The Effect Of Ubiquitin Proteasome Pathway On The Endoplasmic Reticulum Stresse-induced Apoptosis Of AEC? Exposed To Hyperoxia

ObjectiveTo establish a hyperoxia-exposed celluar damange model of primary cultured premature rat AECer?s and investigate the effect of UPP on the ERS –induced apoptosis of AECer?s.MethodsPrimary cultured premature rat AECer?s were randomly exposed to air and 950 m L/L O2 respectively.Some of hyperoxia group was treated with MG132,a proteasome inhibitor,or DMSO as a control.After 24-,48-and 72-hours' exposure,an inverted phase-contrast microscope was applied to observe cell morphology.Then AECer?s were harvested for further experiments.Annexin V-FITC/PI double staining was utilized to detected the apoptosis of AECer?s measured by flow cytometry.Reagent kit with specific fluorescent substrate was utilized to measure the activity of20 s proteasome.Real-time PCR was utilized to detected the m RNA of GRP78,CHOP and ubiquitin.And Western blot was utilized to detected the protein levels of GRP78,CHOP,total ubiquitinated proteins,Bax/Bcl-2 ratio and Caspase-3.Fluorescence microscope was applied to observe the immunofluorescent staining Caspase-3 in AECer?s.Results(1)After 24-,48-and 72-hours' exposure,compaired with air group,the number of AECer?s in hyperoxia group decreased with irregular spreading and vacuolization.Measured by flow cytometry,the apoptosis of AECer?s in hyperoxia group increased(P <0.05).The activity of 20 s proteasome in AECer?s of hyperoxia group decreased.The m RNA expression of GRP78,CHOP,and ubiqutin were significantly increased.Meanwhile,the protein expressions of GRP78,CHOP,total ubiquitinated,Bax/Bcl-2 ratio and Caspase-3 in AECer?s were significantly increased(P<0.05).(2)Compaired with the DMSO group,the apoptosis of AECer?s insreased inhyperoxia group treated with MG132.(P<0.05)The protein expressions of GRP78,ATF4,CHOP,total ubiquitinated,Bax/Bcl-2 ratio and Caspase-3 in AECer?s were significantly increased in hyperoxia group treated with MG132.(P<0.05)And the green fluorescence of Caspase-3 in AECer?s was increased in hyperoxia group treated with MG132.Conclusion(1)Hyperoxia may induce ERS,which decreases the activity of 20 s proteasome,and lead to the apoptosis of AECer?s.(2)MG132 may enhance ERS –induced apoptosis of AECer?s exposed to hyperoxia.