The Role And Mechanisms Of ATⅢ And A2BAR In OA-induced Acute Respiratory Distress Syndrome

Objective:To identify the changes of proteins in lung tissue in OA-incuced actue respiratory distress syndrome in mice.To investigate the role of ATⅢ in OA-induced human microvascular endothelial cells injury and the effect of A2BAR on OA-induced ARDS in mice and the role and mechanism of A2BAR in H₂O₂-induced A549 cells apoptosis.Methods:（1）After mice were injected OA through caudal vein,we identify proteins expression levels in lung tissue using isobaric tags for relative and absolute quantification（iTRAQ）technology.Western blot was used to determine the level of 12-LO,Plasminogen,ATⅢ,A2BAR,Polycystin-2 and DOCK-2 in lung tissue.（2）To establish OA-induced HMEC-1s injury in vitro,western blot was used to detect the level of ATⅢ;small interference RNAs（siRNAs）to silence the level of ATⅢ in HMEC-1s.After HMVECs exposed to 0.5mmol/LOA,ELISA assay was used to detect the level of IL-6,IL-1β,TNF-αand TGF-βin supernatants.Western blot was used to measure the level of ZO-1 and phosphorylation of MLC in HMEC-1s.Immunofluoresence staining of F-actin in HMEC-1s was performed.FITC-dextran transwell assay was used to detect the effect of OA on the HMEC-1s permeability.（3）Mice were pretreated with A2BAR agonist or A2BAR antagonist intraperitoneally before OA administration.To assess the degree of lung injury using pathological technology.Western blot was used to detect the level of A2BAR and cleaved caspase-3 in lung tissue;TUNEL assay was used to determine lung epithelial cell apoptosis;ELISA assay was used to detect the MPO activity in lung tissue;To measure the level of PaO₂和PaCO₂ in arterial blood by blood-gas analyzer（4）Before A549 cells exposed with H₂O₂ were pretreated with A2BAR agonist or antagonist pretreatment.Flow cytometry was used to detect the cell apoptosis and mitochondrial membrane potential;western blot was used to determine the level of mitochondrial apoptosis pathway and MAPKs associated pathway proteins.（5）Using siRNA technology to silence the level of A2BAR in A549 cells,cells were preincubated with JNK,ERK and P38 inhibitors before exposed to H₂O₂.Flow cytometry was used to detect cells apoptosis and western blot was used to determine the level of cleaved caspase-9.Results:（1）A total of 5152 proteins were found to be expressed in lung tissues using iTRAQ technology.Specially,545proteins were upregulated and 304 proteins were downregulated.The level of 12-LO,Plasminogen,ATⅢ,A2BAR,Polycystin-2 and DOCK-2 were significantly elevated.（2）The expression of ATⅢ in HMEC-1s was significantly increased.The level of IL-6,IL-1β,TNF-αand TGF-βin supernatants was obviously elevated;the level of ZO-1,phosphorylation of MLC and endothelial cell permeability were significantly increased and the level of F-actin was significantly increased in ATⅢ-siRNA HMEC-1s group.（3）After BAY60-6583 pretreatment,OA-induced lung tissue injury decreased;lung Wet/Dry ratio decreased;the level of cleaved caspase-3 in lung tissue downregulated;TUNEL-positive cells ratio reduced;MPO activity in lung tissue decreased;the level of Pao₂ increased and the level of Paco₂ decreased in arterial blood;Pretreatment with PSB1115 can reverse the protective effect of BAY60-6583 on OA-induced lung injury（4）After cells pretreated with BAY60-6583,H₂O₂-induced cell apoptosis rates decreased;mitochondrial membrane potential increased;the level of Bax decreased in mitonchondia and the level of Bcl-2 and cytochrome C decreased in cytoplasm.（5）H₂O₂ induced p38,ERK1/2 and JNK signaling pathway activated in A549 cellls,pretreatment with BAY60-6583 decreased the phosphorylation levels,pretreatment with PSB1115 can block the inhibitory role of BAY60-6583 in the phosphorylation levels of p38,ERK1/2 and JNK.However,p38 and ERK1/2 inhibitor markedly reduced apoptosis in A549 cells with A2BAR-siRNA.Conclusion:（1）OA significantly induced the changes of proteins in lung tissue.For example,the level of 12-LO,Plasminogen,ATⅢ,A2BAR,Polycystin-2 and DOCK-2 were significantly increased.（2）The expression of ATⅢ and A2BAR was upregulated in OA-induced acute respiratory distress syndrome in mice;ATⅢ can attenuate lung injury through inhibiting OA-induced HMEC-1s hyperpemeability and inflammation.（3）Activation of A2BAR attenuated OA-induced lung injury through inhibiting lung epithelial cell apoptosis.（4）Activation of A2BAR has a crucial role in preventing H₂O₂-induced A549 cells apoptosis by inhibiting P38 and ERK1/2 signaling pathway.