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

ObjectiveTo establish a hyperoxia-exposed celluar damange model of primary cultured premature rat AECerⅡs and investigate the effect of UPP on the ERS –induced apoptosis of AECerⅡs.MethodsPrimary cultured premature rat AECerⅡs 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 AECerⅡs were harvested for further experiments.Annexin V-FITC/PI double staining was utilized to detected the apoptosis of AECerⅡs 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 AECerⅡs.Results(1)After 24-,48-and 72-hours’ exposure,compaired with air group,the number of AECerⅡs in hyperoxia group decreased with irregular spreading and vacuolization.Measured by flow cytometry,the apoptosis of AECerⅡs in hyperoxia group increased(P <0.05).The activity of 20 s proteasome in AECerⅡs 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 AECerⅡs were significantly increased(P<0.05).(2)Compaired with the DMSO group,the apoptosis of AECerⅡs 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 AECerⅡs were significantly increased in hyperoxia group treated with MG132.(P<0.05)And the green fluorescence of Caspase-3 in AECerⅡs 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 AECerⅡs.(2)MG132 may enhance ERS –induced apoptosis of AECerⅡs exposed to hyperoxia.