| Lipopolysaccharide(LPS[endotoxin]) is the principal component of the outer membrane of Gram-negative bacteria. Acute lung injury (ALI), Acute respiratory dysfunction syndrome (ARDS) and multiple organ dysfunction syndrome induced by endotoxemia are major causes of the death of patients with infection. Previous studies have focused on the role of alveolar macrophages (AM) and polymorphonuclear leukocyte (PMN) in initiating and regulating endotoxin-induced inflammatory responses in the lung, but the role of alveolar typeⅡepithelial cells (ATⅡcells) have been paid little attention to. As a matter of fact, like AM and PMN, ATⅡcells can also be strongly activated by lipopolysaccharides and may contribute to the occurrence and development of ALI. As the LPS signal transducers, Toll-like receptor-4 (TLR4) and MD2 control the entry of LPS signal into cells. It is not yet known whether TLR4 and MD2 are expressed in ATⅡcells. The effects of TLR4 and MD2 on the activation of ATⅡcells induced by LPS are not yet clear. The main objective of our study is to investigate the effects and mechanisms of TLR4 and MD2 on the activation of ATⅡcells induced by LPS and to provide new ideas for preventing and treating ALI.Methods: 1. The expressions of TLR4 and MD2 mRNA in ATⅡcells were detected by semi-quantitative RT-PCR. The activity of NF-κB in ATⅡcells was measured with EMSA. The TNF-( and IL-6 concentrations in the supernatant of cultured ATⅡcells were tested with ELISA . 2. To construct TLR4 and MD2 recombinant plasmids, TLR4 and MD2 gene fragments were cloned with PCR and were reversely inserted into eukaryotic expression plasmid pEFBOS respectively. The positive recombinants were identified with restriction enzyme analysis and DNA sequencing. 3. After the recombinant being transferred into cultured ATⅡcells with liposome, the expressions of TLR4 and MD2 mRNA in ATⅡcells were detected with Northern blot. Theprotein expressions of TLR4 and MD2 in ATⅡcells were determined with Western blot. The activity of NF-κB in ATⅡcells was tested with EMSA. The concentrations of TNF-( and IL-6 in the supernatant of cultured ATⅡcells were also assayed with ELISA .Results: (1) The expressions of TLR4 and MD2 mRNA could be detected in normal ATⅡcells. After being stimulated with a 1 ng/ml LPS, their expressions significantly increased (P<0.01), and this trend continues with increasing LPS concentration. When the concentration of LPS is higher than 103 ng/ml, TLR4 and MD2 mRNA expressions still increase but less significantly. When ATⅡcells were incubated with 103 ng/ml LPS, the expressions of TLR4 and MD2 mRNA showed similar kinetic change. The expressions significantly increased at 1 h, then peaked at 2 ~ 4 h before decreasing substantially. (2) After being stimulated with LPS at different time point, the activity of NF-κΒ was higher than that of the control group (P<0.001). It started to increase at 1 h, peaked at 2 h - 4 h, then gradually decreased. After the stimulations with different LPS concentrations, NF-κΒ activity of ATⅡcells increased significantly (P<0.001) in a dose-dependent manner. (3) After the stimulations with LPS at different time points, the levels of TNF-( and IL-6 were higher than those of the control group (P<0.01). TNF-( started to increase quickly at 1 h, to a peak value at 2 h, and then gradually decreased. IL-6 started to increase quickly at 4 h, peaked at 8 h, and then gradually decreased. After the stimulations with different LPS concentrations, the levels of TNF-( and IL-6 increased significantly (P<0.01). The increases were in a dose-dependent manner. (4) Identified with restriction enzyme analysis and DNA sequencing, antisense expression vectors of TLR4 and MD2 were constructed successfully. Confirmed with Northern blot and Western blot analysis, the recombinants were successfully transferred into ATⅡcells. (5) Compared with the cells without LPS stimulation, in the ATⅡcells after LPS stimulation, without transfection or transfected with empty vector,...
|
PDF Full Text Request