Effect Of Notch1/hes1 On Proliferation And Differentiation Of AEC? By Regulating Expression Of C/EBP?

ObjectiveBronchopulmonary dysplasia(BPD)is one of the serious complications of premature infants,especially in very low birth weight preterm infants,and hyperoxia is one of the most important and most common factors leading to BPD in preterm infants.Notchl/Hesl signaling and CCAAT enhancer binding protein alpha(C/EBPa)play an important role in lung development and lung injury.In this study,Notchl pathway activators and inhibitors were used to stimulate Type II alveolar epithelial cell(AEC ?)respectively,to investigate whether Notchl/Hesl signaling pathway regulates the expression of C/EBPa,affecting the proliferation and differentiation of AEC ? under normal and hyperoxia conditionsMethodsHuman AEC ? were cultured in vitro,and randomly divided into control group,activator group and inhibitor group.The control group was added with an equal volume of PBS buffer,the activation group was added with Notch pathway activator Jaggedl protein 500 ng/ml,and the inhibitor group was added with Notch pathway inhibitor DAPT 10?mol/L.A model of hyperoxia exposure to cell injury was established and AEC? were randomly divided into air group,air activator group,hyperoxia group and hyperoxia activator group.The air group was added with an equal volume of PBS buffer,the air activator group was added with Notch pathway activator Jaggedl protein 500 ng/ml,the hyperoxia group was added with an equal volume of PBS buffer,added with 95%O2 and 5%CO2 mixture gas at the speed 3 L/minute for 10 minutes,and hyperoxia activator group was added with Notch pathway activator Jaggedl protein 500 ng/ml and added at 30 minutes before oxygen administration.After successful modeling,they were placed in a 37?,50 ml/L CO2 incubator,and each group of cells was harvested 24 hours after successful modeling The expressions of Notchl,Hesl and C/EBPa at mRNA and protein levels were determined by RT-qPCR and Western blot.The proliferation of AEC ? was measured by CCK-8 assay and living cells counting.Flow cytometry was used to detect the differentiation of AEC ? by Type I alveolar epithelial cell(AECI)specific surface marker protein aquaporin 5(AQP5).Results(1)Compared with the control group,the mRNA and protein expressions of Notch1,Hes1 and C/EBPa in the activator group were significantly increased(P<0.05),the CCK-8 value and the viable cell count were increased,the AQP5 positive cell ratio was decreased,the proliferation of AEC II was increased,and the differentiation was decreased(P<0.05);the mRNA and protein expressions of Notchl,Hes1 and C/EBP? in the inhibitor group were significantly decreased(P<0.05),the CCK-8 value and viable cell count were decreased,the AQP5 positive cell ratio was increased,the proliferation of AEC II was decreased,and the differentiation was increased(P<0.05).(2)Compared with the air group,the mRNA and protein expressions of Notchl,Hes1 and C/EBPa in the air activator group were increased significantly(P<0.05),CCK-8 value and viable cell count were increased,AQP5 positive cell ratio was decreased,the proliferation of AEC ? was increased,and the differentiation was decreased(P<0.05);the mRNA and protein expressions of Notchl,Hesl and C/EBPa were significantly decreased in hyperoxia group and hyperoxia activator group(P<0.05),CCK-8 value and viable cell count were decreased,the AQP5 positive cell ratio was increased,,the proliferation of AEC ? was decreased and the differentiation was increased(P<0.05).Compared with the hyperoxia group,the mRNA and protein expressions of Notchl,Hes1 and C/EBPa were increased in the hyperoxia activator group(P<0.05),CCK-8 value and viable cell count were increased,the AQP5 positive cell ratio was decreased,the proliferation of AEC ? was increased and differentiation was decreased(P<0.05).ConclusionNotch1/Hes1 signal regulates the expression of C/EBPa and affects the proliferation and differentiation of AEC ?.Activating Notchl/Hesl signal can up-regulate the expression of C/EBPa and regulate the proliferation and differentiation of AEC ? under hyperoxia.