| Objective: At present,BPD is believed to be the result of multiple factors,and long-term inhalation of high concentration oxygen,premature delivery,intrauterine infection,etc.are the main inducing factors of BPD.The main mechanism of high concentration oxygen damage to premature infants is the damage of oxygen radicals to the body.The imbalance of oxidation/antioxidation will lead to excessive production of reactive oxygen species and insufficient scavenging capacity in children.At the same time,it will cause imbalance of protein and cytokine in the body,aggravate inflammatory reaction and lead to lung injury.Animal experiments suggest that hyperoxia is one of the factors causing BPD as well.In addition,studies have shown that the level of IL-1β and TNF-α in bronchoalveolar lavage fluid of children with BPD have increased significantly.IL-1β And TNF-α are the earliest cytokines appearing after infection or trauma,and the classic proinflammatory factors as well.Domestic and foreign scholars generally believe that IL-1β and TNF-α in BALF should be administered early for premature infants,because of its high or low concentration can be used as one of the early prediction and diagnosis means of BPD.In this study,we analyzed the changes of gene expression profiles in neonatal rat lung tissues before and after hyperoxia treatment by using three-generation transcriptome sequencing technology,and obtained corresponding differentially expressed genes.Through GO function cluster analysis of these differential genes,it was found that the differential genes in the lung cell were enriched in the gene subset related to inflammatory reaction under hyperoxia stimulation.Further KEGG analysis showed that it was related to AKT signal activation.Using String database,PPI analysis was performed on these differential genes to screen out candidate factors –PLGF(placental growth factor).This study simulated bronchopulmonary dysplasia by establishing a 90% hyperoxic lung injury model in neonatal rats,and injected PLGF lentivirus interference plasmid into rats with hyperoxia-induced lung injury to observe the effect of PLGF gene silencing on inflammatory reaction and AKT protein expression in lung tissue of neonatal rats under hyperoxia.Methods:1.The transcriptome sequencing technology was used to analyze the changes of gene expression profile in lung tissues of neonatal rats before and after hyperoxia treatment.2.The rats of cesarean section on the 21 st day of pregnancy were randomly divided into: 1)normal control group,2)hyperoxia model group,3)control lentivirus group under hyperoxia treatment,4)lentivirus-mediated PLGF group under hyperoxia treatment。3.A 90% hyperoxia rat model was established to simulate hyperoxia-induced lung injury,and the PLGF gene was silenced by injecting lentivirus interference plasmid into the tail vein for 3 days in a row。4.HE staining was used to observe the pathological changes of lung tissue in rats with hyperoxia lung injury silenced by PLGF gene。5.Immunohistochemistry was used to detect the expression of PLGF in lung tissue of rats with hyperoxia lung injury after PLGF gene silencing.6.Real-time PCR was used to detect the expression of inflammatory factor TNF-α、 IL-1β、 IL-6 expressionin lung tissue of rats with hyperoxia-induced lung injury silenced by PLGF gene.7.Western blot was used to detect the expression of PLGF,AKT/p-AKT protein in lung tissue of rats with hyperoxia-induced lung injury silenced by PLGF gene.Results:1.Three generation transcriptome sequencing technology was used to analyze the changes of gene expression profile in neonatal rat lung tissues before and after hyperoxia treatment,and the factors related to hyperoxia lung injury,placental growth factor(PLGF),were screened.2.HE staining results showed that,compared with the normal control group,the alveolar epithelial cells of the control model group and the control lentivirus group swelled,and a large amount of exudation appeared in the alveolar cavity,inflammatory cell infiltration,and tissue structure disorder were observed as well,while the inflammatory cell infiltration in the lentivirus-mediated PLGF interference group was significantly reduced,and the lung tissue structure was more clear.3.The immunohistochemical results of PLGF showed that no light brown stained positive cells were found in the normal control group,and a large number of light brown stained positive cells were found in the control model group and the control lentivirus group under hyperoxia,while the positive staining cells in the lentivirus-mediated PLGF interference group were significantly reduced.4.Western blot analysis and quantification results showed that compared with normoxia group,a significantly increasing expression of PLGF was detected in hyperoxia group and hyperoxia/shrna-nc group after PLGF gene silencing by lentivirus transfected sh RNA.The expression of PLGF in hyperoxia/shrna-PLGF group was remarkably decreased compared with hyperoxia/shrna-nc group.The expression of p-AKT demonstrated a significant increase in hyperoxia group and hyperoxia/shrna-nc group compared with normoxia group,and exhibited the opposite way in hyperoxia/shrna-PLGF group.No significant changes can be seen in the content of AKT with the viariation of intervening measures.5.The results of Real-time PCR analysis showed that compared with the normal control group,the inflammatory related factors,including TNF-α,IL-1β IL-6 was significantly increased,but after the intervention of the expression level of PLGF in lung tissue,the expression level of the above inflammatory factors was significantly decreased.Conclusions: Inhibiting the expression level of PLGF in lung tissue can inhibit the inflammatory reaction in lung tissue,mainly by inhibiting the expression of inflammatory cytokine TNF-α,IL-1β and IL-6,which can play a certain role in the intervention of hyperoxia-induced lung injury.The possible mechanism is to inhibit the inflammatory reaction of lung tissue by inhibiting the level of AKT phosphorylation and therefore alleviate hyperoxia-induced lung injury. |
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