Effect And Mechanism Of MTOR Signaling Pathway On Hyperoxia-induced Lung Injury In Juvenile SD Rats

BackgroundOxygen therapy,as one of the most common treatment methods in clinical work,can effectively improve hypoxia symptoms,stabilize and maintain the normal function of organs and tissues.In some cases,in order to save the life of critically ill patients and maintain the oxygen saturation required for life,long-term and high-concentration oxygen therapy is inevitable.However,oxygen therapy with high concentration for a long period of time often causes damage to tissues and organs,especially for babies born,except retina,lungs that are most in direct contact with oxygen are the most affected organs.The degree of injury is related to the concentration,duration and external air pressure of inhaled oxygen.Such injury is usually irreversible pathological change.Our previous studies have shown that continuous inhalation of 40%oxygen for more than 24hours can cause irreversible damage to the lungs.Especially in children with mechanical ventilation,the performance is more obvious.Early pathological manifestations of hyperoxia-induced lung injury are increased capillary permeability,inflammatory exudation of lung tissue and pulmonary interstitial edema.Late manifestations are irreversible pulmonary interstitial fibrosis and obstruction of alveolar process,which are the most important risk factors and final pathological features,and are also the focus and difficulty of research.At present,there is no definite and effective treatment for hyperoxia-induced lung injury and fibrosis at home and abroad.The existing alveolar surfactant,hormone therapy and inflammatory mediator antagonism are still under exploration with little effect.This requires us to further study its pathology,pathogenesis and prevention measures.Abnormal repair process of hyperoxia-induced lung injury will eventually lead to lung fibrosis.The important pathological feature of lung fibrosis is the formation of fibroblast foci in lung tissue,which is directly related to abnormal proliferation and transformation of lung fibroblasts.The transformation of lung fibroblasts into myofibroblasts is considered to be one of the important sources of lung myofibroblasts during pulmonary fibrosis.Since the mammalian target protein of rapamycin（mTOR）signaling pathway is involved in the regulation of proliferation and transformation of Cell cycle and cells,and mTOR inhibitors can effectively exert anti-proliferation effect,we hypothesized that mTOR inhibitors negatively regulate abnormal proliferation and transformation of fibroblasts to block the process of hyperoxia-induced lung injury fibrosis,thus improving the quality of life and prognosis of hyperoxia-induced lung injury patients.Objective1.Three-week old SD rats were used as the research object to replicate the animal model of hyperoxia-induced lung injury,and the expression of mTOR signaling pathway was observed.2.To investigate the inhibitory effects of the mTORC1 inhibitor Rapamycin and the mTORC1/2 dual inhibitor OSI-027 on lung injury and fibrosis induced by high volume oxygen（hyperoxia）in juvenile SD rats.3.To investigate the effect and significance of inhibiting mammalian target of rapamycin（mTOR）signaling pathway on p-AKT1 in lung injury induced by hyperoxygen in the juvenile SD rats.Methods72 three-week-old juvenile SD rats were randomly divided into air+physiological saline,hyperoxia+saline,hyperoxia+rapamycin and hyperoxia+OSI-027 groups.Animal models were established（n=18 for each group）.Hyperoxia intervention was performed with 90%oxygen,saline,rapamycin,and OSI-027 interventions were administered intraperitoneally on days 1,3,6,8,10,and 13 of the observation period,respectively.On days3,7 and 14,weight changes,wet/dry ratio（W/D）,lung histopathological examination,lung injury scores,alveolar septal thickness measurements,lung tissue immunity were measured in groups of rats.The distribution and expression of mTOR and phosphorylated S6K1 and AKT1 protein in lung tissues were detected by Western blot.Results1.From the time factor,the body weight of each group of juvenile rats(F_time=297.098,P=0.000),immunohistochemistry of mTOR(F_time=379.978,P=0.000),mTOR(F_time=166.991,P=0.000)and pS6K1(F_time=122.676,P=0.000)protein levels increased with time,Except the air group,the other groups had lung injury scores(F_time=1410.362,P=0.000),alveolar septum thickness(F_time=356.312,P=0.000),and pS6K1 immunohistochemistry(F_time=57.992,P=0.000)increased with time,and the lung W/D(F_time=28.915,P=0.000)increased on 3 and 7 d,and decreased on the 14th.From the grouping factor,body weight(F_group=176.597,P=0.000)was significantly higher in the air group than in the other groups,lung W/D(F_group=28.484,P=0.000)and alveolar septum thickness(F_group=296.223,P=0.000).The air group is significantly lower than the other groups.Except for the third day,mTOR immunohistochemistry(F_group=134.100,P=0.000)was significantly higher in the hyperoxia group than in the other groups,PS6K1immunohistochemistry(F_group=234.697,P=0.000),mTOR(F_group=59.377,P=0.000)and PS6K1(F_group=101.837,P=0.000).The Western blot hyperoxia group was significantly higher than the other groups,and the lung injury score(F_group=2420.76,P=0.000)was significantly higher in the high oxygen rapa group than in the other groups.The hyperoxia OSI-027 group was significantly lower than the hyperoxia group and the hyperoxia rapa group.2.Compared with air group,the body weight of the rats in hyperoxia group was significantly decreased（P<0.05）,the lung W/D was increased in the acute phase of lung injury（P<0.05）,and the alveolar septal width and lung injury scores were significantly increased（P<0.05）.The pS6K1 positive cells in the lung tissues were increased（P<0.05）,p-AKT1 positive cells were decreased（P<0.05）,pS6K1 protein was increased significantly（P<0.001）,and p-AKT1 protein was decreased significantly（P<0.001）.Compared with hyperoxia group,the lung tissue injury in hyperoxic+OSI-027 group was alleviated（P<0.05）,pS6K1 positive cells in the lung tissues was decreased（P<0.05）,p-AKT1 positive cells was increased（P<0.05）,and pS6K1 protein level was significantly decreased（P<0.05）,p-AKT1 protein level was increased（P<0.05）.Hyperoxia+rapamycin further aggravated lung injury（P<0.05）,pS6K1 positive cells decreased（P<0.05）,p-AKT1 positive cells increased（P<0.05）,pS6K1 protein levels decreased significantly（P<0.05）,and p-AKT1 protein levels increased significantly（P<0.05）.Compared with hyperoxia+rapamycin group,the lung tissue damage was alleviated in hyperoxic+OSI-027 group（P<0.05）,p-AKT1 positive cells in the lung tissues were decreased（P<0.05）,and p-AKT1 protein level was decreased（P<0.05）.Conclusion1.High concentration of oxygen can activate mTOR signaling pathway in lung tissue;mTOR may promote the occurrence and development of hyperoxia-induced pulmonary fibrosis,and its regulation mechanism may be related to the inhibition of the activation of mTOR signaling pathway.2.p-AKT1 may be involved in the development of hyperoxia-induced lung injury,and its regulation mechanism may be related to the mTOR signaling pathway.In hyperoxia-induced lung injury,the protein level of p-AKT1 is decreased,and mTOR inhibitors increases the p-AKT1 protein.3.The mTORC1 inhibitor Rapamycin aggravated the lung injury and fibrosis induced by 90%oxygen in juvenile SD rats.The mTORC1/2 dual inhibitor OSI-027 alleviated the effect of hyperoxia-induced lung injury and fibrosis induced by 90%oxygen in juvenile SD rats.