The purpose of this study was to investigate the effect on the expression of NF-kappaBp65 of osteoblast-like cell under stretch load with the same amplitude but different daily loading times. The osteoblast-like cells MG-63 were passage cultured and stretched by the four-point-bend loading device; based on the daily loading times, the osteoblast-like cells were randomly divided into four groups. The first was the control, the others were stretched with mechanical tension with the same amplitude of 2,000 mu strain and at the same frequency of 0.5 Hz., but the daily loading times were 1 time/d, 2 times/d, 4 times/d differently for each group, the periods of mechanical tension applied to the cells of the three groups were all 60 min/d and lasted for 2d total. After the cells being streteched, the expression levels of NF-kappaBp65 of the osteoblast-like cells of the three groups and control group were investigated by using the techniques of immunohistochemistry, and were compared with each other. The results showed that the positive expression ratios of the four groups were different significantly; the positive expression ratio of the control was lower than those of the other three groups; the positive expression ratio of the 4 times/d group was higher than those of the other two stretched groups; the positive expression ratio of the 2 times/d group was higher than that of the 1 time/d group. The results suggested that when the osteoblast-like cell was under the stretch load with different daily loading times but the same amplitude, the expression ratio of NF-kappaBp65 in the cell increased with the rising of the stimulating times. It means that the mechanical strain with high daily loading times could promote the transcriptional level of osteoblast-like cell more effectively.
Cell Line ; Humans ; Mechanotransduction, Cellular ; Osteoblasts ; cytology ; metabolism ; Stress, Mechanical ; Time Factors ; Transcription Factor RelA ; biosynthesis

OBJECTIVETo investigate the effects of different X-ray doses on the expression of nuclear factor-kappaB (NF-kappaB) P65 in human oral squamous cell carcinoma cell (OSCC) line and the relationship between NF-kappaB P65 and radiation-induced OSCC cell line apoptosis.

METHODSThe squamous cell carcinoma of Tca8113 cell was cultivated in the 37 degrees C, 5% CO2 incubator after recovery. The experiment samples were divided into six groups (control group, 2, 4, 6, 8, 10 Gy). After growing to logarithm period, Tca8113 cells were irradiated using above-mentioned X-ray doses. The immunocyteochemistry and Western blot were used to detect the expression of NF-kappaB P65 after irradiation in various times (1, 3, 6, 10, 24, 48 h). The apoptosis rates under different radiotherapy dose were detected by flow cytometer and TDT-mediated dUTP-biotin nick end labeling (TUNEL).

RESULTSCompared with the control group, cytoplasm expression of P65 under different X-ray doses had statistically significant differences (P < 0.05). While the cytoplasm P65 protein expression at different time were compared each other, the 3 h group demonstrated significant difference (P < 0.05). Apoptosis rates in various groups, compared with control group, had statistically significant differences (P < 0.05). While the groups at different time points were compared each other, the apoptosis rates of 3 h group had significant differences (P < 0.05).

CONCLUSIONX-ray can activate the NF-kappaB P65 in oral squmaous cell carcinoma cell lines. The correlation between expressional quantity of P65 and radiotherapy induced apoptosis of oral squamous cell carcinoma cell lines possesses positive correlation. The activated and intranuclear P65 may have radiotherapy resistant effect.

Apoptosis ; Carcinoma, Squamous Cell ; Cell Line, Tumor ; Humans ; Mouth Neoplasms ; Transcription Factor RelA

OBJECTIVE: To study the expression of nuclear factor kappa B p65 in hyperlipemia model of mice, and the relationship between hyperlipemia and deaf. METHOD: Twenty mice were divided into two group. The hyperlipemia diet group was established in ten mice,and the normal diet group was served as normal control. Six weeks later, immunohistostaining was used to detected the express of NF-kappa B p65 in all mouse cochlear. ABR threshold was obtained from both normal group and hyperlipemia group. RESULT: Immunoreactivity NF-kappa B p65 in mouse cochlea of hyperlipemia was localized in the organ of Corti, tectorial membrane, stria vascularis, spiral ligament, spiral ganglion and nerve fibers. The NF-kappa B p65 expression was markedly increased in mouse cochlea of hyperlipemia ABR threshold was significant difference between hyperlipemia group mice and control group mice (P < 0.01). CONCLUSION The expression of NF-kappa B p65 in mouse cochlea can be induced by hyperlipemia. And ABR threshold increased in hyperlipemia group mice. This shows that hyperlipidemia can damage acouesthesia of mice.
Animals ; Cochlea ; metabolism ; Disease Models, Animal ; Hyperlipidemias ; metabolism ; Mice ; Mice, Inbred Strains ; Transcription Factor RelA ; metabolism

OBJECTIVE: To study siRNA inhibited the expression of mRNA and protein of NF-kappaBp65 in the Hep -2 cell line. METHOD: Hep-2 were transfected with p65SiRNA. Western blotting was used to examine the protein levels of NF-kappaBp65. RT-PCR method was adopted to determine the mRNA expression of NF-kappaBp65. MTT method was adopted to investigate the proliferation of the Hep-2 cells after the transfection of p65siRNA. RESULT: The western blotting result showed that the level of NF-kappaBp65 protein was gradually declined after transfection of p65siRNA. The RT-PCR result showed that transfection with p65siRNA caused special degradation of the p65mRNA in Hep-2 cells at 24, 48 and 72 h. After transfection with p65siRNA. CONCLUSION p65siRNA has significant inhibition effects on the proliferation of the Hep-2 cells and expression of purpose gene mRNA and protein. The inhibition effects are time depended.
Cell Line, Tumor ; Cell Proliferation ; Humans ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Transcription Factor RelA ; genetics

OBJECTIVETo investigate the expression of nuclear factor kappa B (NF-kgr;B) and tumor necrosis factor α (TNF-α) in lung tissue of acute paraquat poisoned rats.

METHODS68 male Wistar rats were randomly divided into 2 groups: the control group (n = 8), the intoxication group (n = 60). On the 1st, the 3rd, the 7th, the 14th and the 28th day after intoxication, the expression of NF-κB p65 and TNF-α in lung tissue were detected by LSAB immunohistochemistry (IH) staining. Meanwhile, the level of malondialdehyde (MDA) in plasma, and lung homogenate, the content of malondialdehyde (HPY) in lung homogenate were detected.

RESULTSThe levels of MDA in plasma on the 1st, the 3rd, the 7th day and in lung homogenate on the 1st, the 3rd day of the intoxication group [in plasma: (10.15 ± 3.15), (6.97 ± 1.65) and (5.44 ± 0.66) nmol/ml; in lung homogenate: (10.20 ± 2.43), (10.71 ± 171) nmol/ml] were significantly higher than that of the control group [in plasma: (3.84 ± 1.04) nmol/ml, in lung homogenate: (7.66 ± 0.66) nmol/ml]. The content of HPY in lung homogenate on the 14th and the 28th day after intoxication [(19.98 ± 2.86), (26.06 ± 4.06) µg/0.1 g lung homogenate] were higher than that of the control group [(8.80 ± 1.26) µg/0.1 g lung homogenate] significantly. The expression of NF-κB p65 and TNF-α in lung tissue were both significantly increased on the first day and the 3rd day of the intoxication group compared with the control group and weakened obviously after the 7th day.

CONCLUSIONAcute paraquat poisoning can induce increased expression of both NF-κB p65 and TNF-α in lung tissue; the enhanced activity of NF-κB may take part in the process of pulmonary injury in PQ poisoning.

Animals ; Lung ; metabolism ; pathology ; Male ; Paraquat ; poisoning ; Rats ; Rats, Wistar ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

PURPOSE: This study was performed to determine the role of nuclear factor kappa B (NF-kappa B) on the lens epithelial cell death after ultraviolet (UV) irradiation. METHODS: Simian virus 40 transfected human lens epithelial cells (HLE B-3 cells) were used in this study. UVB located at 10cm from the bottom was irradiated during 1, 2, 3 and 4 minutes. To measure the cytotoxicity MTT assay was used. Translocation of NF-kappa B was examined by immunocytochemistry with anti NF-kappa B p65 antibody and electrophoretic mobility shift assay (EMSA). To confirm the role of NF-kappa B, the cells were pretreated with sulfasalazine, a specific inhibitor of NF-kappa B, for 30 minutes before irradiation, and cytotoxicity and translocation of NF-kappa B were evaluated. RESULTS: UV irradiation produced a progressive cytotoxic effect in cultured HLE B-3 cells after 1 minute and maximum cytotoxicity was reached after 3 minutes irradiation. When HLE B-3 cells were irradiated with UVB, the translocation of NF-kappa B was observed in immunocytochemistry. These translocations were peaked 6 hours after UV irradiation in EMSA. In HLE B-3 cells pretreated with sulfasalazine, the translocation of NF-kappa B was blocked. The cellular death after UV irradiation was markedly blocked by sulfasalazine. UV irradiation can translocate NF-kappa B and sulfasalazine is a useful blocking agent in this pathway. In addition, sulfasalazine can prevent cellular death after UV irradiation. CONCLUSIONS: These findings suggest that NF-kappa B plays an important role in cellular death after UV irradiation.
Electrophoretic Mobility Shift Assay ; Epithelial Cells* ; Humans ; Immunohistochemistry ; NF-kappa B* ; Simian virus 40 ; Sulfasalazine ; Transcription Factor RelA

OBJECTIVE: To investigate the subcellular localization of nuclear factor kappa B (NF-kappa B) in term human fetal membranes and myometrium during labor. METHODS: Fetal membranes and myometrial tissue were collected from term pregnant women undergoing cesarean delivery after labor (n=4) and before labor (n=4). An immunostaining was done with NF-kappa B p65 antibody. The intensity and distribution of nuclear immunostaining of NF-kappa B p65 subunit were evaluated visually using a semiquantitative analysis. RESULTS: NF-kappa B p65 was present in all tissues studied, and it was localized principally in the cytoplasm of cells of amnion and chorion. But, NF-kappa B p65 was localized more abundant in the nucleus than in the cytoplasm in myometrial cells. In amnion, chorion and myometrium, the staining scores of nuclear NF-kappa B did not show any difference between the after-labor group and before-labor group. CONCLUSION: In human term gestational tissues, subcellular localization of NF-kappa B showed cytoplasmic predominance in amnion and chorion, and nuclear predominance in myometrium. But these subcellular localizations did not change during labor.
Amnion ; Animals ; Chorion ; Cytoplasm ; Extraembryonic Membranes* ; Female ; Humans* ; Mice ; Myometrium* ; NF-kappa B* ; Pregnant Women ; Transcription Factor RelA

OBJECTIVETo examine the relationship between NF-kappaB signal transduction way and the sensitivity of Tca8113 carcinoma cell to Pingyangmycin chemotherapy.

METHODS2 mg/L antisense p65 oligodeoxynucle-otides (5'-GAACAGTTCGTCCATGGCCG-3') was transfected into Tca8113 cells through Lepofectin vectin, then the cells were treated with 8 mg/L Pingyangmycin. After 3 h and 6 h, the expression of p65 in nucleus was studied with immunohistochemical methods and Western blot analyses. After 48 h, the inhibitory rate of cell growth was detected with MTT assay.

RESULTSThe NF-kappaB/p65 signal transduction way in Tca8113 cells was activated after treated with Pingyangmycin. But the transfection of antisense p65 oligodeoxynucle-otides inhibited the activation of the signal transduction, the p65 expression in nucleus was decreased significantly (P < 0.05) at 6 h, and the inhibitory rate of cell growth was increased significantly (P < 0.05) at 48 h.

CONCLUSIONThe results suggested the chemotherapeutic sensitivity of Tca8113 carcinoma cell to Pingyangmycin was improved by the inhibition of NF-kappaB/p65 signal transduction.

Bleomycin ; analogs & derivatives ; Cell Line, Tumor ; Cell Proliferation ; Humans ; NF-kappa B ; Signal Transduction ; Tongue Neoplasms ; Transcription Factor RelA ; Transfection

AIMTo elucidate the location and effects of transcription factor-nuclear factor-kappaB (NF-kappaB) in lung tissues of rats with chronic obstructive pulmonary disease (COPD).

METHODSFourteen male Wistar rats were randomly divided into COPD model and control groups equally. The COPD model was established by intratracheal instillation of lipopolysaccharide twice and exposure to cigarette smoke daily. We detected the NF-kappaB p65 protein in lung by immunohistochemical method, and the expression of NF-kappaB p65 mRNA in lung by in situ hybridization.

RESULTSImmunohistochemistry, the expression of NF-kappaB p65 protein in alveolar, bronchiolar epithelium and arteriolar endothelium was significantly higher in the COPD group (0.426 +/- 0.007, 0.434 +/- 0.012 and 0.313 +/- 0.007, respectively) than those of the control group (0.115 +/- 0.006, 0.116 +/- 0.005 and 0.095 +/- 0.007, respectively, all P < 0.01). In situ hybridization showed that the expressions of NF-kappaB p65 mRNA in alveolar epithelium (0.203 +/- 0.008), bronchiolar and arteriolar smooth muscle cell (0.208 + 0. 010 and 0.206 + 0.007) of rats in the COPD group were stronger than those in the control group (0.100 +/- 0.006, 0.102 +/- 0.002 and 0.103 +/- 0.003 respectively) by semiquantitative analysis (all P < 0.01).

CONCLUSIONThe expression and nuclear translocation of NF-kappaB may be the basis event of gene expression of many cytokines and inflammatory mediators, which may positively regulate gene expression of many cytokines and inflammatory mediators in various cell lines.

Animals ; Lung ; metabolism ; pathology ; Male ; Pulmonary Disease, Chronic Obstructive ; metabolism ; pathology ; Rats ; Rats, Wistar ; Transcription Factor RelA ; metabolism

To investigate the expression of nuclear transcription factor kappaB (NF-kappaB) in childhood acute lymphoblastic leukemia (ALL) and its significance, the biotin-streptavidin method and microscopy were used to detect NF-kappaB P65 protein in cells from 32 childhood ALL patients and 40 children without hematologic malignancies as control. The results showed that the positive expression rate of NF-kappaB P65 protein in cells from 32 childhood ALL patients was 87.50%, obviously higher than that in control group (12.50%) (chi(2) = 40.56, p < 0.01). In 28 childhood ALL patients with positive expression, the ratio of weakly positive (+) cases to all positive cases was 10.71% (3/28); the ratio of generally positive (++) case was 42.86% (12/28), and the ratio of strongly positive (+++) cases was 46.43% (13/28). While in the control group the of NF-kappaB P65 protein showed low expression with 100% (5/5). There was significant difference in the level of NF-kappaB P65 protein between ALL patients and control group. While the level of NF-kappaB P65 protein had no significent difference in morphology, immunophenotype (T-lineage ALL and B-lineage ALL) and the courses in the de novo and the relaspsed cases. It is concluded that NF-kappaB P65 protein expresses in cells of childhood ALL, the inhibition of NF-kappaB transduction pathway may have significant value in childhood ALL treatment. This study provides experimental basis concerning clinical treatment for ALL, when NF-kappaB is taken as a target.
Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism