Lipopolysaccharide On The A549 Cells SP-B Expression And Its Mechanism

Surfactant protein B (SP-B) is an essential component of surfactant. It plays a role of diminishing alveolar surface tension, and is required for postnatal respiratory adaptation. At present more than that, the deficiency of surface active material leads to neonatal respiratory distress syndrome (NRDS) that is characterized by shaping the alveoli of lung hyaloid membrane. And SP-B is one of the most important surfactant. In the premature infant hyaline membrane disease, SP-B is absent because of the immaturity of alveolar typeⅡepithelial cells. But it is very complex for the mechanism of the full-term newborn and adult respiratory distress syndrome, and to this day, has not yet been clarified. It is possible that gene mutation, serious infect and oxygen deficiency all play roles in NRDS.There are many factors that can affect the transcription of SP-B, for example, many signal iter, hormone and cytokine. In recent years the research suggests that it plays an important role for retinoic acid receptor-α(RAR-α) in promoting the growth of pneumonic, control the gene expression of surfactant protein B (SP-B).The research reports that tumor nectosis factor-α(TNF-α)is a proinflammatory cytokine, which can reach a high level in the pulmonic inflammation diease. TNF-αalso can restrain the combine of surface activity phospholipids and surface activity protein in lung, and repress the activity of SP-B promoter in NCI-H441 cell. These hint TNF-αaffects the expression of SP-B gene on the transcriptional level. But the role of TNF-αis mediated by the activated nuclear transcription fator-κB (NFкB).The infection is a common cause that leads injury of lungs and respiratory dysfunction. LPS is a major component of bacterial endotoxin, which stimulus rhagiocrine cell and leads to a kind of inflammatory factor (include cell factor) release, guides inflammatory reaction. The clinical data make clear that severity infection can play a role in newborn and adult respiratory distress syndrome. To adopt the suitable medicine in order to carry out aim directly at therapy, it is very important that make clear the exact pathogenesy. In domestic or abroad, there is not clear-cut report that the infection affects the expression of SP-B by changing NFкB or the other mechanisms.ObjectiveExploring the relationship between infection and RDS and its relevant mechanisms by detecting the expression and distribution of RAR-α,NFкB and SP-B after A549 cells were incubated with lipopolysaccharide (LPS), which provides the original thinking and experimental base to the clinical precaution and therapy of the Hyaline membrane disease.MethodsAfter A549 cell have been cultivated for a long time, it have been divided into the control and the experimental group. A549 Cells were incubated with lipopolysaccharide (LPS) at different concentration(s5μg/ml;10μg/ml;15μg/ml) during 24 hour. We observe the morphology change of the cultured cell. We detect the expression and distribution change of RAR-αand SP-B in all groups by immunocytochemical method, and the expression and distribution change of NFкB in all group by fluorescent labeling and immunofluorescence copolymerization microscope.Results1. Morphology of A549 cellIn the common group, the shape of A549 cell is polygon, abundant kytoplasm, cell process manifest, adherence grow, be integrated into film, not karyopyknosis . After A549 cells were incubated with lipopolysaccharide (LPS) at 10μg/ml concentration, the shape of A549 cell change round, the endochylema lucency, ecphyma reduce and appear inequality of size thickness grain in the kytoplasm.2. The immunocytochemical staining results are as the followings:①Compared with control group(0.2252±0.05345),the expression of SP-B was decreased in three experimental group(5μg/ml:0.1256±0.01898;10μg/ml:0.0894±0.02230;15μg/ml:0.0553±0.01466, P<0.05).②Compared with control group(0.3692±0.03096), the expression of RAR-α(nuclei) was decreased in three experimental group(5μg/ml:0.2931±0.01362;10μg/ml :0.2068±0.01458;15μg/ml:0.0946±0.05506, P<0.05).③Compared with control group(0.0206±0.01546), the expression of RAR-α(cytoplasm) was increase in three experimental group(5μg/ml:0.0888±0.1023;10μg/ml:0.1499±0.1323;15μg/ml:0.2152±0.01531, P<0.05).3. The immunofluorescence Copolymerization results are as the followings:In the control group, the expression of NFкB located in the cytoplasm of A549. There is not expressed in the caryon .After A549 cell have been cultivated by lipopolysaccharide (concentration:10μg/ml) during 24h, the red fluor grains of NFкB metabasis to the nuclei.Conclusion1. LPS can cause the cell morphology change of A549 cell, and also may provoke the reduced expression of SP-B.2. In the process that LPS provoke the reduced expression of SP-B, it is possible that LPS induce the nuclear translocation of NFκB, the nuclear down regulation and kytoplasm transposition RAR-α.3. Through NFκB-mediated inflammatory reaction iter and RA-RAR signal iter , the affectoi interfere with the shape of alveolar surfactant, and cause RDS.