| Objective:Survival rates have improved significantly in recent years,in premature babies at the same time,bronchopulmonary dysplasia incidence is also on the rise.By establishing animal models and grouping experiments,this study observed and detected lung development in rats,and studied the effect of intermittent hyperxia or hypoxia on lung development of preterm rats under low-concentration oxygen treatment and the protective effect of Tempol from the aspects of morphology and function as well as from the molecular biological level of HIF-1? and VEGF.Methods:Sprague-Dawley rats with gestation of 15 days were selected to establish an animal model.(1)S:ix preterm rat neonates in each group were anesthetized with intraperitoneal injection of pentobarbital(50ml/kg,Sigma,USA)and exsanguinated by severing the femoral aorta.The lung tissue was exposed and the trachea and lung were isolated.The whole lung was removed and weighted after removing the non-lung tissue gently.Lung coefficient = wet lung weight(mg)/body weight(g).(2)The number of alveoli at the vertical line from the vertical bronchial center to the nearest fibrous or pleural was observed in the HE staining slices under lowmagnification.Each slice was counted for 10 times and the average number was defined as the RAC value,an index to reflect the lung development.(3)As an indicator of protein oxidation and lipid peroxidation,MDA in homogenized lung tissues was detected using the MDA assay kit according to the user manual.(4)The TAOC of lung homogenates was detected using the ferric reducing antioxidant power(FRAP)kit according to the user manual.(5)Immunohistochemical staining of lung tissues was performed to detect the expression of hif-1a and VEGF proteins in rats in each group.Quantitative real-time PCR(qPCR)was used to detect the mRNA expression of hif-1 and VEGF in lung tissues.(6)Intubation was performed through the opened airway connecting to a BuxCo small animal lung function experimental platform.(7)Western blot was used to detect the expression of HIF-1 a and VEGF proteins under intermittent hyperxia or hypoxia and continuous oxygen supply.(8)Hematoxylin and eosin(HE)staining was used to observe the lung tissue,and the morphological differences between groups with and without tempol injection were compared.(9)Radiated alveoli were counted in HE staining sections under low power microscope,and the number of radiated alveoli was compared between groups with and without tempol injection.Results:(1)At day 7,14 and 21,body weight in constant oxygen group is heavier than that in the air control group,intermittent hyperoxia group,and hypoxia groups.The body weight of premature rats in the intermittent hyperoxia and hypoxia group was significantly lighter than that in the control and constant oxygen group on day 14 and day 21 after birth.(2)On histological examination,adverse lung histopathology occurred in intermittent hyperoxia and hypoxia groups,including a slight pulmonary epithelial thickening and less branched septi and alveoli.(3)In this study,the lung coefficient index in intermittent hyperoxia and hypoxia premature rats was significantly higher than that in the air control group from day 7 to day 21 after birth.Moreover,the index of lung coefficient was significantly lower in constant oxygen rats compared with control group on day 14 and day 21 after birth.There was no significant difference in lung coefficient between intermittent hyperoxia and hypoxia group from day 7 to day 21.The number of RAC was significantly less in the intermittent hyperoxia and hypoxia group than that in the control and constant oxygen group from day 14 to day 21.(4)The TV,MV,PIIF,and Cdyn in rats with intermittent hyperoxia and hypoxia were significantly lower than those in control preterm rat neonates and rats received constant oxygen therapy.In contrast,constant oxygen therapy significantly increased TV,MV,PIF,and Cdyn compared to control preterm rat neonates.(5)The TAOC in lung tissue of preterm rat neonates in intermittent hyperoxia and hypoxia group was significantly decreased compared to the air control and constant oxygen group from day 14 to day 21.(6)lung tissue MDA in the intermittent hyperoxia and hypoxia groups was significantly higher from day 7 to day 21 compared to the control and constant oxygen group.There was no significant difference in lung tissue TAOC and MDA concentrations between air control and constant oxygen group.(7)HIF-la mRNA expression showed a decreasing trend in the constant oxygen and intermittent hyperoxia groups while increased in intermittent hyperoxia groups compared to the air control group at day 14 and 21.Similarly,at each age point,HIF-la protein levels in constant oxygen and intermittent hyperoxia groups were decreased.Significant upregulation was observed in the hypoxia group compared to control group.The levels of HIF-la protein in the constant oxygen and intermittent hyperoxia groups were significantly lower than those in the air control group,and the intermittent hyperoxia group was lower than the constant oxygen group.From day 7 to day 21 after birth,VEGF mRNA levels were increased in the intermittent hypoxic group while decreased in intermittent hyperoxia groups.Similarly,the VEGF protein expression significantly increased in intermittent hyperoxia groups and decreased and intermittent hyperoxia groups at day 7,day 14 and day 21 after birth.While no significant difference between air control and constant oxygen group.(8)The lung coefficient index of the two groups of Tempol preterm infants from day 14 to day 21 after birth was significantly lower than that of intermittent hyperoxic and intermittent hypoxia preterm infants.(9)At 21 days of birth,tidal volume,respiratory ventilation per minute,PIF,and Cdyn in the groups injected with tempol were significantly higher than those in the intermittent hyperoxic and hypoxic groups injected with normal saline.At 21 days of birth,TV,MV,PIF,and Cdyn in the two groups injected with tempol were significantly higher than those in the intermittent hyperoxic and hypoxic groups injected with normal saline.(10)At 14 to 21 days,TAOC in lung tissue was significantly higher in intermittent hyperoxic injection of tempol group and intermittent hypoxic injection of tempol group than in intermittent hyperoxic and hypoxic groups · MDA concentrations in the two groups injected with tempol were significantly lower than those in the intermittent hyperoxic and hypoxic groups.Conclusion:Premature rats with intermittent hyperoxia or hypoxic stimulation during low concentration oxygen therapy may result in the decrease of total antioxidant capacity and lung function,and showe poor pathological morphology of lung tissue.Intermittent hypoxia during low concentration oxygen therapy can also lead to over-expression of HIF-la/VEGF in lung tissue and affect lung development.Tempol can restore to a certain extent the total antioxidant capacity of premature rats reduced under intermittent hyperoxia and hypoxia during low concentration oxygen therapy,and improve their poor lung histopathology and lung function. |
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