PURPOSE: The nuclear factor-kappa B (NF-kappa B) has been known to regulate the inflammatory and immune process by transcription of inflammatory intermediates. The purpose of the present study is to show the difference in activity of NF-kappa B and its inhibitory factor-I kappa B alpha in patients with rheumatoid arthritis, osteoarthritis and normal control subjects. MATERIALS AND METHODS: Synovial membrane samples were obtained at the time of orthopedic surgery from the knees of 7 patients with RA and 7 patients with OA. Two control samples were obtained from an amputee with no history of arthritis. We designed the primer of the subunit p65 of NF-kappa B and I kappa B alpha, measured the activity of them by RT-PCR, and analyzed the expression of NF-kappa B by immunohistochemical staining. RESULTS: From the results of RT-PCR, the expression levels of NF-kappa B was found to be higher in synovial tissues obtained from patients with RA than from synovial tissue obtained from patients with OA, and the least from the control group. The expression levels of I kappa B alpha were not different statistically among the three groups. Immunohistochemical staining for the NF-kappa B was dominant in synovial tissue from patients with RA. The result of immunohistochemical staining was similar to the results of RT-PCR for NF-kappa B. The localization of the staining was predominantly nuclear. CONCLUSION: In this study, activity of NF-kappa B of rheumatoid arthritis was higher than the other group, but expressions of I kappa B alpha were no different between the diseases. Further studies about specific inhibitors of NF-kappa B will benefit the development of rheumatoid arthritis regimens with greater efficacy.
Amputees ; Arthritis ; Arthritis, Rheumatoid* ; Humans ; I-kappa B Proteins* ; Knee ; NF-kappa B* ; Orthopedics ; Osteoarthritis* ; Synovial Membrane

BACKGROUND: Sepsis still has a high mortality rate despite adequate supportive care. Newer therapeutic modalities have been developed but they have generally ended in failure. Recently, insulin was reported to have an anti-inflammatory effect by inhibiting the IkappaB/NF-kappaB pathway, and may have therapeutic potential in sepsis. However, the precise mechanism of the anti-inflammatory effect of insulin is unclear. This study examined the role of insulin in activating IkappaB/NF-kappaB in macrophage. METHODS: Raw 264.7 cells, a murine macrophage cell line, were used in this experiment. Western blotting using IkappaB Ab and phosphor-specific IkappaB Ab was performed to evaluate the degradation and phosphorylation of IkappaB cells. For the IkappaB Kinase (IKK) activity, an immune complex kinase assay was performed. The level of interleukin-6 (IL-6) was measured by ELISA to determine the level of proinflammatory cytokine. RESULTS: IkappaBalpha degradation began 30 min after lipopolysaccharide (LPS) treatment. However, an insulin pretreatment suppressed the IkappaBalpha degradation caused by the LPS treatment. The phosphorylation of IkappaBalpha and IKK activity was also inhibited by the insulin pretreatment. Finally, the insulin pretreatment showed a tendency to suppress the induction of IL-6 by LPS. CONCLUSION: Insulin might have an anti-inflammatory effect though partial inhibition of the IkappaB/NFkappaB pathway in macrophage cell lines.
Antigen-Antibody Complex ; Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; I-kappa B Kinase ; I-kappa B Proteins ; Inflammation ; Insulin ; Interleukin-6 ; Macrophages ; Phosphorylation ; Phosphotransferases ; Sepsis

PURPOSE: To analyze the action mechanism of NF-kappaB, IkappaB-alpha and effect of the Dexamethasone (DEXA) in mediating this inflammation, after stimulating cultured herniated intervertebral disc cells with TNF-alpha. MATERIALS AND METHODS: After cultured human intervertebral disc cells passaged three times, they were divided into four groups: A control group (A), DEXA treatment group (B), TNF-alpha treated group (C), TNF-alpha and DEXA were treated at the same time (D). IL-6 and IL-1beta gene expression were measured with semi-quantitative RT-PCR. Western blot analysis was performed to measure protein expression of IkappaB-alpha in the above groups for 10 minutes, 1 hour, 2 hours. In addition, in order to explain the mechanism of NF-kappaB nuclear binding for each group, the nuclear amount of NF-kappaB binding in the nucleus is measured by EMSA. RESULTS: In RT-PCR, expression of IL-6 and IL-1beta was greatest in group C, followed by group D, group A. IkappaB-alpha expression of the group treated with DEXA was not detected in Western blot results within 10 minutes. However, if stimulated by TNF-alpha, the DEXA was not inhibited of IkappaB-alpha concentration. After 1 hour and 2 hours, IkappaB-alpha levels were expressed by cells autonomously (autoregulatory induction). EMSA results expression levels in nuclear protein was maintained in accordance with protein expression. CONCLUSIONS: Our study shows that DEXA inhibits the production of mediators such as inflammatory IL-6 and IL-1beta, however, may not inhibit the transcription of NF-kappaB stimulated by TNF-alpha.
Blotting, Western ; Dexamethasone ; Gene Expression ; Humans ; I-kappa B Proteins ; Inflammation ; Interleukin-6 ; Intervertebral Disc ; Negotiating ; NF-kappa B ; Nuclear Proteins ; Tumor Necrosis Factor-alpha

Intestinal epithelial cells are known to up-regulate the expression of several chemokines in response to bacterial toxins. Since there has been little understanding on the cellular mechanisms of C. difficile toxin A-induced mucosal inflammation, we investigated whether nuclear factor-kappa B (NF-kappaB) could regulate chemokine gene expression in HT-29 intestinal epithelial cells stimulated with C. difficile toxin A. C. difficile toxin A rapidly increased signals of NF-kappaB composed with p65 and p50 subunits in HT-29 cells, whereas it decreased the signals of IkappaBalpha. Blocking the NF-kB activation by transfection with dominant negative I kappa B alpha-containing retrovirus attenuated the upregulated expression of IL-8, GRO-alpha, and MCP-1 induced by C. difficile toxin A. These results suggest that NF-kappaB is a major regulator of chemokine gene expression in C. difficile toxin A-stimulated intestinal epithelial cells.
Bacterial Toxins ; Chemokines ; Clostridium difficile* ; Clostridium* ; Epithelial Cells* ; Gene Expression ; HT29 Cells ; Humans ; I-kappa B Proteins ; Inflammation ; Interleukin-8 ; NF-kappa B ; Retroviridae ; Transfection

Resveratrol (trans-3,4'-trihydroxystillbene), a naturally occurring polyphenolic antioxidant found in grapes and red wine, elicits diverse biochemical responses and demonstrates anti-aging, anti-inflammatory, and anti-proliferative effects in several cell types. Previously, resveratrol was shown to regulate differentiation and inflammation in rabbit articular chondrocytes, while the direct production of nitric oxide (NO) in these cells by treatment with the NO donor sodium nitroprusside (SNP) led to apoptosis. In this study, the effect of resveratrol on NO-induced apoptosis in rabbit articular chondrocytes was investigated. Resveratrol dramatically reduced NO-induced apoptosis in chondrocytes, as determined by phase-contrast microscopy, the MTT assay, FACS analysis, and DAPI staining. Treatment with resveratrol inhibited the SNP-induced expression of p53 and p21 and reduced the expression of procaspase-3 in chondrocytes, as detected by western blot analysis. SNP-induced degradation of I-kappa B alpha (IkappaB-alpha) was rescued by resveratrol treatment, and the SN50 peptide-mediated inhibition of NF-kappa B (NF-kappaB) activity potently blocked SNP-induced caspase-3 activation and apoptosis. Our results suggest that resveratrol inhibits NO-induced apoptosis through the NF-kappaB pathway in articular chondrocytes.
Apoptosis* ; Blotting, Western ; Caspase 3 ; Chondrocytes* ; Humans ; I-kappa B Proteins ; Inflammation ; Microscopy, Phase-Contrast ; NF-kappa B* ; Nitric Oxide ; Nitroprusside ; Tissue Donors ; Vitis ; Wine

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

<b>METHODSb>IkappaBalphaM gene (203 - 1003 bp) encoding 267 amino acids, acquired by site-directed deleting N-terminal phosphorylation sites of serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further polymerase chain reaction (PCR), restriction digestion, deoxyribonucleic acid (DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IkappaBalphaM, containing cytomegalovirus (CMV) promoter, IkappaBalphaM complementary DNA (cDNA) and polyadenylic acid (PolyA) signals, into the type 5 adenovirus (Ad5) vector, the resultant AdIkappaBalphaM was packaged in human embryonic kidney (HEK) 293 cells by cotransfection with lipofectamine. Western blot analysis and electrophoretic mobility shift assay were utilized to detect the AdIkappaBalphaM-mediated overexpression of IkappaBalphaM in HEK293 cells and its suppressive effect on phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in human umbilical vein endothelial (ECV304) cells, respectively.

<b>RESULTSb>The relevant nucleotides and deduced amino acids of 801 bp IkappaBalphaM gene were consistent with those of IkappaBalpha gene (GenBank accession number: M69043). The titer of the prepared AdIkappaBalphaM was 4.0 x 10 (12) plaque-forming units (pfu)/L. Moreover, the IkappaBalphaM gene was overexpressed in HEK293 cells, and potently inhibited the PMA-induced NF-kappaB activation in ECV304 cells dose-dependently.

<b>CONCLUSIONSb>AdIkappaBalphaM is a novel vector for both efficient transfer and specific overexpression of IkappaBalphaM gene, as well as potent inhibition of NF-kappaB activity, providing a promising strategy for gene therapy of asthma.

Adenoviridae ; genetics ; Cell Line ; Endothelial Cells ; metabolism ; Genetic Therapy ; Humans ; I-kappa B Proteins ; genetics ; Mutation ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Tetradecanoylphorbol Acetate ; pharmacology

PURPOSE: Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action. METHODS: LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. IkappaB-alpha degradation, nuclear translocation of NF-kappaB subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-kappaB was also analyzed. RESULTS: Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of IkappaB-alpha induced by P. intermedia LPS. Curcumin blocked NF-kappaB signaling through the inhibition of nuclear translocation of NF-kappaB p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation. CONCLUSIONS: Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease.
Curcumin ; DNA ; I-kappa B Proteins ; Immunoblotting ; Interleukin-6 ; Lipopolysaccharides ; NF-kappa B ; Periodontal Diseases ; Phosphorylation ; Prevotella ; Prevotella intermedia ; Real-Time Polymerase Chain Reaction ; RNA, Messenger

NF-kappaB, a collective name of dimeric transcription factors, is composed of members of the Rel family proteins that recognize and bind a specific DNA sequence. It is normally sequestered in the cytoplasm of non-stimulated cells by associating with a family of inhibitor proteins called IkappaBs. Exposure of cells to a variety of extra-and intra-cellular stimuli leads to the rapid proteolytic degradation of IkappaBs, which frees NF-kappaBs allowing them to translocate to the nucleus where it regulates gene transcription. NF-kappaB is involved in a lot of physiological processes such as immunity, inflammation, cell proliferation, apoptosis and even tumorigenesis by regulating the transcription of a larger number of genes. This review introduces the various mechanisms of NF-kappaB activation including a recently reported alternative activation pathway mediated by lymphotoxin alpha/beta, B cell activating factor and CD40 ligand. The signal transduction pathway leading to NF-kappaB activation via IKK in response to proinflammatory factors like TNF-alpha and IL-1 is addressed in more detail concerning the regulation of IKK activity, mechanism of IkappaB degradation and regulation of transactivation activity of NF-kappaB on different levels. Considering the important role of NF-kappaB in cell proliferation and regulation of various genes participating in apoptosis, the involvement of NF-kappaB in tumorigenesis and drug screening is also discussed.
Animals ; Drug Evaluation, Preclinical ; Humans ; I-kappa B Proteins ; metabolism ; NF-kappa B ; antagonists & inhibitors ; metabolism ; physiology ; Neoplasms ; etiology ; Signal Transduction ; Transcription, Genetic ; physiology

<b>OBJECTIVEb>To investigate the cytotoxicity of bromoxynil on SH-SY5Y cells and its effect on the expression of nuclear factor-kappa B (NF-&kappa;B) and I kappa B alpha (I&kappa;Bα) in SH-SY5Y cells.

<b>METHODSb>SH-SY5Y cells were exposed to bromoxynil (10, 50, or 100 µmol/L) for 24 and 48 h, and other SH-SY5Y cells, which were used as a control, were exposed only to dimethyl sulfoxide. After 24 and 48 h of exposure, the morphological changes of these cells were observed under an inverted microscope, and the cytotoxicity of bromoxynil was measured by MTT assay. The cellular proliferation was examined by cell counting after 12, 24, 48, 72, and 96 h of exposure. After 24 h of exposure, the expression of NF-&kappa;B was evaluated by Western blot and immunocytochemistry, and the expression of I&kappa;Bα was evaluated by Western blot.

<b>RESULTSb>The cellular proliferation inhibition rates (CPIRs) of 50 and 100 µmol/L groups were significantly higher than that of the control group after 24 and 48 h of exposure (P < 0.05); the CPIR was significantly higher after 48 h than after 24 h in the two groups (P < 0.05). The growth curve revealed that these groups began to show differences in cell count at the 24th of exposure and that the differences were even more marked as the exposure went on (F = 17.15, P < 0.05). The control group had a significantly increased cell count at the 48th, 72nd, and 96th h of exposure (P < 0.05); the 10 and 50 µmol/L groups had a significantly increased cell count at the 72nd and 96th h of exposure (P < 0.05); the 100 µmol/L group showed no significant change in cell count during 96h of exposure. The 50 and 100 µmol/L groups hada significantly longer cell doubling time than the control group (P < 0.05). The immunocytochemistry showed that as the dose of bromoxynil increased, the brownish yellow particles in the cytoplasm and nuclei became darker, the expression of NF-&kappa;B was upregulated, and the nuclear translocation of NF-&kappa;B was increased. The Western blot showed that the 100 µmol/L group had significantly higher expression of NF-&kappa;B in the nuclei than the control group (P < 0.05) and that the 50 and 100 µmol/L groups had significantly lower expression of I&kappa;Bα in total proteins than the control group (P < 0.05).

<b>CONCLUSIONb>Bromoxynil can inhibit the proliferation of SH-SY5Y cells under this experimental condition, which may be related to activation of NF-&kappa;B.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; I-kappa B Proteins ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Nitriles ; toxicity

Constitutive activation of nuclear transcription factor-&kappa;B (NF-&kappa;B) exists in a variety of leukemia, and induction of apoptosis through blocking NF-&kappa;B activation may be an alternative strategy for leukemia treatment. The aim of this study was to investigate the inducing effect of modified adenovirus 5-based adenovirus vector (i.e. chimeric Ad5F35 Vec)-mediated expression of mutant I&kappa;Bα (I&kappa;BαDN) on apoptosis of HL-60 cells. The recombinant Ad5F35-I&kappa;BαDN Vec carrying I&kappa;BαDN cDNA which deleted the first 1-70 amino acids coding sequences at 5' terminal of human I&kappa;Bα was transfected into HL-60 cells. The apoptosis, NF-&kappa;B DNA binding activity, the expressions of I&kappa;Bα, cIAP-2 and xIAP in HL-60 cells were detected by DNA binding assay, flow cytometry, real-time quantitative polymerase chain reaction and Western blot respectively. The results showed that apoptosis rates were 22.53 ± 2.999%, 6.08 ± 2.464% and 4.86 ± 1.366% for Ad5F35-I&kappa;BαDN Vec-infected or blank vector of Ad5F35-EGFP Vec-transfected and untransfected HL-60 cells respectively, which showed a significant difference between Ad5F35-I&kappa;BαDN Vec-transfected and untransfected cells (p < 0.001) and between Ad5F35-I&kappa;BαDN Vec-transfected and Ad5F35-EGFP Vec-transfected cells (p < 0.001, p < 0.002), while NF-&kappa;B DNA binding activity was decreased, the truncated I&kappa;Bα was expressed, and I&kappa;Bα mRNA expression was up-regulated, but the expression of cIAP-2 and xIAP mRNA was down-regulated after transduction for 48 hours. It is concluded that the chimeric Ad5F35 Vec can effectively mediate the expression of I&kappa;BαDN cDNA in HL-60 cells, leading to the inhibition of NF-&kappa;B DNA binding activity and inducing apoptosis of HL-60 cells.
Adenoviridae ; genetics ; Apoptosis ; Genetic Vectors ; HL-60 Cells ; Humans ; I-kappa B Proteins ; genetics ; NF-KappaB Inhibitor alpha ; NF-kappa B ; genetics ; Transfection