Role Of SIRT1 Protein Nucleoplasm Transposition In Mediating Apoptosis Of HPAEpiC Induced By Hyperoxia

Objectives: High oxygen damage alveolar epithelial cells model is set up,then observing the changes in cell morphology,apoptosis,SIRT1 protein transposition,SENP1 protein and acetyl-P53 protein expression changes,and explore the SIRT1 protein in hyperoxia damage the meaning of the alveolar epithelial cells,as well as to explore the protective effect of SIRT1 ucleoplasm transposition inhibitor leptomycin B on HPAEpiC.Methods:This experiment is based on HPAEpiC in vitro subculture as the research object of the experiment.The high volume fraction of oxygen which were used to induce human alveolar epithelial cells(HPAEpiC)injury model were the mixted gas.The gas contained O2(900ml/L)and CO2(50ml/h)and were inleted at a speed of 3 litres a minute.The cell were exposed for 10 minutes.HPAEpiC was divided into three groups,including control group,hyperoxia group,hyperoxia and Leptomycin B group.Control group was added DMEM sugar medium containing 10% fetal bovine serum and 1% streptomycin,After joining medium would cell culture in temperature was 37 ?,humidity was 5% CO2 incubator.After joining the same medium hyperoxia group were inleted at a speed of 3L/min to access cell contains 900 ml/L O2 and 50 ml/LCO2 highly homozygous mixture,the processing time for 10 min,then let the cells were cultured in an incubator.After joining the same medium,hyperoxia and Leptomycin B groupwas added the final concentration of 5 ng/ml Leptomycin B,and then was passed into the same mixture at the same rate,the same processing time for 10 min,then let the cells were cultured in an incubator.After 12 h,24h and 48 h,Cells could be Collected and then we could detect the following indicators:Using inverted phase contrast microscope to observe the morphological change.Using flow cytometry technique to detect apoptosis rate of control group,hyperoxia group,hyperoxia and Leptomycin B group After 24 h.Immunofluorescence test transposition of SIRT1 of control group,hyperoxia group,Leptomycin B group After 24 h.The expression of SENP1 and acetylated P53lys(382)protein were measured by Western Blot of these three groups after24 h.Results: Using inverted phase contrast microscope observeed that HPAEpiC cells showed fusiform or polygon grow well in control group,better stick wall,uniform distribution,Observed the less visible suspended cells,the cells spacing was narrow.HPAEpiC cells grew poor in hyperoxia group and the growth situation was bad,most of the cells had changed its forms and became round or oval.meanwhile,more suspended cells appeared,and the spacing between cells had also increased.Compared with hyperoxia group,hyperoxia and Leptomycin B group growth condition is good,most of the cells form were long fusiform or polygon.Given the visible suspended cells.The hyperoxia group however had the larger number of suspension cells.The gap between cells were also increased.According to the result of flow cytometry instrument the apoptosis of hyperoxia group far more than control group and hyperoxiaand Leptomycin B group obviously(P < 0.05).The apoptosis of control group was lowest than other groups,Three groups were compared,the difference had statistical significance.Immunohistochemical results showed that Nuclei appear blue fluorescence,SIRT1 protein present red fluorescence,fusion and a purple color fluorescent.Red fluorescence concentrated in the nucleus of control group,the hyperoxia groups of red fluorescence are distribution in the nucleus and cytoplasm,red fluorescence in the cytoplasm of hyperoxia groups was more obviously than the control group.Compared with other groups the transfer rate of SIRT1 increased in hyperoxia group,with significant difference(P<0.05).The transfer rate of SIRT1 in hyperoxia and Leptomycin B group was higher than the control group,but lower than that of hyperoxia group,with significant difference(P < 0.05).Compared with control group,the cells of hyperoxia group,after processing 24 h,had more expression of SENP1 protein,and was significantly higher than the control group,while the difference was statistically significant(P < 0.05).Cells of hyperoxia group,after processing 24 h,had also increased its own expression of acetyl-P53 which was higher than the control group and hyperoxia and Leptomycin B group,the expression of acetyl-P53 of hyperoxia and Leptomycin B group was more than control group however less than hyperoxia group,the difference was statistically significant(P < 0.05).Conclusion: SIRT1 as an important protein involved in the resistance to oxidative stress in hyperoxia induced apoptosis of alveolar epithelial cells and lung damage.SIRT1 mediated mechanism was the alveolar epithelial cells ofalveolar epithelial cell apoptosis after exposed in hyperoxia.Hyperoxia could induce the expression of SENP1 protein.Then de-SUMOylation of SIRT1 could be increased to promote SIRT1 protein transfer from the nucleus to the cytoplasm.which reduced the deacetylase activity of SIRT1 protein.On the contrary the expression of acetyl-P53 protein could be increased and lead to cell apoptosis.Leptomycin B could reduce lung injury induced by hyperoxia.