Objective To establish a cell culture model in vitro of acute lung injury and investigate the effects of NF-κB p65 on the inflammation and oxidative stress in TNF-α-activated type Ⅱ alveolar epithelial cells. Methods A549 cells were treated with TNF-α ( 10 ng/mL, 24 h) in the absence or presence of NF-κB p65 siRNA ( 50 nmol /L) . RT-PCR and Western blot were performed to analyze the silence efficiency of RNAi targeting NF-κB p65. The contents of IL-1β, IL-4, and IL-6 in the culture supernatant were measured by ELISA. The concentration of MDA and SOD were detected by colorimetric method. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium ( MTT) assay. Results P65 RNAi significantly decreased the transcription and translation of NF-κB p65 induced by TNF-α( P lt; 0. 05) . The levels of IL-1β, IL-4, and IL-6 were significantly lower in the supernatants of A549 cells pretransfected with NF-κB p65 siRNA ( P lt;0. 05) , while the concentration of MDA markedly decreased ( P lt; 0. 05) , and the activation of SOD increased dramatically ( P lt; 0. 05) . Consequently, the survival rate of A549 in the p65 siRNA group improved( P lt; 0. 05) . Conclusions NF-κB p65 plays a key role in the oxidative stress induced by TNF-α. NF-κB p65 silencing can down-regulate the inflammation and oxidative stress induced by TNF-αand enhance the proliferation of alveolar epithelial cells.
ObjectiveTo compare two different ways to establish mouse model with acute lung injury (ALI) via intratracheal instillation or intraperitoneal injection of lipopolysaccharide (LPS). MethodsBALB/c mice received intraperitoneal/intratracheal administration of LPS or sham operation. Wet/dry lung weight ratio, protein concentration in bronchoalveolar lavage fluid (BALF), and lung tissue histology were examined at 0, 1, 2, 6, 12, 18, 24, 48 h after LPS administration. Tumor necrosis factor-α (TNF-α) in BALF and serum was assayed with ELISA method. ResultsLPS treatment significantly increased wet/dry lung weight ratio, BALF protein concentration and TNF-α concentration in serum and BALF. Lung tissue was damaged after LPS challenge. The mice received LPS intraperitoneal injection got a more significant lung edema than those received LPS intratracheal instillation. Inversely, LPS intratracheal instillation induced more severed microstructure destruction. ConclusionsALI animal model by LPS intratracheal instillation or intraperitoneal injection induces inflammation and tissue damage in lung. However, the degree of tissue damage or self-healing induced by two methods is different. Therefore the decision of which way to establish ALI model will depend on the study purpose.
ObjectiveTo establish 16HBE cell lines stably expressing glutathione S-transferase mu 5 (GSTM5) gene, and explore the mechanism of GSTM5 nuclear translocation. MethodsRecombinant lentiviral expression vector containing GSTM5 gene was constructed and lentivirus was produced. After lentivirus infection of 16HBE cells, 16HBE-GSTM5 cell lines were obtained by screening with puromycin. Expression of GSTM5 in different cells was examined by RT-qPCR and Western blot. The nuclear translocation of GSTM5 was observed by confocal laser scanning microscope, after the 16HBE-GSTM5 cell lines were treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) for 0.5 hour. ResultsLentiviral expression plasmids, PLVX-puro-3*flag-SBP-GSTM5-C and PLVX-puro-GSTM5-SBP-3*flag-N, were constructed and lentiviral particles were successfully packed. After infected with lentivirus and screened by puromycin, two cell lines, 16HBE-GSTM5-SBP-3*flag-N and 16HBE-3*flag-SBP-GSTM5-C, were obtained. GSTM5 expression in these two cell lines was significantly higher compared with the control group and parental cells. After treated with TNF-α for 0.5 hour, the nuclear translocation of GSTM5 in 16HBE-GSTM5-SBP-3*flag-N was much more obviously than that in 16HBE-3*flag-SBP-GSTM5-C. ConclusionThe N-terminal region of GSTM5 is critical for nuclear translocation induced by TNF-α, which is mediated by a novel and non-classical nuclear localization signal.