The Study On MDM2 Regulating The Apoptosis Of H₂O₂-induced AEC? Of Rats Through P53

Objective:The purpose of this study was to investigate the expression of MDM2 gene in rat AEC II induced by H₂O₂,to explore the effect of MDM2 gene on the apoptosis of AEC II induced by H₂O₂ and the mechanism of regulating apoptosis.Methods:After 48 hours of isolation,purification and culture,AEC?of healthy male SD rats were randomly divided into 7 groups for model construction:group A?normal control group:normal culture?,group B?H₂O₂ group:0.5mmol/L H₂O₂?,group C?empty lentivirus vector group:empty lentivirus vector infection?,group D?MDM2 group:MDM2 lentivirus overexpression vector infection?,group E?MDM2 silence group:MDM2 lentivirus silence vector infection?,group F?MDM2+H₂O₂ group:MDM2 lentivirus overexpression vector infection+0.5mmol/L H₂O₂ group?,group G?MDM2 silencing+H₂O₂ group:MDM2lentivirus silence vector infection+0.5mmol/L H₂O₂ group?.The growth morphology and density of AEC II were observed by inverted phase contrast microscopy;the characteristic structure of AEC II was identified by transmission electron microscopy?TEM?;the proliferation activity of AEC II was detected by CCK-8;MDM2 mRNA expression level was detected by real-time fluorescence quantitative PCR?RT-qPCR?;the purity of AEC II and the apoptosis of AEC II were detected by flow cytometry?FCM?.The early apoptosis rate of AEC II was detected by Mitochondrial membrane potential?MMP?;Immunocoprecipitation?COIP?was used to verify the interaction between MDM2 and p53protein in AEC II;Immunofluorescence staining?IF?was used to observe MDM2 protein expression by fluorescence microscopy;The expression of MDM2,p53,Bcl-2,Bax and cleaved-caspase3 protein was detected by Western blot.Results:1.Cell morphology:AEC II began to adhere at 24h by inverted phase contrast microscopy,and the adherence rate reached more than 95%at 48h.The specific microvilli and osmiophilic lamellar bodies of AEC II were observed by transmission electron microscopy,and the purity of AEC II was 91.75%+0.74%by flow cytometry.2.There was a low expression of MDM2 in AEC II induced by H₂O₂.RT-qPCR and Western blotting showed that compared with group A,the level of MDM2 mRNA and protein in group B decreased?P<0.05?.3.Model identification:At 48h the results showed that compared with group A,the expression of MDM2 mRNA was increased in group D and decreased in group E?P<0.05?.4.CCK-8:At 24h and 48h,compared with group A,the proliferation ability of group B and E decreased,while that of group D increased?P<0.05?.The proliferation ability of group D was the strongest,while that of group G was the weakest.5.Apoptotic rate:At 24h,48h time points:compared with group A,the apoptotic rate of group B was higher?P<0.05?;compared with group C,the apoptotic rate of group D was lower and that of group E was higher?P<0.05?;compared with group B,the apoptotic rate of group F was lower and that of group G was higher?P<0.05?,and the apoptotic rate of group D was the lowest among groups,and the apoptotic rate of group G was the highest.6.Early apoptotic rate:At 48h the results showed that compared with group A,the early apoptotic rate of group B was higher?P<0.05?;compared with group B,the early apoptotic rate of group G was higher?P<0.05?.7.COIP:COIP validation results showed that there was a binding relationship between MDM2 and p53 protein.8.Detection of mitochondrial apoptotic pathway-related proteins:At 48 hours the results showed that compared with group C,the levels of MDM2 protein in group D increased,while p53,Bax/Bcl-2,cleaved-caspase 3 protein in group D decreased;the level of MDM2 protein in group E decreased,while the levels of p53,Bax/Bcl-2,cleaved-caspase3 protein in group E increased?P<0.05?.Conclusion:1.MDM2 can inhibit the apoptosis of AEC II induced by H₂O₂ through p53.2.One of the mechanisms of MDM2 inhibiting AEC II apoptosis is through up-regulation the level of Bcl-2 protein and down-regulation the level of Bax and cleaved-caspase 3 protein expression through mitochondrial apoptotic pathway.