The Changes Of SIRT1 Nuclear-plasma Shuttle In Pin1 Mediated Hyperoxia Induced Apoptosis Of Human Alveolar Epithelial Cells

Objective The peptidyl prolyl cis/trans isomerase(Pin1) has been found that play an important role in the oxidative stress pathway, which could reduce the hyperoxia lung injury. SIRT1 is known as silent mating type information regulation 2 homolog 1, is mainly localized in the nucleus to involve in regulating oxidative stress. The experiment based on the stable Pin1 expression of A549 cell. When it is exposed to hyperoxia, the SIRT1 can nuclear-plasma shuttle, which is lead to decline the activity of SIRT1,and reduce SIRT1 nuclear-plasma shuttle, and then reducing the hyperoxia lung injury. Methods: The A549-Pin1 sh RNA group was obtained by the preliminary experiments.The study was divided into a control group, a hyperoxia group, an A549-Pin1 sh RNA group. The hyperoxia group, A549-Pin1 sh RNA group were exposed to a mixture of O2(900ml/L)and CO2(50ml/L) for 10 minutes, then cultured in a closed environment for 24 h. The control group was cultured with 10% FBS and 1% streptomycin 1640 medium. The change of morphology were observed under inverted microscope after exposure to oxygen or room air for 24 hours; the expression of Caspase 3 and p53 were detected by immunohistochemistry; the localization of SIRT1 nuclear-plasma shuttle by fluorescence microscopy. Results:(1) Under the inverted microscopy, A549 cells in the control group were grew in good condition and sticked to each other tightly. The suspension cells were less. Compared with the control group, the hyperoxia group were grew showly, the living cells decreased and had a large number of suspension cells. The gap between cells were increased. However, the changes in morphology of A549 were obviously improved in the A549-Pin1 sh RNA hyperoxia group.(2) Immunohistochemistry results showed the followings, the expression of Caspase 3 and p53 in the hyperoxia group were significantly increased(P<0.05); But the expression Caspase 3 and p53 of the A549-Pin1 sh RNA hyperoxia group were between them(P<0.05).(3)Fluorescence microscopy showed that the nuclei were blue fluorescence, and p53 were red fluorescence, which was expressed both in cytoplasm and nucleus, but mainly play a role in the nucleus. Only a faint red fluorescence in the control group. Compared with the control group, the red fluorescence were increased significantly in the the hyperoxia group(P<0.05). The red fluorescence in the A549-Pin1 sh RNA group were decreased(P<0.05), but not reached the control group(P<0.05).Conclusion: The hyperoxia is turn activates the Pin1 resulting in isomerization of p66 shc and chondrosome transposition. It could increase the ROS production. In the A549-Pin1 sh RNA group, we found it can reduce the SIRT1 nuclear-plasma shuttle, which is lead to decline the activity of SIRT1,so it can dampen oxidative stress. It is expected to become a new treatment of hyperoxia-induced acute lung injury.