<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

<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

<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

<b>OBJECTIVEb>To explore the relationship between drug resistance of leukemic cells and the expression of both IkappaB-alpha and NF-kappaB associated with apoptosis induced by arsenic trioxide (As2O3) in K562 and K562/ADR cells.

<b>METHODSb>Apoptosis was induced in K562 and K562/ADR cells cultured with As2O3 in different concentrations. Western blot was used to analyze the expression of NF-kappaB in nuclear and IkappaB-alpha in cytoplasm of these cells. Apoptosis and degradation of IkappaB-alpha protein were also observed by flow cytometry.

<b>RESULTSb>After exposure to As2O3, the ratio of apoptosis cells in K562/ADR was significantly lower than that in K562 cells. K562/ADR [(6.33+/-1.51)%] and K562 cells [(13.25+/-1.83)%] cultured with 1 micromol/L As2O3 were in apoptosis. When cultured with 4 micromol/L As2O3, the apoptosis cells increased to (8.00+/-1.47)% and (50.56+/-8.62)%, respectively. The level of IkappaB-alpha in K562 cytoplasm was down-regulated from 88.07% to 49.21% after As2O3 stimulation, while NF-kappaB in nuclear was up-regulated, that was not found in K562/ADR cells.

<b>CONCLUSIONb>As2O3 could induce apoptosis of K562 cells, associated with the degradation of IkappaB-alpha and the activation of NF-kappaB. There are an elevated expression of NF-kappaB and resistance to apoptosis induced by As2O3 in K562/ADR cells.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; I-kappa B Proteins ; metabolism ; K562 Cells ; NF-kappa B ; metabolism ; Oxides ; pharmacology

<b>BACKGROUNDb>Previous studies have showed that photooxidative stress can lead to down-modulation of nuclear factor-kappa B (NF-kappaB) activity causing apoptosis of cultured photoreceptor cells. This study aimed at investigating whether NF-kappaB was involved in photoreceptor cells apoptosis induced by N-methyl-N-nitrosourea (MNU) in rats.

<b>METHODSb>A single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats. At different intervals after MNU treatment, the animals were sacrificed. Retinal damage was examined by a light microscope. The apoptotic index of the photoreceptor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). NF-kappaB was analysed by Western blot and Transcriptin Factor Assay Kits.

<b>RESULTSb>The pyknosis of the photoreceptor nuclei and the disorientation of the outer segment of the photoreceptor layer was seen after MNU treatment for 24 hours. The outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. Photoreceptor cells apoptosis reached the peaked value at 24 hours. In apoptotic cascade, the protein levels of NF-kappaB p65 were only detected after MNU treatment for 12 and 24 hours in the nucleus. Conversely, the amounts of IkappaBalpha were markedly increased in the cytoplasm as well as in the nucleus. The activity of NF-kappaB p65 in the nucleus was down-modulated in the end.

<b>CONCLUSIONSb>MNU-induced photoreceptor cell destruction was attributed to the apoptotic process by down-regulating the activation of NF-kappaB p65.

Animals ; Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Female ; I-kappa B Proteins ; analysis ; physiology ; Methylnitrosourea ; toxicity ; NF-KappaB Inhibitor alpha ; NF-kappa B ; analysis ; physiology ; Photoreceptor Cells ; chemistry ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; pathology

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

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H2O2 leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)-induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF-kappaB activation, HeLa cells were incubated with H2O2 or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H2O2 and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H2O2 alone, nuclear NF-kappaB was induced after 2 h without detectible degradation of cytosolic IkBa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappaB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.
Cell Nucleus/drug effects/metabolism ; Cytosol/drug effects/metabolism ; Diamide/pharmacology ; Hela Cells/drug effects/metabolism ; Human ; Hydrogen Peroxide/pharmacology ; I-kappa B/drug effects/metabolism ; NF-kappa B/drug effects/genetics/*metabolism ; Oxidants/pharmacology ; *Oxidative Stress ; Protein-Serine-Threonine Kinases/metabolism ; Signal Transduction/drug effects ; Time Factors ; Transcription, Genetic ; Tumor Necrosis Factor/pharmacology

<b>OBJECTIVEb>To investigate the effects of ursolic acid (UA) on T-cell proliferation and activation, as well as to examine its effect on nuclear factor-&kappa;B (NF-&kappa;B) signaling pathway in T cells.

<b>METHODSb>T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30 μmol/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 I&kappa;B-α (p-I&kappa;B-α) in total protein and p65, a subunit of NF-&kappa;B, nuclear translocation were measured by Western blot analysis.

<b>RESULTSb>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 (P<0.01). Significant reduction of IL-2 production was found in activated T cells treated with UA (P<0.01). The PMA-induced increase in p-I&kappa;B-α protein was inhibited, and nuclear translocation of p65 from the cytoplasm was blocked by UA.

<b>CONCLUSIONb>UA is a potent inhibitor for T cell activation and proliferation; these effects are associated with the inhibition of NF-&kappa;B signaling pathway.

Animals ; Cell Nucleus ; drug effects ; metabolism ; Cell Proliferation ; drug effects ; I-kappa B Proteins ; metabolism ; Interleukin-2 ; secretion ; Ionomycin ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Inbred BALB C ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Phosphorylation ; drug effects ; Protein Transport ; drug effects ; Signal Transduction ; drug effects ; T-Lymphocytes ; cytology ; drug effects ; immunology ; secretion ; Tetradecanoylphorbol Acetate ; pharmacology ; Triterpenes ; pharmacology

<b>OBJECTIVEb>To observe the changes of nuclear factor-kappa B (NF-kappaB) in the course of N-methyl-N-nitrosourea (MNU)-induced apoptosis of rat retinal photoreceptor cells and investigate the mechanism of MNU-induced retinal damage.

<b>METHODSb>A single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats, which were sacrificed at different intervals after MNU treatment. The retinal damage was examined with optical microscopy and photoreceptor cell apoptosis detected by TUNEL assay. Western blotting was performed to analyze the changes in NF-kappaB.

<b>RESULTSb>Pyknosis of the photoreceptor cell nuclei and disorientation of the outer segment of the photoreceptor layer was observed 24 h after MNU treatment, and the outer nuclear layer and photoreceptor layer were almost completely lost on day 7. Photoreceptor cell apoptosis peaked at 24 h, and in the apoptotic cascade, NF-kappaB p65 protein was only detected 12 and 24 h after MNU treatment, whereas the amount of I kappa B alpha, in contrast, markedly increased in the cytoplasm as well as in the nuclei.

<b>CONCLUSIONb>MNU-induced retinal damage might be mediated through the signaling pathway of NF-kappaB/I kappa B alpha.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Female ; I-kappa B Proteins ; metabolism ; In Situ Nick-End Labeling ; Methylnitrosourea ; toxicity ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retinal Diseases ; chemically induced ; metabolism ; pathology

The aim of this study was to explore the effect of emodin on lipid accumulation and inflammation in hepatocytes. The cell morphology was observed by microscopy. LDH release was detected by the kit. Levels of intracellular lipid droplets were observed by oil red O staining. The contents of TC and TG in cells were detected by the kit. Western blot was used to determine protein expressions of FASN,SREBF2,APOB,IL-6 and p-NF-κB in hepatocytes. The results showed that the levels of L02 cell LDH were significantly increased after being treated with emodin,and the cells showed shrinkage,volume reduction,decrease in quantity with the increase of dose. Red lipid droplets were observed in L02 hepatocytes. Intracellular TC and TG contents of L02 cell increased in a concentrationdependent manner,with significant differences between medium and high-dose groups( P < 0. 05). Protein expressions of FASN,SREBF2,IL-6 and p-NF-κB were significantly higher than those of the control group,and the expression level of APOB was significantly lower than that of the control group( P<0. 05). In conclusion,emodin could induce lipid accumulation and inflammatory damage in hepatocytes in a dose-dependent manner,which in turn could damage liver cells. This process was related to the up-regulation of FASN,SREBF2,IL-6,p-NF-κB,as well as the down-regulation of the protein expression of APOB.
Apolipoprotein B-100 ; metabolism ; Cells, Cultured ; Emodin ; pharmacology ; Fatty Acid Synthase, Type I ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Inflammation ; Interleukin-6 ; metabolism ; Lipid Metabolism ; Lipids ; NF-kappa B ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism