Adenovirus latent infection enhances the oxidant/antioxidant imbalance in rat alveolar epithelial cells

AIM: To observe the influence of adenovirus latent infection on the oxidant/antioxidant balance in rat alveolar epithelial cells. METHODS: The rat alveolar epithelial cells were stably transfected with the plasmid pE1Aneo and control plasmid pneo. GSH and MDA contents, the activities of major anti-oxidative enzymes including SOD, CAT, GPx, GST and γ-GCS were detected in oxidant stress. RESULTS: Adenovirus E1A expression repressed the activity of γ-GCS, and decreased GSH contents in oxidant stress. As a result, the activity of GPx and GST was decreased. The contents of MDA maintained high in oxidant stress. CONCLUSION: Adenovirus latent infection amplifies the oxidant/antioxidant imbalance in rat alveolar epithelial cells in oxidants stress, and adenovirus E1A protein decreases the activity of γ-GCS, which plays an important role in this process.     

Effect of dominant negative IκBα transfection on NF-κB pathway and cell motility in breast cancer

AIM: To observe the effect on NF-κB pathway and cell motility in breast cancer cell lines after transfection of dominant negative IκBα plasmid.METHODS: After stable transfection of mutant IκBα plasmid into highly metastatic breast cancer lines MDA-MB-231 and MDA-MB-435, we detected NF-κB binding activity by EMSA, cell growth ability by cell growth curve, colony forming test, and cell motility by millicell-PCF chamber.RESULTS: Constitutive activities in MDA-MB-231 and MDA-MB-435 were observed. Stable transfection of a dominant negative ⅠκBα resulted in downregulation of NF-κB binding activity, thus inhibited cell mobility without significant effect on cell growth.CONCLUSION: Cell migration ability is inhibited in highly invasive breast cancer cells by inhibition of NF-κB pathway in vitro.     

Effect of erythromycin on transforming growth factor-β1 and γ-glutamylcysteine synthetase in lung of smoking rats

AIM: To investigate the effect of erythromycin on the level of transforming growth factor-β₁ (TGF-β₁) and γ-glutaglutamylcysteine synthetase (γ-GCS) in smoking rats,and to explore the antioxidate therapeutic role of erythromycin in chronic obstructive pulmonary disease.METHODS: Wistar rats were exposed to cigarettes smoking to establish the model.After passive smoking for 4 weeks,erythromycin intragastric intervention was administered continuously for 8 weeks.The expiratory airway resistance and lung compliance were assessed and the expression levels of TGF-β₁ and γ-GCS proteins (and the mRNA) in airway endothelial cells and alveolar macrophages were observed respectively by immunohistochemical,immunocytochemical and (in situ) hybridization.RESULTS: The expiratory airway resistance was increased and the lung compliance was degraded significantly in smoking group and erythromycin group,compared to control group.In erythromycin group,the airway resistance was lower and the lung compliance was higher than that in smoking group (P<0.05).The levels of TGF-β₁ and γ-GCS in smoking group and erythromycin group was obviously increased in airway endothelial cells and alveolar macrophages in comparison with control group (P<0.05).The levels of TGF-β₁ and γ-GCS were inhibited by erythromycin (P<0.05).TGF-β₁ was obviously positive correlated with γ-GCS in smoking group,but this was not found in erythromycin group.CONCLUSION: Erythromycin therapy improves pulmonary function and relieves emphysema change induced by smoking in rats,and decreases the expression of TGF-β₁ and γ-GCS in alveolar macrophages and airway endothelial cells,suggesting that erythromycin may play a role in the antioxidate therapeutic in COPD.     

uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Nontypeable haemophilus influenzae induces IL-8 expression in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner

AIM:To investigate the key molecular mechanism of inflammatory response in alveolar epithelial cells induced by nontypeable haemophilus influenzae (NTHi). METHODS: A549 cells were co-cultured with NTHi (multiplicity of infection, MOI: 10) and harvested 15 min and 30 min after stimulation. The phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) in A549 cells was detected by Western blotting. The intracellular expression of nuclear factor-κB (NF-κB) p65 was examined by flow cytometry 4 h after stimulation. A549 cells were preincubated with p38 inhibitor (SB203580) or NF-κB inhibitor (PDTC) for 1 h and then stimulated with NTHi for 24 h. The level of interleukin 8 (IL-8) in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The phosphorylation of p38 MAPK was rapidly induced by NTHi stimulation. The expression of NF-κB p65 in A549 cells after NTHi stimulation was significantly up-regulated compared with control group (P<0.05). The level of IL-8 in the supernatants was increased 24 h after bacterial stimulation compared with control group (P<0.05). Blockage of p38 MAPK or NF-κB remarkably decreased IL-8 secretion in A549 cells (P<0.05). CONCLUSION:NTHi induces inflammatory response in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner.     

Cucurbitin E relieves airway inflammation by inhibiting MAPKs/NF-κB signaling pathways in asthmatic mice

AIMTo investigate the effects of cucurbitacin E on airway inflammation and the signaling pathways of MAPKs and NF-κB in asthmatic mice. METHODSHealthy mice (n=40) were randomly divided into control group, model group, low-dose cucurbitacin E group, high-dose cucurbitacin E group and dexamethasone group. Ovalbumin sensitization was used to induce asthma in the mice. The protein levels of p-JNK, p-ERK1/2, p-p38 MAPK and p-p65 in the lung tissues were determined by Western blot. RESULTSCompared with control group, the numbers of inflammatory cells, such as eosinophils, lymphocytes and neutrophils, were significantly increased in model group, and the activity of MAPKs and NF-κB signaling pathway-related proteins was significantly enhanced. Cucurbitin E at high dose attenuated airway inflammation in asthmatic mice, and significantly inhibited the activity of MAPKs and NF-κB signaling pathway-related proteins. Histopathological results showed proliferation of goblet cells and bronchial mucosal epithelial cells, infiltration of inflammatory cells in the alveoli, and narrow alveolar cavity in model group, while the pathological changes were significantly alleviated in cucurbitin E treatment groups. CONCLUSION Cucurbitin E improves airway inflammation in asthmatic mice, and its mechanism may be related to the inhibition of MAPKs and NF-κB signaling pathways.     

Nontypeable Hemophilus influenzae induces inflammatory response of bronchial epithelial cells via NF-κB activation and histone deacetylase activity reduction

AIM: To investigate the effect of nontypeable Hemophilus influenzae (NTHi) on the inflammatory response of bronchial epithelial cells, and to preliminarily explore its mechanism. METHODS: The human normal bronchial epithelial BEAS-2B cells were cultured, and the expression levels of interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant were measured by ELISA and RT-qPCR after infection with NTHi at MOI=10. The levels of IκBα and the phosphoacetylation of histones were determined by Western blot, and then the expression of histone deacetylase (HDAC) and the enzyme activity of HDAC were detected. The binding activity of NF-κB and IL-8 was measured by electrophoretic mobility shift assay and chromatin immunoprecipitation. Finally, the expression levels of GM-CSF and IL-8 were measured after pretreatment with NF-κB and HDAC inhibitors. RESULTS: The expression levels of IL-8 and GM-CSF in the culture supernatant of BEAS-2B cells were significantly increased (P<0.05) after infection with NTHi at MOI=10. In addition, infection with NTHi significantly down-regulated the expression of cytoplasmic IκBα and enhanced the DNA-binding activity of NF-κB. The phosphoacetylation of histones H3 and H4 and the binding of IL-8 and RNA polymerase II were also significantly increased after infection with NTHi. The expression level and enzyme activity of intracellular HDAC were also significantly reduced (P<0.05) after infection with NTHi. The expression levels of GM-CSF and IL-8 were significantly reduced after pretreatment with NF-κB inhibitor (P<0.05), while the secretion of IL-8 was significantly increased after pretreatment with HDAC inhibitor (P<0.05). CONCLUSION: NTHi inhibits the expression and activity of HDAC by activating the NF-κB signaling pathway, and promotes the secretion of IL-8 and GM-CSF in bronchial epithelial cells, thereby aggravating the inflammatory response.