Effects Of C8-ceramide On The Pulmonary Epithelial Barrier Function In Mice

Objective To study the effects of C8-ceramide on the alveolar epithelial barrier, the tight junction proteins of alveolar epithelial type II cells(AEC II), and the induction of apoptosis in AECII.Methods The primarily AECII were isolated from 18 days gestational age ICR mice,and were purified by differential adherence method. The morphology and growth characteristics of AECII were observed by an inverted microscope. The cells were identified with alkaline phosphatase staining and under transmission electron microscopy. The cells were seeded on Transwell polyester membranes to construct in vitro models of AEC II monolayers with different concentration of C8-ceramide(0, 25,50, 100 μmol/L) for 24 hours when they became fused. The monolayer permeability was measured by transepithelial electrical resistance(TEER) and horseradish peroxidase notation(HRP). The total protein and RNA were extracted from AEC II.Western blot and real-time quantitative polymerase chain reaction(RT-PCR) were used to detect the protein and m RNA levels of ZO-1, Occludin and Claudin-4 respectively.Annexin V-FITC/PI were performed to determine apoptosis after the cells were treated with different concentrations(0, 25, 50, and 100 μmol/L) of C8-ceramide for 24 h.Results Stimulated with different concentration of C8-ceramide for 24 h, the TEER of cells decreased(F = 38.780, P < 0.001) and the OD470 increased(F = 19.780, P < 0.001)significantly. Results from Western blot and RT-PCR demonstrated significant decrease of ZO-1(F = 71.150, P<0.01; F = 4.887, P = 0.014) and Occludin(F = 7.703, P = 0.002;F = 3.637, P = 0.048) expression and no significant change in Claudin-4 in the AEC II with C8-ceramide. After stimulation with different dose of C8-ceramide, Annexin V externalization assay proved that the rate of apoptosis increased significantly(P < 0.05)Conclusion Exogenous C8-ceramide may increase the pulmonary epithelial permeability by downregulation the protein and m RNA levels of ZO-1, Occludin rather than Claudin-4. Moreover, ceramide has been found to induce AECII apoptosis which further to disrupt the pulmonary epithelial barrier function.