Function And Mechanism Of ADP Receptor P2Y13 In LPS-induced Acute Lung Injury Mice

Acute lung injury?ALI?is an acute inflammatory disease caused by multiple factors such as pneumonia,sepsis or trauma,as well as epithelial cells,endothelial cell damage and capillary wall destruction in the whole lung.Neutrophils and inflammatory cytokines constantly permeate,infiltrate and accumulate in alveolar spaces and interstitium,causing pulmonary edema.which damages the lungs and obstructs exchange of oxygen and carbon dioxide.Thus ability to cause respiratory failure has a20-50%mortality.However,no effective treatment been established so far.This thesis try to study a new target for the treatment of ALI.Because of the important role of neutrophils in immune regulation,it is difficult to directly treat the disease as a therapeutic target.Current drug development on ALI is mainly focused on how to suppress excessive inflammation and reduce neutrophil infiltration.Recent studies have demonstrated that the extracellular nucleotide ADP released during inflammation contributes to the inflammatory response through NF-k B and inflammatory cytokines such as TNF?and IL-1?.In addition,platelets are the instigators of neutrophil migration and inflammation.Studies have shown that inhibitors of the ADP receptor P2Y13 can directly reduce megakaryocyte production of platelets.In 2017,it subverted the findings of traditional cognition that the lung is a hematopoietic organ.This paper is to study the possibility of ADP receptor P2Y13 as an ALI target.We first constructed an LPS-induced ALI mouse model,and verified two key pathogenic factors of ALI mice,massive release of inflammatory factors and excessive infiltration of neutrophils.HE staining histological observation directly confirmed the construction of ALI mouse model.Subsequently,we examined the release of ADP and the expression of P2Y13 receptors in lungs from bronchoalveolar lavage fluid from ALI mice and found that both ligand ADP and its receptor P2Y13 were abnormally elevated in lungs of ALI mice.In addition,we have found that P2y13^-/-mice can inhibit LPS-induced acute inflammatory responses.After 8 hours of LPS stimulation,P2y13^-/-mice were found to be able to reduce the pulmonary inflammatory cytokines TNF-?,IL-6,IL-1?and neutrophil chemotactic factor CXCL-2 expression and release by real-time PCR and Elisa kits.Moreover,the mortality of P2y13^-/-mice was significantly reduced in the high-dose LPS-induced peritonitis-induced death model.We demonstrated for the first time on the animal level that platelet counts were reduced in P2y13^-/-mice and platelets were the instigators of inflammation and neutrophil migration.Finally,we found that P2y13^-/-mice can significantly reduce LPS-induced infiltration of ALI neutrophils and significantly improve pulmonary edema and lung tissue injury.After 24 hours of LPS induction,we extracted the fluorescein antibody from the bronchoalveolar lavage fluid and examined the neutrophil infiltration on a flow cytometer.We also detected neutrophil signature myeloperoxidase?MPO?.,P2y13^-/-mice were found to have significantly reduced neutrophil infiltration.The occurrence and development of pulmonary edema is the key to the patient's pulmonary ventilation and ventilation dysfunction and the occurrence of respiratory distress.We examined pulmonary capillary permeability by the wet-to-weight ratio of the lungs and Evans blue and found that P2y13^-/-mice can significantly improve pulmonary edema.Later,we observed and scored histological observations by HE staining,immunohistochemistry,and immunofluorescence,and found that P2y13^-/-can effectively reduce lung injury.In summary,our study found that P2y13^-/-can inhibit LPS-induced acute inflammatory responses,reduce LPS-induced ALI neutrophil infiltration,and significantly improve pulmonary edema and lung tissue injury.It was confirmed that ADP receptor P2Y13 can be used as a potential target for the treatment of ALI.