| Objective:1.To establishing a neonatal rats model of hyperoxia-induced lung injury;2.To observe the general condition and the pathologic changes in lung tissue of rats in each group and the levels of protein and mRNA of AQP1,α-ENaC,SP-A in the pathogenesis of acute hyperoxia-induced lung injury;3.To investigate whether furosemide Inhalation could protect against hyperoxia-induced lung injury and the changes of AQP1,a-ENaC,SP-A expression.And providing information for the prevention and treatment of hyperoxic lung injury in neonates.Methods:One hundred and ten neonatal rats that the age less than6hours after birth were randomly assigned to three groups:Group I:control group(n=37);Group Ⅱ:hyperoxia group(hyperoxia+NS,n=37);Group Ⅲ:furosemide group(hyperoxia+furosemide,n=36);The rats of Group Ⅰ were exposed to normoxia(room air) and the rats of group and Ⅲ were exposed to hyperoxia (more than95%oxygen)in a specially constructed boxes.The rats in the group Ⅲ were inhalation of furosemide (lmg/kg), once the next day,a total of seven times from the beginning of hyperoxia exposure.The rats in the group Ⅱ were inhalation with the same volume of normal saline at the same time.Each group was divided into3subgroups,the3day,7day and14day.Twelve rats from each subgroup were randomly selected, and killed to measure the following indices:(1).The total protein concentration in BALF;(2).Lung wet-to-dry ratio:The left lung were isolated and weighed,then the dried lungs were weighed,and calculated the lung wet/dry ratio;(3).Lung histology:Lung sections from the right lung were stained with HE,and examined for the histological changes;(4).The protein and mRNA expression of AQP1,α-ENaC,SP-A:Western blotting and RT-PCR were used to detect the protein and mRNA expression of AQP1, a-ENaC,SP-A;Results:1.The general condition of the rats in each group:Control group, the growth of rats was in normal,and no death. After three days of hyperoxia group,the respiratory rate of rats accelerated without hyperoxia.After seven days,the experimental animals’spirit was dispirited,showed dyspnoea,cyanotic without hyperoxia,After fourteen days,the rats appears dull, poor mental reactions,and irritability, dyspnea obviously, lip cyanosis without hyperoxia, individual rats died in the absence of hyperoxia.The general condition of rats in furosemide group compared with hyperoxia group was improved.2. The pathological morphology of lung tissue:No pathological change were found in the lung of rats in normoxic controls,the alveolar contains no inflammatory cells and exudation. The hyperoxia group, at3th day, vascular congestion, interstitial edema, inflammatory infiltrates were found. At7th day, pulmonary edema, structural disorder alveolar hemorrhage, infiltration of inflammatory cells were significantly increased, widening the interval deformation of lung, the lung tissue structural disorder.At14th day, the changes were more obvious, the structure of lung tissue was more disorder, the pulmonary septal area were wider,significant fibrosis,the numbers of alveoli reduced evidently.The exudation,cell infiltration in alveolar cavity were obviously decreased in furosemide group compared with hyperoxia group.3.The lung wet/dry weight ratio (W/D):at3th day,7th day,14th day, the lung W/D ratio was higher in the hyperoxia groups than the control group (P<0.05), and the lung W/D ration in the furosemide group was lower than that in the hyperoxia group (P<0.05).4.The total protein concentration in BALF:At3th day,7th day,14th day,,the TP concentration in the BLAF of rats exposed to hyperoxia was significantly higher than that in the normoxia control group(p<0.05),and the TP of BALF in the furosemide group was lower than that in the hyperoxia group(P<0.05).5.The expression of AQP1, a-ENaC, SP-A protein in lung tissue:(1). The expression level of AQP1protein in the hyperoxia group and furosemide group were increased by the time.At3th day,7th day,AQP1protein expression in the hyperoxia group was significantly decreased compared with that in the control group(P<0.01). At14th day,the expression level in the hyperoxia group was increased but still less than the control group.The level of AQP1protein expression in furosemide group was higher than that in hyperoxia group, with statistical significance (P<0.05).(2). The expression level of a-ENaC protein in each group were increased by the time.At3th days,7th days,14th day,a-ENaC protein expression in the hyperoxia group was decreased compared with that in the control group (P<0.05). The level of a-ENaC protein expression in furosemide group was higher than that in the hyperoxia group (P<0.05).(3). The expression level of SP-A protein in each were increased by the time.At3th day, the expression level of SP-A protein in the hyperoxia group is higher than that in the control group(P<0.05), and on the7th and14th day the expression level is significantly lower than that in the control group (P<0.05). On the3th day, the expression level of SP-A protein in furosemide group is between the hyperoxia group and the control group (P<0.05), and on the7th and14th day the expression level is significantly increased comparing with the control group (P<0.05).6. The expression of AQP1, a-ENaC, SP-A mRNA in lung tissue:(1). At3th day,7th day,14th day,AQP0mRNA and,a-ENaC mRNA expression in the hyperoxia group were significantly decreased compared with that in the control group(P<0.05). The level of AQP1mRNA and a-ENaC protein expression in furosemide group was higher than that in hyperoxia group, with statistical significance (P<0.05).Correlation analysis on the expression level between AQP1mRNA and a-ENaC protein suggests a positive correlation and the linear correlation coefficient was0.451(p<0.05).(2).At the3th day, the expression level of SP-A mRNA in the hyperoxia group is higher than that in the control group(P<0.05), and at the7th and14th day the expression level is significantly lower than that in the control group (P<0.05). On the3th day, the expression level of SP-A mRNA in furosemide group was between the level of the hyperoxia group and the control group (P<0.05), and on the7th and14th day the expression level is significantly increased comparing with that in the control group (P<0.05).Conclusions:1. Exposed to hyperoxygen will cause lung injury in rats, and the injury will increase with the extension of hyperoxia time.The main histopathological of lung tissue are local hyperemia, hemorrhage, inflammatory exudate, structure disorder,development disturbance, decrease in the number of alveolar and interstitial pulmonary fibrosis.2.The expression of AQP1and a-ENaC in the lung of rats with hyperoxia-induced lung injury decreased significantly compared with those in room air,that coincided with the degrees of pulmonary edema.The results suggests that the changes in the expression level of AQP1and a-ENaC may be responsible for pulmonary edema in the rats with acute hyperoxia-induced lung injury.3.The expression level of SP-A increased firstly and then decreased. The level is parallel with the degree of lung injury. These findings suggest that the decrease or dysfunction of pulmonary surfactant protein is an important factor.4. Inhaled furosemide may inhibit sodium-potassium chloride channel, relaxing blood vessels, reduce inflammation, and thus increase the expression levels of AQP1, aENaC, SP-A,so that Inhaled furosemide can improve lung function, and play a protective role in hyperoxia-induced lung injury.. |
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