| Intrauterine infection can lead to intrauterine inflammation which not only triggers premature birth as an important independent risk factor but also plays an important role in the development of bronchopulmonary dysplasia(BPD).However,the precise molecular biological mechanisms of intrauterine infection/inflammation on fetal and neonatal lung development are unclear.In recent years,although the studies on the mechanisms of respiratory diseases have found that microRNAs(miRNAs)participate in the regulation of a variety of pathophysiological processes including respiratory system development,infection,inflammation,fibrosis and tumorigenesis,the studies on the role of miRNAs in intrauterine infection/inflammation induced fetal and neonatal lung injury are very rare.The research of the characteristics of miRNAs expression in developmental phases of lungs with injury after intrauterine infection/inflammation has not been reported yet.ObjectivesTo study effects and mechanisms of intrauterine infection/inflammation on lung development at different time in fetal rats and neonatal rats,and to investigate the characteristics of miRNAs expression in neonatal rat lungs to identify miRNAs related to lung injury in developmental period and to predict their target genes.Methods1.An animal model of intrauterine infection/inflammation was established with pregnant SD rats.In the intrauterine infection group,pregnant SD rats were endocervically inoculated with E.coli suspension.While in the control group,pregnant SD rats were endocervically injected with sterile normal saline instead.2.The fetal and neonatal rats were observed at 17,19,21 days of gestation and 1,3,7,14 days after birth,respectively.Body weight,lung weight and lung/body weight ratio of the fetal and neonatal rats were measured at each observation time point.The fetal and neonatal rats lung tissues histomorphology were observed by microscopy after HE staining.The expression levels of NLRP3,TNF-?,IL-1??IL-6,VEGF,Collagen ?,SP-A,SP-B and SP-C in the lung tissues of fetal and neonatal rats were qualified by immunohistochemical staining technique,quantitative real-time PCR technique,and Western blot technique at each observation time point.3.MicroRNA microarray technique was applied to detect the lung tissues of neonatal rats after intrauterine infection/inflammation at different observation time points.Expression profiles of 1218 kinds of miRNAs were detected and analyzed,and the differential expression miRNAs were screened compared to the control group.4.The miRNAs with significant differential expression between two groups were qualified by fluorescence quantitative PCR technique to verify the results of miRNAs microarray assays.5.Target genes of the miRNAs screened were predicted through online software of TargetScan.Results1.There were 24 pregnant SD rats endocervically inoculated with E.coli suspension or sterile normal saline at 15 days of gestation,which had no significant changes in food intake or activity after inoculation.The gestation period of the pregnant SD rats was 21-22 days in this study.There were 77(77/79)(live rat pups/total rat pups)fetal rats and 48(48/51)neonatal rats in the intrauterine infection group,and 76(76/78)fetal rats and 52(52/53)neonatal rats in the control group.There was no significant difference in the death rates of rat pups between two groups.Vascular congestion,edema,and marked neutrophil infiltration was manifested in the placenta and uterine wall in intrauterine infection group.While in the control group,there was no manifestation of inflammation in the placenta or uterine.2.The mean body weights of the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3 days after birth in intrauterine infection group were significantly lower compared to the control group.The mean lung weights of the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3,7 days after birth in intrauterine infection group were significantly lower compared to the control group.The mean lung/body weight ratios at 17,19,21 days of gestation in intrauterine infection group were significantly lower compared to the control group.3.The fetal and neonatal rats lung tissues histomorphological examination showed inflammatory infiltration,reduced alveolar vesicular structure,less alveolar numbers and thickened alveolar septa in intrauterine infection group compared to the control group.4.The immunohistochemical examination and quantitative real-time PCR analyses of the fetal and neonatal rats lung tissues showed significantly increased expression of NLRP3 in the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3,14 days after birth in intrauterine infection group compared to the control group.Western blot assays of the fetal and neonatal rats lung tissues showed significantly increased expression of NLRP3 in the fetal rats at 19,21 days of gestation and the neonatal rats at 1,3,14 days after birth in intrauterine infection group compared to the control group.5.Quantitative real-time PCR analyses of the fetal and neonatal rats lung tissues showed significantly increased expression of inflammatory cytokines including TNF-?(in the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3 days after birth),IL-1?(in the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3,7 days after birth),and IL-6(in the fetal rats at 17,21 days of gestation and the neonatal rats at 1 day after birth)in intrauterine infection group compared to the control group.6.Quantitative real-time PCR analyses of the fetal and neonatal rats lung tissues showed significantly decreased expression of pulmonary surfactant proteins including SP-A(in the fetal rats at 19,21 days of gestation and the neonatal rats at 1,3,14 days after birth),SP-B(in the fetal rats at 19,21 days of gestation and the neonatal rats at 1,3 days after birth)and SP-C(in the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,7 days after birth)in intrauterine infection group compared to the control group.Western blot assays of the fetal and neonatal rats lung tissues showed significantly decreased expression of pulmonary surfactant proteins including SP-A(in the fetal rats at 17,19 days of gestation and the neonatal rats at 1,3,14 days after birth),SP-B(in the fetal rats at 19,21 days of gestation and the neonatal rats at 1,3 days after birth)and SP-C(in the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3,7 days after birth)in intrauterine infection group compared to the control group.7.The immunohistochemical examination and analyses of the fetal and neonatal rats lung tissues revealed significantly decreased expression of VEGF in the fetal rats at 17,19,21 days of gestation and the neonatal rats at 1,3 days after birth in intrauterine infection group compared to the control group.Both quantitative real-time PCR analysis and Westernblot assays of the fetal and neonatal rats lung tissues showed significantly decreased expression of VEGF in the fetal rats at 19,21 days of gestation and the neonatal rats at 1,3,7 days after birth in intrauterine infection group compared to the control group.8.The immunohistochemical examination and analyses of the fetal and neonatal rats lung tissues revealed significantly increased expression of Collagen I in the fetal rats at 21 days of gestation and the neonatal rats at 1,3,7,14 days after birth in intrauterine infection group compared to the control group.Both quantitative real-time PCR analyses and Westernblot assays of the fetal and neonatal rats lung tissues showed significantly increased expression of Collagen I in the fetal rats at 19,21 days of gestation and the neonatal rats at 1,3,7,14 days after birth in intrauterine infection group compared to the control group.9.MicroRNA microarray technique analysis of neonatal rats lung tissues revealed that out of the 1218 miRNAs,16 miRNAs(6 significantly upregulated and 10 significantly downregulated)with significant differential expression were screened in the neonatal rats at 1 day after birth in intrauterine infection group compared to the control group;14 miRNAs(1 significantly upregulated and 13 significantly downregulated)with significant differential expression were screened in the neonatal rats at 3 days after birth in intrauterine infection group compared to the control group;22 miRNAs(2 significantly upregulated and 20 significantly downregulated)with significant differential expression were screened in the neonatal rats at 14 days after birth in intrauterine infection group compared to the control group.The miRNAs of rno-miR-122-5p,rno-miR-204-5p,rno-miR-6215,rno-miR-490-5p,rno-miR-3559-3p,rno-miR-466b-2-3p,rno-miR-208a-5p and rno-miR-1-3p showed differential expression in intrauterine infection group at different time points compared to the control group.Changing trends of miRNAs with significant differential expression were various.10.Quantitative real-time PCR analyses of the fetal and neonatal rats lung tissues verified the results of miRNAs microarray assays,which showed that rno-miR-3559-3p was significantly upregulated at 3 days after birth in intrauterine infection group compared to the control group,and rno-miR-1-3p was significantly downregulated at 14 days after birth in intrauterine infection group compared to the control group.Both results were consistent with the results of miRNAs microarray assays.11.Target genes of the 43 miRNAs screened were predicted through online bioinformatic software of TargetScan.The predicted target genes that probably express the most effectively targeted mRINAs are very rich and providing very useful clues for further study on these miRNAs.Conclusions1.Intrauterine infection/inflammation may result in significantly decreased body weight,lung weight and lung/body weight ratio in the fetal and neonatal rats.It may also result in reduced alveolar vesicular structure,less alveolar numbers and thickened alveolar septa,which are very similar to the changes observed in the new BPD.2.Possible mechanisms of intrauterine infection/inflammation on fetal and neonatal lung development are very complex,which may include NLRP3 inflammasome activation followed by inflammatory cytokines expression upregulated,inflammatory damage,inhibiting the expression of pulmonary surfactant proteins,interfering with lung interstitial development.3.The expression profiles of miRNAs are changed in neonatal rat lung tissues after intrauterine infection/inflammation.There were 43 miRNAs identified by miRNAs microarray assays,among which were rno-miR-122-5p,rno-miR-204-5p,rno-miR-6215,rno-miR-490-5p,rno-miR-3 5 59-3p,rno-miR-466b-2-3p,rno-miR-208a-5p and rno-miR-1-3p,that may play an important role in regulating the processes of lung development injuries after intrauterine infection/inflammation.4.The miRNAs with regulatory effects on neonatal rats lung tissues after intrauterine infection/inflammation vary with time.The results of identified miRNAs and their rich predicted target genes that probably express the most effectively targeted mRNAs have suggested a wide range of complicated effects of identified miRNAs in neonatal lung injury induced by intrauterine infection/inflammation. |
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