<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>OBJECTIVEb>To observe the inhibitory effect of oligodeoxynucleotides (ODN) on nuclear translocation and nuclear binding activity of nuclear factor (NF)-kappaB in THP-1 cells.

<b>METHODSb>Oligodeoxynucleotides were transfected via liposome into THP-1 cells followed by stimulation of the cells with lipopolysaccharide (LPS). Immunocytochemistry, electrophoretic mobility shift assay (EMSA) and reverse transcription (RT)-PCR were performed to detect the nuclear translocation and nuclear binding activity of NF-kappaB.

<b>RESULTSb>Immunocytochemical results showed that after LPS stimulation of the ODN-transfected cells, NF-kappaB expression was still localized in cytoplasma. EMSA demonstrated inhibited nuclear binding activity of NF-kappaB in the ODN-transfected cells, and ODN inhibited the mRNA expression of tumor necrosis factor (TNF)-alpha, a NF-kappaB-associated inflammatory factor, as shown by RT-PCR.

<b>CONCLUSIONb>ODN can inhibit the nuclear translocation and binding activity of NF-kappaB in THP-1 cells, whereby the transcription and expression of the related inflammation factor genes is suppressed, which shed light on a new solution for clinical treatment of acute pancreatitis.

Cell Line ; Humans ; Lipopolysaccharides ; Macrophages ; cytology ; drug effects ; metabolism ; NF-kappa B ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; Transcription Factors ; metabolism ; Transfection

<b>OBJECTIVEb>To observe the histopathological changes in rat hippocampus at different maturational stages after repeated kindled seizures, and to explore their underlying epileptogenesis processes.

<b>METHODSb>Three groups of Wistar rats (postnatal days: P10, P20, P60) were given pentylenetetrazol (PTZ) intraperitoneal injection for 5 days to induce repeated kindled seizures, and the age-matched rats in control group were injected with normal saline. The behavioral changes, the morphology and the neurons counting in hippocampus, as well as the expression of NF-kappaB were observed.

<b>RESULTSb>(1) In the three groups, the latency of seizure and the latency of IV/V grade were significantly lower in the rats of group P10 and P20 [(1.2 +/- 0.6) min and (14.4 +/- 2.3) min vs. (4.7 +/- 1.6) min and (24.5 +/- 4.5) min] than group P60 [(8.6 +/- 2.0) min and (41.9 +/- 4.5) min], whereas the duration of convulsion in group P10 and P20 [(46.2 +/- 4.8) min and (29.8 +/- 5.9) min] was longer than those of group P60 [(17.1 +/- 5.0) min]. (2) The neuron counting of CA(1), CA(3) and hilar in the P10 and P20 groups showed no differences as compared to their controls, whereas adult rats (P60) had a significant neuron loss in CA(1) and CA(3) pyramidal cells, compared with the control group [(6.3 +/- 1.5)/250 microm(2), (3.6 +/- 1.4)/250 microm(2) vs. (8.2 +/- 1.9)/250 microm(2), (5.6 +/- 1.7)/250 microm(2)]. However, the dentate granule cells in immature rats (P10) with daily seizures had a significant increase as compared with the controls [(23.3 +/- 3.1)/250 microm(2) vs. (16.3 +/- 1.6)/250 microm(2)]. (3) Prominent sprouting was seen in the CA(3) stratum pyramidal layer in all experimental rats with 5 daily seizures, regardless of the age. But the degree of sprouting had significant differences among the experimental groups (P < 0.05). (4) NF-kappaB was expressed significantly in CA(3), CA(1) and dentate granule cells 24 hours after PTZ-kindling when compared with the control groups, with the spectral density decreased with age.

<b>CONCLUSIONb>(1) There were great differences in the vulnerability to the repeated seizure-induced brain damage at different maturational stages in rats. The immature brain appeared to be less vulnerable to the repeated seizures. (2) There was less hippocampus neuron loss and milder mossy fiber sprouting after repeated seizures in the developing rats than mature ones, which may be a pathological evidence underlying the prospect that the immature brain was more resistant to the seizure-induced neuronal injury. (3) The high expression of NF-kappaB may exert a certain biological effects in the seizure-induced neuronal injury.

Age Factors ; Animals ; Hippocampus ; drug effects ; pathology ; NF-kappa B ; metabolism ; Pentylenetetrazole ; adverse effects ; Rats ; Rats, Wistar ; Seizures ; chemically induced

This study was aimed to investigate the effect of proteasome inhibitor MG-132 on apoptosis of L1210 cells and its mechanism. L1210 cells were treated with MG-132 of different concentrations (0, 2.5, 5, 10, 10 micromol/L). Cell viability was tested by MTT assay, apoptosis rate was detected by using flow cytometry, activity of caspase 3 was detected by colorimetry, the expression of NF-kappaB nuclear protein was detected by Western blot. The results showed that the growth inhibition of L1210 cells treated for a same time (24 hours) was enhanced along with increasing of MG-132 concentrations (0, 2.5, 5, 10, 20 micromol/L); the inhibitory rate, apoptosis rate and activity of caspase 3 increased also along with raising of MG-132 concentrations; while the expression of NF-kappaB nuclear protein decreased along with raising of GM-132 concentrations. It is concluded that MG-132 can induce the apoptosis of L1210. The mechanism of apoptosis may be related to the down-regulation of the expression of NF-kappaB and the activation of caspase 3.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Enzyme Inhibitors ; pharmacology ; Humans ; Leupeptins ; pharmacology ; NF-kappa B ; metabolism

<b>OBJECTIVEb>To investigate the TNF-alpha induced apoptosis of U937 cells, the expression, degradation and subcellular localization of IkappaB-alpha, and its degradation mechanism.

<b>METHODb>Changes and subcellular loca-lization of IkappaB-alpha were observed by fluorescence microscopy, expression and degradation of IkappaB-alpha protein with N-tosyl-L-phenylalanylchloromethyl ketone (TPCK protease inhibitor) blocking test and apoptosis of U937 cell by flow cytometry.

<b>RESULTSb>(1) immunolfluorescence assay showed that IkappaB-alpha localized in cytoplasm only. (2) The level of IkappaB-alpha protein was downregulated after TNF-alpha stimulation, flow cytometry also confirmed the downregulation. (3) The downregulation of IkappaB-alpha protein levels in TNF-alpha induced apoptosis was partially inhibited by TPCK. (4) The apoptosis rate of U937 cells induced by TNF-alpha was (60.73 +/- 1.61)%.

<b>CONCLUSIONb>(1) Degradation of IkappaB-alpha protein during TNF-alpha induced apoptosis of U937 cells suggested the activation of NF-kappaB. (2) TPCK sensitive protease plays an important role in the degradation of IkappaB-alpha protein. (3) TPCK sensitive protease also involved in the apoptosis of U937 cells induced by TNF-alpha.

Apoptosis ; drug effects ; Down-Regulation ; Humans ; NF-kappa B ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; U937 Cells

This study explored the protective effect of atractylenolide Ⅰ(AO-Ⅰ) against acetaminophen(APAP)-induced acute liver injury(ALI) in mice and its underlying mechanism. C57 BL/6 J mice were randomly divided into a control group, an APAP group(500 mg·kg~(-1)), a low-dose combination group(500 mg·kg~(-1) APAP + 60 mg·kg~(-1) AO-Ⅰ), and a high-dose combination group(500 mg·kg~(-1) APAP + 120 mg·kg~(-1) AO-Ⅰ). ALI was induced by intraperitoneal injection of APAP(500 mg·kg~(-1)). AO-Ⅰ by intragastric administration was performed 2 hours before APAP treatment, and the control group received the same dose of solvent by intragastric administration or intraperitoneal injection. The protective effect of AO-Ⅰ against APAP-induced ALI was evaluated by detecting alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels in the plasma and H&E staining in liver tissues of mice. The malondialdehyde(MDA) and glutathione(GSH) content and catalase(CAT) activity in mouse liver tissues were detected to evaluate the effect of AO-Ⅰ on APAP-induced oxidative stress in the liver. The proteins in the liver p38 mitogen-activated protein kinase(p38 MAPK), c-jun N-terminal kinase(JNK), and nuclear factor kappa-B p65(NF-κB p65) signaling pathways were measured by Western blot, and the liver inflammatory cytokines interleukin-1β(IL-1β) and interleukin-6(IL-6) were detected by real-time PCR. Compared with the APAP group, the combination groups showed reduced APAP-induced ALT level and liver MDA content, potentiated liver CAT activity, and elevated GSH content. Mechanistically, AO-Ⅰ treatment significantly inhibited APAP-up-regulated MAPK phosphorylation and NF-κB p65, and significantly reduced the transcriptional activities of IL-1β and IL-6, downstream targets of NF-κB p65. AO-Ⅰ can improve APAP-induced ALI and the underlying mechanism is related to the inhibition of the MAPK/NF-κB p65 signaling pathway in APAP-challenged mice.
Acetaminophen/adverse effects* ; Animals ; Chemical and Drug Induced Liver Injury/drug therapy* ; Lactones ; Mice ; NF-kappa B/metabolism* ; Sesquiterpenes ; Signal Transduction

<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

<b>OBJECTIVEb>To investigate the effect of nuclear receptor inhibitor importazole (IPZ) on cell cycle and apoptosis of multiple myeloma (MM) cells and its regulatory mechanisms.

<b>METHODSb>MM cell lines RPMI 8226 and NCI-H929 cells were treated with different concentrations of IPZ. Cell viability was detected through MTT method. Cell cycle and apoptosis were measured by flow cytometry (FCM). Nuclear NF-&kappa;Bprotein expression was tested by Western blot. Electrophoretic mobility shift assay (EMSA) was used to analyze the DNA binding activity.

<b>RESULTSb>IPZ induced a dose- and time- dependent inhibition of myeloma cells growth. And the IC50 values of IPZ on RPMI 8226 and NCI-H929 after 48 hours incubation were (4.43±0.41) and (4.78±0.35) μmol/L, respectively, and the percentages of S phase cells decreased from (54.95±4.34)％ and (51.38±2.43)％ to (42.77±3.19)％ and (40.98±6.46)％, respectively. After treatment with IPZ at 8, 12 and 16 μmol/L, the apoptosis rate significantly increased from (2.47±0.60)％ of the control group to (14.53±0.90)%, (32.57±1.80)% and (58.3±1.9)% (P<0.05) in RPMI 8226 and from (2.37±0.70)％ of the control group to (19.46±0.70) %, (46.02±1.10) % and (60.63±1.60)% in NCI-H929, respectively. Treatment of RPMI 8226 and NCI-H929 cells with 8 μmol/L IPZ for 24 h could inhibit NF-&kappa;B import to nucleus and reduce its DNA binding activity.

<b>CONCLUSIONb>The nuclear receptor inhibitor importazole inhibits proliferation and induces apoptosis of multiple myeloma cells by blocking the NF-&kappa;B signal pathway in vitro.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Multiple Myeloma ; metabolism ; pathology ; NF-kappa B ; metabolism ; Quinazolines ; pharmacology ; Signal Transduction ; drug effects

<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>BACKGROUNDb>Currently, no medicine is available that can prevent or treat neural damage associated with optic nerve injury. Minocycline is recently reported to have a neuroprotective function. The aims of this study were to exarmine the neuroprotective effect of minocycline on retinal ganglion cells (RGCs) and determine its underlying mechanisms, using a mouse model of optic nerve crush (ONC).

<b>METHODSb>ONC was performed in the left eye of adult male mice, and the mice were randomly divided into minocycline-treated group and saline-treated control group. The mice without receiving ONC injury were used as positive controls. RGC densities were assessed in retinal whole mounts with immunofluorescence labeling of βIII-tubulin. Transmission electron microscopy was used to detect RGC morphologies, and Western blotting and real-time PCR were applied to investigate the expression of autophagy markers LC3-I, LC3-II, and transcriptional factors nuclear factor-&kappa;B1 (NF-&kappa;B1), NF-&kappa;B2.

<b>RESULTSb>In the early stage after ONC (at Days 4 and 7), the density of RGCs in the minocycline-treated group was higher than that of the saline-treated group. Electron micrographs showed that minocycline prevented nuclei and mitochondria injuries at Day 4. Western blotting analysis demonstrated that the conversion of LC3-I to LC3-II was reduced in the minocycline-treated group at Days 4 and 7, which meant autophagy process was inhibited by minocycline. In addition, the gene expression of NF-&kappa;B2 was upregulated by minocycline at Day 4.

<b>CONCLUSIONb>The neuroprotective effect of minocycline is generated in the early stage after ONC in mice, partly through delaying autophagy process and regulating NF-&kappa;B2 pathway.

Animals ; Autophagy ; drug effects ; Male ; Mice ; Minocycline ; therapeutic use ; NF-kappa B p52 Subunit ; metabolism ; Optic Nerve Injuries ; drug therapy ; metabolism ; Retinal Ganglion Cells ; drug effects ; metabolism