Effect Of RA Signaling Pathway On The Expression Of SP-B In A549 Cells Treated With LPS

Respiratory distress syndrome (RDS) is a severe disease which can lead to neonatal and adult respiratory failure and the form of the hyaline membrane is the main feature of it. It has been known that NRDS is caused by a deficiency of pulmonary surfactant (PS), especially its important ingredient-surfactant protein B (SP-B). The mechanism of the full-term newborns and adults in RDS is very complicated, which has not yet been clarified. Gene mutation, serious infections, hypoxia and other factors may have caused RDS. SP-B secreted by alveolar type II epithelial cells, which is an important component of the pulmonary surfactant, plays an important role in the function, composition and metabolism of the PS. Previous studies have indicated that the transcription of SP-B gene is regulated by retinoic acid (RA).RA belongs to vitamin A compounds, which plays a wide range of biological effects in vertebrate development, cell differentiation and the maintenance of balance in the body. Recent studies have found that retinoic acid plays an important role in the promotion of lung development, regulation and control of SP-B gene expression. Research shows that RA can enhance the transcriptional activity of the SP-B promoter and increases the content of the SP-BmRNA and the former SP-B protein in the human lung adenocarcinoma cell line (H411) and cultured fetal lung tissue.Infection is the common cause that caused lung damage and respiratory dysfunction. LPS(lipopolysaccharide, LPS) is the main ingredients of bacterial endotoxin , which may stimulate the cells and lead to a variety of inflammatory substances (including cytokines) release and induced inflammatory response. Clinical data indicates that severe infection are closely related to neonatal and adult respiratory distress syndrome, then ascertain the exact pathogenesis of it is very important for our appropriate choice of targeted drug treatment. Our preliminary studies have confirmed that LPS inhibited SP-B expression in human lung adenocarcinoma cell (A549), however, there has been no definite report that effect of lipopolysaccharide on the expression of the SP-B whether related to the retinoic acid pathway and whether RA can prevent or treat the down-regulation of SP-B caused by LPS. It was surmised that LPS caused the down-regulation of SP-B in A549 cells, which may be involved in retinoic acid pathway.Objective1. By detecting the expression and distribution changes of the RARαin A549 cells treated with LPS, to understand the mechanism of the retinoic acid receptor abnormalities may be involved in infection-related hyaline membrane disease. 2. Treatment of A549 cells with retinoic acid before and after LPS and observed the expression and distribution changes of SP-B, RARαin A549 cells, so as to explore whether trans retinoic acid has intervention role in the down-regulation of SP-B in A549 cells caused by LPS, which maybe provide new ideas and experimental basis for the prevention and treatment of infection-related hyaline membrane disease.MethodsCultured human lung adenocarcinoma cell line A549 cells and then divided them into control and experimental groups. The group incubated with culture medium was seen as a control group. There were three experimental groups: LPS group, A549 cells were incubated with final concentration of 10μg/ml LPS for 24 h; ATRA+LPS group, A549 cells were incubated with 1×10-6mol/ml of retinoic acid for 24 h, then incubated with final concentration of 10μg/ml LPS for 24 h; LPS+ATRA group, A549 cells were incubated with final concentration of 10μg/ml LPS for 24 h, then incubated with 1×10-6mol/ml of retinoic acid for 24 h. The cell morphological changes of the control group and experimental group were observed by inverted microscope. RARαand SP-B expression was detected by immunocytochemical method.Results1. Morphology of A549 cellIn control group, the shape of A549 cell is polygon, abundant cytoplasm, adherence grow, not karyopyknosis. In LPS group, the shape of A549 cell change round and appear inequality of size thickness grain in cytoplasm; In LPS+ATRA group, cytoplasm translucent of which is clearer than LPS group and particle in cytoplasm is fewer than the LPS group; In ATRA+LPS group, compared with the LPS group,cell morphology did not change significantly.2. The immunocytochemical staining results are as the followings:①The expression of SP-B: SP-B expresses diffusely in cytoplasm of A549 cells in control group; Compared with control group, the expression of SP-B was decreased in LPS group(P<0.05); Compared with control group, the expression of SP-B had no changes in LPS+ATRA group(P>0.05), while compared with LPS group, the expression of SP-B was increased in LPS+ATRA group (P<0.05); Compared with control group, the expression of SP-B was decreased in ATRA+LPS group(P<0.05),while compared with LPS group, the expression of SP-B had no changes in ATRA+LPS group (P>0.05).②The expression of RARαin nuclei: RARαexpresses mostly in nuclei in control group; Compared with control group, the expression of RARαin nuclei was decreased in LPS group (P<0.05); Compared with control group, the expression of RARαin nuclei had no significant changes in LPS+ATRA group (P>0.05), while compared with LPS group, the expression of RARαin nuclei was increased in LPS+ATRA group (P<0.05); Compared with control group, the expression intensity of RARαin nuclei was decreased in ATRA+LPS group (P<0.05), while compared with LPS group, the expression of RARαin nuclei had no significant changes in ATRA+LPS group (P>0.05).③The expression of RARαin cytoplasm: The RARαin cytoplasm almost had no expression in control group ; Compared with control group, the expression of RARαin cytoplasm was increased in LPS group(P<0.05); Compared with control group, the expression intensity of RARαin cytoplasm had no significant changes in LPS+ATRA group (P>0.05), while compared with LPS group, the expression of RARαin cytoplasm was decreased in LPS+ATRA group (P<0.05); Compared with control group, the expression intensity of RARαin cytoplasm was increased in ATRA+LPS group(P<0.05), while compared with LPS group, the expression of RARαin cytoplasm had no significant changes in ATRA+LPS group (P>0.05).Conclusion1. LPS can lead to the down-regulation of SP-B in A549 cells, the mechanism of which may be related to the abnormal expression and cellular distribution of the RARα.2. By inducing the expression of RARαin nuclei, RA may play a role in treatment of the down-regulation of SP-B in A549 cells caused by LPS.3. Pretreatment of A549 cells with RA may be not reduce the down-regulation of SP-B caused by LPS.