<b>OBJECTIVEb>To investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (I&kappa;B) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).

<b>METHODSb>(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-&kappa;B/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-&kappa;B/p65 in nuclei, and the protein levels of I&kappa;Bα, phosphorylated I&kappa;Bα (p-I&kappa;Bα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-&kappa;B/p65 in the nuclear and target gene was determined by NF-&kappa;B/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.

<b>RESULTSb>(1) High expression of NF-&kappa;B/p65 was observed in cytoplasm of control group. High expression of NF-&kappa;B/p65 was observed in nuclei of TNF-α group. The expression of NF-&kappa;B/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-&kappa;B/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-&kappa;B/p65 protein expressed in nuclei of control group. The expression of NF-&kappa;B/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-&kappa;B/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of I&kappa;Bα in TNF-α group was significantly decreased but that of p-I&kappa;Bα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of I&kappa;Bα gradually increased and the levels of p-I&kappa;Bα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).

<b>CONCLUSIONSb>LaCl3 can block the activation of NF-&kappa;B signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.

Culture Media ; HeLa Cells ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Lanthanum ; pharmacology ; NF-KappaB Inhibitor alpha ; Signal Transduction ; drug effects ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

Berberine (BBR) is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus (T2DM) in China. The development of T2DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in HepG2 cells. Cellular glucose uptake, quantified by the 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxy-D-glucose (2-NBDG), was inhibited by 21% after HepG2 cells were incubated with insulin (10(-6) mol/L) for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor (α7nAChR) protein was reduced without the change of acetylcholinesterase (AChE) activity. The level of interleukin-6 (IL-6) in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β (IK&kappa;β) Ser181/IKKβ and the expression of nuclear factor-kappa B (NF-&kappa;B) p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7nAChR protein and inhibited AChE activity. These changes were also accompanied with the decrease of the ratio of pIKKβ Ser181/IKKβ, NF-&kappa;B p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in HepG2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of AChE activity.
Berberine ; pharmacology ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; pharmacology ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Insulin ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Transcription Factor RelA ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

The NF-kappaB pathway regulates the expression of over 150 target genes, e.g., cytokines, chemokines, leukocyte adhesion molecules and inducible effector enzymes. Consequently, it plays a crucial role in innate and adaptive immune responses, inflammatory response, stress responses, apoptosis and so on. IkappaB kinase (IKK) is the key of this pathway, and it owns a special structure which consists of catalytic subunit and regulatory subunit. Naturally, the activation of IKK needs the interaction of the two subunits and phosphorylation by its upstream kinases. Actually, there are two methods of activation of the NF-kappaB pathway, and both of the methods need the IKK complex. Given to the crucial role of IKK, researchers have isolated and synthesized amounts of IKK inhibitors, and these provide a great convenience to develop novel anti-inflammatory and anti-tumor drugs.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents ; pharmacology ; Enzyme Activation ; Enzyme Inhibitors ; metabolism ; pharmacology ; Humans ; I-kappa B Kinase ; antagonists & inhibitors ; chemistry ; metabolism ; physiology ; NF-kappa B ; metabolism ; Phosphorylation ; Signal Transduction

Angiotensin II ; pharmacology ; Blotting, Western ; Cells, Cultured ; Glomerular Mesangium ; cytology ; drug effects ; metabolism ; Humans ; I-kappa B Kinase ; NF-kappa B ; analysis ; Peptide Fragments ; pharmacology ; Protein-Serine-Threonine Kinases ; analysis

<b>BACKGROUNDb>Lipoxins (LXs), endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells (DCs) play crucial roles in the initiation and maintenance of immune response, we determined whether LXs could modulate the maturation process of DCs and investigated the effects of lipoxin A(4) (LXA(4)) on lipopolysaccharide (LPS)-induced differentiation of RAW264.7 cells into dendritic-like cells.

<b>METHODSb>RAW264.7 cells were cultured in vitro with 1 microg/ml LPS in the absence or presence of LXA(4) for 24 hours, and cellular surface markers (MHC-II, CD80 (B7-1), CD86 (B7-2)) were measured by flow cytometry (FCM). Mixed lymphocyte reaction was performed to evaluate the allostimulatory activity. Cytoplastic IkappaB degradation and nuclear factor kappa B (NF-kappaB) translocation were detected by Western blotting. Luciferase reporter plasmid was transiently transfected into RAW264.7 cells, and luciferase activity was determined to measure the transcriptional activity of NF-kappaB.

<b>RESULTSb>LXA(4) reduced the ratio of LPS-treated RAW264.7 cells to DCs with morphological characteristics and inhibited the expression of MHC II. LPS-induced up-regulation of CD86 was moderately suppressed by LXA(4) but no obvious change of CD80 was observed. Moreover, LXA(4) weakened the allostimulatory activity of LPS-treated RAW264.7 cells. These alterations of LPS+LXA(4)-treated cells were associated with a marked inhibition of IkappaB degradation, NF-kappaB translocation and then the transcriptional activity of NF-kappaB.

<b>CONCLUSIONSb>LXA(4) negatively regulates LPS-induced differentiation of RAW264.7 cells into dendritic-like cells. This activity reveals an undescribed mechanism of LXA(4) to prevent excessive and sustained immune reaction by regulating maturation of DCs.

Animals ; Biological Transport ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; I-kappa B Kinase ; metabolism ; Lipopolysaccharides ; pharmacology ; Lipoxins ; pharmacology ; Macrophages ; cytology ; drug effects ; Mice ; NF-kappa B ; metabolism ; Phenotype ; Transcription, Genetic ; drug effects

Human immunodeficiency virus type 1 (HIV-1) transcription is a crucial step in the viral replication cycle, which is considered to be a potential target for inhibition of HIV-1 replication. Among the factors involved in this step, the cellular protein nuclear factor NF-kappaB is the most powerful inducer of HIV-1 transcription. HIV-1 transcription is initiated by the binding of NF-kappaB to the enhancer region in the long terminal repeat (LTR) of HIV-1. Several compounds suppress HIV-1 transcription through the inhibition of NF-kappaB activation. The mechanisms of NF-kappaB in the transcription of HIV-1 and progress of the current inhibitors of NF-kappaB are reviewed.
Anti-HIV Agents ; pharmacology ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Nicotinic Acids ; pharmacology ; Nitriles ; pharmacology ; Transcription, Genetic ; drug effects ; Virus Replication

This study is to investigate the inhibitory effect of kaempferol on inflammatory response of lipopolysaccharide(LPS)-stimulated HMC-1 mast cells. The cytotoxicity of kaempferol to HMC-1 mast cells were analyzed by using MTT assay and then the administration concentrations of kaempferol were established. Histamine, IL-6, IL-8, IL-1β and TNF-α were measured using ELISA assay in activated HMC-1 mast cells after incubation with various concentrations of kaempferol (10, 20 and 40 µmol.L-1). Western blot was used to test the protein expression of p-IKKβ, I&kappa;Bα, p-I&kappa;Bα and nucleus NF-&kappa;B of LPS-induced HMC-1 mast cells after incubation with different concentrations of kaempferol. The optimal concentrations of kaempferol were defined as the range from 5 µmol.L-1 to 40 µmol.L-1. Kaempferol significantly decreased the release of histamine, IL-6, IL-8, IL-1β and TNF-α of activated HMC-1 mast cells (P<0.01). After incubation with kaempferol, the protein expression of p-IKKβ, p-IKBa and nucleus NF-&kappa;B (p65) markedly reduced in LPS-stimulated HMC-1 mast cells (P<0.01). Taken together, we concluded that kaempferol markedly inhibit mast cell-mediated inflammatory response. At the same time, kaempferol can inhibit the activation of IKKβ, block the phosphorylation of I&kappa;Bα, prevent NF-KB entering into the nucleus, and then decrease the release of inflammatory mediators.
Cells, Cultured ; Histamine ; metabolism ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Inflammation ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Kaempferols ; pharmacology ; Lipopolysaccharides ; Mast Cells ; drug effects ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Emerging evidence indicates that aldosterone and mineralocorticoid receptors (MRs) are associated with the pathogenesis of erectile dysfunction. However, the molecular mechanisms remain largely unknown. In this study, freshly isolated penile corpus cavernosum tissue from rats was treated with aldosterone, with or without MRs inhibitors. Nuclear factor (NF)-kappa B (NF-κB) activity was evaluated by real-time quantitative PCR, luciferase assay, and immunoblot. The results demonstrated that mRNA levels of the NF-κB target genes, including inhibitor of NF-κB alpha (IκB-α), NF-κB1, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), were higher after aldosterone treatment. Accordingly, phosphorylation of p65/RelA, IκB-α, and inhibitor of NF-κB kinase-β was markedly increased by aldosterone. Furthermore, knockdown of MRs prevented activation of the NF-κB canonical pathway by aldosterone. Consistent with this finding, ectopic overexpression of MRs enhanced the transcriptional activation of NF-κB by aldosterone. More importantly, the MRs antagonist, spironolactone blocked aldosterone-mediated activation of the canonical NF-κB pathway. In conclusion, aldosterone has an inflammatory effect in the corpus cavernosum penis, inducing NF-κB activation via an MRs-dependent pathway, which may be prevented by selective MRs antagonists. These data reveal the possible role of aldosterone in erectile dysfunction as well as its potential as a novel pharmacologic target for treatment.
Aldosterone/pharmacology* ; Animals ; Cytokines/biosynthesis* ; Gene Knockdown Techniques ; I-kappa B Kinase/antagonists & inhibitors* ; Interleukin-6/genetics* ; Male ; Mineralocorticoid Receptor Antagonists/pharmacology* ; NF-kappa B/genetics* ; Penis/metabolism* ; Protein Serine-Threonine Kinases/antagonists & inhibitors* ; RNA, Messenger/biosynthesis* ; Rats ; Rats, Inbred WKY ; Receptors, Mineralocorticoid/genetics* ; Signal Transduction/drug effects* ; Spironolactone/pharmacology* ; Transcriptional Activation ; Tumor Necrosis Factor-alpha/biosynthesis* ; NF-kappaB-Inducing Kinase

Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.
Animals ; Anticoagulants/*pharmacology ; Genistein/pharmacology ; I-kappa B/metabolism ; Macrophages/drug effects/*enzymology/metabolism ; Mice ; NF-kappa B/antagonists & inhibitors/metabolism ; Nitric Oxide/biosynthesis ; Nitric-Oxide Synthase/antagonists & inhibitors/*biosynthesis ; Phosphorylation ; Plant Extracts/pharmacology ; Polysaccharides/*pharmacology ; Protein Transport/drug effects ; Protein-Tyrosine Kinase/antagonists & inhibitors/physiology ; Pyrrolidines/pharmacology ; Research Support, Non-U.S. Gov't ; Thiocarbamates/pharmacology ; Zea mays/*chemistry

<b>OBJECTIVEb>To investigate the way of nuclear factor kappa B (NF-kappaB) activation and the mechanism of NF-kappaB-promoted proliferation in estrogen receptor (ER)-negative breast cancer cells.

<b>METHODSb>The protein of IkappaB kinase alpha (IKKalpha) was measured by Western blot and the influence on cell-cycle was assayed by flow cytometry (FCM).

<b>RESULTSb>The IKKalpha was tested higher in three ER-negative breast cancer cell lines than in MCF-7. The influence caused by epidermal growth factor (EGF), tumor necrosis factor (TNF)-alpha and E(2) to tumor cells' proliferation was tested. EGF could remarkably enhance cyclin D(1) expression about 83% more in EGF group than that in control group and proliferation index from 0.22 to 0.31 (P < 0.01). On the other hand, TNF-alpha inhibited cyclin D(1) expression. Protein kinase C inhibitor, Go6976, could peculiarly prevent NF-kappaB over-expression caused by EGF. The cell-cycle was assayed by FCM in phase G(0)/G(1) 69.36% and in phase S 22.77% when adding EGF and in phase G(0)/G(1) 91.54% and in phase S 7.81% when adding EGF and Go6976. The proliferation index decreased from 0.31 to 0.09 (P < 0.01).

<b>CONCLUSIONSb>EGF-EGFR pathway can provide ER-negative breast cancer cells the signal for the autonomous growth. This signal promoted tumor cells' proliferation is transmitted by activating NF-kappaB and expressing more cyclin D(1). In this pathway, NF-kappaB play an important role as signal transmitting. The strategy to NF-kappaB activating may provide new way to treat ER-negative breast cancers.

Breast Neoplasms ; metabolism ; pathology ; physiopathology ; Carbazoles ; pharmacology ; Cell Proliferation ; drug effects ; Cyclin D1 ; biosynthesis ; Epidermal Growth Factor ; pharmacology ; Estradiol ; pharmacology ; Female ; Humans ; I-kappa B Kinase ; metabolism ; Indoles ; pharmacology ; NF-kappa B ; metabolism ; physiology ; Receptors, Estrogen ; metabolism ; Signal Transduction ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; pharmacology