Effects And Mechanisms Of Sphingosine 1-phosphate Receptor 1 On Lipopolysaccharide-induced Acute Lung Injury In Mice

ObjectivesrTo investigate the effect and mechanism of S1P1 agonist FTY720 on LPS-induced acute lung injury in mice.Methods:140 healthy male BALB/c mice(weighing about 20 g)were fed for one week.The experiment was divided into four groups:Normal saline control group(NS group),LPS model group(LPS group),FTY720 control group(NF group),FTY720 pretreatment group(FL group).H&E staining observed pathological changes of lung tissue;Detected wet/dry weight(W/D)ratio of lung tissue;BCA determined the total protein concentration in bronchoalveolar lavage fluid(BALF);Automatic cell counting analyzer detected the total cell number in BALF;Wright-Giemsa staining cell smears,detected the neutrophils in BALF;ELISA detected the concentrations of NF-κB、TNF-α、TGF-β、IL-1β and IL-6;Immunohistochemical staining detected the expressions of NF-κB、TNF-α、TGF-β and IL-6 in lung tissue;Western blotting detected the protein expressions of NF-κB、AKT、p38MAPK、IκBα and IKKα/β;The mice were orally administered with AKT inhibitor AZD5363(100mg/kg)and intraperitoneal injection of p38MAPK inhibitor SB203580(25mg/kg).Western blotting detected the related protein expressions of NF-κB、iκBα and IKKα/β.Results:L H&E staining showed that compared with NS group,the lung tissue of LPS group have obviously inflammatory cell infiltration and alveolar wall thickening;No obvious pathological changes in lung tissue in NF group;While compared with the LPS group,the pathological state of lung tissue inflammatory cell infiltration and alveolar wall thickening was significantly improved in the FL group.2.W/D ratio of lung tissue:Compared with NS group,W/D ratio of LPS group was increased significantly(P<0.01);There was no significant difference between NS group and NF group;While the W/D ratio of FL group was significantly lower than that of LPS group(P<0.01).3.BCA detected the total protein concentration in BALF:The total protein concentration in BALF of LPS group was significantly increased than NS group(P<0.01);There was no significant difference between the NF group and the NS group;While the FL group was significantly lower than the LPS group(P<0.01).4.Automatic cell counting analyzer detected total cell count in BALF:Compared with NS group,total cell count in BALF in LPS group was increased significantly(P<0.01);There was no significant difference between the NF group and the NS group;While the number of neutrophils in BALF in FL group was significantly lower than that in LPS group(P<0.01).5.Wright-Giemsa staining detected the number of neutrophils in BALF:Compared with NS group,the number of neutrophils in BALF in LPS group was significantly increased(P<0.01);There was almost no difference between the NS group and the NF group;The number of neutrophils in the FL group was decreased significantly than in the LPS group(P<0.01).6.ELISA detected the expression of inflammatory factors in BALF:Compared with NS group,the expression of NF-κB、TNF-α、TGF-β、IL-1β and IL-6 in BALF in LPS group was significantly higher(P<0.01);There was almost no difference between the NS group and the NF group;Compared with LPS group,the expression of inflammatory factors in FL group was significantly lower(P<0.01).7.Immunohistochemistry detected the expression of cytokines in lung tissue:Compared with NS group,the expression levels of NF-κB、TNF-α、TGF-β and IL-6 cytokines in LPS group was significantly increased;The expression of cytokines in NF group and NS group are low;While compared with LPS group,those cytokines expression in FL group was significantly decreased.8.Western blotting detected the expression of related proteins:Compared with NS group,the expression levels of NF-κB、IκBα、IKKα/β、AKT phosphorylated protein and p38MAPK protein in LPS group were significantly increased(P<0.01);There was almost no difference between NS group and NF group;While compared with LPS group,the expression of the above proteins were significantly decreased in FL group(P<0.01).9.Detected relate protein expression after administrated AKT inhibitor AZD5363 and p38MAPK inhibitor SB203580:Compared with NS group,the expression levels of NF-κB、IκBα and IKKα/β phosphorylated protein in LPS group were significantly increased(P<0.01);The expression levels of NF-κB、IκBα and IKKα/β phosphorylated protein in FL group were significantly decreased than in LPS group(P<0.01);Meanwhile,the expression levels of NF-κB、IκBα and IKKα/βphosphorylated protein in saline+AZD5363+LPS group、FTY720+AZD5363+LPS group、saline+SB203580+LPS group and FTY720+SB203580+LPS group were significantly lower than in LPS group(P<0.01),there was no significant difference with NS group;However,not only in FL group、saline+AZD5363+LPS group、FTY720+AZD5363+LPS group which administrated with AKT inhibitor,but also in FL group、saline+SB203580+LPS group、FTY720+SB203580+LPS group which administrated with p38MAPK inhibitor the expression levels of NF-κB、IκBα and IKKα/β phosphorylated proteins in the lung tissues were similar,and there was no statistical significance(P>0.05).Conclusions:Sphingosine 1-phosphate receptor 1 inhibits the production of pro-inflammatory cytokines by inhibiting PI3K/AKT/NF-κB signaling pathway and p38MAPK/IKKα/p/IκBα/NF-κB signal transduction pathway,and inhibits LPS-induced acute lung injury.