The Role And Regulation Mechanism Of Autophagy In Bronchopulmonary Dysplasia Of Neonatal Rats

Objective: Bronchopulmonary dysplasia(BPD)is one of the most common complications of respiratory system in preterm infants,especially those with very low birth weight and those with very low birth weight.As the survival rate of premature infants improves,the occurrence of BPD The rate also increased year by year.Children with BPD are often accompanied by a series of cardiovascular and neurological complications due to pulmonary developmental disorders that may persist into adulthood.So far,there is no safe and effective way to treat BPD.Therefore,to clarify the pathogenesis of BPD and explore new treatments has become the focus of neonatal work.Type II alveolar epithelial cells(AT-II)are the key target cells of BPD lung epithelial injury.The study found that the mechanism of injury involves membrane lipid peroxidation,proliferation inhibition,excessive apoptosis and so on.Apoptosis of AT-II is considered to be a key link in alveolar dysplasia of BPD.However,it remains unclear whether there are other regulatory mechanisms after apoptosis as a consequence of AT-II injury in BPD.Autophagy is a biological phenomenon that exists widely in mammalian nervous system,respiratory system,cardiovascular system and blood system.It is a common mechanism in the process of development and aging to maintain the stability of intracellular environment.However,autophagy Abnormalities can also affect the process of apoptosis or death.In order to confirm whether the autophagy process is impaired in BPD and where the target of the injury is,we tested the markers of autophagy and other proteins involved in the autophagy pathway.To confirm the role of autophagy in the development of BPD,We administered BPD model autophagy to drugs or autophagy inhibitory drugs to observe the alveolar development and apoptosis levels;we also established a hyperoxic exposure cell model to observe changes in cell autophagy,and the application of autophagy intervention Drug,to clarify the role of autophagy on oxidative stress cell injury;further,we look for possible changes in the level of autophagy caused BPD key proteins,to explore the new mechanism of BPD and found a new experimental basis for the treatment of BPD.Methods: One hundred newborn Sprague-Dawley(SD)rats were randomly divided into model group(exposed to O2,Fi O2 0.8)and control group(exposed to air,Fi O2 0.21)according to our established method.At 1,3,7,14 and 21 days after the experiment,10 rabbits in each group were selected and the autophagosomes and apoptotic bodies were observed under transmission electron microscope.TUNEL staining was used to observe the apoptotic cells.LC3 B,P62,The expression and localization of LC1 B,Lamp1 and Stx17 were detected by immunofluorescence double staining.LC3 B and p180(alveolar type II epithelial cell marker)were co-localized and expressed.The m RNA expression of LC3 B,P62,Lamp1 and St17 were detected by real-P62,lamp1,caspase3-cleaved and Stx17 protein expression levels.The other 140 newborn SD rats were divided into three groups,the newborn rats were treated with autophagy intervention,each group of 10 mice at each dose.A group of BPD model rats were treated with rapamycin at doses of 2.5mg / kg,5mg / kg,10 mg / kg and 20 mg / kg once every other day.The second group was given lithium chloride(BPD)kg,50 / kg,100 mg / kg and 200 mg / kg once a day respectively.The third group was given chloroquine to normal neonatal rats at doses of 1 / kg,10 / kg and 20 mg / kg once daily.Each group were given placebo group,usage are intraperitoneal injection,a total of two weeks.Lung tissue samples were collected to observe the development of lung tissue and the levels of autophagy-related proteins and their m RNAs.80 newborn SD rats were selected again.After birth,BPD model group and control group were established.After 3 and 7 days,20 rats in each group were separated and purified according to the past adherent selection method of our group,Cultured AT-Ⅱ cells,and LC3 B,P62,Lamp1 protein and m RNA detection.In addition,the establishment of hyperoxic exposure cell model,the AT-Ⅱ primary cells were cultured in Fi O2 0.8 incubator,respectively,after 0h,6h,12 h,24h,48 hours after the cells were collected,and the application of autophagy intervention drugs,application of MTT.The survival of cells was detected.Western-blot was used to detect the levels of autophagy and apoptosis-related proteins.Results:Apoptotic bodies and autophagosomes of AT-Ⅱ cells in lung of newborns with BPD were observed.Under transmission electron microscope(TEM),apoptotic cells increased in the model group at 7 days,and found that apoptotic bodies and autophagy coexisted in the same AT-Ⅱ cells.Further observation,a large number of autophagosomes were observed in the model group 7d,significantly increased compared with the control group.Changes of lung tissue protein and m RNA levels in neonatal rats with BPD in vivo.Western-blot results showed that LC3B-II protein peaked on the 7th day in the model group,which was significantly higher than that in the control group.The level of LC3B-II protein decreased in the first 14 days and lowest in the 21 st day,lower than that in the control group.The protein expression of P62 in model group was the highest at 7 days,which was significantly higher than that in control group,and the expression of P62 in model group persisted.The expression of Lamp1 protein gradually increased with the prolongation of hyperoxia exposure,which was the highest at 7 days,significantly higher than that of the control group and maintained at a high level afterwards.Immunohistochemistry showed that almost all the cells in lung tissue were expressed,and the staining of cytoplasm was the main.LC3 B staining showed that the number of positive cells in model group increased more than that in control group at 7 days,and there was an increase of dot-like aggregates.Immunohistochemical staining of p62 and Lamp1 showed that in the 7-day model group,the positive staining was significantly increased compared with the control group,and cytoplasm staining.LC3 B and p180 double immunofluorescence staining shows that LC3 B and p180 co-localized in AT-Ⅱ cells,and 7 days model group double staining positive cells increased significantly compared with the control group.Rrealtime-PCR results showed that there was no significant difference in m RNA expression of LC3 B,p62 and Lamp1 between the model group and the control group.The changes of autophagy-related protein and m RNA in primary lung cancer AT-Ⅱ cells of BPD neonatal rats.Western-blot results showed that the changes of primary lung AT-II apoptosis and autophagy in BPD neonatal rats were similar to those of BPD model lung tissues.The expression of LC3B-II and p62 protein peaked on the 7th day,which was significantly higher than that of the control group.Lamp1 protein expression was also not highest in the 7th day,which was significantly higher than that of the control group.Three kinds of protein m RNA expression no significant statistical difference.4,in vivo experiments using autophagy intervention drug BPD neonatal rat lung tissue autophagy protein levels.The results of Western-blot showed that after RAPA or lithium chloride treatment,the value of RAC was increased,the thickness of the alveolar septum was decreased,and the expression of p62,LC3B-Ⅱ and caspase-3-cleaved was decreased in the newborn rats of BPD.Normal newborn rats with chloroquine,BPD showed similar performance,RAC decreased alveolar septum thickening,Western-blot shows p62,LC3B-Ⅱ,Caspase-3-cleaved increased.5,in vitro experiments of primary AT-Ⅱ cells in autoxidation model of autoxidation protein levels.Western-blot results showed that LC3B-II and p62 gradually increased with hyperoxia exposure time,the most obvious at 12 h,gradually recovered at 24 h and 48 h,Stx17 at 6h and restored at 12 h.6,in vitro experiments using autophagy intervention drug primary AT-Ⅱ cell hyperoxia exposure model cell activity.The results of MTT assay showed that the survival rate of cells increased with autophagy promoter rapamycin and lithium chloride,and the survival rate of cells decreased with 3-MA and chloroquine.7,in vivo experimental BPD neonatal rat lung tissue Stx17 expression.Western-blot results showed that Stx17 protein expression began to decrease at 3 days and increased after 14 days in BPD.The results of Rrealtime-PCR showed that the expression of Stx17 m RNA in BPD group was significantly lower than that in control group at 3 days.In vitro experiments The expression of Stx17 in primary AT-II cells exposed to rapamycin after hyperoxia exposure was measured.Western-blot results showed that the expression of Stx17 increased with rapamycin 6 h after hyperoxia exposure.Conclusion: 1.During the early stage of rapid lung development in normal newborn rats,the levels of LC3B-II,p62 protein and gene expression increase synchronously,suggesting that the development of lung tissue of normal neonatal rats needs to maintain slightly higher levels of autophagy.2.In the early stage of BPD,although the levels of LC3B-II and p62 protein were abnormally increased,the corresponding m RNA was not increased,suggesting that the increase of autophagy-associated protein is caused by degradation disorders,and the obstruction of autophagy may be involved in the development of BPD.3.At the cellular level,it was verified that the increase of LC3B-II and p62 protein levels in AT-II cells at the time of BPD was not accompanied by an increase in the corresponding m RNA expression.The trend was similar to the tissue level,suggesting that autophagy impairment of AT-II cells exist during the development of BPD.4.After autophagy enhancer(rapamycin,lithium chloride)was applied,the level of autophagy was restored and the levels of LC3B-II and p62 protein were decreased.The results of alveolar development in BPD neonatal mice were significantly improved;after autophagy inhibitor(chloroquine)was used,the autophagic flow was blocked,and the LC3B-II and p62 proteins accumulated.The normal neonatal rats showed developmental dysplasia in the alveoli,and lung tissue changes similar to BPD were observed.5.At the tissue and cell level,it was verified that the decrease of Stx17 was closely related to the increase of LC3B-II and p62 protein levels in the autophagic process of BPD,suggesting that the impaired expression of Stx17 may be an important mechanism for the development of BPD.