The Effects Of AngⅡ And Its Receptor Inhibition On Inflammatory Injurys In Meconium-exposed S-D Rat Lungs

Objective Meconium aspiration syndrome(MAS) is a severe emergency as well as a very important cause of death in neonate.Lung tissue inflammation is implicated in the pathogenesis of meconium aspiration-induced lung injury in the newborn,but the mechanisms of the reactions are still poorly known.We used the younger S-D rats as the MAS model,investigated the inflammatory injuries(taking the wet/dry weight ratio,the lung injury score,the content of TNF-α,the MPO activity as the indexes) and the expression of angiotensin(ANGⅡ)in the lungs by immunohistochemistry. Meanwhile,we used the microsoft system to get the PU value of the ANGⅡamong different groups.Then,we evaluated the contribution of angiotensin(ANG)Ⅱaction on these processes in the meconium-induced lung injury by studying the correlationship between the PU value of ANGⅡand the the content of TNF-α,the MPO activity.At the same time,we used an unspecific ANGⅡreceptor inhibitor saralasin to study whether pretreatment of the rat lungs by saralasin inhibits meconium-induced lung inflammatory injury.Through this study,we could know more about the mechanisms of the lung injuries and may provide a new treatment for the MAS.Methods Experimental meconium aspiration was induced by intratracheal instillation of human meconium(20%)1ml/kg in Sprague-Dewley rats(weighing 100～120g).There were 30 SD rats which were divided into 3 groups(A,B and C, n=10 rats each group).Group A,saline(1ml/kg) instilled rats;group B, meconium-instilled rats;group C,.saralasin—pretreated(0.75mg/kg) and then meconiun-instilled rats.These rats were allowed to have normal activity and feeding after instillation of meconium.Then,24 hours later,they were killed.The right upper lung was obtained for the measurement of the wet/dry weight ratio;Pulmonary tissue samples from the right lower lobe were used for the determination of the severity of lung injury and for the immunohistochemistry which can indicate the expression of AngⅡin the lung,.Then,we used the Leica Q55IW imaging analysistic system to get the PU values of AngⅡ;at the same time,a few of lung tissue samples were studied for the content of TNF-αand the myeloperoxidase(MPO) activity.A score from 0 to 4 represented the percentile of affected area of the lung section(0=0%;1=1-25%; 2=26-50%;3=51-75%;4=76-100%) and was assigned for three different characteristics:1) extension of pulmonary leukocyte infiltration,2) amount of intra-alveolar leukocytes,and 3) amount of exudative debris and edema fluid.The calculated total injury score represents the sum of these scores.The content of TNF-αin the lung tissue was measured by ELISA(Kits bought from Shenzhen Jingmei company).The myeloperoxidase(MPO) activity was measured by colorimetric method(Kits bought from NanJing Jiancheng biological research).Welch test,one-way ANOVA test(if equal variances assumed,using the LSD test to analyze the multiple comparisons;if equal variances not assumed,using the Games—Howell test to analyze the multiple comparisons),Pearson relative analysis were used to analyze the differences among various group.P＜0.05 was considered to have significant difference.Result From the outcomes of the study,we can get this conclusion:Compared with the saline-instilled group(group A) and the Saralasin-pretreated and then meconium-instilled group(group C),the inflammatory injuries of meconium-instilled group(group B)were more serious.Pathological changes were examined under optical microscope.Histologically,alveolar edema, hemorrhage,and massive inflammatory cell infilltration were observed in group B. The pathological changes of group A were not serious;and the pathological changes of group C were more serious than group A but less serious than group B.Using the one-way ANOVA test,we can get that:there were signifcant differences in the injury score(F=6.520,P=0.005)among the three groups,the scor for group A,B and C were 8.44±1.20,10.00±1.01 and 8.57±1.02.Furthemor,we analyzed the the multiple comparisons between each group.The differences of group A and B,group B and C were both significant(P=0.003and P=0.006),.The mean difference(B-A) and(B-C) were 1.57 and 1.44.But the difference of group B and C was not significant(P=0.790). The mean difference(C-B) was 0.13.The differences in wet/dry weight ratio among the three groups were also significant(Statistic~a=15.163,P=0.000) which were analyzed by Welch test.The ratios for group A,B and C were 4.47±0.40,5.63±0.53 and 4.76±0.19.Because the equal variances were not assumed,we use the Games-Howell test to analyze the multiple comparisons:the differences of group A and B,group B and C were both significant(P=0.000and P=0.001),the mean difference(B-A) and(B-C) were 1.16 and 0.29.But the difference of group B and C was not significant(P=0.126).The mean difference(C-B) was 0.13.The expreesion of ANGⅡwhich can be indicated by the immunohistochemistry were different in the three groups.The PU value of group A,B and C were 16.31±2.20,28.75±4.01 and 24.26±3.65 respectively.We used one-way ANOVA test to compared the PU values.From the outcome we could conluded that there were significant difference between each group(F=15.255,P=0.000).Because the equal variances were assumed,we use the LSD test to analyze the multiple comparisons:the differences of group A and B,group B and C,group A and C were all significant(P=0.000, P=0.035 and P=0.030 respectively),the mean difference(B-A),(B-C) and(C-A)were 12.44,4.49 and 7.95.In the three groups,group B had the highest value and group A had the lowest value.At the same time,the TNF-αcontent and the myeloperoxidase(MPO) activity between each group were also significantly different which were both analyzed by Welch test.(Statistic~a=46.407,P=0.000 and Statistic~a=49.434,P=0.000).The TNF-αcontent of group A,B and C were 56.16±16.75,204.97±49.01 and 106.17±24.29 respectively.The myeloperoxidase(MPO) activity of group A,B and C were0.15±0.04,0.61±0.18 and 0.32±0.06 respectively.The outcomes of the multiple comparisons which were about the TNF-αcontent were that the differences of group A and B,group B and C,group A and C were all significant(P=0.000 all of them).The mean difference of TNF-αcontent(B-A),(B-C) and(C-A)were 148.81,98.80 and 50.01.The outcomes of the multiple comparisons which were about the MPO activity were that the differences of group A and B,group B and C,group A and C were also significant(P=0.000,P=0.001 and P=0.000 respectively).The mean difference of MPO activity(B-A),(B-C) and(C-A) were 0.47,0.29 and 0.18.Furthermore.there were a positive relationship between the PU value of AngⅡand the TNF-αcontent (rTNF-α=0.744,P=0.000),the same as the PU value of AngⅡand the myeloperoxidase(MPO) activity(rMPO=0.747,P=0.000).Conclusion The meconuim which was intratracheal instilled can induce the rats lung injurys and the inflammation is one important reason for the injurys.AngⅡplays an important role in the meconium-exposed lung inflammation,which can directly induce the injury or indirectly increase the inflammatory factors such as TNF-αand the inflammatory cells. ANGⅡreceptor inhibitor saralasin can inhibits the combination of AngⅡand its receptor.Through this way,saralasin can prevent meconium-induced lung inflammatory injury and can decreace the the inflammatory factors and cells.So saralasin has a protective effect on the inflammatory Injurys in meconium-exposed S-D rat lungs.