Experimental Study Of Venovenous Extracorporeal Membrane Pulmonary Oxygenation Supporting Acute Respiratory Distress Syndrome In Rats

Objective:Acute respiratory distress syndrome（ARDS）is a severe acute,diffuse and inflammatory lung injury.Although progress has been made in the treatment of mechanical ventilation in recent years,the morbidity and mortality of ARDS are still high.At present,venovenous extracorporeal membrane oxygenation（VV-ECMO）is an effective method for the treatment of ARDS,which can replace pulmonary function and maintain systemic oxygen supply.However,the mechanism of VV ECMO on lung is not clear.The purpose of this study is to explore the mechanism of lung protection related to VV ECMO through rat ARDS model,and to provide theoretical basis for clinical treatment.Methods:1.Oleic acid-induced ARDS rat model was established with the support of VV ECMO.The effect of VV ECMO on rat ARDS lung was observed by gross specimen,blood gas analysis,histopathology,enzyme-linked immunosorbent assay（ELISA）,Western Blot（WB）and immunofluorescence.2.The potential molecular mechanism of VV ECMO supporting lung injury repair in ARDS was screened by transcriptome sequencing and analysis,and verified by molecular agonist and inhibitor intervention,dexamethasone intervention,WB,immunofluorescence and quantitative reverse transcription PCR（RT-q PCR）.3.A549 alveolar type II epithelial cell injury model induced by oleic acid was established and cultured in different oxygen environments.The above molecular mechanism was verified in vitro experiments by small interfering RNA（Si RNA）and other techniques.Results:1.One hour after the injection of oleic acid into the femoral vein of rats,the oxygenation index was≤300 mm Hg,meeting the diagnostic criteria of ARDS,and then mechanical ventilation（IMV group）and VV ECMO（ECMO group）treatment were performed.The oxygenation index of ECMO group was significantly higher than that of IMV group,while the lung injury score,lung tissue Wet/Dry weight ratio and inflammatory factor content were lower than those of IMV group.The results of immunofluorescence and WB showed that the contents of markers of alveolar type I cells（AQP5）and alveolar type II cells（SPC）in ECMO group were significantly higher than those in IMV group.2.Transcriptome sequencing showed that 339 genes in ECMO group were significantly up-regulated and 840 genes were significantly down-regulated compared with IMV group.KEGG enrichment analysis showed that the genes up-regulated in ECMO group were significantly enriched in Hippo/Yap signal pathway.The results of immunofluorescence and WB showed that the expression of Yap and Tead4 in ECMO group was higher than that in IMV group.At the same time,RT-q PCR confirmed that the expression of Yap and its target genes Cyr61 and Ctgf in ECMO group were higher than those in IMV group.3.Yap inhibitor（VP group）and Yap agonist（LPA group）were used in ECMO group to observe the effect of Yap on alveolar regeneration.WB results showed that the content of AQP5 and SPC in LPA group was significantly higher than that in VP group.The results of immunofluorescence showed that the expression of Yap in ATⅡcells in VP group was significantly lower than that in LPA group,while the differentiation of ATⅡto ATⅠin LPA group was significantly higher than that in LPA group.4.In the ECMO group,the oxygenator received oxygen therapy（O₂group）and no oxygen therapy（No-O₂group）to evaluate the effect of oxygen on Yap expression.The oxygenation index of O₂group was significantly higher than that of No-O₂group.The results of WB showed that the contents of Yap and Tead4 in O₂group were significantly higher than those in No-O₂group.Immunofluorescence results showed that the expression of Yap in ATⅡcells and the differentiation of ATⅡto ATⅠin O₂group were significantly higher than those in No-O₂group.In order to simulate the effect of oxygen concentration on alveolar typeⅡcells,oleic acid-induced alveolar typeⅡepithelial A549 cells were cultured in normoxic（21%O₂group）and anoxic（1%O₂group）environment in vitro.Scratch test showed that the wound healing rate of 1%O₂group was significantly lower than that of 21%O₂group.WB results showed that the expression of Yap in 21%O₂group was higher than that in 1%O₂group,and hypoxia（1%O₂group）could significantly inhibit the expression of interstate markers（Claudin 4 and Keratin 8）in A549 cells.5.ECMO group was treated with dexamethasone（Dex group）and saline（Vehicle group）respectively to evaluate the protective effect of dexamethasone on lung.The results of WB showed that the expression of Yap,AQP5 and SPC in Dex group was significantly higher than that in Vehicle group.The results of immunofluorescence showed that the expression of Yap in ATⅡcells in Dex group was significantly higher than that in Vehicle group,and the differentiation of ATⅡto ATⅠin Dex group was also significantly enhanced.Dexamethasone also significantly promoted the expression of Yap,Claudin 4 and Keratin 8 in A549 induced by oleic acid.Conclusion:1.VV ECMO can alleviate lung injury and promote the proliferation and differentiation of alveolar typeⅡcells,thus maintaining alveolar regeneration.2.VV ECMO promotes the repair of damaged alveolar epithelium by activating Hippo/Yap signal pathway,which provides a new therapeutic target for perioperative application of VV ECMO.3.The combined application of VV ECMO and dexamethasone can further activate Hippo/Yap signal pathway and reduce lung injury and inflammation,which provides a theoretical basis for drug repurposing.