Protective Effect And Mechanism Of GLP-1 Receptor On Pulmonary Vascular Endothelial Cells In Acute Lung Injury

Acute respiratory distress syndrome(ARDS)is characterized by pulmonary vascular endothelial cells and alveolar epithelial cells damaged induced by a variety of pathogenic factors,which causes inflammatory cell infiltration,plasma protein exudation,pulmonary interstitial and alveolar edema,leading to acute hypoxic respiratory insufficiency.The pathophysiological manifestations were decreased lung compliance and dysmotility blood flow imbalance.Acute lung injury(ALI)is the pathogenesis of ARDS.The incidence and mortality of ARDS are very high.In addition to controlling the primary disease,respiratory support,controlling infection,and strengthening nursing,there are no specific treatment drugs.Therefore,it is necessary to further study the pathogenesis of ARDS and find safer and more effective therapeutic drugs.Glucagon-like peptide-1 is a peptide hormone secreted by intestinal L cells.GLP-1 receptor(GLP-1R)is a natural target of GLP-1,distributed in various tissues and organs,and has a wide range of biological functions.Liraglutide,a GLP-1analogue,is commonly used in the treatment of type 2 diabetes mellitus which can bind and activate GLP-1R.It has been found that GLP-1R activation can protect endothelial cell barrier function and regulate inflammatory response,thus protecting multiple organs from functional damage caused by multiple injury factors.Considering the anti-inflammatory and endothelial protective effects of GLP-1R,activation of GLP-1R may be beneficial to the treatment of ALI.Therefore,in the first part of this study,the protective effect of liraglutide-activated GLP-1R on ALI in mice was investigated by intratracheal instillation of lipopolysaccharides(LPS)induced ALI model.In the second part,the effects of liraglutide-activated GLP-1R on LPS-induced human pulmonary microvascular endothelial barrier dysfunction were further studied,and the mechanisms of liraglutide-activated GLP-1R on LPS-induced human pulmonary microvascular endothelial barrier dysfunction were explored by establishing a co-culture system of neutrophils and endothelial cells.Part ? The Protective Effects of GLP-1R activation on Acute Lung Injury induced by lipopolysaccharide in miceObjection:To investigate the protective effect of liraglutide-activated GLP-1R on LPS induced ALI in mice.Methods:Forty C57BL/6 mice were randomly divided into five groups:control group,LPS group,LPS+liraglutide(0.2,0.8 mg/kg)group and liraglutide(0.8mg/kg)alone group,with 8 mice in each group.The ALI model of mice was established by intratracheal injection of LPS(15 mg/kg)with a subcutaneous injection of liraglutide.Twenty-four hours later,the mice were sacrificed and peripheral blood,bronchoalveolar lavage fluid(BALF),lung tissue were collected.Serum and BALF GLP-1 levels were detected by ELISA.mRNA levels of GLP-1R,TLR4 and MIF,IL-6,CXCL1,CXCL2 in lung tissue were detected by RT-qPCR.GLP-1,TLR4 and intercellular junction protein ZO-1,Occludin,VE-cadherin protein expressions were analyzed by Western blot.HE staining and quantitative lung injury score were used to evaluate lung injury.Immunohistochemical staining was used to evaluate neutrophil infiltration in lung tissue.Cell sorting count was used to calculate the number of neutrophils and macrophages in BALF.TUNEL was used to detect cell apoptosis in lung.Evans blue dye exudation test and BALF protein content determination were used to evaluate pulmonary vascular permeability.Results:HE staining showed that liraglutide(0.2,0.8 mg/kg)attenuated LPS-induced pathological damage in lung tissue.Immunohistochemistry showed that there was a large amount of Ly6G~+neutrophil infiltration in the lung tissue of ALI mice,and the administration of liraglutide(0.2,0.8 mg/Kg)significantly reduced neutrophil infiltration.The cell sorting results in BALF showed that liraglutide(0.8 mg/Kg)inhibited LPS-induced alveolar neutrophil and macrophage exudation.HE staining showed that liraglutide(0.2,0.8 mg/kg)attenuated LPS-induced pathological damage in lung tissue.TUNEL staining showed that administration of liraglutide(0.2,0.8mg/kg)significantly reduced LPS-induced apoptosis.Compared with the control group,LPS can decrease serum GLP-1 level in ALI mice,and increase GLP-1 level in BALF.GLP-1R,TLR-4 mRNA and GLP-1,TLR-4 proteins in lung tissue are also caused by LPS.LPS stimulates increased expression and liraglutide inhibits the above effects.Western blot analysis showed that LPS stimulation reduced the expression of VE-cadherin,ZO-1 and Occludin in lung tissue,which were restored by the treatment of liraglutide(0.8 mg/kg).Conclusion:GLP-1R activation by liraglutide can alleviate LPS-induced lung pathological damage,reduce the number of apoptotic cells,reduce inflammatory cell infiltration and release of inflammatory factors,and reduce pulmonary capillary permeability,thereby improving LPS-induced ALI in mice.Part ? The Effect and Mechanism of GLP-1R activation on LPS induced human pulmonary microvascular endothelial cell dysfunctionObjective:To explore the protective effect of liraglutide-activated GLP-1R on LPS-induced human pulmonary microvascular endothelial cells function disorder and the mechanisms underlying.Methods:human pulmonary microvascular endothelial cells were cultured and divided into control group,LPS group,LPS+liraglutide(200,400,800nM)group and liraglutide(800nM)alone group.The levels of ZO-1 and Occludin mRNA were detected by RT-qPCR.The expression of ZO-1 and VE-cadherin was detected by immunofluorescence and Western blot.Neutrophils and LPS-stimulated human pulmonary microvascular endothelial cells were co-cultured.The expression of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule-1(VCAM-1)were detected by RT-qPCR and Western blot.The adhesion of neutrophils on human pulmonary microvascular endothelial cells were detected by immunofluorescence.Transwell analysis was applied to investigate the transendothelial layer migration of neutrophils.Rho-GTP protein,MYPT1 and p65phosphorylation levels were detected by Western blot.Results:Liraglutide reversed LPS-induced down-regulation of mRNA of ZO-1 and Occludin.as well as protein expressions of ZO-1 and VE-cadherin in human pulmonary microvascular endothelial cells.Immunofluorescence staining showed that liraglutide restored the expression of VE-cadherin and ZO-1.The results from co-culture system showed that liraglutide inhibited the expression of ICAM-1 and VCAM-1 in human pulmonary microvascular endothelial cells induced by LPS,and decreased the adhesion and transendothelial migration of neutrophils to activated human pulmonary microvascular endothelial cells.In addition,liraglutide inhibits LPS-induced expression of Rho-GTP and phosphorylated MYPT1 and p65 proteins.Conclusion:Activation of GLP-1R by liraglutide can maintain the intercellular junction of human pulmonary microvascular endothelial cells and inhibit the expression of adhesion molecule,alleviating LPS-induced dysfunction of human pulmonary microvascular endothelial barrier and reducing the adhesion and transendothelial migration of neutrophils to endothelial cells.The protective effect of liraglutide on LPS-induced dysfunction of human pulmonary microvascular endothelial barrier may be related to the inhibition of Rho and NF-kappa B signaling pathway by activating GLP-1R.