Objective To observe the effect of epidermal growth factor (EGF) on the proliferation, adhesion, invasiveness and the activation of nuclear factor-κB (NF-κB), matrix metalloproteinases (MMPs) expression and explore related mechanisms in pancreatic cancer cells. Methods Cell invasion assay, proliferation assay and adhesion assay were used to examine the proliferation, adhesion and invasiveness of pancreatic cancer cells, respectively. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA), and MMPs protein and mRNA expressions were investigated by gelatin zymography, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Results EGF increased the invasiveness of pancreatic cancer cell in a dose-dependent manner (P＜0.05), but did not affect cell proliferation or adhesion. The expressions of MMP-9 mRNA and protein significantly increased after induction by EGF and were highest when EGF concentration was 50 ng/ml, while there was no effect on the expressions of MMP-2 mRNA and protein. Furthermore, NF-κB activity increased with increased concentration of EGF in a concentration-dependent manner (P＜0.05). In addition, NF-κB activity and the expressions of MMP-9 mRNA and protein by pretreatment with both pyrrolidine dithiocarbamate (PDTC) and EGF decreased when compared that by pretreatment with EGF alone. The invasiveness of pancreatic cancer cell by pretreatment with both PDTC and EGF decreased when compared that by pretreatment with EGF alone and nothing (P＜0.05).Conclusion The findings indicate that the NF-κB-mediated MMP-9 induction is essential for EGF-induced invasiveness in pancreatic cancer cells, which can be inhibited by PDTC.
ObjectiveTo identify the expression functions of human NF-κBp65 nuclear localization signals' deletion mutant plasmids(namely pcDNA3.1(+)-NF-κBp65ΔNLS, NF-κBp65ΔNLS, for short) and the changes of proliferation, migration and adhesion ability of A549 lung cancer cells with low expression of NF-κBp65 (namely A549/NF-κBp65 shRNA cells). MethodsHuman A549/NF-κBp65 shRNA cells were cultivated and divided into a control group, a transfection pcDNA3.1 (+) group, and a transfection NF-κBp65ΔNLS group. Indirect immunofluorescence, real-time fluorescent quantitative PCR and Western blot techniques were used to detect the NF-κBp65 intracellular localization and the change of NF-κBp65 mRNA and protein expression level. MTT, Transwell and cell adhesion experiments were used to analyze the changes of proliferation, migration and adhesion ability of A549/NF-κBp65 shRNA cells. ResultsThe human NF-κBp65ΔNLS eukaryotic expression plasmid was successfully constructed. Compared with the control group and the transfection pcDNA3.1(+) group, NF-κBp65 mRNA expression level in A549/NF-κBp65 shRNA cells was increased in the transfection NF-κBp65ΔNLS group(10.63±0.84 vs. 1.04±0.21 and 1.23±0.22, P < 0.01) and NF-κBp65 protein expression level was also increased (1.07±0.06 vs. 0.53±0.02 and 0.59±0.04, P < 0.01). NF-κBp65 protein mainly located in the cytoplasm, and did not significantly transferred into the nucleus after stimulated by TNF-α. At the same time, A549/NF-κBp65 shRNA cells' proliferation, migration and adhesion ability were enhanced compared with the control group and the transfection pcDNA3.1(+) group. ConclusionsThrough gene mutation technology to build the human NF-κBp65ΔNLS eukaryotic expression plasmid and transfect into A549/NF-κBp65 shRNA lung cancer cell lines, both mRNA and protein expression levels of NF-κBp65 were increased significantly, and NF-κBp65 protein mainly located in the cytoplasm. The overexpressed NF-κBp65 in cytoplasm can obviously enhance the A549/NF-κBp65 shRNA cell's proliferation, migration and adhesion ability. It suggests that NF-κBp65 stranded in the cytoplasm can still regulate biological behavior of lung cancer cells by influencing the NF-κB signaling pathway related proteins.
【 Abstract 】 Objective To investigate the protective effect of peroxisome proliferator-activated receptor γ (PPAR γ ) activator 15-deoxyprostaglandin J2 (15d-PGJ2) in rat hepatic ischemia-reperfusion injury and its mechanism. Methods The models of 70% warm ischemia-reperfusion injury were established in SD rats, rats were randomly divided into 4 groups: sham operation group, ischemia-reperfusion group, 15d-PGJ2 group and 15d-PGJ2+GW9662 group. After reperfusion, serum AST and ALT levels were determined; the liver tissues were removed for measurement of activity of NF-κB and myeloperoxidase (MPO), TNF-α content and expression of ICAM-1. Results Compared with sham operation group, the serum levels of ALT and AST, and the activities of MPO and NF- κ B, TNF- α content and expression of ICAM-1 in ischemia-reperfusion group, 15d-PGJ2 group and 15d-PGJ2+GW9662 group were greatly improved (P ＜ 0.05). Compared with ischemia-reperfusion group, the serum levels of ALT and AST and the activities of MPO and NF- κ B, TNF- α content and expression of ICAM-1 in 15d-PGJ2 group were significantly decreased (P ＜ 0.05). Compared with 15d-PGJ2 group, the serum levels of ALT and AST, and the activities of MPO and NF- κ B, TNF- α content and the expression of ICAM-1 in 15d-PGJ2+GW9662 group were obviously increased (P ＜ 0.05). Conclusion PPAR γ activator 15d-PGJ2 could protect against ischemia-reperfusion injury in rats, with its possible mechanism of inhibiting NF-κB activation and down-regulating TNF-α content and ICAM-1 expression in a PPARγ dependent fashion.
Objective To investigate the expression of transcription factors including nuclear factor-κB （NF-κB） and activator protein-1 （AP-1） in vascular endothelial cells （ECs） in different flow fields， and provide experimental evidence for mechanical signal effects on gene regulation pattern of ECs. Methods Cultured human umbilical vein ECs were loaded into steady flow chambers of laminar flow or turbulent flow and observed at 6 time points （0.5 h， 1 h， 2 h， 3 h， 4 h and 5 h） based on different load time. Spacial and temporal characteristics of NF-κB and AP-1 expression in ECs in different flow chambers were detected at a protein level by laser confocal microscope. Results In laminar flow， NF-κB expression rose to peak at 1 hour （26.49±1.63， P＜0.05）and then declined. In turbulent flow， NF-κB expression rose to peak at 3 hours （34.41±6.43， P＜0.05）. In laminar flow， c-Jun/AP-1 expression was transiently elevated， reached its peak at 0.5 hour （18.95±5.38，P＜0.05）and then fell to its baseline level. In turbulent flow， c-Jun/AP-1 expression rose slowly but steady to peak（P＜0.05） . Conclusion The effects of turbulent flow on NF-κB and AP-1 expression in ECs are different from those of laminar flow. Up-regulation and activation of NF-κB and AP-1 expression in ECs induced by turbulent flow may cause pathological changes in morphological structure and functional behavior of ECs.
ObjectiveTo explore the mechanism of lung injury in Sprague-Dawley (SD) rats induced by acute organic phosphorus pesticides (AOPP) by observing the changes of the blood serum nuclear factor (NF)-κB consistence, NF-κB level of lung tissue and lung coefficient. MethodNinety-six healthy male SD rats (six weeks old) were randomly divided into group A (control, n=48) and group B (poison, n=48). The rats of group B were given omethoate by gavage (45 mg/kg), and the rats of group A accepted normal saline. Then the rats were killed at designated observing points (30 minutes; 3, 6, 12, 24, and 48 hours), and the lung coefficient, blood serum NF-κB consistence and NF-κB level of lung tissue were measured. At the same time, we observed the pathological changes of the rats' lung tissue. ResultsCompared with group A, blood serum NF-κB consistence, NF-κB level of lung tissue and the level of lung coefficient in group B were significantly higher (P<0.01). The lung tissues of group A were normal at each time point, but in group B, the lung pathological changes gradually appeared 30 minutes later with pulmonary interstitial engorging, alveolar septum widening and some alveolus being full of red blood cells, and this situation reached its peak at hour 12. Then it gradually mitigated from 24 to 48 hours. ConclusionThere are significant increases in blood serum NF-κB consistence and NF-κB level in lung tissues in rats with lung injury induced by omethoate poisoning. The NF-κB may play a role in the process of lung injury induced by organophosphorus pesticide.
【Abstract】 Objective To study the effects of ischemic preconditioning (IP) on the activity of nuclear factor-κB （NF-κB） and the expressions of TNF-α and intercellular adhesion molecule-1 (ICAM-1) during early reperfusion following liver transplantation in rats. Methods The models of rat orthotopic liver transplantation were established. The donor livers were stored for 2 hours in Ringers solution at 4 ℃ before transplantation. All rats were randomly divided into sham operation group （SO group）， control group and IP group. IP group was achieved by clamping the portal vein and hepatic artery of donor liver for 10 minutes followed by reperfusion for 10 minutes before harvesting. The activity of NF-κB and expressions of TNF-α and ICAM-1 at 1 h, 2 h, 4 h and 6 h after reperfusion were measured. Serum ALT, LDH were also determined. Results The liver function of recipients with IP were significantly improved. Compared with SO group， the graft NF-κB activity increased after transplantation in control group and IP group (P＜0.05), while compared with control group that was significantly attenuated at 1 h and 2 h in IP group. Similarly, hepatic levels of TNF-α and ICAM-1 were significantly elevated in control group and were reduced in IP group. Conclusion IP might down-regulated TNF-α and ICAM-1 expression in the grafts after orthotopic liver transplantation through depressed NF-κB activation， and attenuate neutrophil infiltration in the grafts after reperfusion.
Objective To investigate the effects of glutathione S-transferase M5 (GSTM5) on the inflammation in human bronchial epithelial 16HBE cells and its possible molecular mechanisms. Methods Acute lung injury cell model was constructed with 16HBE cells induced by tumour necrosis factorα (TNF-α, 10 ng/mL). The cells were devided into a control group, a TNF-α group (TNF-α), a GSTM5 group (GSTM5+TNF-α), a negative control group (negative control plasmid+TNF-α). GSTM5-GFP plasmid and negative control plasmid were respectively transfected to the cells of the GSTM5 group and the negative control group using Lipofectamine2000. The contents of interleukin-6(IL-6), IL-8, IL-10 in the cell supernatant were measured by ELISA.The expression of nuclear factor-κB (NF-κB) mRNA was detected by RT-PCR, and the expression of NF-κB, phospho-NF-κB, p38, phospho-p38 protein were detected by Western blot. Results The GSTM5-GFP eukaryotic expression vector was successfully constructed and transfected successfully confirmed by fluorescence microscope. The contents of IL-6, IL-8, IL-10 in the TNF-α-induced cell supernatant were significantly higher than those in the control group(P < 0.05), and the contents of IL-6, IL-8, IL-10 in the GSTM5 group were lower than those in the TNF-α group (P < 0.05)with statistically significant difference. At the same time, the total NF-κB mRNA, phospho -NF-κB and phospho-p38 protein were increased in TNF-α stimulated cells compared with the control group (P < 0.05), while the GSTM5 group was lower than that in the TNF-α group and the negative control group (P < 0.05). Conclusion Overexpression of GSTM5 inhibits the phosphorylation of p38MAPK and NF-κB and down-regulates the inflammation of TNF-α-induced human bronchial epithelial 16HBE cells.
Objective To explore the effect on apoptotic genes of pancreatic adenocarcinoma cell BxPC-3 from subcutaneous transplantation tumor in nude mice induced by 5-FU and sulfasalazine (SZ).Methods Changes of apoptosis-related genes 〔bcl-2, cyclinD1, Bax and NF-κB (p65)〕 in subcutaneous transplantation tumor treated by 5-FU, SZ alone or both at the levels of mRNA and protein were measured by RT-PCR and Western blot. Results NF-κB (p65) at mRNA relative content and protein expression in subcutaneously heterotopic transplantation tumor treated by 5-FU (7.5, 15 mg/kg), SZ (10, 20 mg/kg) alone or both showed significant difference, except for two subsets in SZ group, respectively, in comparison with each control group (P＜0.01). Meanwhile bcl-2 and cyclinD1 at the levels of mRNA and protein, and Bax protein level were significantly different from each control group (P＜0.01). The above-mentioned indexes were show obvious interaction of both by multiple factor analysis of variance. Conclusion Up-regulated level of Bax, down-regulated levels of bcl-2, cyclinD1 and NF-κB (p65) might be one of apoptotic mechanisms that SZ synergistically enhanced apoptotic effect on pancreatic adenocarcinoma cell BxPC-3 of subcutaneous transplantation tumor in nude mice induced by 5-FU.
ObjectiveTo observe the effect of rosiglitazone on cognitive function, serum high sensitive C reactive protein (hs-CRP) and expression of nuclear factor-κB (NF-κB), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in hippocampal tissues of senile diabetic rats. MethodsThirty aged Wistar rats (20-22 months) were randomly divided into normal control group (n=6), diabetic model group (n=12), and rosiglitazone treatment group (n=12). Streptozotocin-induced diabetic rat model was established. In the rosiglitazone treatment group, the rats were treated with rosiglitazone 4mg/kg/d for 8 weeks. The cognitive function of rats was evaluated with the Morris water maze test. Serum hs-CRP was detected by ELISA. The expression of NF-κB in hippocampal tissues was detected by western blot and IL-6 and TNF-α by Real-time PCR. ResultsThe Morris water maze test showed that escape latency was longer in the rosiglitazone treatment group and the diabetic model group than that in the control group (P<0. 05). Compared with the diabetic model group, the rosiglitazone treatment group showed a significant decrease in the average time of escape latencies (P<0.05), and an increased percentage of time spent in the central area and the more times navigating the original platform position (P<0.05). Serum hs-CRP and the expression of NF-κB, IL-6 and TNF-α in the rosiglitazone treatment group and the diabetic model group was significantly higher than those in the control group (P<0.01). Compared with the diabetic model group, serum hs-CRP and the expression of NF-κB, IL-6 and TNF-α in the rosiglitazone treatment group was decreased (P<0.05). ConclusionCognitive impairment in senile diabetic rats is associated with serum hs-CRP. The cognitive function can be improved with rosiglitazone treatment. The protective mechanisms may be related to the decrease of serum hs-CRP, inhibition of NF-κB signal and down-regulation of the expression of IL-6 and TNF-α in hippocampal tissues.
ObjectiveTo explore the protective mechanism and effect of the resveratrol for kidney injury of obstructive jaundice. MethodsThe rats were randomly divided into three groups: sham operation group receiving laparotomy without bile duct ligation (BDL), the obstructive jaundice group with BDL, and the obstructive jaundice + resveratrol group given resveratrol following BDL. The levels of total bilirubin (TBIL), direct bilirubin (DBIL), blood urea nitrogen (BUN), and creatinine (Cr) in the serum were tested. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, glutathione (GSH) level in the renal tissues were detected. The expressions of the silent information regulator 1 (SIRT1) and nuclear factor-κB (NF-κB) proteins were tested by Western blot. The expression of SIRT1 mRNA was detected by RT-PCR and the renal cell apoptosis was examined by TUNEL staining. Results①Compared with the sham operation group, the levels of serum TBIL, DBIL, BUN and Cr were significantly higher (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were signi-ficantly lower (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly higher (P < 0.05) in the obstructive jaundice group.②Compared with the obstructive jaundice group, the levels of serum TBIL, DBIL, BUN and Cr were significantly lower (P < 0.05); the activity of SOD and level of GSH, and the expressions of SIRT1 mRNA and SIRT1 protein in the renal tissues were significantly higher (P < 0.05); the content of MDA, the expression of NF-κB protein, and the rate of cell apoptosis in the renal tissues were significantly lower (P < 0.05) in the obstructive jaundice+resveratrol group. ConclusionThe resveratrol could alleviate renal damage and play a beneficial role to resist inflammation, oxidation, and apoptosis by activating the SIRT1 which probably inhibits the expression of NF-κB protein and promotes the activity of SOD in cholestatic kidney injury.