Study On AECⅡ Autophagy And Apoptosis Regulated By MTOR In HALI

Objective:The PI3K/Akt/mTOR signaling pathway in HALI is involved in regulating AECⅡautophagy and apoptosis.However,the relationship between mTOR-regulated apoptosis and autophagy is unclear.The aim of this study was to up-regulate and down-regulate autophagy via mTOR target and to assess the changes in AECⅡapoptosis levels to explore the relationship between mTOR-regulated AECⅡautophagy and apoptosis in HALI.Methods:1.In vivo experiments:SD rats were randomly divided into 4 groups,isolated and purified after 0 h,24 h,48 h and 72 h in hyperoxide,TEM and SP-C identified type II alveolar epithelial cells,and performed WB to assess the change of autophagy level;HE and Tunel stained lung tissue were taken to assess the degree of lung injury and the change of lung cell apoptosis level,and the modeling time point was selected.2.In vitro experiments:H₂O₂treated MLE-12 for 2 h.The level of ROS change was assessed in flow and the effect of modeling was evaluated at a final concentration of 0.5mmol/l H₂O₂.CCK8 mapped CQ modeling concentration,time.WB assessed the change in autophagy protein level induced by CQ+H₂O₂treatment versus H₂O₂treatment of MLE-12 at different times and selected modeling time points.CCK8,WB mapped Rapa,MHY1485 modeling concentration,time.MLE-12 was divided into 5 groups:NC group,H₂O₂group,Rapa+H₂O₂group,MHY1485+H₂O₂group,DMSO+H₂O₂group.Autophagy double-labeled to assess the change of autophagy level,flow and WB to assess the change of autophagy and apoptosis level of MLE-12.3.In vivo experiments:SD rats were randomly divided into 5 groups:NC group,Hyperoxia group,Rapa+Hyperoxia group,MHY1485+Hyperoxia group,DMSO+Hyperoxia group,and the purified AECII was isolated for WB,and lung tissues were taken for HE and Tunel staining to assess the degree of lung injury and the level of apoptosis of lung cells.Results:1.Hyperoxia induces AECⅡautophagy,lung cell apoptosis and lung injury in ratsHyperoxia induced modeling at 0 h,24 h,48 h,and 72 h,respectively,and compared with 0 h,LC3 expression was highest at 24 h,^***P<0.001;Lower at 48 h than at 24 h,**P<0.01,^**P<0.01;the longer the hyperoxia exposure time,the higher the apoptosis rate of lung cells and the heavier the tissue damage;the apoptosis rate at 48 h compared with the control group,^**P<0.01,and HE staining lung tissue damage is significantly more severe,with lung The septal thickening,inflammatory cell infiltration and bullae formation were observed.2.H₂O₂can induce MLE-12 autophagy and apoptosis,and the mTOR inhibitor Rapa can further upregulate MLE-12 autophagy to aggravate apoptosis,and mTOR activator MHY1485 downregulate MLE-12 autophagy without significantly reducing apoptosisH₂O₂can induce MLE-12 autophagy,the highest at 8 h,12 h lower than 8 h,^***P<0.001;modeled after Rapa pretreatment,autophagy lysosomes are more than in the H₂O₂group,**P<0.01;LC3,cleaved-caspase3 relative expression is higher than that of H₂O₂group,*P<0.05;early apoptosis rate is higher than that of H₂O₂group,***P<0.001;MHY1485 pretreatment modeling,autophagosomes are modeled compared with DMSO+The H₂O₂group was less,*P<0.05;the relative expression of LC3 was lower than that of DMSO+H₂O₂group,and the relative expression of cleaved-caspase3 was lower than that of DMSO+H₂O₂group,but there was no significant difference,and the ^nsP>0.05.3.Under the condition of hyperoxia,rat intraperitoneal injection of mTOR inhibitor Rapa further upregulated rat AECⅡautophagy can aggravate apoptosis and lung damage,and mTOR activator MHY1485 downregulates AECⅡautophagy can alleviate apoptosis and lung damageHyperoxia modeling after intraperitoneal injection of Rapa in rats,the relative expression of LC3 was higher than that of hyperoxia group,^**P<0.01;cleaved-caspase3was higher than that of Hyperoxia group,^***P<0.001;AECⅡapoptosis rate was more serious than that of Hyperoxia group,*P<0.05;HE staining lung injury worsened;after intraperitoneal injection of MHY1485 in rats,the relative expression of LC3 was lower than that of Hyperoxia group,**P<0.01;the relative expression of cleaved-caspase3 and AECⅡapoptosis rate were lower than those in DMSO+Hyperoxia group,**P<0.01;HE staining lung injury was lighter,but not obvious.Conclusion:1.Hyperoxia can induce AECⅡautophagy and apoptosis in both in vivo and in vitro.2.Under hyperoxia,inhibition of mTOR and activation of autophagy can induce AECⅡapoptosis and lung injury;activation of mTOR and inhibition of autophagy can reduce AECⅡapoptosis and lung injury.