, were all abolished by CCR-7 siRNA transfection. CCR-7 siRNA also diminished CCL-19-induced NF-kappaB activation and nuclear translocation.

CONCLUSIONSCCR-7 siRNA could inhibit expression of CCR-7 and diminish the increased proliferation and invasion of SCCHN induced by CCL-19 in vitro. CCR-7 siRNA may provide a potential treatment strategy for SCCHN.

Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Head and Neck Neoplasms ; genetics ; pathology ; Humans ; Neoplasm Invasiveness ; RNA, Small Interfering ; Receptors, CCR7 ; genetics ; Transcription Factor RelA ; metabolism

Animals ; Disease Models, Animal ; Liver ; pathology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; metabolism

OBJECTIVETo study the effect of melatonin on the expressions of glial fibrillary acidic protein (GFAP), nuclear factor-κB (NF-κB p65) and synaptophysin in mice of different ages.

METHODSTwenty young male B6C3F1 mice (5.5 months) and 20 aged mice (26 months) were both divided into control and melatonin treatment (daily dose of 0.04 mg/kg) groups. After 2.5 months of treatment, the brain tissues of the mice were collected to examine the expressions of GFAP, NF-κB and SYN by immunohistochemistry.

RESULTSIn the control groups, the expression of NF-κB p65 in the brain tissue increased with age, whereas a reverse change was found in melatonin-treated aged rats (P<0.05). Synaptophysin expression also decreased with age, but melatonin treatment significantly enhanced its expression in aged mice (P<0.05). GFAP expression in the brain tissue increased with age regardless of melatonin treatment (P>0.05).

CONCLUSIONGFAP expression is almost not affected by melatonin treatment in aged mice. Melatonin can reduce the expression levels of NF-κB p65 and synaptophysin in the brain tissue to protect the brain and slow down the aging process.

Aging ; metabolism ; Animals ; Brain ; metabolism ; Chimera ; Glial Fibrillary Acidic Protein ; Male ; Melatonin ; pharmacology ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; NF-kappa B ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Synaptophysin ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism

OBJECTIVE: To investigate the spatiotemporal expression of neuromedin B receptor (NMBR) in mice myometrium at different pregnant stages, as well as its mechanism and relation with parturition. METHODS: The pregnant mice were divided into no-pregnancy (NP), early pregnancy (EP), mid-pregnancy (MP), late-pregnancy (LP), parturition (PT) and postpartum (PP) groups (12 mice in each group), according to pregnant stage. The mRNA and protein expression of NMBR, HSP70 and IL-6 were detected in myometrium in pregnant mice by semi-quantitative RT-PCR and Western blot, while NF-kappaB-P65 DNA binding activity was determined by NoShift transcription factor assay kits, respectively. Their relation with parturition was analyzed. RESULTS: The mRNA expression level of NMBR in the PT group was significantly higher than that in the NP, EP, LP and PP groups (P<0.05), but this difference was not observed in the MP group (P>0.05). The NMBR protein in PT group was significantly higher than that in the other 5 groups (P<0.01). NF-kappaB-P65 DNA binding activity at PT group was remarkably higher than that in the NP, LP and PP groups (P<0.05). The expression of IL-6 mRNA was significantly higher than that in the NP, LP and PP groups (P<0.05), its protein expression in PT and LP groups was significantly higher than that in the NP and PP groups (P<0.05). The expression of HSP70 mRNA in the PT group was significantly higher than that in the NP and PP groups (P<0.05), and the protein of HSP70 was significantly up-regulated in PT and PP groups compared with in NP and LP groups (P<0.05). The DNA-binding activity of P65 was positively correlated to the mRNA expression of NMBR and IL-6 (r=0.40, P<0.01; r=0.30, P<0.05), so were positively correlated to DNA-binding activity of P65, mRNA expression of HSP70 and NMBR ( r=0.40, P<0.01; r=0.49, P<0.01). DNA-binding activity of P65 did not correlate with the mRNA expression of HSP70. CONCLUSION The mRNA and protein expressions of NMBR reach a peak at the onset of labor. NMBR may play an important role in the parturition via NF-kappaB P65-IL-6 signal transduction pathway. It may also influence the onset of labor by regulating HSP70, but this role does not rely on P65 pathway.
Animals ; Female ; Gestational Age ; HSP70 Heat-Shock Proteins ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Myometrium ; metabolism ; Parturition ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Receptors, Bombesin ; genetics ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism

Small heterodimer partner (SHP) is an atypical member of nuclear receptor superfamily that lacks a DNA-binding domain. In previous study, we showed that SHP, c-jun, p65 of NF-kappaB subunits, and p21WAF1 expression was increased during monocytic differentiaton with the exposure of human leukemia cells to a differentiation agent, PMA. In this study, c-Jun and p65 were shown to mediate the transcriptional activation of the SHP promoter. In addition, SHP induced the cell cycle regulatory protein levels and cooperatively increased an induction of p21WAF1 expression with p65. Furthermore, SHP protected differentiated cells from etoposide-induced cellular apoptosis through the induction and cytoplasmic sequestration of p21WAF1. Complex formation between SHP and p21WAF1 was demonstrated by means of coimmunoprecipitation. These results suggest that SHP prolongs a cellular survival of differentiating monocytes through the transcriptional regulation of target genes of cell survival and differentiation.
*Apoptosis ; Cell Differentiation ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/genetics/*metabolism ; Gene Expression Regulation ; Humans ; Monocytes/cytology ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-jun/genetics/metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Transcription Factor RelA/genetics/metabolism

OBJECTIVETo study the effects of nuclear factor (NF)-kappa B p65 ASODN on transforming growth factor beta-1 (TGF beta 1) and intercellular adhesion molecule-1 (ICAM-1) of rat hepatic stellate cells (HSC) and the mechanisms of NF-kappa B p65 ASODN in treating liver fibrosis.

METHODSType IV collagen enzyme digestion and density centrifugation methods were used to separate rat hepatic stellate cells. NF-kappa B p65 ASODN was manually synthesized and completely phosphorothioate-modified. The changes of TGF beta 1 and ICAM-1 mRNA were detected by RT-PCR and albumen of TGF beta 1 and ICAM-1 were detected by ELISA. The changes of NF-kappa B activity were determined by ELISA.

RESULTSNF-kappa B activity and the expressions of ICAM-1 and TGF beta 1 increased after the HSC were treated by TNF alpha. NF-kappa B activity weakened after being treated with NF-kappa B p65 ASODN (0.001-1.000 micromol/L), P less than 0.05 in a dose dependent manner. Transferring NF-kappa B p65 ASODN (0.001-1.000 micromol/L) also weakened the expression of ICAM-1 and TGF beta 1 mRNA and the protein induced by TNF alpha in HSC. It was also in a dose dependent manner, P less than 0.05.

CONCLUSIONSAfter transferring NF-kappa B p65 ASODN into HSC, their NF-kappa B activity decreased, and their mRNA and protein expressions of ICAM-1 and TGF beta 1 also decreased. This may serve as a new way in treating hepatic fibrosis.

Animals ; Cell Line ; Hepatic Stellate Cells ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Oligonucleotides, Antisense ; Rats ; Rats, Sprague-Dawley ; Transcription Factor RelA ; genetics ; Transforming Growth Factor beta1 ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo explore how NF-κB family members regulate maspin expression in prostate cancer cells.

METHODSThe expression of NF-κB subunits and maspin was detected by Western blot analysis in prostate cancer DU145, PC-3, and LNCaP cell lines. RNA interference was performed to analyze whether RelB- or RelA-deletion affectes cell death as well as the expression of NF-κB subunits and maspin. The impact of RelB-silencing in DU145 cells was investigated by flow cytometry. The regulation of RelB on maspin expression in the prostate cancer PC-3 cells was also examined via stable transfection of RelB expression plasmid.

RESULTSRelA, p50, RelB, and p52 were constitutively expressed in androgen-independent prostate cancer DU145 and PC-3 cells, while RelB had the highest expression in DU145 cells. Low expression of maspin was detected in LNCaP and DU145 cells, but elevated expression in PC-3 cells. RelB-silencing in DU145 cells by siRNA interference upregulated the endogenous expression of maspin and induced cell apoptosis (13.3±4.2)%. Overexpression of RelB in PC-3 cells inhibited the endogenous expression of maspin. RelA-silecing had no significant influence on the endogenous expression of maspin.

CONCLUSIONSThe classical and alternative NF-κB activitions are sustained in androgen-independent prostate cancer cell lines. The expressions of RelB and maspin are inversely correlated in these cancer cells. The expression of RelB negatively regulates the endogenous expression of maspin, then interferes the cell survival. RelA is not involved in the regulation of maspin expression.

Apoptosis ; Cell Line, Tumor ; Gene Silencing ; Humans ; Male ; NF-kappa B ; genetics ; metabolism ; NF-kappa B p50 Subunit ; genetics ; metabolism ; NF-kappa B p52 Subunit ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Serpins ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Transcription Factor RelB ; genetics ; metabolism ; Transfection