Changes Of Pulmonary SP-A And SP-B After Severe Blast Lung Injury In Adult And Infancy Rats

Objective:By means of establishing the severe lung blast injury models of adult and infancy SD rats,and observing the changes of SP-A and SP-B transcription and expression in their lungs after severe blast injury,the characteristic and relationship between the adult and infancy lung blast injuries,and the role and mechanism of SP-A and SP-B on the pathophysiology of blast lung injury were studied to explore and provide a new theoretical basis and clinical guidance for the blast lung injury medical cares.Methods:1.Establish the model of the same severe lung blast injury in the SD rats about adult and juvenile: to select 40 healthy adult(250±15)g and infancy(20-days old)male SD rats randomly and respectively,and through the BST-I type biological shock tube to set up the different driving pressure value,then the experimental animals were injured.After the different driving pressure injury,comparative analysis of general physiology and pathology,Lung Group SD rat anatomy,pathological classification of lung tissue and lung injury score,and then determined the best severe pulmonary injury model.Adult SD rats were randomly divided into 4 groups: 4.5Mpa,4.8Mpa,5.0Mpa and 5.5Mpa,each group of 10 rats;infancy SD rats were randomly divided into 4 groups: 4.5Mpa,4.8Mpa,5.0Mpa and 5.5Mpa,each group of 10 rats;The survival of SD rats were recorded after injury,the anatomy of lung tissue were observed under light microscope,and classified the degree of lung injury(OIS)and abbreviated injury score(AIS)to determine the eventual establishment of stable of adult and infancy SD rats severe lung blast injury models.2.Study on SP-A and SP-B in adult and infancy SD rats after severe lung blast injury: according to the adult and young,selecting 48 healthy adult and infancy male SD rats randomly and respectively,and then divide the SD rats into 6 groups,each group of 8 rats,and randomly selected one group as the normal control group.The driving pressure value of adult SD rats was 5.0Mpa,and the driving pressure value of young SD mice was 5.5Mpa.The shock wave were injured,then after injury,0h,2h,4h,6h,12 h,24h,48 h and 72 h were collected and extracted from the lung tissue alveolar lavage fluid,then the extracted samples were analyzed and the results of correlation detection.(1)Using Real-Time PCR method,and first to grind treatment of lung tissue,then according to kit instructions,total RNA were extracted from the samples by centrifugation.The Cq value of the target protein SP-A and SP-B was processed by data processing,and the levels of SP-A and SP-B mRNA in the lung were analyzed at different time points.(2)The ELISA method was used to collect the bronchoalveolar lavage fluid at each time point in SD rats groups and normal control groups.And added the samples to 96 well plates,each sample set of 3 holes,SP-A and SP-B detect OD value in bronchoalveolar lavage fluid and analysis the protein concentration level.(3)Western Bolt method was used to program the extracted lung tissues,and to detect the expression of SP-A and SP-B in lung tissues and semi quantitative analysis.Results:1.The establishment of young and adult SD rats lung severe blast injury model: adult SD rats in the driving pressure value is 5.0Mpa,the degree of injury of the lung in grade IV(60%),the AIS score was(3.00±0.47),reaching severe injury;Young SD rats in the driving pressure value is 5.5Mpa,more concentrated in lung injury IV(70%),the AIS score was(3.10±0.56).The degree of lung injury was approximately the same,and there was no significant difference(p<0.05).2.Study of pulmonary SP-A and SP-B after severe blast lung injury in adult and infancy rats:(1)The characteristics and changes of the injury: infancy and adult rats showed severe lung blast injury,no statistically significant difference between the two groups of injury;but the peak degree of lung injury in young rats most appeared at 4h after injury,and adult rats after injury subsequent to 6h.(2)The transcription level of intracellular surfactant protein A,B mRNA: by RT-PCR analysis,the expression of mRNA relative to SP-A in young rats was first increased and then decreased,the relative amount of young SD rats SP-A mRNA,reached the highest at 4h after injury,398% of the normal control group(P<0.05).Then the expression of the trend is decreased.However,the relative amount of mRNA in SP-B appeared to divide between 6h and 12 h after injury: After injury,0h after injury to 6 hour after injury showed a sharp increase in the expression,accounted for the normal control group 576%,823%,and 1061% respectively,and there was a significant statistical difference(p<0.01).Subsequently,12 h returned to normal range after injury,then 24 h to 72 h increased slowly,which was 291%,301% and 163% of the normal control group,respectively.The relative amount of SP-A mRNA in adult SD rats after injury tended to decrease slowly and then increased.Then slow down to 4h after injury and reached the minimum,accounted for the normal control group 76%,then with the time increased,and at 48 h after injury to 72 h showed high expression,which was 418% and 415% of the normal control group respectively.And there was a significant statistical difference(p<0.01).However,the relative amount of in adult did not change significantly after 0h,but the expression of SP-B mRNA from 2h to 72 h after injury is low,and the lowest was 25% of the control group at the time of 12 h after injury,and then increased slowly.(3)The expression results of pulmonary surfactant protein A and B 3 in lung tissue by Western Bolt analysis: the expression of SP-A in juvenile SD rats after shock injury first decreased rapidly,and then reached the bottom at the time of 4h after injury,which was 74.7% of the control group and have statistically significant(p<0.05).And then gradually recovered,especially in 24 h after injury to 72 h,accounting for 129.1%,152.7% and 188.9 respectively.However,the content of SP-B protein in the lung tissue of young rats showed a trend of first increase and then decrease,and the peak value reached 137.2% of 4h after injury(p<0.05).Then gradually declined,and 24 h,48h and 72 h after injury were not significantly different from those in the control group(p>0.05).The changes of SP-A and SP-B in adult SD rats were similar to those in young SD rats.(4)Bronchoalveolar lavage results expression of pulmonary surfactant protein A,B solution: detected by ELISA analysis,the expression of bronchoalveolar fluid SP-A in young SD rats after shock injury decreased rapidly after injury,and reached the lowest 4h after injury,which was 43.4%,significantly lower than the normal value(P<0.05).Then gradually picked up,the injury after 12 h was still significantly lower than normal,about 63.1% of normal,24 h returned to normal levels,48 h and 72 h were higher than normal(P<0.05),respectively,for the normal group of 123.7% and 132.8%.Young SD rats express SP-B in bronchoalveolar lavage fluid was gradually increased after injury,and the peak value of 4H reached 170.6% after injury,which was significantly higher than that of normal(P<0.05);and then gradually fall back to normal level after 24h(P>0.05).Whether it is SP-A or SP-B,the adult SD rats after blast injury at different time points and the expression trend of juvenile SD rats groups showed no significant difference(p>0.05),but the expression of adult SD rats groups of the SP-A and SP-B expression level at each time points is higher than young SD rat’s,and there were statistical significance(p<0.05).Conclusion:1.The lung blast injury of adult and young rats showed different degrees of pulmonary hemorrhage,edema,atelectasis,lung tissue rupture.The higher the stress value,the more serious the injury.Under the same condition of blast injury,the lung injury of adult rats was significantly higher than that of young rats.2.Through the BST-Ⅰtype biological shock tube,the 5.0Mpa driving pressure can be established from adult SD rats lung with blast injury model,and use the 5.5Mpa driving pressure established in young SD rats lung severe blast injury model,the two models of injury consistent and stable,reproducible,and can be used to explore the occurrence and development of treatment study on the mechanism of immature lung and mature lung blast injury.3.The same consistent severe pulmonary injury,SP-A and SP-B mRNA transcription trend in young and adult SD rats respective is the opposite:(1)the relative amount of SP-A mRNA in young SD rats,reached the highest at 4h after injury(398% of the control group),the expression trend then decreased;SP-A mRNA in adult SD rats relative to after the injury rose after the first drop,reached the lowest at 4h after injury(76% of the control group)and high expression at 48 h to 72 h after injury.(2)The relative amount of SP-B mRNA in youth SD rats is roughly from high to low,and reach the high expression at 6h after injury,12 h after injury returned to normal range,then 24 h to 72 h after injury increased slowly;the relative amount of adult SP-B mRNA was lower than that of 72 h after injury,and reached a minimum at 12 h after injury,accounting for about 25% of the normal control group.4.Whether it is in the bronchoalveolar lavage fluid or lung tissue,the expression trend of SP-A and SP-B in the adult and young SD rats was similar.The expression of SP-A was first decreased and then recovered,and the lowest value was 4h after injury.However,the expression of SP-B increased gradually from 2h after injury,and reached the highest value at 4h after injury,then decreased slowly until 12 h after injury,and then was no longer affected by time,and the expression level was maintained in the normal control group.5.Under the condition of the same severe lung blast injury,the peak of lung injury in young rats was earlier than that in adult rats.Although the changes of SP-A and SP-B protein in lung tissue and bronchoalveolar lavage fluid were similar in young and adult rats,but the variation of mRNA transcription is very different(In the later stage,the decrease of SP in juvenile rats was more significant,p<0.05).The mechanism and significance of the system is very worthy of further study and analysis.