Effect Of C8-Ceramide On Apoptosis Of Alveolar Type â…¡ Epithelial Cells

Objective To investigate the effects of exogenous C8-ceramide on survival of primary alveolar type II epithelial cell(AECⅡ) in vitro, and the induction of apoptosis in type II epithelial cells. Methods AECⅡ were isolated from 18 days gestational age ICR mice, and cultured in Dulbecco’s Modified Eagle Medium with 95% air and 5% CO2 at 37°C, cells were identificated by immunocytochemistry and transmission electron microscopy. The cells were treated with different concentrations(0, 20, 40 and 80μmol/L) of C8-ceramide for 24 h and treated with 80μmol/L of C8-ceramide for different time(3, 6, 12, and 24 h) to observe the livability of cells by MTT assay. Then, the flow cytometry and TUNEL assay were performed to determine apoptosis after the cells were treated with different concentrations(0, 20, 40, and 80μmol/L) of C8-ceramide for different time(12 and 24 h). Results Cell identification results proved that cells isolated is AECⅡ, the characteristic structure of AECⅡ was observed. The results of MTT assay showed that cell viability decreased in a dose- and time- dependent manner. In PI staining assay, the cells showed a distribution of(5.80±0.30)%、(7.00±0.10)%、(9.97±0.50)%、(12.67±0.67)% and(5.67±1.39)%、(8.87±0.49)%、(13.97±1.33)%、(18.80±0.46)% cells in apoptosis after treated with 0, 20, 40, 80 μmol/L of C8-ceramide for 12 and 24 h, the apoptosis rate of AECⅡ is increasing when the C8-ceramide concentration and time increased(P<0.05, compared with the control group). In Annexin V externalization assay, the cells showed a distribution of(20.63±0.86)%,(22.67±1.72)%,(42.03±1.34)%,(50.40±1.30)% and(20.93±2.51)%,(28.93±3.19)%,(47.00±1.08),(57.77±3.04)% cells in apoptosis after treated with 0, 20, 40, 80 μmol/L of C8-ceramide for 12 and 24 h(It is significant difference from untreated cells except for 20μmol/L in 12 h point, all P<0.05). In TUNEL assay, the cells showed a distribution of(5.51±1.41)%,(13.24±2.62)%,(35.10±4.59)%,(75.07±4.37)% and(5.56±1.36)%,(21.34±2.09)%,(37.12±2.11)%,(91.33±0.72)% cells in apoptosis after treated with 0, 20, 40, 80 μmol/L of C8-ceramide for 12 and 24 h(It is significant difference from untreated cells at the same time point, all P<0.05). Conclusion The results demonstrate that C8-ceramide can decrease the viability of AECⅡ and induce apoptosis.