| ObjectiveAfter cardiac surgery performed under deep hypothermia with low-flow (DHLF) cardiopulmonary bypass (CPB) in infants, the lung injury often becomes more severe than elder children driven by diverse pathogenic etiologies, which has always been a major cause of infant patients’ death during the post-operation time. Previous studies reported the potential advantages of additional lung protective solution perfusion on alleviate lung injury during cardiac surgery with CPB. For now, the mechanism of DHLF-CPB induced lung injury is inconclusive, a suitable lung preservation solution is still required. Due to their pleiotropic actions, microRNAs (miRNAs) are potential candidates involved in a diverse pathophysiological processes and diseases via regulating gene expression.In this study, we mimicked clinical procedure and established a stable piglet model undergoing deep hypothermia with low-flow (DHLF) cardiopulmonary bypass (CPB) with pulmonary artery perfusion as a lung protective strategy, aimed to evaluate the protective efficacy of pulmonary artery perfusion with urinary trypsin inhibitor (UTI) on immature lung. Furthermore, we investigated the changed miRNAs and their potential target genes and relevant function in neonatal piglet lungs in response to DHLF-CPB and lung protection with pulmonary perfusion with ulinastatin, in order to provide theoretical basis for CPB related immature lung injury and lung protection solution improvement.Methods15piglets aged14-21day, weighting2.4-7.0kg were randomly divided into3groups, with5piglets in each group:DHLF-CPB related lung injury group (Group C), the pulmonary artery perfusion without ulinastatin group (Group P) and the pulmonary infusion with ulinastatin group (Group U). A systemic CPB flow was established via aortic cannula and venous cannula in the right atrial appendage via median sternotomy. In group P and U, lung protective solution without or with ulinastatin was perfused via pulmonary artery respectively after cardiac arrest was obtained by aortic cross clamp and antegrade St. Thomas’cardioplegia through the aortic root. The DHLF-CPB was performed for60minutes (25℃,50ml/kg/min) during in the2hours aortic cross-clamping. After the piglets weaned from CPB, then received another120min observation. The hemodynamics and respiratory indices, blood gas analysis were recorded at four defined time-points:before CPB, at the end of CPB, and60minutes,120minutes after CPB. Pulmonary venous blood was sampled at the same preceding time interval. Right lung lower lobe was harvested at the end of the study. The levels of cytokines (TNF-α, IL-6, MPO, MDA and SOD) in pulmonary venous serum and lung tissue and the activity of NF-kappa B in lung tissue were measured by enzyme-linked immunosorbent assay (ELISA) and elctrophoresis mobility shift assay (EMSA), respectively. Morphological comparison was assessed by light and electronic microscopy. A TUNEL technique was performed to evaluate the state of cell apoptosis in the lungs. The wet to dry ratio of lung tissue is calculated to assess the extent of tissue edema. We applied miRNA microarray and qRT-PCR analysis to compare the miRNA expression of group C and group U with normal piglets in lung tissue. Meanwhile, predicted mRNA targets were obtained from miRanda. Then the predicted mRNA target genes had a Gene Ontology analysis and KEGG analysis performed by DAVID to obtain the functional annotation and pathway mapping. qRT-PCR and ELISA are used for further validation for the chosen target genes.ResultsAll the15piglets were accomplished CPB successfully with stable hemodynamics, and demonstrated a state of lung injury. While pulmonary artery perfusion with UTI significantly ameliorated lung function and histopathological changes and we found a decrease in W/D ratio and the number of TUNEL-positive apoptotic cells in group U lungs when compared with that in the other two group lungs. There is a great decrease in the serum levels of TNF-α,IL-6, MPO and lung tissue levels of IL-6, MPO in group U compared to group C and group P. Also, we found an increase in the level of IL-10, SOD in group U lungs compared with that in lungs and serum of group C and group P which correlated with a strongly inhibition in the activity of NF-κB. We found no significant difference between group U and group P in the concentration of MDA in pulmonary venous serum and level of TNF-α and MDA in lung tissue.Using miRNA microarray we identified that when compared to normal piglets, the expression of16miRNAs in group C,8miRNAs in group U were significantly changed (P<0.05). The qRT-PCR analyses verified up-regulation of miR-21and down-regulation of miR-127, miR-145miR-204in group C. The same trend was also found in group U except miR-204. But there are up-regulation of miR-127, miR-204and down-regulation of miR-21in group U when compared to group C.Target genes were cross-referenced to the molecular pathways after obtained from miRanda, suggesting that the differentially expressed miRNAs were related to series of important biology pathway related to CPB-induced lung injury, including T Cell receptor signaling pathway, PI3K-Akt-NF-κB signaling pathway and RAS signaling pathway. We performed qRT-PCR and ELISA of3putatively target genes ACE, PTGS2and PIK3CG for further validation. The results revealed that the expression of the3target genes were up-regulated in group C. And there is an up-regulation of ACE and PTGS2in group U. And the extent of up-regulation of the3target genes in group C was higher than group U. Conclusion Deep hypothermic low-flow cardiopulmonary bypass surgery can induce severe lung damage. Pulmonary artery perfusion with UTI ameliorated immature pulmonary injury in the lungs via inhibiting the activity of NF-κB to reduce the extent of oxidative shock, inflammatory response and apoptosis of cells.Our results showed that dynamic changes of miRNA expression in piglet lungs of DHLF-CPB induced immature lung injury and pulmonary perfusion with ulinastatin lung protective solution. The level of putative anti-inflammatory miRNA:miR-127, miR-145and miR-204down-regulated and putative pro-inflammatory miRNA:miR-21up-regulated in group C and group U indicated that miRNA involved in the process of lung injury with close correlation to inflammatory response and oxidative stress. While the degree of miRNA level change was lower in group U suggested that ulinastatin alleviated DHLF-CPB induced lung injury by regulate relevant miRNA. Moreover, by understanding how the changed miRNAs regulate lung injury may provide a basis for understanding the pathogenesis of DHLF-CPB induced immature lung injury and pave a way to develop a novel therapeutic approach for lung protection. |
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