The Regulatory Mechanism Of MiR-138 Targeting MLK3 In The Formation Of High-altitude Pulmonary Hypertension And The Predictive Value Of NLR In Pediatric Congenital Heart Disease Associated With Pulmonary Arterial Hypertension During Perioperative Period

Part one:The regulatory mechanism of miR-138 targeting MLK3 in the formation of high-altitude pulmonary hypertensionObjective:High-altitude pulmonary hypertension(HAPH)is a progressive disease characterized by persistent increase of pulmonary vascular resistance and pulmonary vascular remodeling caused by high-altitude hypobaric hypoxia.The detailed pathogenesis of HAPH is still not completely clear,and there is no effective clinical treatment at present.It has been reported that some microRNAs are closely related to hypoxic pulmonary vascular remodeling.MiR-138 has many effects,such as promoting cell proliferation,inhibiting cell apoptosis and anti-inflammation.This study aims to explore the regulatory role of miR-138 and MLK3 in the formation HAPH in mice.Methods:32 C57BL/6 male mice based on the weight were randomly divided into 5000m high-altitude group and control group.The high-altitude group was subdivided into hypoxia+NS group,hypoxia+antagomir-NC group and hypoxia+antagomir-138 group,with 8 mice in each group.The mice in the high-altitude group were placed in a hypobaric oxygen chamber to simulate 5000 meters of hypobaric hypoxia environment for 3 weeks and were injected with NS,antagomir-NC and antagomir-138 according to 8μg/g respectively through the tail vein on the 3rd,6th,9th,12th,15th,18th and 21st day.The mice in control group,fed with normal oxygen concentration in the plain,were injected with the same dose of NS.On the 22nd day of feeding,the echocardiographic indexes of mice were collected,the pulmonary artery pressures were measured,the heart and lung tissues of mice were gathered.The mRNA expressions of miR-138 and MLK3 in lung tissues of mice in each group were detected by QRT-PCR.Immunohistochemistry was used to localize the level of MLK3 protein in mice pulmonary artery.The pathological changes of pulmonary artery and right ventricular muscle were evaluated by HE staining,modified Masson staining and Sirius red staining.The protein changes of MLK3 as well as its downstream signaling pathway JNK/c-jun were detected by western blot.The level of apoptosis in lung tissues of mice was tested by Tunel,the expression of relevant apoptosis proteins in lung tissues were detected by western blot and immunohistochemistry.Results:1.Compared with the control group,the expression level of miR-138,right ventricular diameter,right ventricular diameter/left ventricular diameter,interventricular septum thickness,pulmonary artery systolic pressure and right ventricular hypertrophy index in the hypoxia+NS group were significantly increased,while all the above indexes in the hypoxia+antagomir-138 group were significantly decreased than those in the hypoxia+antagomir-NC group.Pathological staining suggested that compared with the control group,the pulmonary vascular wall thickened,lumen narrowed and pulmonary vascular remodeling occurred in hypoxia+NS group,and the fibrosis degree of collagen fibers in right ventricular muscle and pulmonary vessels was significantly increased in hypoxia+ NS group,while in hypoxia+antagomir-138 group,the pulmonary vascular remodeling was alleviated and the fibrosis degree of collagen fibers in right ventricular muscle and pulmonary vessels was significantly improved compared to hypoxia+antagomir-NC group.2.QRT-PCR,western blot and immunohistochemistry demonstrated that the mRNA and protein levels of MLK3 in hypoxia+antagomir-138 group were significantly higher than those in hypoxia+antagomir-NC group.Further by using western blot,we perceived that the phosphorylation levels of JNK and c-jun,the downstream molecule of MLK3,also increased significantly.3.The results of Tunel staining indicated that the level of apoptosis in lung tissue in hypoxia+NS group was lower than that in control group,however,after antagonizing miR-138,the level of apoptosis in lung tissue in hypoxia+antagomir-138 group was significantly higher than that in hypoxia+antagomir-NC group.In addition,the results of western blot and immunohistochemistry revealed that compared with the control group,the pro-apoptotic protein cleaved caspase-3 was significantly decreased and the anti-apoptotic protein bcl-2 was significantly increased in the peripheral pulmonary artery in the hypoxia+ NS group,while the pro-apoptotic protein cleaved caspase-3 was significantly increased and the anti-apoptotic protein bcl-2 was significantly decreased in the hypoxia+antagomir-13 8 group compared to the hypoxia+antagomir-NC group.Conclusion:1.The mice HAPH model was successfully established by simulating 5000 meters high altitude for three weeks,and the expression of miR-138 in the lung tissues of HAPH mice was significantly up-regulated compared to the control group.2.MLK3 is the target protein of negative regulation of miR-138.Intravenous injection of miR-138 antagonist could significantly activate MLK3/JKN/c-jun signaling pathway and improve pulmonary vascular remodeling induced by hypoxia,thus preventing the formation of HAPH in mice.3.Antagonizing the expression of miR-138 could promote hypoxia-induced pulmonary vascular apoptosis in mice,suggesting that miR-138 may be a key regulatory molecule in the formation of HAPH.Part two:The predictive value of NLR in pediatric congenital heart disease associated with pulmonary arterial hypertension during perioperative periodObjective:To explore the relationship between the neutrophil to lymphocyte ratio(NLR)and early clinical prognosis of children diagnosed as congenital heart disease associated with pulmonary arterial hypertension(CHD-PAH)after cardiopulmonary bypass(CPB)surgery and to investigate their clinical value in predicting prolonged mechanical ventilation.Methods:A total of 190 children with CHD-PAH who underwent CPB open heart surgery in the Department of Cardiovascular Surgery of the General Hospital of the Western Theater Command from January 2013 to August 2019 were analyzed retrospectively.All clinical data during the perioperative period were collected.The correlations between NLR and perioperative clinical indexes were analyzed,and the reliability of preoperative and postoperative NLR levels as predictive indexes of prolonged mechanical ventilation were evaluated by ROC curves.Results:We found that preoperative NLR was significantly correlated with AST,STB,CR,UA(all P<0.05),while postoperative NLR was significantly correlated with ALT,AST,BUN(all P<0.05).Increased postoperative neutrophil count and NLR as well as decreased lymphocyte count could be observed after cardiac surgery(all P<0.05).Level of preoperative NLR was significantly correlated with mechanical ventilation time,ICU stay time and total length of stay(all P<0.05),while the level of postoperative NLR was only significantly correlated to mechanical ventilation time and ICU stay time(all P<0.05).By using ROC curves analysis,relevant areas under the curve for predicting prolonged mechanical ventilation time beyond 24h,48h and 72h by preoperative and postoperative NLR were statistically significant(all P<0.05).Conclusion:1.NLR was significantly correlated with organ function reservation during perioperative period in children with CHD-PAH,and was closely related to early clinical outcomes after cardiac surgery.2.NLR could act as a novel marker to predict the occurrence of prolonged mechanical ventilation in children with CHD-PAH.