To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Chemiluminescent Measurements ; DNA-Binding Proteins/*analysis ; DNA-Binding Proteins/genetics ; Electrophoretic Mobility Shift Assay ; NF-kappa B/*analysis ; NF-kappa B/genetics ; NF-kappa B/metabolism ; Rats, Sprague-Dawley ; *Trans-Activation (Genetics) ; Trans-Activators/analysis ; Trans-Activators/genetics ; *Transcription, Genetic

<b>OBJECTIVEb>To investigate the effects of burn sera on the nuclear translocation of endothelial NF-kappaB heterodimers p50/p65 and on the degradation of inhibiting kappaB (IkappaBalpha), in order to explore the role of burn sera on activation of the endothelium.

<b>METHODSb>Cultured human umbilical vein endothelial cells (HUVECs) (ECV-304 strain) were employed as the target cells. The cells were stimulated by sera from healthy volunteers and from burn patients and burn sera together with PDTC (pyrrolidine dithiocarbarnate). The normal cultured cells were taken as the control. The nuclear translocation of endothelial p50/p65 at 30, 60, 120 and 480 mins after the stimulation was observed with laser confocal microscopy, and the endothelial IkappaBalpha protein degradation at 30, 60, 90 and 120 mins after the stimulation was determined by Western blotting.

<b>RESULTSb>When compared to that in control group, the nuclear translocation of p50/p65 took place 30 mins after the endothelial cells were stimulated by burn sera, and it reached the summit at 30 - 60 mins, but recovered to pre-stimulation state at 2hrs. In addition, IkBalpha degradation occurred 30 mins after the cells were stimulated by burn sera (P < 0.01) and peaking at 45 - 60 mins after the stimulation and recovered at 2hrs after the stimulation. The nuclear translocation of endothelial p50/p65 and IkBalpha degradation at 30 and 60 mins after the stimulation by burn sera could be effectively inhibited by PDTC.

<b>CONCLUSIONb>Burn sera might induce the nuclear translocation of endothelial NF-kappaB p50/p65 and IkappaBalpha degradation and activate NF-kappaB, which ultimately lead to the secretion of cytokines from the endothelium.

Active Transport, Cell Nucleus ; Adolescent ; Adult ; Burns ; blood ; Female ; Humans ; I-kappa B Proteins ; analysis ; Male ; Middle Aged ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; NF-kappa B p50 Subunit ; Transcription Factor RelA

<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

Humans ; In Situ Hybridization, Fluorescence ; Membrane Proteins ; analysis ; NF-kappa B ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Tissue Array Analysis ; methods ; Tumor Cells, Cultured

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>OBJECTIVEb>To study the correlation between obesity and hypersensitive C-reactive protein (hsCRP), leptin, and insulin sensitive index (ISI) in children.

<b>METHODSb>The subjects included 69 obese volunteers and 30 age and gender-matched normal volunteers who were recruited from 13702 children aged 2 to 18 years in Xiangtan City by sampling survey. The body mass index (BMI), hsCRP, leptin, fasting plasma glucose (FPG), and fasting insulin (INS) were tested, and then the ISI was calculated. The results between the obese and normal children were compared. The correlation between the parameters was evaluated.

<b>RESULTSb>The values of hsCRP, leptin and INS in obese children were significantly higher than those in the normal controls (P < 0.01), but the ISI in obese children was significantly lower than that in normal controls (P < 0.01). The BMI was significantly positively correlated with the values of hsCRP, leptin and INS (r=0.225, P < 0.05; r=0.776, P < 0.01; r=0.568, P < 0.01), but was significantly negatively correlated with the ISI (r=-0.889, P < 0.01). There was a positive correlation between the value of hsCRP and the values of FPG and INS (r=0.429, P < 0.01; r=0.206, P < 0.05), and there was a negative correlation between the value of hsCRP and the ISI (r=-0.889, P < 0.01). The value of leptin significantly positively correlated with the values of INS and BMI, and significantly negatively correlated with the ISI.

<b>CONCLUSIONSb>Insulin resistance and leptin resistance exist in obese children. The inflammatory factors such as CRP and leptin may be involved in the pathogenesis of obesity.

Adolescent ; Blood Glucose ; analysis ; Body Mass Index ; C-Reactive Protein ; analysis ; Child ; Female ; Humans ; Insulin Resistance ; Leptin ; blood ; Male ; NF-kappa B ; physiology ; Obesity ; blood

<b>OBJECTIVEb>To study the role of serum from asphyxiated neonates in the inducement of human renal proximal tubular epithelial cells (HK-2) adhesion to neutrophils and possible mechanisms.

<b>METHODSb>HK-2 cells were cultured randomly with 20% serum from neonates (1, 3, and 7 days after asphyxia), pyrrolidine dithiocarbamate (PDTC) or placebo. The activity of myeloperoxidase (MPO), an indicator of adhesion ability of HK-2 cells to neutrophils in suspensions, was detected by the biochemistry assay. Intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-kappaB (NF-kappaB) of HK-2 cells were examined with the immunohistochemical staining.

<b>RESULTSb>The expression of MPO in the post-asphyxial 1-day serum treatment group were significantly higher than that in the PDTC treatment and the control groups as well as the post-asphyxial 3 and 7-day serum treatment groups (P<0.01). The expression of ICAM-1 and NF-kappaB in the post-asphyxial 1-day serum treatment group was also significantly higher than that in the other groups (P<0.01).

<b>CONCLUSIONSb>Serum from asphyxiated neonates can induce HK-2 cell adhesion to neutrophils, possibly through activating NF-kappaB and increasing the synthesis and expression of ICAM-1 on the surface of renal tubular epithelial cells.

Asphyxia Neonatorum ; blood ; complications ; Cell Adhesion ; Cells, Cultured ; Humans ; Infant, Newborn ; Intercellular Adhesion Molecule-1 ; analysis ; biosynthesis ; Kidney Tubules ; pathology ; NF-kappa B ; analysis ; metabolism ; Neutrophils ; physiology

<b>OBJECTIVEb>To study the effect of budesonide aerosol inhalation on the expression of glucocorticoid receptor (GR) and nuclear factor (NF)-&kappa;B in asthmatic mice.

<b>METHODSb>Twenty-four healthy male BALB/c mice aged 6 to 8 weeks were randomly divided into three groups (n=8 each): normal saline (control group), asthma model (asthma group) and budesonide-treated asthma (BUD group). Asthma was induced by intraperitoneal injection of ovalbumin (OVA) and aluminium hydroxide suspension and aerosol inhalation of OVA solution. Mice were sacrificed 24 hours after the last challenge. Eosinophil count in the bronchoalveolar lavage fluid (BALF) was determined. Pathological examination of the lung tissues was performed and the expression levels of GR and NF-&kappa;B were measured by immunohistochemical analysis.

<b>RESULTSb>Eosinophil count in the BALF was significantly higher in the asthma and BUD groups than in the control group (P<0.05). BUD treatment decreased eosinophil count in the BALF compared with the asthma group (P<0.05). The lung tissues in the BUD group showed a less severe infiltration of eosinophils and lymphocytes compared with the asthma group. The percentage of GR-positive cells in the asthma group decreased significantly compared with the control group (P<0.05), and the percentage of GR-positive cells in the BUD group increased significantly compared with the asthma group (P<0.05). Compared with the control group, the percentage of NF-&kappa;B-positive cells increased significantly in the asthma group (P<0.05), and the percentage of NF-&kappa;B positive cells in the BUD group was significantly reduced compared with the asthma group (P<0.05).

<b>CONCLUSIONSb>The action mechanism of budesonide in treating asthmatic mice may be related to the upregulation of GR expression and the inhibition of NF-&kappa;B activity.

Aerosols ; Animals ; Asthma ; drug therapy ; metabolism ; Budesonide ; administration & dosage ; Eosinophils ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; analysis ; Receptors, Glucocorticoid ; analysis

<b>OBJECTIVEb>To study the expression of basic fibroblast growth factor (b-FGF) and nuclear factor-kappaB (NF-kappaB) in the airway and the effect of budesonide on their expression in rats with asthma.

<b>METHODSb>Forty-five Sprague-Dawley male rats were randomly divided into three group: placebo control, untreated asthma, and budesonide-treated asthma. Asthma was induced by intraperitoneal injection of 10% ovalbumin (OVA) on days 1 and 8 and then challenged by inhalation of 1% OVA aerosol. The budesonide-treated asthma group received an inhalation of budesonide (1 mg) 30 minutes after OVA challenge. The pathological changes of the airway were assessed, and the expression of b-FGF and NF-kappaB in the airway was assayed by hematoxylin and eosin staining and immunohistochemistry.

<b>RESULTSb>Budesonide treatment alleviated airway injuries. Compared with the control group, b-FGF and NF-kappaB expression in the airway in the untreated asthma group increased significantly (P< 0.05). The budesonide-treated asthma group demonstrated significantly decreased b-FGF (111.61+/- 5.52 vs 126.21+/- 6.46; P< 0.05) and NF-kappaB expression (110.65+/- 8.71 vs 134.15+/- 9.42; P< 0.05) in the airway as compared with the untreated asthma group. B-FGF expression was positively correlated to NF-kappaB expression in the budesonide-treated group.

<b>CONCLUSIONSb>b-FGF and NF-kappaB may be associated with airway remodeling in rats with asthma. Budesonide can improve airway remodeling, possibly by decreasing the expression of b-FGF and NF-kappaB.

Animals ; Asthma ; metabolism ; pathology ; Bronchi ; drug effects ; pathology ; Budesonide ; pharmacology ; Fibroblast Growth Factor 2 ; analysis ; Immunohistochemistry ; Male ; NF-kappa B ; analysis ; Rats ; Rats, Sprague-Dawley

Aeriscardovia aeriphila, also known as Bifidobacterium aerophilum, was first isolated from the caecal contents of pigs and the faeces of cotton-top tamarin. Bifidobacterium species play important roles in preventing intestinal infections, decreasing cholesterol levels, and stimulating the immune system. In this study, we isolated a strain of bacteria from the duodenal contents of broiler chickens, which was identified as A. aeriphila, and then evaluated the effects of A. aeriphila on growth performance, antioxidant functions, immune functions, and gut microbiota in commercial broiler chickens. Chickens were orally gavaged with A. aeriphila (1×10⁹ CFU/mL) for 21 d. The results showed that A. aeriphila treatment significantly increased the average daily gain and reduced the feed conversion ratio (P<0.001). The levels of serum growth hormone (GH) and insulin-like growth factor 1 (IGF-1) were significantly increased following A. aeriphila treatment (P<0.05). Blood urea nitrogen and aspartate aminotransferase levels were decreased, whereas glucose and creatinine levels increased as a result of A. aeriphila treatment. Furthermore, the levels of serum antioxidant enzymes, including catalase (P<0.01), superoxide dismutase (P<0.001), and glutathione peroxidase (P<0.05), and total antioxidant capacity (P<0.05) were enhanced following A. aeriphila treatment. A. aeriphila treatment significantly increased the levels of serum immunoglobulin A (IgA) (P<0.05), IgG (P<0.01), IgM (P<0.05), interleukin-1 (IL-1) (P<0.05), IL-4 (P<0.05), and IL-10 (P<0.05). The broiler chickens in the A. aeriphila group had higher secretory IgA (SIgA) levels in the duodenum (P<0.01), jejunum (P<0.001), and cecum (P<0.001) than those in the control group. The messenger RNA (mRNA) relative expression levels of IL-10 (P<0.05) and IL-4 (P<0.001) in the intestinal mucosa of chickens were increased, while nuclear factor-‍κB (NF‍-‍κB) (P<0.001) expression was decreased in the A. aeriphila group compared to the control group. Phylum-level analysis revealed Firmicutes as the main phylum, followed by Bacteroidetes, in both groups. The data also found that Phascolarctobacterium and Barnesiella were increased in A. aeriphila-treated group. In conclusion, oral administration of A. aeriphila could improve the growth performance, serum antioxidant capacity, immune modulation, and gut health of broilers. Our findings may provide important information for the application of A. aeriphila in poultry production.
Animals ; Swine ; Antioxidants/pharmacology* ; Chickens ; Gastrointestinal Microbiome ; Interleukin-10/pharmacology* ; Interleukin-4/pharmacology* ; NF-kappa B/metabolism* ; Immunity ; Diet/veterinary* ; Animal Feed/analysis* ; Dietary Supplements/analysis*