| Objective: Bronchopulmonary dysplasia(BPD)is a chronic lung disease of premature infants characterized by delayed alveolar development and capillary dysplasia.High concentration oxygen therapy is the main pathogenic factor.There have been reports of changes in pulmonary vascular permeability in BPD,however,its specific mechanism has not been fully clarified.Twist1 can control pulmonary vascular permeability through Ang-Tie2 system,but whether it is involved in the regulation of BPD vascular permeability is unclear.Therefore,the purpose of this research is to observe the expression of Twist1 and Ang-Tie signaling pathway related molecules in hyperoxia-induced acute lung injury of the newborn rats,and to explore the relationship and mechanism between Twist1 signaling pathway and the change of pulmonary microvascular permeability in BPD.Methods: The hyperoxia-induced acute lung injury model of the newborn SD rats was established.The newborn SD rats were randomly divided into hyperoxia group and air group within 12 hours after birth.The high oxygen group was placed in a closed oxygen tank,and 2L/min of oxygen was continuously introduced to maintain the oxygen concentration at 85~90%.Sodium lime was used to absorb excessive carbon dioxide.The air group was placed in the same room air,and the oxygen concentration was about 21%.Exchange the female rats of the two groups,change the litter and feed,and observed and recorded the general growth and development of the newborn SD rats every day.The newborn SD rats of the two groups were killed on the first day,the seventh day and the fourteenth day after birth respectively.The lung tissues were collected and lung weight was recorded.The lung/body weight ratio was used to reflect the degree of lung injury.The lung tissue sections were stained with HE to observe the pathological changes of the lung tissues.The immunofluorescence technology was used to detect the expression of CD31,Twist1,Tie1,and Tie2 in lung tissues.Lung microvascular permeability was evaluated by Evans blue staining.The mRNA levels of Twist1,Tie1,Tie2,Ang1 and Ang2 were detected by real-time fluorescence quantitative PCR(qRT-PCR).The protein levels of the Twist1,Tie2,pTie2,pAkt,Akt,pFoxo1 and Foxo1 were measured by western blot.Results:(1)General growth and development: the newborn SD rats in the hyperoxia group had the symptoms of hair lusterless,small size,head shaking,gait instability since the 7th day of oxygen exposure,while the growth and development of the newborn SD rats in the air group was good.With the extension of the hyperoxia time,the survival rate of newborn rats in the hyperoxia group was significantly lower than that in the air group.(2)Body weight and lung weight/body weight ratio(LW/BW): with the prolongation of the hyperoxia exposure time,the body weight of the hyperoxia group was significantly lower than that of the air group on the 7th and 14 th day after birth.The LW/BW of the hyperoxia group was significantly lower than that of the air group on the 7th day while significantly higher than that of the air group on the 14 th day.(3)The pathological manifestations of the lung tissues showed that the enlarged alveolar cavity,thinned alveolar wall,simplified alveolar structure appeared in the hyperoxia group since the 7th day after birth,and the inflammatory cells and red blood cells exuded in the alveolar cavity.The mean linear intercept(MLI)of the alveolar development indicators showed that the MLI value of the newborn SD rats in the hyperoxia group was significantly increased compared with the air group on the 7th day and 14 th day.(4)Immunofluorescence of the lung tissues showed that the expression levels of the Twist1,Tie1,and Tie2 in CD31-positive vascular endothelial cells of the hyperoxia group was lower than that of the air group on the 7th and 14 th day after birth.(5)The Evans blue staining of the lungs showed that the lung staining of the hyperoxia group was darker than that of the air group,and the absorbance of Evans blue was significantly higher than that of the air group on the 7th and 14 th day after birth.(6)The qRT-PCR showed that the level of Twist1 mRNA in the lung tissues of the hyperoxia group was significantly lower than that in the air group on the 14 th day.The level of Tie1,Tie2,and Ang1 mRNA in the lung tissues of the hyperoxia group were significantly lower than those in the air group,while the level of Ang2 mRNA was significantly higher than that in the air group on the 7th and 14 th day.(7)Western blot showed that the Twist1 protein level in lung tissues of the hyperoxia group was significantly lower than that in the air group on the 14 th day.The protein level of Tie1,Tie2,and Ang1 in the lung tissues of the hyperoxia group were significantly lower than those in the air group,while the protein level of Ang2 was significantly higher than that in the air group on the 7th and 14 th day.Conclusion: Hyperoxia induced microvascular dysplasia and increased microvascular permeability of the lung tissue.The expression of Twist1 decreased,which may be involved in the increase of pulmonary vascular permeability induced by hyperoxia through down-regulating Ang-Tie-Akt-Foxo1 signaling pathway,and this lays an experimental foundation for early intervention of BPD. |
PDF Full Text Request